key: cord- - wuqz authors: balinsky, corey a.; schmeisser, hana; wells, alexandra i.; ganesan, sundar; jin, tengchuan; singh, kavita; zoon, kathryn c. title: irav (flj ), an interferon-stimulated gene with antiviral activity against dengue virus, interacts with mov date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: wuqz dengue virus (denv) is a member of the genus flavivirus and can cause severe febrile illness. here, we show that flj , which we refer to as irav, is induced by denv in an interferon-dependent manner, displays antiviral activity against denv, and localizes to the denv replication complex. irav is an rna binding protein and localizes to cytoplasmic processing bodies (p bodies) in uninfected cells, where it interacts with the mov risc complex rna helicase, suggesting a role for irav in the processing of viral rna. after denv infection, irav, along with mov and xrn , localizes to the denv replication complex and associates with denv proteins. depletion of irav or mov results in an increase in viral rna. these data serve to characterize an interferon-stimulated gene with antiviral activity against denv, as well as to propose a mechanism of activity involving the processing of viral rna. importance dengue virus, a member of the family flaviviridae, can result in a life-threatening illness and has a significant impact on global health. dengue virus has been shown to be particularly sensitive to the effects of type i interferon; however, little is known about the mechanisms by which interferon-stimulated genes function to inhibit viral replication. a better understanding of the interferon-mediated antiviral response to dengue virus may aid in the development of novel therapeutics. here, we examine the influence of the interferon-stimulated gene irav (flj ) on dengue virus replication. we show that irav associates with p bodies in uninfected cells and with the dengue virus replication complex after infection. irav also interacts with mov , depletion of which is associated with increased viral replication. our results provide insight into a newly identified antiviral gene, as well as broadening our understanding of the innate immune response to dengue virus infection. importance to public health. dengue virus infects an estimated million people each year, with more than . billion people living in regions where they are at risk of dengue virus transmission ( ) ( ) ( ) ( ) . activation of interferon (ifn) signaling pathways results in the upregulation of hundreds of interferon-stimulated genes (isgs) ( ) ( ) ( ) . interferon-stimulated genes encoding viperin, trail, ifitm , ifitm , ifitm , isg , and trim have all been shown to inhibit denv replication through a variety of mechanisms ( ) ( ) ( ) ( ) ( ) . in addition, a number of isgs, including ifit , ifit , isg , trim , and trim ␣ genes, have been found to have activity against other flaviviruses ( ) ( ) ( ) ( ) ( ) ( ) . high-throughput screens have implicated a number of other isgs in inhibition of flavivirus replication ( , ) . previously, we identified flj as one of a number of genes that were upregulated in daudi cells in response to treatment with ifn ( ) and showed that they displayed antiviral activity against encephalomyocarditis virus (emcv) ( ) . flj has also been shown by our laboratory and others to have antiviral activity against denv ( , ) . flj , which we refer to here as irav (interferon-regulated antiviral gene) (also annotated as c orf , upf , or ryden), encodes a protein amino acids (aa) in length with a calculated molecular mass of . kda. analysis of published microarray data suggests that irav (flj ) is upregulated in response to type i and type ii ifns ( , , ( ) ( ) ( ) . irav (flj ) has been shown in microarray screens to be upregulated in response to the yellow fever virus vaccine ( ) , as well as after infection with a number of different pathogens, including adenovirus ( ) , influenza virus ( ) , lassa virus ( ) , and ebola and marburg viruses ( ) , as well as after infection with human herpesvirus and human herpesvirus ( , ) . in addition, proteomic analysis of the hepatitis c virus (hcv) interactome identified irav (flj ) as one of human proteins interacting with the hcv ns protein ( ) , suggesting a role for irav in the host pathogen response. here, we show that irav is upregulated in response to denv infection in an ifn-dependent manner. upregulation of irav in response to ifn-␤ treatment can be blocked by disrupting the canonical isgf pathway. crispr/cas -mediated knockout of irav resulted in increased titers of denv, as well as of emcv. we also demonstrate that irav associates with denv proteins and localizes to the viral replication complex. irav is an rna binding protein and localizes to p bodies in uninfected cells. irav also associates with the host rna binding proteins upf and hur (elav ) and interacts with mov (a risc complex rna helicase), suggesting a role for irav in processing or stability of rna. furthermore, we propose a mechanism of action for irav that utilizes intrinsic rna decay pathways. these pathways have been shown to be of increasing importance to the life cycles of multiple viruses, as well as in an array of cellular processes. to determine if irav is upregulated in response to denv infection, a cells were infected with denv for h. quantitative real-time pcr (qrt-pcr) analysis showed upregulation of irav starting at h postinfection, corresponding to increased expression of denv rna, ifn-␤, and isgs, including ifit (fig. a) . to determine if irav is regulated through the canonical ifn (isgf ) signaling pathway, irf knockouts (ko) were generated in a cells using crispr/cas . knockout of irf resulted in decreased expression of irav after denv infection (fig. b) , as well as after ifn-␤ treatment (fig. c) , indicating that the canonical isgf pathway plays a role in induction of irav. to further characterize irav induction in response to ifn, hela cells were treated with various concentrations of ifn-␤ for h or were treated with ifn-␤ at a concentration of ng/ml and collected at the indicated time points. samples were analyzed by western blotting for expression of irav and ifit . analysis showed irav to be detectable in unstimulated hela cells and to be upregulated in response to treatment with ifn-␤ in both dose-dependent (fig. d ) and time-dependent (fig. e) that monocytes and u and daudi cells produced an additional low-molecular-mass band of approximately kda that matched the predicted molecular mass of an alternate irav isoform (fig. f ). crispr-mediated knockout of irav results in increased titers of denv and emcv. the crispr/cas system was used to generate a irav ko cells. treatment of a cells with ifn-␤ resulted in upregulation of irav, as well as of other isgs. however, in the ko cells, irav was not detected at either the rna ( fig. a) or protein (fig. b) level, while expression of other isgs remained unaffected. infection of a or irav ko cells with denv ( fig. c ) resulted in increased titers of denv (fig. d) , as well as significant increases in denv rna relative to control cells (fig. e ). similar effects were observed after infection with emcv ( fig. f and g) . when denv-infected irav ko cells were examined for expression of other genes (fig. h) , we observed increased expression of the ifit , mx , irf , and ifn-␤ isgs in irav ko cells relative to the control a cells. these results suggest that irav directly affects denv replication rather than exerting its effects via regulation of other isgs. irav associates with the dengue virus replication complex. to determine if irav has a direct influence on virus replication, hek cells were stably transfected with an expression vector encoding irav with an amino-terminal (n-terminal) green fluorescent protein (gfp) fusion or with a negative-control vector with an n-terminal gfp tag. because irav is poorly expressed in hek cells (fig. f ), they are an ideal cell line for overexpression experiments. cells were infected with denv (multiplicity of infection [moi], . ), and supernatants were collected at time zero and at h postinfection, and the titer was determined by plaque assay. while detectable in the negative-control group, no virus was detected in cells stably expressing irav (fig. a) . the iravexpressing hek cells were also treated with a small interfering rna (sirna) targeting irav or with a negative-control sirna. the cells were then infected with denv at an moi of . for h. virus titration showed a significant increase in virus titers in irav-expressing hek cells after knockdown of irav (fig. b ), demonstrating that irav has a significant influence on denv replication even in the absence of interferon treatment. because irav was previously identified by mass spectrometry (ms) as an interaction partner for hcv ns ( ), we examined whether irav was interacting with denv proteins. coimmunoprecipitation (co-ip) experiments were performed on denvinfected cells using gfp-irav as bait and gfp-chloramphenicol acetyltransferase (cat) as a negative control. western blotting showed that the denv protease/helicase ns and the replication complex-associated protein ns a interacted with irav, while the denv envelope (e) protein did not (fig. c ). interactions between irav and ns were not affected by rnase a treatment (data not shown). to further substantiate our findings, colocalization experiments were performed in a cells. here, a cells were infected with denv serotype (denv- ) for h, followed by fixation and immunostaining for irav and either denv ns (fig. d ) or ns a (fig. e) protein. while irav was localized to small puncta in mock-infected cells, colocalization between irav and ns or ns a was observed in perinuclear regions of denv-infected cells (fig. f ). this suggests that irav relocalizes to the denv replication complex after infection. in order to demonstrate association of irav with the denv replication complex in primary human cells, monocyte-derived macrophages were infected with denv- as described above. as shown for a cells, colocalization was observed between irav and denv ns (fig. g ) or ns a (fig. h ) in denv-infected monocyte-derived macrophages. irav associates with host rna binding proteins. to determine if irav interacted with host proteins in denv-infected hek cells, co-ip experiments were performed using overexpressed irav as bait. the immunoprecipitated proteins were then analyzed by ms. gene ontology (go) analysis was performed using the panther classification system (http://pantherdb.org) ( ) on ms hits with a spectral count of Ͼ . ontology derived based on the protein class revealed a strong preference ( %) for interactions with nucleic acid binding proteins (fig. a) . to characterize the nucleic acid binding properties of irav, fluorescence polarization (fp) assays were performed using here, dilutions of irav were incubated with either synthetic single-stranded (ssrna) or double-stranded (dsrna) rna or single-or double-stranded dna containing complementary nucleic acid sequences. fp showed irav to be a nucleic acid binding protein with a higher affinity for ssrna and ssdna than for dsdna (fig. b ). to further explore interactions between irav and proteins associated with rna processing, ms hits were ranked based on the spectral count, and the top-scoring proteins were further characterized (see table s in the supplemental material). we were able to verify interactions between irav and mov , upf , and hur by co-ip of overexpressed irav, followed by western blotting (fig. c ). because mov , upf , and hur are all known to be rna binding proteins, we examined the influence of rnase a treatment on these interactions. rnase treatment resulted in either complete or partial loss of interactions with irav (fig. d ). partial loss of interactions due to rnase a treatment was previously described for interactions between mov and upf ( ) . irav colocalizes with p bodies. because many of the rna binding proteins shown to interact with irav are associated with ribonucleoprotein (rnp) granules, we next investigated whether irav colocalized with specific rnp granule markers. here, we examined a cells for the localization of p body markers (xrn , dcp a, and ddx / rck), as well as the stress granule marker g bp (fig. a ). we observed colocalization between irav and all three p body markers in ifn-treated cells; however, no statistically significant colocalization was observed between irav and g bp , indicating that irav associated with p bodies in the cytoplasm of ifn-treated cells (fig. b) . to characterize the association of irav with p body components after denv infection, a cells were infected with denv, followed by staining for irav and xrn ( fig. a and b). as previously described for xrn ( ) , irav relocalized to the denv replication complex, colocalizing with denv ns . irav interacts with mov . due to its known role as a restriction factor for retroviruses ( ) ( ) ( ) ( ) , hepatitis c virus ( ) , and hepatitis b virus ( ), we further characterized interactions between mov and irav. co-ips and reciprocal co-ips were performed using hek cells transfected with either irav (gfp-irav) or a negative control (gfp-cat). co-ips were then performed using an antibody either to overexpressed irav or to endogenous mov , and western blots were analyzed for the presence of either mov or irav (fig. a ). in addition, confocal microscopy and fluorescence resonance energy transfer (fret) by fluorescence lifetime imaging (flim) analyses were used to characterize interactions between endogenous irav and mov in a cells. confocal microscopy revealed colocalization between irav and mov , as determined by pearson's linear correlation coefficient ( fig. b and c) . moreover, fret-by-flim analysis demonstrated interactions between irav and mov , with lifetime values of . to . compared to values of . to . for the donor control, representing % to % fret efficiency. the endosomal marker rab was used as a negative control. rab did not demonstrate significant colocalization with irav or a shift in lifetime values ( fig. d and e) . confocal microscopy was also used to examine localization of irav and mov in denv-infected and mock-infected a cells ( fig. a and b) . here, it was observed that, similar to xrn , both irav and mov relocalized to the viral replication complex, colocalizing with denv ns . to further substantiate a role for mov in restriction of (continued on next page) denv replication, sirna-mediated knockdown experiments were performed in a and irav ko cells (fig. c) . depletion of mov resulted in an increase in virus replication for denv, as well as for emcv, which was significantly greater in irav ko cells than in irav-expressing a cells (fig. d and e) , indicating that both irav and mov play roles in the restriction of virus replication. interferon-stimulated genes are key mediators of antiviral immunity and play a pivotal role in the immune response to denv infection. while the ifn response is an innate and broad-spectrum response to many pathogens, isgs have been demonstrated to have remarkable specificity both for the viruses they target and the pathways through which they inhibit viral replication ( ) . hundreds of isgs have been shown to be upregulated in response to induction of ifn signaling pathways, illustrating the scope and complexity of the ifn response. to date, only a fraction of these genes have been characterized. here, we show that irav is an ifn-stimulated gene with antiviral activity against denv. knockout of the ifn signaling component irf results in a significant reduction of irav expression after ifn treatment, suggesting regulation through the canonical isgf complex; however, it should be noted that there appears to be some baseline constitutive expression of irav. in addition, irav homologues were identified in hemichordates and echinoderms, organisms that lack a "classical" ifn response, suggesting the possibility of ifn-independent functions for irav-like proteins. irav associates with p bodies, as evidenced by interactions with p body-associated proteins (mov and upf ) and colocalization with p body markers (dcp a, xrn , and ddx ). p bodies are discrete cytoplasmic structures composed of rna and proteins that are associated with various aspects of rna turnover, including nonsense-mediated decay, adenylate-uridylate-rich element (are)-mediated decay, and gene silencing ( ) . notably, both denv and west nile virus (wnv) have been shown to associate with components of p bodies. subgenomic flavivirus rna (sfrna) inhibits the activity of xrn and trim ( ) ( ) ( ) , and ddx interacts with denv untranslated regions (utrs) and localizes to denv replication complexes, possibly playing a role in virus replication ( ) . furthermore, wnv infection of hela cells results in a decrease of both the size and number of p bodies, along with a relocalization of p body components (including gw , ddx , and xrn ) to wnv replication complexes, colocalizing with ns ( ). this is similar to what we observed with irav during denv infection, with irav puncta becoming more diffuse and colocalizing with ns in distinct perinuclear complexes. a number of other viruses, including hepatitis c virus ( ) , poliovirus ( ) , influenza virus ( ) , and bunyaviruses ( , ) , have been shown to interact with p bodies or with p body components ( ) . mov and upf are both members of the sf family of helicases and have been previously shown to interact with one another and to form complexes with apobec g and argonaute ( , ) , as well as with the antiviral protein zap ( ) . interactions between mov and upf are partially sensitive to rnase treatment ( ) , as we have also demonstrated for irav interactions with mov or upf . knockdown of mov results in an increase in the half-lives of mrna targets ( ) . mov has been shown to be involved in retrotransposition of line elements ( ) ( ) ( ) , as well as in microrna pathways ( , ) . mov may also play a role in mediating antiviral response ( ) and restricts replication of retroviruses ( ) ( ) ( ) ( ) , hepatitis c virus ( ), hepatitis b virus ( ) , and influenza virus ( ) . upf is a key component of nonsense-mediated rna decay pathways ( ) and has been demonstrated to play a role in viral replication cycles ( , ) . irav also associates with the rna binding protein hur. hur has been shown to be involved in rna decay pathways ( ) and has been linked to viral replication and modulation of the host response ( ) ( ) ( ) ( ) . in addition, irav has been previously shown to interact with rna binding proteins larp and pabpc , both of which have also been shown to be involved in rna decay ( ) . taken together, irav's role as an rna binding protein, its association with proteins linked to rna decay, and its localization to p bodies (sites of rna decay) and the marked increase in viral rna observed after knockout of irav all suggest a role for irav in degradation of viral rna. the role of mov in irav-mediated antiviral activity remains unclear; however, given mov 's role as an rna helicase involved in rna decay pathways, as well as its localization at the viral replication complex and the increase in viral rna observed after its knockdown, mov may function in conjunction with irav and other proteins to aid in destabilization of viral rna. in conclusion, we show that irav is an isg that is regulated through the canonical type i interferon signaling pathway. irav displays antiviral activity against denv and emcv and interacts with denv proteins. irav is an rna binding protein that localizes to p bodies, sites of rna decay. additionally, irav interacts with mov and upf , two proteins previously shown to interact with each other and to be involved in rna decay pathways. these data serve to identify an isg with antiviral activity against denv, as well as to suggest a mechanism of action involving the destabilization of viral rna. viruses and cell culture. the denv serotype isolate tonga/ was generously provided by s. whitehead (laboratory of infectious diseases [lid], niaid) and passaged in c / cells. the titer of virus was determined on vero cells as previously described ( , were obtained from the american type culture collection (atcc) (manassas, va) and passaged in vero cells. virus infections were performed at the indicated moi, and virus was allowed to adsorb to the cells for hour. unbound virus was then removed, and the cells were washed with phosphate-buffered saline (pbs), followed by the addition of rpmi medium containing % fetal bovine serum. primary human monocytes were obtained using the gambro elutra method from the nih clinical research center department of transfusion. monocyte-derived macrophages were cultured as previously described ( ) . were performed using the sensifast probe no-rox one-step kit (bioline usa inc., taunton, ma), and reactions were run on a stratagene mx p real-time thermocycler (agilent technologies, santa clara, ca). primers and probes were obtained from integrated dna technologies, inc. (coralville, ia), and were as follows: denv- (forward, =-tgc cta cag ttc tac gtc tcc- =; reverse, =-tcg ttt cct aac aat ccc acc- =; probe, =- -carboxyfluorescein (fam)-cct tcc aat-zen-ctc ttt cct gaa gcc tct c-iowa black fluorescence quencher [iabkfq]- =), emcv (forward, =-ttc agc gtt ttc tac tcc ctg- =; reverse, =-tca ctc ccc tca ctt acc c- =; probe, =-fam-aga aat cct-zen-tcc ctg cgc tca cc-iabkfq- =) for fp assays, the following fam-labeled -mer rna probes were used: =-rargru rurgru rurarg rurcru rarcrg rurgrg rarc/ -fam- =, with or without the reverse complement =-rgrurc rcrarc rgrura rgrarc rurara rcrara rcru- = (where "r" refers to ribose); or dna probes =-agt tgt tag tct acg tgg ac-fam- =, with or without the reverse complement =-gtc cac gta gac taa caa ct- =. oligomers were dissolved in binding buffer ( mm kcl, mm hepes, ph . ), heated to °c for min, and allowed to cool slowly. the oligomers were then added to serial dilutions of protein solution in triplicate, and fp was measured on a corning black -well, half-area assay plate (corning, ny) using a hidex sense microplate reader (turku, finland). confocal microscopy and fluorescence resonance energy transfer by fluorescence lifetime imaging. a cells were plated on poly-d-lysine-coated -mm culture dishes (mattek, ashland, ma) and either treated with ifn-␤ ( ng/ml) for h or infected with denv- (moi, ) for h. the cells were fixed with % paraformaldehyde in pbs and permeabilized with . % or . % triton x- , followed by blocking with % bovine serum albumin (bsa) for min. primary antibodies were diluted in % normal goat serum and incubated at room temperature for h, followed by three washes in pbs and staining with fluorescently labeled secondary antibody ( : ) and nuclear dapi ( =, -diamidino- -phenylindole) stain (life technologies). primary labeled antibodies were diluted in % normal goat serum and incubated at room temperature for h, followed by three washes in pbs, and postfixed with % paraformaldehyde in pbs. images were collected on a leica sp inverted confocal microscope with a ϫ oil immersion objective (leica microsystems, buffalo grove, il). colocalization analysis was performed using imaris software (bitplane inc., south windsor, ct). fret-by-flim analysis was performed as previously described ( ) . gene ontology and statistical analyses. go analysis was performed using proteins identified by ms with spectral counts above . selected protein accessions were analyzed using the panther classification system (http://pantherdb.org) ( ) . all statistical analyses were performed on prism (graphpad software, inc.), using one-way analysis of variance (anova) with tukey's post hoc test and a p value of . . supplemental material for this article may be found at https://doi.org/ . / jvi. - . text s , pdf file, . mb. we thank tsan xiao and david garboczi for their insights into protein structure; ming zhao and renee olano from the research technologies branch, niaid, nih, for excellent technical assistance; yajuan li from the school of life sciences, university of science and technology of china, for assistance with protein purification; daniel green for providing human monocytes; xavier ambroggio and vijayaraj nagarajan for their help with computational analysis; emerito amaro-carambot for technical expertise; stephen whitehead for providing reagents and support; and sonja best and ted pierson for their valuable insight and critical readings of the manuscript. this work was supported by the intramural research program of the niaid, nih. danvers, ma; gapdh (glyceraldehyde- -phosphate dehydrogenase) (d h ) and e (sab ) fluorescent labeling of primary antibodies for colocalization and fret-by-flim of irav and mov , rab , denv ns , and denv ns a were performed using zenon antibody-labeling kits (life technologies) according to the manufacturer's specifications the irav gene sequence was then transferred to a pcdna . /n-emgfp-dest vector (life technologies) via lr recombination using gateway lr clonase enzyme mix (life technologies) according to the manufacturer's protocol. the constructs were sequenced to verify correct sequence and orientation. negative-control vectors were obtained from life technologies. transfections were performed in hek cells using opti-mem and x-tremegene hp according to the manufacturer crispr/cas knockout and sirna knockdown experiments. crispr/cas knockout cell lines were generated using a cells. an flj /c orf (irav) crispr/cas knockout plasmid (sc- ), containing the cas nuclease and guide rna, and its complementary homologydirected repair plasmid (sc- ), containing red fluorescent protein and a puromycin resistance gene flanked by = and = arms homologous to regions flanking the cas target site a cells were cotransfected with crispr/cas and homology-directed repair plasmids using genmute transfection reagent (signagen laboratories, ijamsville, md), and knockouts were screened for puromycin resistance ( g/l). individual subclones were then assayed for gene expression by both pcr and western blotting the chromatography was performed in line with an ltq-velos orbitrap mass spectrometer (thermofisher scientific, west palm beach, fl), and the mobile phase consisted of a linear gradient prepared from solvent a and solvent b ( . % formic acid, % water, and . % acetonitrile) at room temperature. nano-liquid chromatography (lc)-ms (lc-tandem ms [ms-ms]) was performed with a proxeon easy-nlc ii multidimensional liquid chromatograph and a temperature-controlled ion max nanospray source (thermofisher scientific) in line with the ltq-velos orbitrap mass spectrometer. a mascot (matrix science, beachwood, oh) search of the data was performed against a concatenated sequence file containing dengue virus proteins found in the uniprotkb trembl database, human protein uniprotkb swiss-prot, and the common contaminant proteins found in the gpm.org's crap database, all downloaded in march . the data were searched with allowed missed cleavages and mass tolerances of ppm and . da for the precursor and fragment ions, respectively. carbamidomethylation of cysteine was set as a fixed modification, while oxidation of methionine and deamidation of asparagine and glutamine were searched as dynamic modifications. the resulting search files were reclustered against the same sequence database for further analysis using proteoiq software the global distribution and burden of dengue dengue: a continuing global threat global spread and persistence of dengue who scientific working group report on dengue interferon-stimulated genes: a complex web of host defenses a diverse range of gene products are effectors of the type i interferon antiviral response interferon-stimulated genes: roles in viral pathogenesis trail is a novel antiviral protein against dengue virus the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus viperin is induced following dengue virus type- (denv- ) infection and has anti-viral actions requiring the c-terminal end of viperin identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections overlapping and distinct molecular determinants dictating the antiviral activities of trim against flaviviruses and coronavirus a role for ifit in restricting west nile virus infection in the brain isg and ifitm proteins inhibit hepatitis c virus replication isg , a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment associations between human trim gene expression and the response to combination therapy with peg-ifn␣- a and ribavirin in iranian patients with chronic hepatitis c trim ␣, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral rna polymerase =-o methylation of the viral mrna cap evades host restriction by ifit family members a short hairpin rna screen of interferon-stimulated genes identifies a novel negative regulator of the cellular antiviral response identification of alpha interferon-induced genes associated with antiviral activity in daudi cells and characterization of ifit as a novel antiviral gene characterization of a novel interferon stimulated gene with antiviral activity characterization of a novel interferon stimulated gene with antiviral activity against dengue virus characterization of ryden (c orf ) as an interferon-stimulated cellular inhibitor against dengue virus replication gene expression changes in peripheral blood mononuclear cells from multiple sclerosis patients undergoing beta-interferon therapy cyclic changes in gene expression induced by peg-interferon alfa- b plus ribavirin in peripheral blood monocytes (pbmc) of hepatitis c patients during the first weeks of treatment gene expression profiling reveals similarities between the in vitro and in vivo responses of immune effector cells to interferon-alpha yellow fever vaccine induces integrated multilineage and polyfunctional immune responses adenovirusdirected ocular innate immunity: the role of conjunctival defensin-like chemokines (ip- , i-tac) and phagocytic human defensin-alpha innate immune response to influenza a virus in differentiated human alveolar type ii cells transcriptome analysis of human peripheral blood mononuclear cells exposed to lassa virus and to the attenuated mopeia/lassa reassortant (ml ), a vaccine candidate global suppression of the host antiviral response by ebola-and marburg viruses: increased antagonism of the type i interferon response is associated with enhanced virulence human herpesvirus- infection of primary pulmonary microvascular endothelial cells herpes simplex virus type -induced transcriptional networks of corneal endothelial cells indicate antigen presentation function hepatitis c virus infection protein network panther in : modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees mov is a = to = rna helicase contributing to upf mrna target degradation by translocation along = utrs p-body components lsm , gw , ddx , ddx and xrn are recruited to wnv replication sites and positively regulate viral replication p body-associated protein mov inhibits hiv- replication at multiple stages perturbation of the p-body component mov inhibits hiv- infectivity mov and apobec g localization to processing bodies is not required for virion incorporation and antiviral activity moloney leukemia virus (mov ) protein inhibits retrovirus replication the role of moloney leukemia virus in hepatitis b virus expression in hepatoma cells p-bodies and stress granules: possible roles in the control of translation and mrna degradation rna structures that resist degradation by xrn produce a pathogenic dengue virus rna. elife :e dengue subgenomic rna binds trim to inhibit interferon expression for epidemiological fitness a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn and alters host mrna stability quantitative mass spectrometry of denv- rna-interacting proteins reveals that the dead-box rna helicase ddx binds the db and db = utr structures hepatitis c virus infection inhibits p-body granule formation in human livers poliovirus-mediated disruption of cytoplasmic processing bodies the ns protein of influenza a virus interacts with cellular processing bodies and stress granules through rna-associated protein (rap ) during virus infection signatures of host mrna = terminus for efficient hantavirus cap snatching bunyaviral cap-snatching vs. decapping: recycling cell cycle mrnas regulation of stress granules and p-bodies during rna virus infection apobec g inhibits microrna-mediated repression of translation by interfering with the interaction between argonaute- and mov the broad-spectrum antiviral protein zap restricts human retrotransposition mov rna helicase is a potent inhibitor of retrotransposition in cells the mov helicase inhibits line- mobility affinity proteomics reveals human host factors implicated in discrete stages of line- retrotransposition mov and fmrp regulate ago association with microrna recognition elements mov provides antiviral activity against rna viruses by enhancing rig-i-mavs-independent ifn induction host protein moloney leukemia virus (mov ) acts as a restriction factor of influenza a virus by inhibiting the nuclear import of the viral nucleoprotein up-frameshift protein (upf ): multitalented entertainer in rna decay the host nonsense-mediated mrna decay pathway restricts mammalian rna virus replication upf is crucial for the infectivity of human immunodeficiency virus type progeny virions dysregulation of ttp and hur plays an important role in cancers changes in cellular mrna stability, splicing, and polyadenylation through hur protein sequestration by a cytoplasmic rna virus hur displaces polypyrimidine tract binding protein to facilitate la binding to the = untranslated region and enhances hepatitis c virus replication hepatitis b virus x upregulates hur protein level to stabilize her expression in hepatocellular carcinoma cells hur and ago bind the internal ribosome entry site of enterovirus and promote virus translation and replication vaccine candidates derived from a novel infectious cdna clone of an american genotype dengue virus type epidemiologic, clinical, and virologic observations on dengue in the kingdom of tonga modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons nucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particles key: cord- -e xwb g authors: yamashita, akifumi; sakamoto, tetsuya; sekizuka, tsuyoshi; kato, kengo; takasaki, tomohiko; kuroda, makoto title: dgv: dengue genographic viewer date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: e xwb g dengue viruses (denvs) and their vectors are widely distributed throughout the tropical and subtropical regions of the world. an autochthonous case of denv was reported in tokyo, japan, in , for the first time in years. a comprehensive database of denv sequences containing both serotype and genotype data and epidemiological data is crucial to trace denv outbreak isolates and promptly respond to outbreaks. we constructed a denv database containing the serotype, genotype, year and country/region of collection by collecting all publically available denv sequence information from the national center for biotechnology information (ncbi) and assigning genotype information. we also implemented the web service dengue genographic viewer (dgv), which shows the geographical distribution of each denv genotype in a user-specified time span. dgv also assigns the serotype and genotype to a user-specified sequence by performing a homology search against the curated denv database, and shows its homologous sequences with the geographical position and year of collection. dgv also shows the distribution of denv-infected entrants to japan by plotting epidemiological data from the infectious agents surveillance report (iasr), japan. this overview of the denv genotype distribution may aid in planning for the control of denv infections. dgv is freely available online at: (https://gph.niid.go.jp/geograph/dengue/content/genomemap). dengue viruses (denvs) are members of the genus flavivirus in the family flaviviridae and consist of four serotypes (denv- to - ) (lanciotti et al., ; kuhn et al., ) . each serotype can be divided into five to six genotypes. however, there is no standard genotyping classification [denv- , (goncalvez et al., ) ; denv- , (anez et al., ; khan et al., ) ; denv- , (lanciotti et al., ; wittke et al., ; klungthong et al., ) ; denv- , (abubakar et al., ) ]. denv has a positive-sense, single-stranded rna genome that is ∼ kb in length and encodes a capsid protein (c), premembrane protein (prm), and envelope glycoprotein (e) in addition to seven non-structural proteins (nss, ns , ns a, ns b, ns , ns a, ns b, and ns ) . denv infection causes dengue illness, which may range from dengue fever (a mild illness) to dengue hemorrhagic fever and dengue shock syndrome (the severe forms of the illness) in addition to asymptomatic cases abbreviations: denv, dengue virus; c, capsid protein; prm, premembrane protein; e, envelope glycoprotein; ns, nonstructural protein; ncbi, national center for biotechnology information; iasr, infectious agents surveillance report. (centers for disease control and prevention; http://www. cdc.gov/dengue/clinicallab/clinical.html). infection results in lifetime immunity against the same serotype, but successive exposure to different denvs increases the likelihood of contracting a severe form of dengue illness, such as dengue hemorrhagic fever or dengue shock syndrome (chiappelli et al., ) . denv and its vectors have become widely distributed throughout the tropical and subtropical regions of the world (murray et al., ). an autochthonous case of denv infection was reported in tokyo, japan, in for the first time in years (kutsuna et al., ) . to ensure prompt action in response to a denv outbreak, a comprehensive denv database based on the genotypes would be essential for tracing the outbreak source. to date, only two denv databases provide genotype information. the web service vipr (pickett et al., ) supports genetic analysis based on the viral genome for a tested input sequence, including denv sequences. the second database is the dengue virus genotyping database (yamashita et al., ) , which provides a summary table containing the denv serotype/genotype, year and country of collection and accession number. there are many other denv databases; however, no other sites provide summarized genotype information. the dengue virus resource facilitates the retrieval of denv sequences deposited in genbank according to serotype, disease symptom, host, region/country, genome region, and collection and/or release data (resch et al., ) . denvirdb provides sequence information and computationally curated information of dengue viral proteins (asnet et al., ) . denvdb focuses on the dengue virus sequence database for keyword searches (no publication: http://proline.bic.nus.edu.sg/denvdb/). finally, the dengue virus portal is a sequence collection with metadata (no publication: https://www.broadinstitute.org/ annotation/viral/dengue/home.html). here, we constructed the website dengue genographic viewer (dgv), which presents denv information based on the genotype and epidemiological data by using the geographic tool google maps © to update the recent dissemination of denv genotypes from a global perspective. the denv genotype database was constructed as follows: ( ) all accessible denv nucleotide sequences were collected; ( ) the complete sequences of each protein region (c, prm, e, ns , ns a, ns b, ns , ns a, ns b, and ns ) were extracted from the sequences; ( ) a blastn homology search was performed against the genotype database and the genotype of the most homologous sequence was assigned; ( ) and the genotype data were stored in a database using sqlite (https://www.sqlite.org/). . the denv nucleotide sequences were downloaded from the ncbi database using key words ("dengue virus"[porgn:__txid ]). . a blastx homology search was performed to detect nucleotide regions that corresponded to each mature denv protein; nucleotide regions that exhibited more than % sequence coverage to the protein were used for the subsequent analysis. . to reduce the time required to obtain the most homologous sequences in the genotype database, we reduced the number of sequences used in the blast search by clustering highly homologous sequences. we performed a uclust search (edgar, ) against the nucleotide sequences of each protein region and selected one representative sequence for each homologous sequence group with a clustering threshold of % identity; then, the representative sequences were subjected to a homology search against the genotype database. the original genotype database was constructed according to the method proposed by previous report (yamashita et al., ) . briefly, the representative sequences were aligned by using the mafft (katoh and standley, ) program and neighbor joining (nj) phylogenetic trees were constructed using the mega program (tamura et al., ) . the genotype of each gene was assigned manually according to the previous genotype database (yamashita et al., ) . . sequence id, country/region and year of collection were extracted from the deposited genbank data and integrated into the sql database by using an in house perl script. the above processes except for the original database construction are performed automatically every night to update the recent denv database. we implemented a set of viewer applications on dgv by using google maps © , which shows the data in a temporal and spatial manner. one application presents the geographical distribution of each denv genotype on the map in a userspecified time span. another option is a homology search program that searches for the most homologous denv sequence in the dgv database and show the geographical positions of closely related sequences on the map. the other interface shows the sources of imported dengue cases on the map, according to the infectious agents surveillance report (iasr), japan (http://www.nih.go.jp/niid/en/iasr-e.html). this set of applications is available at the dgv web site (https://gph.niid.go.jp/geograph/dengue/content/genomemap). on march , , dgv included a total of , denv sequences, which consisted of , , , and denv serotype- , - , - , and - sequences, respectively ( table ) . some genotypes have been abundantly sequenced and deposited in the public database, whereas other genotypes have rarely been sequenced (i.e., denv- genotype ii was reported in only seven records from to , denv- genotype iii was also reported in only seven records from to , and denv- genotype iv has not been reported since ). these rare genotypes may have become minor populations or may be undergoing a silent transmission cycle (lanciotti et al., ; chen and vasilakis, ; santiago et al., ) . fifteen years' worth of data from to for all serotypes showed that denv sequences were primarily reported from south to southeast asia, central to south america, and the countries of oceania (figure a) . some biases in denv serotype compositions were observed in several countries. for instance, the dominant serotypes were denv- and - in mexico, denv- and - in polynesian countries with the exception of fiji, and denv- and - in pakistan. in contrast, all serotypes were sampled in brazil and thailand. intriguingly, when focusing on the genotype instead of the serotype, the data from to showed at least three potential geographical genotype distribution border lines in asia (figures b, ) . the first border is between the american continents and other regions (figure b) , the second is located between bangladesh and myanmar for the genotype distributions of denv- and - and india and myanmar for denv- , and the third is located between indochina and the malay peninsula (figure ) . there seem to be differences in the denv- and - distributions between malaysia, singapore and indonesia; however, the border line is not clear because malaysia and indonesia consist of many islands and share kalimantan island and the deposited sequence data do not specify the original island isolation site. although, some boundaries are not clear, these boundaries are roughly conserved among all serotypes except for the bangladesh-myanmar border line for denv- , suggesting potential barriers against the vector mosquitos' movements or human activities between the countries. we also found a timeline change in the predominant genotypes. from to , the dominant genotype in asia was cosmopolitan, although india-pakistan-sri lanka and southeast-oceania belonged to different lineages (khan et al., ) . the major genotypes in the indochina countries were different from those of the other asian countries; genotype figure | a screenshot of the denv sequence similarity search. an env sequence derived from an autochthonous case in japan (lc or gi: ) was used as a sample query. the query was assigned as the env region of denv- genotype i. asian i was predominant in thailand, whereas genotype asian american was predominant in cambodia and vietnam (figure and movie s ). from , asian i increased in cambodia and vietnam until finally in asian i became the predominant genotype in indochina. the genotype asian i viruses in thailand seemed to be widely disseminated into vietnam via cambodia but did not reach malaysia and bangladesh (figure ) . thus, the asian american genotype was replaced by asian i in cambodia and vietnam between and . this example also suggests the idea of genotype transition, which probably reflects the mosquito vector habitat and human activities in the indochinese peninsula. dgv currently does not support the prediction of dengue epidemics, because number of deposited sequence data does not always reflect the actual number of events, in addition, it takes long time to be a public sequence through isolation, sequencing, and publication. dgv provides a search engine for the assignment of the denv serotype, genotype, and origin country according to the most homologous sequence on the basis of a blastn search against the denv database. the search results are shown as text and are also plotted through google maps © . subsequently, the query sequence is divided into mature protein regions and displayed with a serotype/genotype assignment. the homology search results and the divided nucleotide sequences in fasta format can be downloaded. here, we present an example similarity search for an env sequence derived from an autochthonous case in japan (lc or gi: ). dgv assigned the sequence as the env region of the denv- genotype i and identified homologous sequences from japan, china, singapore and indonesia. these results are consistent with those from a previous study (figure ; kutsuna et al., ) . to aid in visualizing the source countries of dengue infection cases imported to japan, the number of annual imported cases was also mapped on google maps © . the serotype (but not genotype), year, and visiting country/area are also indicated based on the infectious agents surveillance report (iasr), which releases monthly data and information obtained from prefectural and municipal public health institutes and quarantine stations to the public (figure ). ay performed the experimental design, participated in the analysis and drafted the manuscript. ts implemented the application, performed the data collection, constructed the original genotype database, and participated in the analysis. ts , kk, and tt reviewed the application and participated in the discussion. mk contributed to the experimental design, performed the analysis and drafted the manuscript. all authors read and approved the final manuscript. this work was supported by a grant for research on emerging and re-emerging infectious diseases (h shinko-ippan- /h shinko-gyosei-shitei- ) from the ministry of health, labor and welfare, japan, and was also supported by the research program on emerging and re-emerging infectious diseases ( fk h and fm h ) from the japan agency for medical research and development, amed. this work was also partially supported by jsps kakenhi grant number k .the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. we are grateful to ms. inamine from niid for drawing the dgv icon. we thank prof. ikuta and prof. yasunaga from biken for allowing us to use the original dataset "dengue virus genotyping database." the supplementary material for this article can be found online at: http://journal.frontiersin.org/article/ . /fmicb. movie s | genotype transition of denv- in asia from - to - . frontiers in microbiology | www.frontiersin.org emergence of dengue virus type genotype iia in malaysia circulation of different lineages of dengue virus type in central america, their evolutionary time-scale and selection pressure analysis denvirdb: a web portal of dengue virus sequence information on asian isolates dengue-quo tu et quo vadis? viruses viral immune evasion in dengue: toward evidence-based revisions of clinical practice guidelines search and clustering orders of magnitude faster than blast diversity and evolution of the envelope gene of dengue virus type mafft: iterative refinement and additional methods emergence and diversification of dengue cosmopolitan genotype in pakistan molecular genotyping of dengue viruses by phylogenetic analysis of the sequences of individual genes structure of dengue virus: implications for flavivirus organization, maturation, and fusion autochthonous dengue fever rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction molecular evolution and epidemiology of dengue- viruses epidemiology of dengue: past, present and future prospects virus pathogen database and analysis resource (vipr): a comprehensive bioinformatics database and analysis resource for the coronavirus research community virus variation resources at the national center for biotechnology information: dengue virus reemergence and decline of dengue virus serotype in puerto rico mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods extinction and rapid emergence of strains of dengue virus during an interepidemic period origin and distribution of divergent dengue virus: novel database construction and phylogenetic analyses key: cord- -zyw aukk authors: wong, ho him; sanyal, sumana title: manipulation of autophagy by (+) rna viruses date: - - journal: semin cell dev biol doi: . /j.semcdb. . . sha: doc_id: cord_uid: zyw aukk autophagy is an evolutionarily conserved process central to host metabolism. among its major functions are conservation of energy during starvation, recycling organelles, and turnover of long-lived proteins. besides, autophagy plays a critical role in removing intracellular pathogens and very likely represents a primordial intrinsic cellular defence mechanism. more recent findings indicate that it has not only retained its ability to degrade intracellular pathogens, but also functions to augment and fine tune antiviral immune responses. interestingly, viruses have also co-evolved strategies to manipulate this pathway and use it to their advantage. particularly intriguing is infection-dependent activation of autophagy with positive stranded (+)rna virus infections, which benefit from the pathway without succumbing to lysosomal degradation. in this review we summarise recent data on viral manipulation of autophagy, with a particular emphasis on +rna viruses and highlight key unanswered questions in the field that we believe merit further attention. rna viruses have evolutionarily constrained genome sizes. at the same time they have co-evolved efficient means to manipulate host cellular processes to acquire nutrients while evading immune detection. multifunctional viral proteins, molecular mimicry of host components, and the intrinsically high mutagenicity of their rna genome converge to dysregulate host cellular pathways, and exploit metabolic processes to their advantage. one such target is the autophagy machinery. while this cellular degradative process has been historically described to restrict intracellular pathogens including bacteria, parasites and viruses, many have evolved mechanisms to circumvent and even actively benefit from it. autophagy is initiated by sequestration of cytoplasmic proteins and damaged organelles into crescent-shaped double-membrane vesicles known as isolation membranes, long-debated on their membrane source [ ] . the best understood trigger for induction of autophagy is amino acid deprivation, whereby autophagy related proteins (atgs) are recruited to nucleate the isolation membrane, which forms a cup-shaped phagophore. current consensus on the source of autophagosomal membranes is the endoplasmic reticulum [ ] . once contents are captured, the immature isolation membranes expand to forming autophagosomes, which subsequently fuse with lysosomes, thus forming autolysosomes. the contents undergo degradation within the autolysosomes to enable recycling during starvation. about genes have been reported to participate in the process of autophagosomal degradation. the core autophagy proteins are broadly categorised into five complexes: (i) the unc- like kinase (ulk ) complex, (ii) atg , (iii) the class iii pi k complex, (iv) wd repeat domain phosphoinositideinteracting proteins (wipi), and (v) ubiquitin-like atg and atg complexes. although a mechanistic understanding of the process is currently incomplete, formation of phagophore is believed to involve a cooperative activity of the ulk and pi k complexes, along with local phosphatidylinositol synthesis. these activities are followed by recruitment of atg -containing vesicles to phagophore assembly sites, which results in membrane expansion to form the autophagosomes (fig. ). detailed analyses of the known regulatory mechanisms have been reviewed elsewhere [ , ] . apart from turnover of organelles and primarily long-lived proteins, autophagy operates to defend host cells against intracellular pathogens -delivering trapped bacterial or viral products to lysosomes for degradation. besides, it is equipped to clear invasive pathogens through induction of cd + t-cell responses, and also initiates a primordial innate immune response by cooperating with pattern recognition receptor signalling to induce interferon production. this was recently described in the context of dna virus infections, where cyclic gmp-amp (cgamp) and sting-dependent activation of autophagy was necessary to remove viral dna from the cytosol [ ] . however, in an ongoing evolutionary arms race, most pathogens have acquired the ability to hijack and subvert autophagy to evade degradation through this pathway. remarkably, +rna viruses have adapted to not only protect themselves from autophagic elimination but even harness the machinery to their own benefit, as will be covered in more detail in the subsequent sections. among the +rna viruses, data on favourable versus detrimental impact of autophagy is particularly confounding [ ] . a link between autophagosomes and virus-induced vesicles was proposed by george palade by em imaging of poliovirus containing vesicles that resembled autophagosomal membranes [ ] . over the past few decades, a growing body of research has defined the critical role of this pathway in facilitating infection by numerous +rna rna viruses, including poliovirus (pv) [ , ] , coxsackievirus b (cvb ) [ , ] , cvb [ ] , enterovirus (ev ) [ ] , human rhinovirus (hrv) [ ] , foot-and-mouth disease virus (fmdv) [ ] , encephalomyocarditis virus (emcv) [ ] , dengue virus (denv) [ , ] , zika virus (zikv) [ , ] , hepatitis c virus (hcv) [ ] , mouse hepatitic virus (mhv), newcastle disease virus (ndv) [ ] , severe and acute respiratory syndrome coronavirus (sars-cov) [ ] , chikungunya virus (chikv) [ ] , and japanese encephalitis virus (jev) [ ] . in many of the above cases, pharmacological or genetic manipulation of autophagy in vitro confirmed an inhibition in replication and/or spread of these viruses, whereas induction of autophagy resulted in increased production of progeny virions [ , ] . current evidence indicates that many, if not all +rna rna viruses depend on the initiation of the autophagic pathway for their optimal production. this is counterintuitive, since these viruses replicate in the cytosol, and autophagy serves to promote degradation of cytosolic contents. therefore, it is evident that +rna rna viruses in particular have evolved sophisticated mechanisms to circumvent or exploit this pathway. not surprisingly, most +rna rna viruses also trigger massive membrane remodelling within infected host cells to create membrane delineated structures, often referred to as replication organelles, vesicle packets, convoluted membranes or double membrane vesicles, depending on their morphology and ultrastructure [ , ] . whether these replication organelles are pseudo-autophagosomes, has long been a point of contention. several genome-wide screens, e.g with crispr/cas libraries, haploid kbm cells, and shrna depletions, as well as proteomic studies have universally indicated the involvement of the autophagy pathway in +rna rna virus infections [ , , ] . however, among the odd autophagy-related genes, the functional contribution of the individual components in virus infection is far from clear. a recent targeted crispr/cas screen uncovered a fairly diverse range of involvement among autophagic components in three +rna rna virus infections -pv, denv and zikv [ ] . the authors reported that all three viruses employed multiple proteins of the autophagy pathway while bypassing others, and each virus used a unique set of initiation components. a common feature among the tested viruses underscored the requirement of the lc protein but not its canonical cellular lipidation process, where lc was recruited to virally induced membranes by alternative means. this study highlights the importance of assessing the pathway in its entirety when seeking to understand how pathogens co-opt it for purposes of genome replication and spread, as well as to identify universal drug targets. many different mechanisms have been proposed on how autophagy is manipulated to facilitate infection while preventing degradation of +rna rna viruses (fig. ). while in no way exhaustive, the following sections cover the salient features that are recurrent among several viral genera: the physical hallmark of the autophagy pathway is formation of lc + cytosolic double-membrane vesicles, also often observed in +rna rna virus infections. one of the long-running debates is whether these replication organelles are themselves immature autophagosomes or take advantage of the same machinery for their biogenesis. however, canonical autophagic vesicles are part of a degradative pathway, where they fuse with lysosomes for their contents to be hydrolysed by proteases and lipases. viruses from different families appear to possess a diverse set of strategies to prevent this from happening. flaviviruses, such as denv and zikv have been reported to trigger autophagy on the one hand, while utilising the er as a focal point for generating their replication organelles and assembly of progeny virions. consequently, both viruses have evolved means to suppress er-turnover via reticulophagy. the er-localised reticulophagy receptor fam b was identified as a restriction factor for both denv fig. . induction of the autophagy pathway. autophagy is initiated typically from cellular stress, such as starvation, whereby unc- -like kinase (ulk ) complex (comprising ulk , autophagy-related protein (atg ), fip and atg are activated. this complex triggers nucleation of the phagophore by phosphorylating components of the class iii pi k (pi kc ) complex i (consisting of class iii pi k, vacuolar protein sorting (vps ), beclin , atg , activating molecule in beclin -regulated autophagy protein (ambra ) and general vesicular transport factor (p ). this in turn activates local phosphatidylinositol- -phosphate (pi p) production at discrete er sites often referred to as omegasomes. wd repeat domain phosphoinositide-interacting proteins (wipis) and zincfinger fyve domain-containing protein (dfcp ) are then recruited to these phagosome assembly sites followed by recruitment of atg ˜atg -atg l complex that enhances atg -mediated conjugation of atg family proteins, including microtubule-associated protein light chain (lc ) proteins to membrane-resident phosphatidylethanolamine (pe), thus forming the membrane-bound, lipidated form lc -ii -the characteristic signature of autophagic membranes. atg s are required for elongation and closure of the phagophore membrane, and in selective autophagy, are involved in sequestration of specific cargo into autophagosomes. several cellular membranes, most likely the er, contribute to elongation of the autophagosomal membrane by serving as membrane reservoir -delivered by atg -containing vesicles. once sealed, autophagosomal membranes give rise to double-layered vesicles called autophagosomes, which mature and fuse with the lysosomes. autophagic cargo is hydrolysed and recycled back to the cytoplasm. and zikv. rnai-depletion of fam b significantly enhanced denv and zikv replication at an early stage of the viral life cycle. the virusencoded ns protease from several flaviviruses directly cleaved fam b at a single site within its reticulon homology domain to selectively suppress er degradation [ ] , underscoring a sophisticated mechanism to differentially regulate specific arms of autophagy. coxsackievirus b (cvb ), an enterovirus belonging to the picornaviridae family, is known to rely on autophagosome formation for optimal replication [ , ] ; however, both in vitro and in vivo evidence suggest that during infection, amphisome maturation and autophagic protein degradation are inhibited. an increase in autophagosomal abundance concomitant with a decrease in autophagic flux was reported with cvb , prompting the hypothesis that infection selectively triggers autophagosome formation while preventing the terminal stages in degradation [ , ] . the molecular determinants and mechanism by which cvb limits autophagic degradation is currently unknown. interestingly, treatment of cvb -infected cells with inhibitors of autophagosome maturation resulted in increased virus production, indicating that canonical autophagy was not completely blocked in virus-infected cells, and at least a population of the virus remained sensitive. a similar finding was reported more recently with rotavirus where virus replication benefited from induction of autophagy while blocking degradation [ ] . as with cvb , the mechanism by which rotaviruses specifically inhibit autolysosomal degradation has not been elucidated, emphasising the importance of identifying the specific virus or host components that prevent degradation to provide fundamental insights on autophagic regulation in general. the case with hcv infection is more convoluted on account of contradictory data: whereas gfp-rfp-lc expressing cells infected with hcv displayed a complete maturation of autophagosomes followed by fusion with lysosomes [ , ] , atleast one other study reported that hcv replication restricted autophagosomal fusion [ ] . yet another study reported that the autophagy pathway in its entirety was necessary during hcv-infection for optimal replication; however, the advantage derived from it was primarily due to suppression of innate immune responses [ ] . blocking fusion between autophagosomes and lysosomes has also been reported to increase denv yield [ ] . however, this effect may be viral serotype-specific, since inhibiting lysosome fusion reduced denv production [ ] . the mechanisms by which autophagy favors denv production was recently described where rather than replication, assembly and release of progeny virions was affected by blocking autophagy mediated lipid droplet hydrolysis [ , , ] . with coronaviruses, initiation of autophagy appears to be through the er-derived, ptdlns p-enriched omegasomes that normally operate during starvation. infectious bronchitis virus (ibv) -an avian coronavirus responsible for major losses to the poultry industry, is one such example where a significant portion of the genome encodes nonstructural proteins (nsp) dedicated to virus replication. expression of nsp proteins resulted in increased levels of ptdlns p on er membranes, recruitment of ptdlns p effector protein wipi and the generation of autophagosomes directly from the er [ , ] . however, when compared to the properties of starvation-induced autophagosomes, those generated by coronavirus infection or nsp proteins presented significant differences. nsp -induced autophagosomes displayed limited ability to undergo expansion, preventing formation of large autolysosomes, and hence circumvented degradation of viral particles through the lysosomal pathway [ ] . results obtained with ibv, sars and mhv nsp was recapitulated with middle eastern respiratory syndrome coronavirus (mers-cov), where a similar phenomenon was observed for nsp [ ] . a distinctive feature shared by +rna rna viruses is to assemble and replicate on intracellular membranes, which have been proposed to offer a two-fold advantage: (a) scaffold for anchoring and concentrating schematic illustration of the different pathways of selective autophagy that are triggered upon +rna rna virus infections. initiation of autophagosomes is through formation of an isolation membrane most likely derived from the er. depending on the molecular composition and function, they may form either omegasomes, edemosomes or amphisomes. flaviviruses such as zikv non-structural protein a (ns a) and ns b activate autophagy by inhibiting akt and mtorc ; autophagosomes generated are subverted to specialized functions to prevent viral degradation. turnover of organelles occur through convergence of specialised autophagosomes with lysosomes for their selective degradation: er via reticulophagy; mitochondria via mitophagy; lipid droplets via lipophagy and virions or viral proteins via virophagy. viruses that are known to upregulation specific autophagosomal pathways are depicted in black, those that suppress specific types or steps of autophagy are depicted in red. seminars in cell and developmental biology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] the replication complexes and (b) to insulate dsrna intermediates from innate sensing by cytosolic pattern recognition receptors [ ] . the replication complexes are typically composed of the viral rna-dependent rna polymerase, accessory non-structural proteins, viral rna, and host factors. specialised autophagosomes, sometimes referred to as amphisomes (formed upon fusion with endosomes), omegasomes, and ede-mosomes (both er-derived) have been hypothesised to function as replication organelles [ , , ] . given the resemblance of virustriggered double membrane vesicles with that of autophagosomes, it is plausible that generation of replication organelles are a result of mechanisms similar to autophagy [ ] [ ] [ ] [ ] . like autophagosomal membranes, many virus-induced vesicles are believed to be er-derived. recent data from several different studies have provided direct evidence of the association between viral replication complexes and autophagosome structures as summarized in table . poliovirus (pv) vesicle clusters were found to contain lc and lysosomal markers, reminiscent of autolysosomes, and colocalised with the pv replication complex [ , ] . furthermore, formation of infectious pv progeny virions was reported to depend on vesicular acidification, prompting the hypothesis that particle assembly, genome replication and virion maturation occurred in bona fide autophagosomal vesicles [ ] . among the flaviviridae family, several non-structural proteins have been observed in lc + vesicles. denv non-structural protein ns and dsrna were reported to co-localise with lc and ribosomal proteins [ , ] . another study described the induction of lc + vesicles, which colocalised with ns a in infected cells, or when transfected with a combination of denv ns a and ns b [ ] . this was independently corroborated by zikv infection in human fetal neural stem cells where expression of ns a and ns b were sufficient to block neurogenesis and promote autophagy, displaying partial colocalisation with lc + vesicles [ ] . ultrastructural analysis of chikv virions also suggested their location in the lumen of autophagome-like vacuoles [ ] . similar to other members of this family, hcv infection induces massive intracellular membrane rearrangements. competing hypotheses have been proposed as to whether autophagosomes themselves serve as sites for hcv replication. by sucrose gradient analysis, lc -ii was found to co-sediment with hcv rna and non-structural proteins ns and ns a [ ] . however, in a separate study confocal microscopy showed little evidence of co-localisation of lc or atg with hcv proteins [ , ] . along the same lines, depletion of either lamp or rab , which allowed accumulation of autophagosomes by preventing fusion with lysosomes inhibited hcv viral replication, also suggesting that they are not the major sites for hcv genome replication [ ] , while multiple reports indicate that lipid droplets are the more likely sites for replication and assembly, as reviewed elsewhere [ , ] . conflicting evidence also exists for mhv-induced replication compartments. on the one hand, mhv replication complexes were found to be associated with lc and atg in embryonic stem cell lines [ ] . on the other hand in primary macrophages and murine embryonic fibroblasts mhv replication did not require the autophagy gene atg [ ] . differences in permisiveness to infection often exists between primary cells and transformed cell lines, as does viral tropism towards distinct cell types, either of which can account for these experimental discrepancies. among other +rna rna viruses, immunoelectron microscopy demonstrated co-localisation of ev capsid protein vp with autophagosomes in virus-infected mouse neurons [ ] . similarly, during emcv infection, colocalisation of non-structural protein a and capsid protein vp was visualised by confocal and immunoelectron microscopy [ ] . colocalisation of non-structural proteins b, c, and a with lc , and structural protein vp with atg were also reported in fmdv-infected cells [ ] . although direct evidence of the association of viral replication complexes with autophogasomes is lacking for cvb , impaired maturation of autolysosomes brought about through pharmacological or genetic inhibition increased the accumulation of autophagosomes in virus-infected cells resulting in enhanced viral replication [ , ] . these data implicated autophagosomes as virus anchoring and replication sites during cvb replication. delineating the process of viral assembly from replication is technically challenging, especially since both processes would very likely induces formation of autophagosome-like double-membrane liposomes [ ] summary of interactions between proteins from positive strand rna viruses and host autophagy machinery. seminars in cell and developmental biology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] occur in concert at the same sites. although utilisation of autophagosomes as assembly sites has been recorded for some dna-viruses e.g., hepatitis b virus (hbv) [ ] , experimental data with +rna rna viruses are scant. however, atleast for denv, recent evidence indicates that autophagy might actually assist in assembly of progeny virions, without them serving as replication organelles. one of the initial studies describing the induction of autophagy in flavivirus infections was performed by lee et al [ ] . the authors demonstrated that denv infection in hepatocytes induced autophagy; targeting with either the inhibitor -methyladenine ( ma) or sirnas against autophagy genes compromised infection. denv-induced autophagosomes colocalised with lamp , a marker of lysosomal fusion, which was independently validated by immunofluorescence assays and pharmacological inhibition. following this initial characterisation, a more mechanistic study emerged describing the role of selective autophagy facilitating hydrolysis of lipid droplets in an infection-specific manner [ ] . apart from the relatively non-specific bulk macroautophagy, cellular organelles are turned over through several types of selective autophagy. this phenomenon occurs under normal physiological conditions and is hypothesised to initiate a physiological response to appropriately address a specific stress. in the context of denv infection, a type of selective autophagy of lipid storage organelles (lipid droplets) referred to as lipophagy was described that hydrolyses neutral fat deposits to free fatty acids and cholesterol, and supplements cellular energy reservoirs [ , , ] . heaton et al performed a targeted sirna screen to identify cellular cofactors of denv replication in hepatocytes, which revealed, among others, genes involved in the induction of autophagy [ ] , and were further characterised to reveal that denv induced autophagosomes not only acquired lamp , but underwent complete maturation to become autolysosomes [ ] . these did not colocalise with markers of the viral replication complex, suggesting that they may play an indirect, non-structural role in denv replication. a subsequent study described the involvement of aup , a type-iii membrane protein, in the initiation of virus-induced autophagy. aup was regulated by monoubiquitin modification, where infection or a combined expression of viral ns a and ns b were necessary and sufficient to generate the unmodified form of aup -a step that was critical in induction of this pathway. interestingly, loss of aup did not affect viral replication; however, impaired autophagy was accompanied by degradation of viral proteins through the proteasomal pathway resulting in significantly reduced production of progeny virions, supporting a specific role of aup -dependent lipophagy in assembly of virus particles [ ] . these results were in agreement with an independent report demonstrating expression of dengue ns a was sufficient to trigger autophagy and protect against cell death. a growing body of evidence indicates that mitochondrial function is altered during flavivirus infections. although the mechanistic underpinnings are currently not well understood, atleast for hcv, parkindependent mitophagy has a significant effect on virus propagation. this was verified by silencing parkin and pink , which inhibited hcvtriggered mitophagy and in turn blocked virus replication. ultrastuctural analyses by electron microscopy and immunoelectron microscopy also confirmed the presence of damaged mitochondria in double-membrane vesicles in hcv-infected cells [ ] . whether this pathway is activated during infection by other flaviviruses is currently not known. several reports on mechanisms of secretion and cell-to-cell transfer of intracellular pathogens indicate non-degradative autophagic vesicles as an efficient mode of transport. an recent study with mycobacterium demonstrated that autophagosomes chaperone an organelle referred to as the "ejectosome", facilitating cell-to-cell spread of cytosolic bacteria [ ] . secretory autophagy is a newly discovered pathway in which autophagosomes fuse with the plasma membrane instead of lysosomes and release single membrane vesicles containing cytosolic content into the extracellular milieu [ ] . non-degradative autophagy has been suggested to facilitate nonlytic egress of some +rna rna viruses. the initial characterisation was with enteroviruses, which appear to exploit this pathway to exit cells, and are released into the extracellular environment as particle populations contained within vesicles [ ] . clusters of enteroviral particles were packaged with phosphatidyl serine into autophagic vesicles, which enabled efficient transfer to primary macrophages, significantly enhancing viral infectivity. this revealed a novel mode of transport where viral genomes were transferred en bloc to recipient cells, facilitating genetic cooperativity and enhancing infection. this mode of transfer had previously also been noted for cvb , where a recombinant fluorescent virus was released into the extracellular medium in microvesicles containing autophagic markers [ ] . poliovirus is often considered a lytic virus; however, non-lytic release of poliovirus has also been reported [ ] . reduced levels of viral particles in extracellular medium in autophagy-deficient cells correlated with inhibition of non-lytic release of autophagic vesicles. more recently, an important role of the secretory autophagy pathway was implicated in zikv vertical transmission as well as cell-to-cell spread of denv. zikv-induced autophagic activity in human trophoblasts restricted by pharmacological inhibition, or by deficiency in an essential autophagy gene, atg l , limited zikv vertical transmission and improved placental and fetal outcomes, which supported a role for autophagic secretion in the process [ ] . along the same lines, it was hypothesised that denv might evade neutralising antibodies and increase viral spread by exploiting autophagic vesicles for delivery to the extracellular medium [ ] . double staining of denv e antigen and lc in a close-contact co-culture experimental set-up verified secretion of denv-containing autophagic vesicles from donor cells, which were subsequently taken up by recipient cells. in a parallel study, maturation of infectious denv virus particles was attributed to this process, when cleavage of pr peptide from prm by the furin protease was prevented upon blocking autophagy [ ] . further investigation is needed to provide more direct evidence on the mechanism, regulation and molecular determinants of secretory autophagy in facilitating viral release. activation of autophagy represents a fairly ubiquitous response to eliminate intracellular pathogens. several studies have described mechanisms where pathogen recognition receptors trigger this response upon detection of microbe-specific pathogen associated molecular patterns. a diverse set of pathogens including bacteria, viruses and parasites have provided corroborating evidence supporting this phenomenon. whether this process occurs in parallel to non-degradative autophagy, or the cross-talk that might exist between the two flavours of autophagy during +rna rna viral infection merits further investigation. recent studies have shed light on how autophagy offers an advantage to hcv infection by suppressing innate immune responses [ , ] . this contrasts with previous data on hcv-induced incomplete autophagy and defined a pathway where the entire process from initiation through lysosomal degradation is necessary for hcv replication largely to suppress anti-viral innate immune response. in hcv-infected cells, interferon-β (ifn-β) production could be modulated by uprmediated autophagy; activation of this pathway reduced ifn-β production and vice-versa [ ] . inhibition of autophagy by suppressing beclin- or atg reduced hcv replication, which was accompanied by the activation of ifn signaling. a similar phenomenon was also proposed for denv where activation of autophagy not only facilitated viral h.h. wong and s. sanyal seminars in cell and developmental biology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] replication, but also suppressed ifn-i production, suggesting that both viruses may share the same mechanism to evade innate immune responses. interestingly, in west nile virus infections, perturbation of intracellular cholesterol levels dictated ifn-i responses [ , ] . although the role of autophagy in wnv infection has been contested, replication was reported to occur independent of autophagy by mutiple groups -a clear difference from other flaviviruses; however, whether it plays a role in regulating free cholesterol and fatty acid levels is yet to be determined. together, these studies indicate a critical mechanism by which flaviviruses may avoid innate immune responses through activating the host autophagy pathway. the link between autophagy and apoptosis has been defined extensively in various contexts including cancer, neurodegenerative disorders, and infectious diseases [ ] . a more comprehensive understanding on circumventing cell death has been recorded for bacterial rather than viral infections, amongst which most are dna viruses. premature cell death can function as an anti-viral host mechanism by providing an unfavorable environment and shorter timeframes for viral propagation. induction of autophagy has often been linked to inhibition of apoptosis [ , ] . both denv and a murine flavivirus-induced autophagy was reported to prevent apoptosis mediated via the viral ns a protein [ ] . knockdown of autophagy-related gene expression abolished the protective role of autophagy against cell death and resulted in reduced viral replication. apart from denv, cross-talk between autophagy and apoptosis was also reported in cvb infection, where suppression of autophagy by ma triggered caspase activation and vice-versa [ ] . an interesting question that arises from virus-triggered induction of autophagy in the context of flavivirus infections is the process of antigen presentation. particularly in major histocompatibility complex (mhc)-ii positive cells, autophagosomes are constitutively generated to deliver viral antigens on mhc-ii molecules for adaptive immune responses. virus infections therefore frequently impede maturation of antigen presenting cells and subsequent adaptive immunity as reviewed in detail elsewhere [ ] . monocytes and monocyte-derived cells are a major target of flaviviruses, where antigen presentation is facilitated by autophagy. this implies that while autophagy favours production of viral progeny, it should simultaneously increase viral antigen presentation and t-cell responses, thus generating neutralising antibodies and promoting cytotoxic t-cell killing. information on these seemingly contradictory processes is currently limited. however, denv-infected human monocyte-derived dcs fail to upregulate mhc and co-stimulatory molecules and have an impaired ability to polarize cd + th type (th ) effector properties [ ] , contributing to inefficient adaptive immune responses observed in patients. in bulk cultures of dendritic cells, exposure to denv augments mhc-i and mhc-ii expression in non-infected bystander cells; however, infected monocyte-derived dendritic cells display an inhibition in this process within the same cultures [ ] . in clinical studies gene expression analyses of denv patients revealed that severe cases expressed lower levels of genes linked to antigen processing, presentation and t-cell activation compared to mild cases. another related flavivirus, japanese encephalitis virus, inhibits expression of mhc-i and induces functional impairment of dcs, resulting in poor cd + t cell responses [ ] . thus impaired antigen presentation and functionality of virus-infected dcs may reflect a viral immune escape strategy to dampen t-cell responses and impact disease severity. one study reported that denv activated autophagy only during the early infection stage, suggesting a biphasic response of autophagy to denv infection, where it shifted from a supporting to an antiviral role at later time points [ ] . these results might enable us to reconcile how flaviviruses have evolved strategies to manipulate this pathway while subverting t-cell based immune responses. a quantitative and time resolved analyses of this process in virus-infected cells might shed light on its utilisation in the benefit versus detriment towards virus production. activation of autophagy in the presence of intracellular pathogens is a fairly universal cellular response. xenophagy as an intrinsic defence mechanism was first described through electron microscopy studies upon visualising hsv- and cytomegalovirus inside autophagosomes [ ] . among viruses, autophagic protection has been recorded in a wideranging species and genera [ ] [ ] [ ] . the mechanism of autophagymediated restriction of +rna rna viruses is less well-documented on account of its proviral influence in most cases. however, there are instances where autophagic degradation of virions (virophagy) or viral proteins has been observed, especially in neurons where it is a critical form of antiviral defence. during sindbis virus (sinv) infection, beclin and p -dependent degradation of the capsid protein protects against sinv-mediated encephalitis [ , ] . moreover, atg deficiency results in delayed sinv clearance and accumulation of the autophagy receptor p . more recently, fanconi anaemia group c protein (fancc) was found to interact with the sinv capsid protein and facilitate virophagy [ ] . picornaviruses, such as pv and hrv permeabilise endosomes to release their genome into the cytosol. this step is detected by galectin , which restricts viral infection by initiating degradation of the viral rna genome [ ] . as counterstrategy, the host protein hras-like suppressor (pla g ) is exploited by the virus to enable genome delivery. cvb , also belonging to the same family, undergoes p -dependent degradation and uses the viral protease a to cleave p and inhibit virophagy [ ] . interestingly, although hcv has been demonstrated to induce autophagy to its own advantage by multiple groups, one study demonstrated that an er transmembrane protein, scotin, interacted with the viral protein ns a, resulting in its autophagic degradation to suppress viral replication [ ] . among flaviviruses, autophagosomal degradation of neurotropic viruses has been recorded. in the drosophila brain, zikv infection triggered nfκb-dependent inflammatory signaling, inducing expression of dsting which subsequently restricted infection by upregulating autophagy. defective or absence of autophagy resulted in increased infection in the fly brain and death [ ] . sting-dependent induction of protective autophagy was independently reported for dna viruses and sea anemone, supporting an evolutionarily conserved role for sting in microbial autophagy [ ] . despite major advances in elucidating molecular determinants of the autophagy pathway, the rules that govern its utilisation during infections are far from obvious. a complex interplay between viral manipulation and host innate immunity dictates disease outcomes. autophagy is expected to restrict viral infections at multiple levels by eliminating viruses, regulating inflammatory responses and promoting antigen presentation. however, +rna viruses have co-evolved to manipulate autophagy for immune evasion, replication, assembly and release from infected cells. the repertoire of universal and distinct mechanisms that these viruses draw on to interfere with autophagy are striking, often targeting the same pathway in unique ways with different functional implications. distinct viral strategies fine-tune the process to simultaneously escape destruction while capitalising on the structural and nutrient benefits that autophagy provides. several gaps remain in our understanding within the remit of viral manipulation of autophagy. first and foremost, more advanced strategies of isolating autophagic vesicles will be imperative for better characterisation of this process. emerging data indicate that many lc -positive vesicles that h.h. wong and s. sanyal seminars in cell and developmental biology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] are induced upon virus infections are not autophagosomes. a combination of ultracentrifugation, density gradient separations and enrichment techniques will be necessary to characterise these vesicle populations. furthermore, in the context of virus infection, the equilibrium between degradative and secretory autophagy versus biogenesis of exosomal vesicles will need to be quantitated to arrive at firm conclusions regarding the functional outcome of autophagy. along the same lines, differences between infected host cells and neighbouring cells will become important to develop a more complete picture of its impact in viral pathogenesis versus immune responses. further understanding of the contribution of autophagy to the different stages in the viral lifecycle, subversion of its antigen presentation function and innate immune responses is therefore necessary to delineate the diverse functions of autophagy in virus pathogenesis. a current perspective of autophagosome biogenesis endoplasmic reticulum and golgi complex: contributions to, and turnover by mechanism and medical implications of mammalian autophagy autophagy during 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promotes the replication of encephalomyocarditis virus in host cells hepatitis b virus subverts the autophagy elongation complex atg - / l and does not require atg /lc lipidation for viral maturation autophagic machinery activated by dengue virus enhances virus replication regulation of lipid stores and metabolism by lipophagy autophagy regulates lipid metabolism dengue virus nonstructural protein redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy the autophagic machinery ensures nonlytic transmission of mycobacteria phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses coxsackievirus b exits the host cell in shed microvesicles displaying autophagosomal markers inhibition of autophagy limits vertical transmission of zika virus in pregnant mice autophagyassociated dengue vesicles promote viral transmission avoiding antibody neutralization inhibition of cellular autophagy deranges dengue virion maturation knockdown of autophagy enhances the innate immune response in hepatitis c virus-infected hepatocytes activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro cholesterol manipulation by west nile virus perturbs the cellular immune response perturbation of intracellular cholesterol and fatty acid homeostasis during flavivirus infections autophagy in cell death: an innocent convict? lysosomes and autophagy in cell death control bcl- family members: dual regulators of apoptosis and autophagy flavivirus ns a-induced autophagy protects cells against death and enhances virus replication impairment of cd + t cell polarization by dengue virus-infected dendritic cells differential effects of dengue virus on infected and bystander dendritic cells multifront assault on antigen presentation by japanese encephalitis virus subverts cd + t cell responses dengue virus inhibition of autophagic flux and dependency of viral replication on proteasomal degradation of the autophagy receptor p herpes simplex virus and human cytomegalovirus replication in wi- cells. iii. cytochemical localization of lysosomal enzymes in infected cells autophagy is an essential component of drosophila immunity against vesicular stomatitis virus hsv- icp . confers neurovirulence by targeting the beclin autophagy protein autophagy pathway intersects with hiv- biosynthesis and regulates viral yields in macrophages protection against fatal sindbis virus encephalitis by beclin, a novel bcl- -interacting protein autophagy protects against sindbis virus infection of the central nervous system pla g represents a switch between entry and clearance of picornaviridae calcoco /ndp and sqstm /p differentially regulate coxsackievirus b propagation interferon-inducible protein scotin interferes with hcv replication through the autolysosomal degradation of ns a inflammation-induced, sting-dependent autophagy restricts zika virus infection in the drosophila brain amino acid substitutions in the non-structural proteins a or b modulate the induction of autophagy in west nile virus infected cells independently of the activation of the unfolded protein response the role of secretory autophagy in zika virus transfer through the placental barrier japanese encephalitis virus replication is negatively regulated by autophagy and occurs on lc -i-and edem -containing membranes rab and class iii phosphoinositide -kinase vps are involved in hepatitis c virus ns b-induced autophagy hepatitis c virus upregulates beclin for induction of autophagy and activates mtor signaling the autophagy elongation complex (atg - / l ) positively regulates hcv replication and is required for wild-type membranous web formation coronavirus membraneassociated papain-like proteases induce autophagy through interacting with beclin to negatively regulate antiviral innate immunity coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication coronavirus nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate cleavage of sequestosome /p by an enteroviral protease results in disrupted selective autophagy and impaired nfkb signaling protein b of coxsackievirus b induces autophagy relying on its transmembrane hydrophobic sequences enteroviruses remodel autophagic trafficking through regulation of host snare proteins to promote virus replication and cell exit foot-and-mouth disease virus capsid protein vp activates the cellular eif s -atf pathway and induces autophagy via hspb viroporin activity of the foot-and-mouth disease virus non-structural b protein modification of cellular autophagy protein lc by poliovirus double-membraned liposomes sculpted by poliovirus ab protein this work was supported by health and medical research funds ( and and ), and partially by research grants council-general research funds ( ). ss is supported by the croucher foundation. key: cord- - e xn kj authors: falcón-lezama, jorge abelardo; santos-luna, rené; román-pérez, susana; martínez-vega, ruth aralí; herrera-valdez, marco arieli; kuri-morales, Ángel fernando; adams, ben; kuri-morales, pablo antonio; lópez-cervantes, malaquías; ramos-castañeda, josé title: analysis of spatial mobility in subjects from a dengue endemic urban locality in morelos state, mexico date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: e xn kj introduction: mathematical models and field data suggest that human mobility is an important driver for dengue virus transmission. nonetheless little is known on this matter due the lack of instruments for precise mobility quantification and study design difficulties. materials and methods: we carried out a cohort-nested, case-control study with individuals ( cases, intradomestic controls and population controls) with the goal of describing human mobility patterns of recently dengue virus-infected subjects, and comparing them with those of non-infected subjects living in an urban endemic locality. mobility was quantified using a gps-data logger registering waypoints at -second intervals for a minimum of natural days. results: although absolute displacement was highly biased towards the intradomestic and peridomestic areas, occasional displacements exceeding a -km radius from the center of the studied locality were recorded for all three study groups and individual displacements were recorded traveling across six states from central mexico. additionally, cases had a larger number of visits out of the municipality´s administrative limits when compared to intradomestic controls (cases: . versus intradomestic controls: . , p = . ). we were able to identify extradomestic places within and out of the locality that were independently visited by apparently non-related infected subjects, consistent with houses, working and leisure places. conclusions: results of this study show that human mobility in a small urban setting exceeded that considered by local health authority’s administrative limits, and was different between recently infected and non-infected subjects living in the same household. these observations provide important insights about the role that human mobility may have in dengue virus transmission and persistence across endemic geographic areas that need to be taken into account when planning preventive and control measures. finally, these results are a valuable reference when setting the parameters for future mathematical modeling studies. a a a a a dengue fever (df) is the most important arthropod-borne viral disease in the world. it is caused by infection with any of the four dengue virus (denv) serotypes. nearly half of the human population inhabits areas with denv transmission. in mexico dengue incidence and severe cases have been increasing in the last decade. to date, there is no specific treatment or vaccine for df and vector control stands as the cornerstone for df prevention [ , ] . df is an important public health problem, especially in urban areas [ ] , where it usually presents in large outbreak. the costs of treatment and management during a df outbreak are a serious burden for health systems, especially when there is a risk for saturation of health facilities [ ] . the actors that are necessary for denv transmission are fairly well understood. nonetheless, some of the dynamical features about these actors still need to be elucidated in order to understand how they impact on transmission. human mobility has been studied in relation to other infectious diseases, where its role as an important driver for disease transmission has been proven [ , ] . mathematical models have suggested that local scale human mobility may play a role in denv transmission, outbreak persistence, and control efficiency [ , ] . however, little information is available on detailed human mobility patterns in geographic areas where df is endemic or on confirmed cases during an outbreak. recently, gps-based technologies have been tested and shown to be a reliable and acceptable tool for quantifying human mobility [ , ] . human mobility has been described in relatively recent reports by using indirect measures [ , ] . for these reasons, we studied the micro and macromobility of dengue virus-infected subjects in an endemic locality. here we present the results of a cohort-nested case-control study on a dengue endemic urban locality in mexico. the protocol of the project was reviewed and approved by the comité de etica y de prevención de conflictos de interes (institutional review board) ci no and departamento de investigación (external review) servicios de salud de morelos, mexico dei/cei/ / . cohort-nested, case-control study. sample: individuals ( cases, intradomestic controls and population controls) with age older than , and residents in axochiapan, morelos state, méxico, were selected from the cohort "peridomestic infection as determinant for dengue virus transmission" [ ] . they were assigned into three study groups: a. cases were individuals with laboratory evidence of recent, symptomatic or asymptomatic, denv infection, and identified as the only persons infected within their households during the study. b. intradomestic controls, were individuals with a negative serological result for recent denv infection, living in the same household with a case; c. population controls were individuals with a negative serological result for recent denv infection, randomly selected from the same locality. all controls tested negative for denv during the same period and using the same validated techniques than cases (igm or igg capture elisa) as reported in the cohort study [ ] . partici-pant´s selection was performed as follows: cases were approached first, if accepted participation an intradomestic control was randomly assigned from the pool of subjects living in the same house that had both baseline and final negative elisa results. for each pair of in-house participants, a randomly selected population control was then assigned (fig ) . given the limited number of available gps loggers and the three-month time frame for the follow-up, the recruitment was limited to a maximum of cases with their respective intradomestic and population controls. between may and september, , with prior signed informed consent, all participants were provided with a portable gps (gps data-logger, transystems mod. a+), programmed for recording its position at -second intervals (variables date, time, latitude, longitude, altitude and speed), during hours a day, for a minimum period of days. participants were instructed to carry their gps at all times whenever they left their homes and to recharge the equipment's battery daily during their in-home resting times. this follow up was performed in cases identified during the immediate previous season, on average one year after diagnosis confirmation, in order to match the activities performed during the high transmission season, and under the assumption that their mobility patterns remained unchanged after disease, and constant through time. a web-based interface was developed to import the text files from gps equipment to a main database. variables were homogenized, waypoints in the initial and final days of each individual gps track (comprising incomplete days) were eliminated in order to standardize the period of time to be analyzed starting at : : hours on the second day of follow up and ending at : : on the last to final day of follow up. data were converted into a feature dataset and projected from the geographic coordinate system to a lambert coordinate system (from hexadecimal to metric units) with the purpose of performing arithmetic operations for distance calculations. origin or routinely residence sites were identified by means of an iterative algorithm (mean center) employing waypoints from : : to : : hours, monday to friday. routine residence coordinates were added to the database. distance to home variable (dhome) was calculated for each extradomestic waypoint using sql applying the following formula: where; x = xhome (x coordinate from home), x = xccl (x coordinate from each waypoint), y = yhome (y coordinate from home) y, y = yccl (y coordinate from each waypoint). waypoints within the peridomestic area (dhome < m) were identified. distance, speed, altitude and time differentials were created: where: xccl = (x coordinate from previous waypoint), xccl = (x coordinate from current waypoint), yccl = (y coordinate from previous waypoint) and, yccl = (y coordinate from current point) displacement and spatial permanency variables for each subject with complete data were generated. visit sites were defined as those areas out of the individual's home with a m radius in which each participant remained static for a period enough to allow a potential effective interaction with local vectors. these sites were identified by generating an algorithm through which visit clusters were formed using the following criteria: stops lasting minutes or longer, a distance from home of m or farther, distance of the current waypoint from previous waypoint < m, and speed for the current waypoint of km / h or less. for each cluster (visit site) a centroid was calculated. common visit sites for cases were identified as hexagonal cells with m radius [ ] which were visited by at least two different cases at a given time, and where the proportion of different visiting cases was at least two thirds of the total visiting population for that cell. common visit sites for controls were identified as hexagonal cells with m radius which were visited by at least two members from each control population and not visited by any of the cases. the geographic universe in the study was divided in five areas ( .-inside the house, .-out of the house but in the locality, .-out of the locality but in the municipality, .-out of the municipality but in the state, .-out of the state), limited by four buffers. a circular buffer with m radius around each participant's home limited the first area and three additional polygonal buffers were drawn according to the administrative limits for the locality, municipality and state. arcgis arcinfo was used for processing and analyzing spatial data, sql server was used to create a geodatabase, arc sde was used as interpreter between entre sql and arcgis. statistical analyses were performed using stata . fifty randomly selected cases were asked to participate in the study from which ( %) accepted participation. all approached controls agreed to participate. in total individuals ( cases, intradomestic controls and population controls) were recruited. our drop-out rate was lower than % ( / ) since one participant (intradomestic control) did not finish the follow-up due to the loss of the assigned gps logger. table describes the main characteristics of the subjects in each group. no statistically significant differences were observed in most of variables except in age, since cases were significantly younger than the intradomestic or population controls (cases mean: . , sd: . ; intradomestic controls mean: . sd: . ; population controls mean: . sd: . . p = . ). of participants, ( . %) participants completed their follow up since one gps used by an intradomestic control went missing. the final database contains , , waypoints from these participants, and all participants were followed by a mean of . continuous days. as for the number of days of follow-up for each group no differences were recorded. as expected, most of the waypoints in the population fell within the intradomestic area (< m radius from home centroid). as distance from home (absolute displacement) increased, we observed a marked decrease in the proportion of waypoints. all three groups presented a small peak when the distance reached the m radius. from this point the proportion of waypoints quickly decayed (fig ) . no differences were noticed for absolute displacement among the groups. the hourly distribution of recorded waypoints out of the participant's homes is shown in fig . as expected, participants usually left their homes early in the morning and returned by the end of the day. although we recorded waypoints out of the participants' homes in every hour of the day, the period comprised between : pm and : pm registered the peak in the number of waypoints recorded out of the homes, and this number decreased steadily as the day progresses. the pattern during weekdays ( fig a) suggests that cases leave their homes and return to them slightly earlier than control groups. as for the weekends (fig b) , both control groups show a similar pattern to that observed for weekdays, nonetheless, cases seem to remain in their homes more often and return earlier. table shows values for different mobility variables. no significant differences were recorded among groups for the following variables: mean distance from home at all times, maximum recorded distance at any given time, and mean time spent in each geographic area at any speed or at static speed. nonetheless, when comparing the number of visits per geographic area, the cases had fewer recorded visits in the area out of the locality but in the municipality ( . vs . , p = . ), and more visits in the area out of the municipality ( . vs . , p = . ), both compared to intradomestic controls. these differences were statistically significant. consistent with this behavior, although non-statistically significant, cases visited more states, municipalities, and regions with high dengue incidence through their follow up, in comparison to both control groups. we next examined the proportion of waypoints recorded by the comparison groups in each area stratified by age (fig ) . there is a notorious difference in the proportion of waypoints among the cases, observing an increase of nearly percentile points in the intradomestic waypoints, recording the highest frequency in cases under age (fig a. however the difference not statistically significant (cases < : median . %, interquartile-range . - . ; cases ! : % iqr - . ; p = . ). we also observed a difference in the area out of the municipality but in the state, where the group of cases aged and older spent the highest proportion of time (age < : % iqr - %; age ! : . % iqr - . ; p = . ). there was no significant difference between cases and population controls, regardless of age. when comparing cases versus intradomestic controls, we observed a statistically significant difference in time spent in area out of the municipality but in the state (cases: . % iqr - . ; ic: % iqr: - . ; p = . ). we found differences in mobility patterns when analyzing data by gender ( fig b) . women had a higher proportion of waypoints within the intradomestic area than men. these differences were statistically significant for intradomestic area (male: . % iqr: . - . ; female: . % iqr . - . ; p = . ), out of their homes but in the locality (male: . % iqr . - ; female: . % iqr . - . ; p = . ) and the area out of the locality but in the municipality (male: . % iqr: . - . ; female: . % iqr: - . ; p< . ). nonetheless, linear mean (p = . ) and maximum (p = . ) distances were not. a) cases vs. intradomestic controls. a conditional logistic regression analysis was performed, including variables identified in the bivariate analysis as having a p value < . , and by data mining techniques using all variables as reported previously [ ] . for bivariate analysis variables were age (continuous and dichotomic [under or and older]) gender, occupation (intra or extradomestic), education (dichotomic), and the proportion of time spent in each geographic area. for the data mining we considered the whole data base. the final model included age (or: . ic % . - . ; p = . ) and the area out of the municipality but in the state (or . ic % . - . ; p = . ). we observed a protective effect in the and older group, and a risk effect when the proportion of time spent in the area out of the municipality but in the state is increased. b) cases vs. population controls. a multiple logistic regression analysis was performed including variables identified with p value < . in the bivariate analysis and data mining techniques using all variables as reported previously [ ] . for bivariate analysis, only the variables age (continuous and dichotomic [under or and older]), gender, occupation (intra and extradomestic), education (dichotomic), proportion of time spent in each area and linear distance were taken into consideration. for the data mining we considered the whole database. the final model included: occupation (or . ic % . - . ; p = . ), proportion of time in the area out of the municipality but in the state ( . ic % . - . ; p = . ), proportion of time in the area out of the locality but in the municipality (or . ic % . - . ; p = . ), and age (or . ic % . - . , p = . ). next we analyzed the geographic distribution of the recorded waypoints for each group, both locally and regionally (fig ) . all three groups recorded waypoints exceeding a km radius from their homes (fig a, b and c ). as expected, most of recorded waypoints were located within the locality. nonetheless, all three groups recorded waypoints exceeding the locality, municipality and state limits. these trajectories were headed mainly to the east, north and west of the locality, and were consistent with the location of the main cities in the area, including cuernavaca (population , ) and cuautla (population , ), the capital city and the second most important city in the state of morelos, respectively. the geographic distribution of the visits performed by cases and df cumulative incidence for the central mexico region, during year , is shown in fig . as seen, this group performed visits to locations with and without df transmission. the number of states, municipalities and regions with high dengue incidence is shown in table . within the locality of axochiapan the most visited areas were identified by dividing the locality in m-radius hexagonal cells (fig a) . the most visited cells were those located in the locality's central area, which correspond to the location of the main market, road junctions and main administrative and / or service offices. the location of the cells considered as common visit sites for cases are shown in fig b. unlike in fig a, the geographical distribution of these cells tends to be peripheral with respect to the locality. using google earth™ we identified the geographic features of each of the cells that was classified as a common visit site for cases. fourteen out of fifteen cells were geographically located within the locality of axochiapan, morelos, and the last one was in the central area of a neighboring small locality (town of tzicatlán) in the state of puebla. as for their typology, xix out of cells clearly corresponded to residential areas (including that in the neighboring state), one cell was a residential area adjacent to a local large business, four cells included small processing plants, a warehouse and a local business, three peripheral cells were crop fields and one cell was clearly a soccer field. previous works have used gps tools for measuring exposure to infectious diseases [ ] [ ] [ ] . in df, recent works in the endemic area of iquitos, peru, have elegantly described human population mobility [ , ] . however, few data are available for infected cases so far. our work builds upon our knowledge of the role played by mobility in denv transmission documenting spatial mobility of subjects from an endemic region that had, or had not been recently infected by denv. our data show that the people from axochiapan stay within their houses or surrounding areas most of the time. this is consistent with previous observations in iquitos, where population rarely moves more than km away from their homes [ ] , however, some individuals recorded movements to very distant locations through the relatively short follow-up period. these movements were present in all three study groups and exceeded a -km radius from the center of the study, covering the neighboring states of morelos, state of mexico, puebla, mexico city, tlaxcala and hidalgo. all of these states are located in the mexican central plateau, which is also the best connected region in the country and therefore it is not difficult to reach those destinations by commute travel [ ] . surprisingly, spatial mobility in humans was not geographically symmetrical in our study, since no movements were recorded to state of guerrero, which is a coastal, highly endemic area for dengue and also a popular destination for leisure activities. as for the reason why the mobility of the individuals is biased towards central plains in mexico and practically absent towards the southern regions, it was a surprising finding also for us, but we think that it has to do with two factors: first the economic activities in axochiapan are mainly related to agriculture, trade and services which are strongly influenced by the needs of mexico city and its metropolitan area, comprised also by the states of morelos, puebla, méxico and hidalgo. the main cities of these states were those that were visited by the cases and in a lesser extent by the controls. secondly, we did not perform any follow-up during summer and christmas holidays, which in mexico are specific periods for leisure. these activities are usually performed in places that might be different that those observed in our study, including the beaches in the southern coast. it is possible that had we performed our follow-up in vacation periods the observed mobility might have been different, and also leave us with a very interesting research question for the future. the large size of the area covered by these few individuals from a small locality (axochiapan has roughly , inhabitants) is of capital importance, given the fact that in mexico, and probably in many other places, epidemiological surveillance, prevention and control activities for df are mainly planned, supported and executed by local health authorities, who rely on the information generated by a number of systems, most of them automated [ ] , but that are usually restricted to their local administrative limits, namely municipality, sanitary jurisdiction or state at best. thus when df outbreaks overcome those limits and a wider coordination is needed, it is probably that the outbreaks are already established and the window of time for effectively applying control measures has been lost. our data show that the cases group had the largest difference on the time spent in the home area with strong age dependence. older cases spent less time in their homes compared to younger cases. as far as the number of visits is concerned, subjects in the cases group, especially those aged over , performed many and more distant visits, than subjects in the intradomestic control group. this difference with the population control group was less marked. this scenario suggests that the population aged over might play an important role in denv persistence and dispersion perhaps working as geographic spreaders. our group previously determined dengue incidence for this age group and proposed a dynamic model which seems to be corroborated by the results presented here [ ] . infected individuals, both symptomatic but also asymptomatic [ ] , may facilitate the infection of extradomestic mosquito populations in a local scale as models have suggested [ ] , or at a regional scale introducing or exporting the virus. given the fact that we performed an uninterrupted -hour follow up, we were capable to register the time when individuals left their homes for whatever activity they performed. to our surprise, we recorded waypoints out of the participant's homes virtually at any hour of the day. although the majority of records show that people in axochiapan have a day-light pattern of activities, we recorded waypoints from cases, between : and : am, which were consistent with participants' declared jobs, which were related to nocturnal activities such as bakers and workers from the local stone processing plant. the dispersion patterns described above, both in space and time, might be of importance in the results obtained in the control of denv transmission in this and similar small localities. the usual schedule considered by local health authorities for applying preventive measures, which favors early hours for insecticide spraying and the visits by entomological control brigades, in a geographically focalized strategy might hinder the efficacy of the actions by the mere fact that people moves from their homes and remain away during the time these actions are normally applied. in our data, the hourly pattern for activity suggests that cases might leave earlier their homes during weekdays, and thus their homes might have a higher probability to remain closed by the time health authorities apply preventive or control measures. it is important to notice the high mean age of the cases in axochiapan, in both the participants in the study and those recorded historically in the state of morelos, in comparison to other endemic areas from mexico and the americas. this is however, consistent with a previous work in the area [ ] , and is probably due to the fact that most ( out of members of the cases group) of the cases that we studied were asymptomatic, also, although not statistically significant, the mean age of the asymptomatic individuals was higher than that from symptomatic individuals (mean age asymptomatic: . vs mean age symptomatic: . , t test p = . ). thus, suggesting a possible stronger role of asymptomatic population with age above as spreaders for denv transmission. previous studies have described that visiting other cases' households is a risk factor for denv dispersion [ ] ; working sites have also been suggested as possible transmission sources outside of cases' households [ ] . models have shown that sites outside homes can play a role in denv transmission and infection [ ] , and a recently published work showed positive correlations in thailand between the aedes spp. house index and specific landscape features [ ] . our findings are consistent with these data since cases coincided in houses different to their own in at least five different geographical locations. additionally we found cases that coincided in four potential working places, and a soccer field. as far as we know, this is the first time that leisure sites have been documented as possible areas for denv transmission. the lack of study of such sites has previously been pointed out as a weakness in the study of human mobility [ ] . as for the common visit sites for controls, we identified cells that were visited only by individuals from both control groups but not by cases. these cells included only one potential working site; whereas the cells commonly visited by cases included several likely workplaces. the identification and study of the extradomestic sites where people coincide is relevant: a recent simulation model has concluded that the selection of areas for df control out of the cases' homes is important not only in terms of the time the subjects spend in them, but also because of the local vector:host ratio, and the other habitual destinations of people that visit the same area [ ] . it is possible that much of the movement in a given society is driven by specific population needs and the possibility to fulfill them within or outside from their own locality. axochiapan is a fairly small and well connected locality to other small cities, all of them endemic for df. it is not unrealistic to think that some of the population needs can be readily fulfilled within the same locality whereas other cannot, compelling the population to move away in a permanent or transitory fashion. if a specific population such as that with age older than becomes infected and effectively play a role as spreaders, then perhaps small and peripheral localities to larger cities might have a key importance in sustaining denv transmission across large geographic areas. the assessment of such situations requires a critical review of existing data and the generation of specific studies that may help us to recast current models and more importantly, to completely understand urban denv transmission [ ] . we have identified some weaknesses in our study. the most relevant is our small sample size which might have hindered our capability to identify clear patterns in the mobility from this mexican community. there is a possibility that any individual belonging to either control group might have get infected during the follow up; thus making her/him eligible to become a case, therefore disqualifying him/her for being a suitable control and consequently introducing an information bias; nonetheless, we believe that, although a possibility, this was negligible due to two reasons: first, the follow-up of days was very short for this event to occur, and secondly because while recovering each gps, we asked all individuals whether they had experienced fever or any other symptom suggesting dengue infection during the follow-up. none of the participants reported any change in their health status. although we understand that a more robust argument to ensure the infected/uninfected status might be performing an elisa to each control after finishing their follow-up in order to be certain about their exposure, financial constrains made impossible this procedure. possible future improvements in our study are: increase the limited sample size, the inclusion of adequate representation for the population under the age of , which probably is a relevant group for transmission during the initial and focalized phases of a df outbreak. although no schools could not be identified in our study as a common site visited by cases, this observation needs to be taken cautiously since our study did not consider the follow-up of children usually studying elementary education. thus, we cannot rule out any role of these sites in dengue transmission at younger ages. additionally, extending the duration of the follow up might improve the chances of successful identification of patterns whose frequencies are longer than a week, such as wage collection, bill payments, and communitarian meetings, among others. this last topic is essential; nonetheless it is limited by technical issues that might be addressed as technology for massive and continuous long-lasting follow up becomes available. finally, the main reasons for which the participants move were not deeply explored in our study, thus we cannot be certain whether the recorded movements indeed depend on non-satisfied needs or on leisure activities. we can only assume that at least those movements performed during the mornings and afternoons between monday and friday correspond to real needs such as employment, education, and supply acquisition, and those performed during weekends are related to leisure. some causes for loss of gps information in field studies have been recently described [ ] . although some of these causes might be present in our study, we believe they did not represent significant sources of bias or information loss, since we took some specific measures. for example, people were prevented of accidentally turning the gps off by strapping a tape in the controls. in order to diminish the probability that the participants could forget their gps units at home we performed a weekly phone call reminding them the importance of the usage attachment according the protocol during each individual follow-up. barriers to signal were not important in the studied area since it is located in a plateau with few elevations, and buildings taller than -stories are practically absent. finally, the gps equipment used in the study had battery autonomy of up to straight hours and enough memory for recording up to five times the mean number of waypoints programmed to collect in each subject. based on our own data and that from recently published works we conclude that gpsbased technology is a solid tool for the study of detailed human mobility in denv transmission or other infectious diseases, which can and must be adopted in public health and epidemiology as a basic instrument. the important geographic dispersion in our results demonstrates the necessity for studying the potential role that human mobility has in denv transmission and outbreak duration and also a strong argument to study and clarify the role that asymptomatic cases might have in dengue virus dispersion. furthermore our data strongly suggest that the size of the areas considered for prevention and control of df outbreaks needs to be revised and that it is necessary to integrate this knowledge into the planning of preventive and control measures, which usually are prone to using basic shapes such as circles or squares as geographic references in order to define limits, ranges, trajectories and points of origin. it is clear that human populations move normally across geographical areas and not only during holidays or vacations. according to our data, the magnitude of these displacements is larger than that considered as an administrative responsibility for local health services providers. this is relevant for denv transmission if a large fraction of that mobile commuting population is also asymptomatic but viremic, facilitating with their movements the exposure of local uninfected mosquito populations with the virus, which might result in an increased geographical dispersion and persistence of the outbreaks due a continuous process of spreading and reintroduction of the virus to susceptible populations. finally, we believe that the data here reported should be valuable for parameterization of mathematical models exploring specific issues in dengue epidemiology such as geographical dispersion of human activities, contact rate among humans in intermediate spots, optimal range for vector control coverage, optimal target places for health promotion activities, impact of coordinated regional collaboration, and transmission dynamics among satellite and large cities. all essential topics that are still to be understood and weighed as drivers in the transmission of this and other mosquito-transmitted diseases. dengue and dengue heamorrhagic fever world health organization. dengue guidelines for diagnosis treatment, prevention and control urbanisation and infectious diseases in a globalised world the global economic burden of dengue: a systematic analysis travel implications of emerging coronaviruses: sars and mers-cov effect of travel on influenza epidemiology man 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and cattle on rangeland in south texas: implications for disease transmission health &demographic surveillance system profile: the kombewa health and demographic surveillance system (kombewa hdss) multiple outbreaks for the same pandemic: local transportation and social distancing explain the different "waves" of a-h n pdm cases observed in méxico during asymptomatic humans transmit dengue virus to mosquitoes house-to-house human movement drives denguevirus transmission epidemiology of dengue and dengue haemorrhagic fever in a cohort of adults living in bandung analyzing the spatio-temporal relationship between dengue vector larval density and land-use using factor analysis and spatial ring mapping population movement and vector-borne disease transmission: differentiating spatial-temporal diffusion patterns of commuting and noncommuting dengue cases recasting the theory of mosquito-borne pathogen transmission dynamics and control strengths and weaknesses of global positioning system (gps) data-loggers and semi-structured interviews for capturing fine-scale human mobility: findings from iquitos authors wish to thank to servicios de salud de morelos for its support to this project. the authors have declare that no competing interests exist. key: cord- - mev otu authors: rathore, abhishek singh; sarker, animesh; gupta, rinkoo devi title: production and immunogenicity of fubc subunit protein redesigned from denv envelope protein date: - - journal: appl microbiol biotechnol doi: . /s - - -y sha: doc_id: cord_uid: mev otu dengue virus (denv) is a vector-borne human pathogen that usually causes dengue fever; however, sometime it leads to deadly complications such as dengue with warning signs (dws+) and severe dengue (sd). several studies have shown that fusion (fu) and bc loop of denv envelope domain ii are highly conserved and consist some of the most dominant antigenic epitopes. therefore, in this study, fu and bc loops were joined together to develop a short recombinant protein as an alternative of whole denv envelope protein, and its immunogenic potential as fusion peptide was estimated. for de novo designing of the antigen, fu and bc peptides were linked with an optimised linker so that the three dimensional conformation was maintained as it is in denv envelope protein. the redesigned fubc protein was expressed in e. coli and purified. subsequently, structural integrity of the purified protein was verified by cd spectroscopy. to characterise immune responses against recombinant fubc protein, balb/c mice were subcutaneously injected with emulsified antigen preparation. it was observed by elisa that fubc fusion protein elicited higher serum igg antibody response either in the presence or in absence of freund’s adjuvant in comparison to the immune response of fu and bc peptides separately. furthermore, the binding of fubc protein with mice antisera was validated by spr analysis. these results suggest that fu and bc epitope-based recombinant fusion protein could be a potential candidate towards the development of the effective subunit vaccine against denv. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. in the recent decades, dengue has emerged as one of the world's most dominant tropical diseases (guzman et al. ) with around million ( % credible interval to million) cases recorded per year, of which only million ( % credible interval to million) cases were manifested clinically (giraldo-garcía and castaño-osorio, ; bhatt et al. ). according to world health organization (who), an estimate of . billion people in countries are living in areas with a high risk of dengue infection (brady et al. ). the recently estimated annual million dengue cases reveals that the dengue disease burden has tripled as compared to previous predictions of to million reported cases without dengue warning signs. nonetheless, , to , patients were hospitalised due to dengue with warning signs (dws+) and severe dengue (sd), and the total annual cost of dengue burden was estimated globally around us$ . billion (giraldo-garcía and castaño-osorio, ; guzman et al. ) . in spite of having paucity of effective vaccines and drugs to expel dengue comprehensively, still it's enduring a big challenge to human (martina et al. ). therefore, next-generation vaccine strategies such as inactivated purified virus, dna or protein-based subunit vaccines, and their fusion chimeras are now going under investigation and some of them even under clinical trials (coller et al. ; danko et al. ; liu et al. ) . recently, dengvaxia (cyd-tdv), a tetravalent live attenuated vaccine has been approved for use in some of the highly dengue endemic areas where the sero-prevalence is higher than % (guy et al. ; pang et al. ). according to abhishek singh rathore and animesh sarker contributed equally to this work. electronic supplementary material the online version of this article (https://doi.org/ . /s - - -y) contains supplementary material, which is available to authorized users. who, it has shown significant efficacy and acceptable safety profile during clinical trials in seropositive individuals; however, it carries a risk of severe dengue infection in seronegative individuals, and also failed to confer subsequent protection in denv- positive people in areas with less exposure of dengue (arredondo-garcía et al. ; durbin et al. ; guy et al. ) . several other vaccine candidates were also formulated based on live-attenuated dengue viruses (e.g. tv , tv ) and inactivated purified virus (e.g. dpiv), of which some have already completed clinical trials and some are currently going under phase ii to iii clinical trials (whitehead et al. ; diaz et al. ). on the other hand, dna or proteinbased alternative subunit vaccines (e.g. tvdv, den- ) and dengue-flavivirus chimeras (e.g. ediii-p k, e-stf , ediii-hbsag) are going either under preclinical study or phase i clinical test (govindarajan et al. ; danko et al. ; castaño-osorio et al., ) . although, still most of them are seemed to be slow immunity booster and require a strong adjuvant and longer immunisation to achieve full protection against each of the dengue serotype, which all make them ill-suited for universal vaccine licence . the dengue envelope (e) protein is composed of three ecto-domains, a membrane-proximal stem and a transmembrane anchor (klein et al. ). various crystal structures have shown that the ecto-domains are arranged into three antiparallel dimer on virus surface in a icosahedral symmetry, where ecto-domain i (ei) is located at the centre holding domain ii (edii) and iii (ediii) in two opposite sites in each of the monomer of a dimeric unit (fibriansah et al. ; kuhn et al. ; modis et al. ). most of the protective antibodies against dengue virus are identified to domain iii, and initially were thought to be a potential subunit vaccine target (murphy and whitehead ; sukupolvi-petty et al. ) . although, recombinant ediii domain injected by plasmid dna or purified from bacterial expression systems was found to be poorly immunogenic, and has shown low protective efficacy in animal model (guzman et al. ). on the other hand, edii has been reported as the dimerisation domain and consists of the most conserved fusion (fu) and bc loop (li et al. ; zhang et al. ). several studies have also shown that the conserved fu loop is highly immunogenic (lai et al. ; cherrier et al. ; smith et al. ) , and induces cross-reactive antibodies, of which some are crosstalk with adjacent bc loop (gallichotte et al., ; cherrier et al. ). moreover, during endocytosis, both of the loops are found to involve in tertiary conformation of membrane fusion (nayak et al. ; sukupolvi-petty et al. ) . therefore, it has been anticipated that both of the loops might have integrated role for boosting cross-neutralizing immunity. in this study, we aim to develop an alternative vaccine by combining fu and bc loop together in a single orf for production of a fusion antigen. although, the recombinant antigen in isolation tends to be poorly immunogenic in vivo; the use of potent immunomodulating compounds, fusion partner or suitable delivery systems improve specific immune response (higgins et al. ; garçon et al. ) . herein, we have expressed the recombinant fubc antigenic protein, optimised its large-scale purification protocol and finally evaluated its protein-specific immune response in balb/c mice. prior to the animal challenge, its secondary structure was checked by cd spectroscopy, and binding specificity was cross-checked with a characterised anti-fusion loop scfv antibody ). all of the male and female balb/c mice were obtained from national institute of nutrition, telangana, hyderabad, india and were maintained in standard light: dark ( : ) cycle with the supplement of adequate standard food, and water was provided from ad libitum. all of the animals were acclimated and randomly distributed into different experimental groups. furthermore, all the in vivo experiments were performed in accordance with the committee for the purpose of control and supervision on experiments on animals (cpcsea) guidelines and were approved by the south asian university institutional animal ethics committee (iaec) that was responsible for the care and use of laboratory animals. the dna sequence of fubc gene was retrieved from fusion (fu) and bc loop of denv serotype envelope protein deposited in the protein data bank (pdb: oan). in order to construct stable and immunogenic protein, highly conserved fusion (fu) and bc loops and their neighbouring residues (amino acid residues to ) were selected by using antigen variability analyser (avana), pblast and multiple sequence alignment tool of ncbi. for convenient expression in bacteria, all of the oligos were codon optimised for e. coli, using codon optimisation tool of integrated dna technology. complete fubc gene was constructed by assembly pcr reactions using four nucleotide long overlapping oligonucleotides. for cloning into pet a expression vector, two unique restriction sites, ecori and xhoi, were also incorporated at the ′ and ′ ends respectively during the final amplification of fubc full-length gene using forward and reverse primers. initially, the full-length fubc gene was cloned into a ta cloning vector using instaclone kit (thermo scientific). positive clones were screened using x-gal bluewhite screening method and digested with ecori and xhoi restriction enzymes. the digested fubc gene was further sub-cloned into a pet a expression vector. the recombinant plasmid (fubc + pet a) was transformed into e. coli xl- gold for cloning, and subsequently into e. coli bl- rosetta (de ) for protein expression. the e. coli bl- (rosetta) cells carrying fubc gene in pet a vector were grown overnight at °c in ml lb broth (luria-bertani medium) containing μg/ml kanamycin (sigma, usa). overnight grown ml primary culture was used to inoculate kanamycin containing ml secondary culture and was further incubated at °c. the incubated secondary culture was induced by . mm isopropyl β-d- thiogalactopyranoside (iptg) when its od reached at around . , and the culture was grown for another h at °c. the cells were harvested by centrifugation at rpm for min. the resulting cell pellet was resuspended in lysis buffer containing mm tris-hcl ph . , mm cacl , with . % triton x- , lysozyme . mg/ml, mm edta and mm pmsf. then, the resuspended cells were kept on a rocker for an hour at room temperature, and sonication was done using % amplitude for five times s on/off pulse. the lysed sample was separated into supernatant and pellet by centrifugation at , rpm for min at °c. finally, fubc expression level in supernatant and pellet were checked on % sds-page. a major fraction of the fubc protein was observed in pellet after a number of efforts made to recover it in a soluble form. then, it was decided to recover soluble protein from pellet fraction. the resulting pellet protein was washed to times with te / ( mm tris ph . and mm edta ) buffer to remove impurities and extra salts. the remaining pellet was re-solubilised using mild denaturing agent such as . m urea and % n-propanol along with pbs buffer (ph . ) and was centrifuged at , rpm for min. the soluble protein fraction was initially purified by using ni-nta agarose beads and was confirmed by western blot using anti-his antibody. however, the purity and yield were not sufficient. therefore, soluble protein fraction was subjected to gel filtration in superose / column by using fast protein liquid chromatography (fplc) for largescale good quality protein production. the column was preequilibrated with pbs ( mm phosphate buffer ph . and mm nacl), and the protein sample was eluted with the same pbs buffer. . ml protein sample was injected in each run by using . ml loop at . ml/min flow rate. the elution profile of injected protein was followed by monitoring uv absorbance at nm on the akta fplc system with u -l uv monitor. different peaks greater than mau were collected in fraction collector and checked using a % sds page. multiple runs of fplc were carried out, and the fraction containing fubc protein was pooled together. finally, fubc was concentrated and desalted by using . ml millipore-amicone filter with a cut-off of kda. concentration of the purified fubc was also measured by using bca protein assay kit. in order to assure the proper folding of purified (> %) fubc protein, secondary structure was analysed by cd spectroscopy at far uv wavelength ranging from to nm. to achieve the best conformational reading, cd spectrum was obtained at the different protein and buffer concentrations, because the cd spectrum of a protein needs to be adequately intense for interpreting the data as the intensity of a cd spectrum directly relies on the protein and buffer concentration (kelly et al. ; miles and wallace ) . the cd spectra of pbs buffer and fubc protein samples at . mg/ml and . mg/ml concentrations (diluted in mm and mm pbs buffer, ph . ) were recorded at far uv spectra ranging from to nm with a step size of nm to bandwidth nm. the measurement was performed at room temperature ( °c), and the uv spectra were recorded for each sample with five scans. the baseline cd spectrum of the buffer was deducted from the spectrum containing the protein to yield the actual fubc protein cd spectrum. the mean residue ellipticity [θ] mrw at wavelength λ was quoted in units of degree cm / dmol, and was calculated as [θ] mrw, λ = mrw × θλ/ × d × c, where θ is the observed ellipticity (degrees) at wavelength λ, d is the path length (cm) and c is the concentration (g/ml). mrw is the mean residual weight for the peptide bond which is given as mrw = m/(n − ); where m is the molecular mass of the polypeptide chain (in da), and n is the number of amino acids in the chain; the number of peptide bonds is n − . complete and incomplete freund's adjuvant (sigma, usa) was used for primary (day ) and booster immunisation (days , and ) respectively. to prepare a primary dose of the immunogen, complete freund's adjuvant (fa) was mixed with equal volume of purified fubc ( μg) in pbs and emulsified by vigorous vortex. similarly, three booster doses of immunogen were prepared with an equal volume of fubc protein ( μg) and incomplete freund's adjuvant. all of the immunogens were prepared according to the protocol "immunization of mice" by maira-litrán, . finally, emulsified fubc immunogens were checked by observing stable droplet on the water surface. healthy balb/c mice ( weeks old, male and female) were randomly distributed into four different groups. each group was immunised with different antigen preparations. out of the four, two groups were immunised with fubc antigen: one group was injected with fubc protein with adjuvant and other was with fubc protein without adjuvant. rest of the two groups were immunised with fu and bc peptide: one group was injected with fu and bc peptides with adjuvant, and other was fu and bc peptide without adjuvant. each of the groups was also subdivided into male and female sets, and with each set, one adjuvant control was used replacing adjuvant with pbs buffer. the mice of each group were subcutaneously immunised with emulsified antigen preparations, first dose (day ) with cfa and three subsequent booster doses (at days , and ) with ifa (according to protocol maira-litrán ). after days of the final booster dose, blood samples were collected by retro-orbital cavity, and all the antisera isolated from blood were stored at − °c for further analysis. recombinant fubc protein-specific igg was measured by indirect elisa. for this, well elisa plates were coated by incubating overnight at °c with fubc protein ( μg/ well) in mm sodium carbonate-bicarbonate buffer ph . . blocking was done for h with % bsa in pbst (pbs plus . % tween ). serum collected from immunised and control mice were incubated for h at °c after making double dilutions in pbst starting from / μl. subsequently, the elisa plate was washed once with pbst, and μl of anti-mouse hrp-conjugated secondary antibody ( : dilution in pbst) was added and incubated for min at room temperature. then, the plate was washed three more times with pbst, and the binding reaction was developed by adding μl/ well opd substrate (prepared in a phosphate-citrate buffer, . m, ph − . plus . % h o ). the reaction was stopped by adding μl of . m h so just after observing an optimum colour, and the absorbance was measured at -nm wavelength using biotek synergy ht microplate reader (winooski, vt). mean absorbance was plotted against anti-sera dilutions, and two-way anova was performed to find a statistically significant difference between two groups. comparison of the immune response in different groups of antisera was made by converting each elisa curve to linear equations (y = mx + c), and computing serum dilutions for a fixed elisa absorbance. the data are expressed as mean ± standard deviation (sd). p values of < . were considered statistically significant. to validate the immune response generated by recombinant fubc, collected mice anti-sera were allowed for spr interaction experiment with a autolab esprit instrument. similar to elisa, here also recombinant fubc protein of denv envelope was immobilised on a gold plate of spr device. for coupling of fubc protein on gold disc, -ethyl- -( dimethylaminopropyl) carbodiimide (edac) and nhydroxysuccinimide (nhs) were applied for activation. nhs activates the carboxymethyl groups by creating a highly reactive succinimide ester on the disc surface, which reacts with amine and other nucleophilic groups on proteins that subsequently help to bind the target protein on activated disc surface. ethanolamine was added to block the remaining activated carboxymethyl group. for the qualitative assay, all of the serum samples (diluted in running buffer) were applied at a flow rate of μl/min over min. in between injections, the surface of the sensor chip was regenerated by injecting m nacl at the same flow rate for s. in addition, surface coupling was done by using buffer ( mm nacl, mm edta, . % surfactant p and mm hepes-naoh) at ph . , and the buffer containing mm phosphate and mm nacl at ph . was used for sample running. initially, a series of serum dilutions were applied as analytes to find an optimum refractive index. it was observed that the refractive index at : dilution was within the detection limit among the series of serial dilutions (fig. a) . therefore, : dilution of different experimental samples was applied as standard for further comparative analysis between fubc immune response with and without freund's adjuvant. serum control (the serum collected before immunisation) sample with the identical dilution was also applied to find the basal (non-specific) immune response of serum. finally, refractive index was then analysed from association and dissociation curve of the spr sensorgram. the fubc synthetic gene sequence was deposited in genbank database with accession number mn . generally, dengue envelope (e) folded into three distinct domains (designated by domain i, ii and iii), membrane proximal stem and a transmembrane anchor (klein et al. ) . throughout these structural element, four highly conserved regions have been identified by in silico sequence analysis, and two of them were found in the domain ii with a very less informational entropy . further studies have revealed that these conserved regions are the part of previously characterised flavivirus fusion (fu) and bc loop (kuhn et al. ) . during the process of endocytosis, this hydrophobic fusion loop remains buried at the dimer interface in the prefusion state and forms cluster into larger hydrophobic surface at one end to form trimer at later state that finally initiates membrane fusion (klein et al. ). in addition to this structural property, fu and bc regions consist some of the most potent antigenic epitopes that were identified by denv neutralising conformation sensitive anti-denv hmabs (goncalvez et al. ; costin et al. ; yamanaka et al. ) . therefore, these two conserved fu and bc loops have been selected to design a recombinant fubc antigenic protein for the development of dengue subunit vaccine. in this study, an alignment of the domain ii amino acid sequences of denv - envelope proteins spanning residues from to was used to find optimum conserved sequence for the development of fubc fusion protein (fig. a) . the structural details of truncated fubc protein compare to whole envelope in dengue and fu, and bc peptides were also analysed by modelling each of their three dimensional structure. it reveals that the presence of three anti-parallel β-strands and one disulphide linkage play a crucial role in preserving the three dimensional conformations of truncated fubc protein same as in original denv envelope protein (fig. b, c) . ironically, due to the absence of anti-parallel β-strands and a single protein frame of fu and bc loops, these two separately expressed peptides fail to form similar three-dimensional conformation as in original denv envelop (fig. c) . the electrostatic and solvent accessible areas of fubc truncated protein are also comparable with original dengue envelope. hence, it is speculated that the recombinant fubc protein would be an alternative vaccine target of whole dengue envelope. fu loop is shown in orange and bc loop is shown in yellow. b in denv envelope, the fu and bc loops are present at the end of domain ii and are linked by the di-sulphide linkage that holds both loops together to form a stable structure that can also work as an epitope. c when the structure of fubc is compared to original denv envelope, it is plausible that the presence of three anti-parallel β-strands present in fubc protein (same as denv protein) plays a crucial role in maintaining the three dimensional conformation of fubc protein as it is in denv protein. another major factor that plays in conformation of fu and bc loops is the disulphide linkage between the two highly conserved loop in the fubc protein that ensures the same conformation of denv envelope as it is shown in b and c, and d due to the lack of anti-parallel β-strands and same protein frame while these peptides are expressed separately, it is unlike to form di-sulphide linkage and fold in the same conformation as reside in denv envelope and fubc proteins for in vitro synthesis of short antigenic protein, a recombinant fubc gene was constructed by assembly pcr using overlapping oligonucleotides, designed from fu and bc loop encoding dna sequences of dengue envelope. therefore, full length of fubc gene was amplified by using and end primers flanked by ecori and xhoi restriction sites. the fubc gene was then cloned into a ta cloning vector, and the clones were screened by colony pcr using fubc gene specific end primers. two positive clones were then confirmed by restriction digestion using ecori and xhoi enzymes (fig. s a-d) . the digested and purified fubc insert gene was further sub-cloned into pet a expression vector, and the positive clones were confirmed by restriction digestion (fig. s b) and sanger sequencing. primarily, the recombinant fubc protein expression was observed only in pellet fraction; there was no such fubc equivalent protein band in supernatant fraction, while they were separated on sds page (fig. s c) . therefore, fubc expression level was checked further by lowering growth temperature and iptg concentration, but no significant change was noticed in supernatant fraction. chaperone-assisted folding system did not help remarkably to express the fubc recombinant protein in the soluble form. therefore, the insoluble pellet fraction of fubc protein was further utilised in mild solubilisation process to recover soluble fubc protein. initially, the recovered soluble fubc protein fraction was purified by using ni-nta agarose beads, and the purified protein band was confirmed by western blot analysis using anti-his tag monoclonal antibody (fig. s ) . although, ni-nta affinity chromatography has not revealed good purification quality and the yield was also not sufficient. furthermore, gel filtration chromatography was used to recover good quality large-scale recombinant protein, and single distinct peak greater than mau was observed at approximately . ml position for all of injected fubc protein samples (fig. a) . after pooling together, the peak fractions were separated on % sds-page, and single bright band was observed at molecular weight . kda (fig. b) . originally, the fu and bc loop of dengue envelope is composed of three anti-parallel β-sheets. and the formation of recombinant fubc secondary structure was characterised here by negative bending at nm and nm wavelength (kumagai et al. ) . the cd spectrum of recombinant fubc protein has showed significantly decreased spectral peaks around and nm that signify the formation of β-sheet (fig. ). in addition, it was noticed that the acquired cd spectrum of fubc at a protein concentration of . mg/ml in mm pbs buffer was adequately intense (fig. ) . therefore, it can be inferred from fig. (lower panel) that fubc at concentrations of . mg/ml and . mg/ml in mm pbs retained the best globular folded state as compared to other conditions. immune response to the purified fubc protein in balb/c mice from indirect elisa, it was observed that the serum igg levels in both male and female groups treated with fubc proteins were significantly higher than those treated with only adjuvant and pbs control (fig. a, b) . in addition, the response with only fubc recombinant protein (without adjuvant) in the female group was also observed higher than their male counterpart group (fig. b) . all of the elisa data were also found statistically significant by two-way anova (table s ) . similarly, by spr assay, high level immune response was also observed for serum injected with recombinant fubc protein along with adjuvant, and without any adjuvant, the response was also higher than the control (serum with pbs only) (fig. b ). in post-dengue infection, most of the circulating antibodies are non-neutralizing and found to be raised against dengue envelope e protein and the prm protein (wahala and de silva ) . due to the absence of highly specific neutralising antibodies in secondary infections, cross-reactive nonneutralising antibodies usually enhance the dengue severity (de alwis et al. ). according to the revised dengue case classification (denco), to % of secondary infections with another serotype causes life threating dengue with warning signs (dws+) and severe dengue (sd). it was reported that serotype-cross-reactive non-neutralising antibodies enhance the entry of dengue genome into fc receptor-bearing monocyte cells and promote disease severity by a process known as antibody-depended enhancement (ade) flipse et al. ) . that means nature of the antibody response to denv is most likely to play a major role in defining disease outcome. therefore, it is predictable that antibodies that recognise specific neutralizing epitopes help in virus clearance and reduce symptoms; however, antibodies that recognise non-neutralising epitopes lead to more severe forms of disease like dws+/sd. hence, there is an urgent need for an advanced vaccine which could generate highly specific and cross-neutralising antibody (costin et al. ). recently, several attempts have been made towards the development of a potent dengue vaccine. the most advanced candidate, dengvaxia (cyd-tdv), was licenced in some of the dengue endemic countries (imai and ferguson ) . however, it was revealed risky in children or naïve dengue patient with severe infection as they were vaccinated (arredondo-garcía et al. ) . therefore, considering safety issues, production of recombinant subunit vaccine with efficient immune protective properties is looking attractive (govindarajan et al. ) . meanwhile, an admixture of four live attenuated recombinant dengue vaccine tv /tv have completed phase iii clinical trial and licenced to several manufacturers including butantan, vabiotech and merk (whitehead et al. ) . moreover, some recombinant tetravalent vaccines (e.g. den- e, tvdv) expressing the prm and e genes of each of the four denv serotypes from plasmid dna, have already completed phase i clinical trial (govindarajan et al. ; danko et al. ). it has also been shown that the recombinant dengue envelope domain iii can inhibit dengue infectivity, and induce dengue-neutralising immunoglobulin in mice (hermida et al. ) . in addition, a number of antibodies which were raised in mice and fig. purification of recombinant fubc protein by size exclusion chromatography. a fplc chromatogram of fubc protein expressed in e. coli inclusion bodies. three distinct peaks at . ml, . ml and . ml position were collected separately. b gel image of unpurified and purified fubc protein sample separated on % sds-page. lanes and represent the un-purified sample and lanes and represent purified protein sample collected from peak at position . ml fraction chimpanzees against dengue domain ii fusion loop were found cross-reactive to other flaviviruses (goncalvez et al. ; stiasny et al. ) . although, the hmabs specific to dengue domain ii fusion loop were not found equally effective on other flaviviruses including wnv, yfv and other denv strain (costin et al. ) . therefore, it was conjectured that adjacent to the fusion loop, additional contact residues of original domain ii surface structure might be involved to raise cross-neutralizing antibodies. several studies have identified that adjacent to the fusion loop, another similar loop exists in most of the flaviviral (e.g. wnv, tbe, jev, yfv) envelope (fibriansah and lok, ; li et al. ) . our previous bioinformatics studies have also shown that the envelope of all denv serotype consists of four conserved regions (> %), and two of them were found in domain ii, in which one is fusion loop (fu) with more than % conservation and another is its nearby bc loop (fig. a ) . therefore, these two highly conserved loops were targeted in this study for the development of a fusion protein. by using reverse dna technology, we have previously shown the structural integrity ( fig. b-d) and binding specificity of fubc protein with an anti-fusion loop scfv antibody, derived from dengue and wnv-specific mab e (rodenhuis-zybert et al. ; rathore et al. ) . the very early challenge of subunit vaccine design is to produce large quantities of functional protein. hereby, we have successfully optimised the expression and purification methods for the production of large-scale high-quality recombinant fubc protein. the e. coli expression system was used here, and the recombinant protein was found to be expressed in higher quantity, though most of the protein was extracted initially in pellet fraction. no significant change was noticed by altering regular growth temperature and inducer concentration. therefore, we have utilised the mild-solubilisation methodology to recover soluble fubc protein from insoluble pellet protein. for purification, firstly, we have used convenient ni- nta affinity purification method, and by adjusting the lysis and elution buffer, we were able to purify recombinant fubc protein to some extent but the quality and quantity was not sufficient. finally, to scale up the quality protein production, size exclusion chromatography was used in akta fplc system. to confirm the recombinant protein expression, simple western blot was performed by using anti-his monoclonal antibody as primary. furthermore, in vitro structural integrity and functional authenticity of the experimental fubc protein were also verified by cd spectroscopy (fig. ) and indirect elisa (fig. ) . however, most of the hmabs identified earlier from dengue patient serum were predominantly cross-reactive and recognise epitopes containing highly conserved residues at the fu loop of domain ii (lai et al. ). however, having such sequence homology in fu loop of all flaviviruses, these hmabs are non-neutralizing against heterologous serotypes (lai et al. ; smith et al. ) and thus was found to be responsible for ade in animals (goncalvez et al. ). previously, it has also been stated that this scenario might happen due to the cryptic nature and poor accessibility of the fusion loop epitope on the surface of the mature virion (lai et al. ; cherrier et al. ). however, in addition to the partially exposed fusion loop on immature flaviviruses, some neutralizing hmabs were also found to bind with bc loop (costin et al. ) . therefore, it is suggestive that along with conserved fusion loop, adjacent less conserved linker sequence and bc loop might be required to be exposed as a whole to generate effective cross-neutralizing immunity. our current mouse immunisation experiment also supports this idea as it was observed that the antibody response to full length fubc protein was stronger than the response elicited by the fu and bc peptides in separate (figs. and ) . moreover, without freund's adjuvant, recombinant fubc protein was found immunogenic, and the response in female mice was stronger than in male (figs. and ) . these observations were also symmetrical with other studies where it was stated that female mice have a better immune response due to the higher number of leukocytes occupying the naive peritoneal and pleural cavities, and also have a number of t-and blymphocytes as well as macrophages (terres et al. ; weinstein et al. ; klein et al. ) . therefore, we have used female mice serum samples for further qualitative elisa and spr test to quantify immune response of recombinant fubc protein. it was noticed that igg antibody response to fu, and bc peptides were significantly lower than the response observed to fubc recombinant protein, and the immune response achieved to fu and bc peptide with freund's adjuvant was significantly higher as compared to the peptides without adjuvant and serum control (fig. ) . two-way anova was also performed to analyse the significance of igg immune responses between fubc + fa and fubc without fa; fu, bc peptide + fa and fu, bc peptide without fa. from f values and p values obtained by anova test, it can be interpreted that the difference between fubc protein with freund's adjuvant and fu, bc peptide with freund's adjuvant were significantly higher than protein and peptides injected without freund's adjuvant (table s ) . also, the igg response to recombinant fubc protein was recorded better than fu and bc peptides in both cases either with or without adjuvant. in addition, the p value of igg immune response between fubc protein with fa and without fa was found insignificant (p = . > . ) that suggests that fubc without any adjuvant is sufficient to elicit significant immune response (table s ) . consistently, it was observed by spr experiment that the refractive index of fubc + fa was significantly higher than fubc + without fa and serum control (fig. b) . since, the newly developed fubc recombinant protein expresses vastly in e. coli and induces significant immune response in mice, it might be a good agent of dengue subunit vaccine development. due to the presence of highly conserved fusion loop epitope, overlapping less conserved linker sequences and also bc epitope, this fusion protein might induce cross-neutralisation immunity against heterologous dengue serotypes. though, still a lot of investigations, like the evaluation of a proper adjuvant to induce robust immune response, the memory immune response generated against the fubc protein both by humoral and cell-mediated immunity, and whether this memory response will provide protection against a secondary encounter with denv, are required before going clinical trial. nevertheless, the production process and immune response of this fusion protein would provide new insight for the development of dengue subunit vaccine. four-year safety follow-up of the tetravalent dengue vaccine efficacy randomized controlled trials in asia and latin america the global distribution and burden of dengue 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immunogenicity and protective efficacy in nonhuman primates from research to phase iii: preclinical, industrial and clinical development of the sanofi pasteur tetravalent dengue vaccine development of the sanofi pasteur tetravalent dengue vaccine: one more step forward dengue infection domain iii of the envelope protein as a dengue vaccine target a recombinant fusion protein containing the domain iii of the dengue- envelope protein is immunogenic and protective in nonhuman primates immunostimulatory dna as a vaccine adjuvant targeting vaccinations for the licensed dengue vaccine: considerations for serosurvey design how to study proteins by circular dichroism structure of a dengue virus envelope protein late-stage fusion intermediate sex-based differences in immune function and responses to vaccination structure of dengue virus: implications for flavivirus organization, maturation, and fusion going deep into protein secondary structure with synchrotron radiation circular dichroism 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a mouse monoclonal antibody against dengue virus type mochizuki strain targeting envelope protein domain ii and displaying strongly neutralizing but not enhancing activity publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -ixiam qr authors: zhu, xun; he, zhenjian; yuan, jie; wen, weitao; huang, xuan; hu, yiwen; lin, cuiji; pan, jing; li, ran; deng, haijing; liao, shaowei; zhou, rui; wu, jueheng; li, jun; li, mengfeng title: ifitm ‐containing exosome as a novel mediator for anti‐viral response in dengue virus infection date: - - journal: cell microbiol doi: . /cmi. sha: doc_id: cord_uid: ixiam qr interferon‐inducible transmembrane proteins , and (ifitm , ifitm and ifitm ) have recently been identified as potent antiviral effectors that function to suppress the entry of a broad range of enveloped viruses and modulate cellular tropism independent of viral receptor expression. however, the antiviral effect and mechanisms of ifitms in response to viral infections remain incompletely understood and characterized. in this work, we focused our investigation on the function of the extracellular ifitm protein. in cell models of denv‐ infection, we found that ifitm contributed to both the baseline and interferon‐induced inhibition of denv entry. most importantly, our study for the first time demonstrated the presence of ifitm‐containing exosome in the extracellular environment, and identified an ability of cellular exosome to intercellularly deliver ifitm and thus transmit its antiviral effect from infected to non‐infected cells. thus, our findings provide new insights in the basic mechanisms underlying the actions of ifitm , which might lead to future development of exosome‐mediated anti‐viral strategies using ifitm as a therapeutic agent. conceivably, variations in the basal and inducible levels of ifitms, as well as in intracellular and extracellular levels of ifitms, might predict the severity of dengue virus infections among individuals or across species. study for the first time demonstrated the presence of ifitm-containing exosome in the extracellular environment, and identified an ability of cellular exosome to intercellularly deliver ifitm and thus transmit its antiviral effect from infected to noninfected cells. thus, our findings provide new insights in the basic mechanisms underlying the actions of ifitm , which might lead to future development of exosome-mediated anti-viral strategies using ifitm as a therapeutic agent. conceivably, variations in the basal and inducible levels of ifitms, as well as in intracellular and extracellular levels of ifitms, might predict the severity of dengue virus infections among individuals or across species. dengue virus is an enveloped, single-stranded positive strand virus, belonging to the flavivirus genus of the family flaviviridae, with four related but distinct serotypes (denv- to ) (halstead, ; simmons et al., ) . denv is the aetiologic agent of dengue fever (df), the most prevalent arthropod-borne viral disease in humans with more than - million cases worldwide annually. denv is transmitted by mosquito aedes aegypti or aedes albopictus in the tropical and subtropical regions, where these mosquitoes are endemic, placing . billion people at risk of infection globally (guzman et al., ; simmons et al., ) . of note, % of infected individuals develop more severe, and often lethal, syndromes, designated dengue haemorrhagic fever (dhf) and dengue shock syndrome (dss), with children bearing most of the disease burden guzman et al., ) . while genotypic differences of denvs have been indicated to be associated with variations in viral virulence, heterotypic dengue virus antibodies are believed to be a risk factor for the development of dhf or dss in secondary infections (guzman et al., ) . despite the formidable toll imposed by denv on world health, no approved vaccine or effective specific anti-denv therapies are currently available for denv infection (halstead, ; guzman et al., ) . as a major host defence system against viral infection, the interferon (ifn) family, notably ifn-alpha and ifnbeta, initiates a potent antiviral response that activates the innate immunity mediated by induction of more than ifn-stimulated genes (isgs), leading to the establishment of an anti-viral state (sadler and williams, ) . ifn inducible transmembrane proteins , and (ifitm , and ) have recently been identified as antiviral mediators induced by ifn, to confer host cells resistance to a variety of pathogenic viruses such as influenza a virus (iav) (brass et al., ; feeley et al., ; huang et al., ; everitt et al., ) , west nile virus (brass et al., ; jiang et al., ) , denv (brass et al., ; jiang et al., ; chan et al., ) , vesicular stomatitis virus , marburg virus (huang et al., ) , ebola virus (huang et al., ) , sars coronavirus (huang et al., ) , human immunodeficiency virus (lu et al., ) and hepatitis c virus (yao et al., ) . mechanistically, ifitms are the only known isg products that act to restrict the entry step of viral infection process (feeley et al., ) . in particular, brass et al. demonstrated that the action of a single intrinsic immune effector, ifitm , profoundly effected as an essential barrier to iav infection in vitro, in a knockout mouse model and in humans, by blocking the virus-cellular membrane fusion and thus preventing cytosolic entry of the virus (brass et al., ; feeley et al., ; everitt et al., ) . interestingly, another study revealed that ifitm proteins could interfere with the antibody-dependent enhancement (ade) effect during secondary dengue virus infection, which bypassed the ifn-mediated restriction (chan et al., ) . taken together, these previous studies have defined ifitm as a mediator required for the anti-viral action of ifn, representing a key component of human anti-viral defence system, probably through targeting an early step of viral infection. open questions, however, remain concerning the detailed biological functions of ifitm in ifn-triggered cascades and the precise mechanisms how the host cellular defence system is involved in ifitm activation in response to viral infections. such knowledge will provide insights in designing new antiviral therapeutics using ifitm proteins. furthermore, it is worth noting that secreted or membrane-bound proteins are of particular interest in drug development, because their extracellular nature renders them more accessible for therapeutic intervention. as a novel alternative secretion system, exosomes are small vesicles ( - nm in diameter) of endocytic origin that are released from cell into the extracellular environment, under both normal and pathological conditions (thery et al., ) . exosomes are formed through the inward budding of late endosomal membrane that gives rise to intracellular multivesicular bodies (mvbs), which involve their fusion with the plasma membrane and release of mvbs into the extracellular environment as exosome (thery et al., ) . prior work has focused on exosome as a new family member of 'bioactive vesi-cles' that function to promote intercellular communication, by shuttling for proteins, lipids and rnas of the cells, and participate in various biological processes including immunomodulatory events (schorey and bhatnagar, ) . while shuttling for components of pathogenic microbes, exosome could stimulate immune responses. for instance, exosome isolated from cells infected with mycobacterium tuberculosis, have been shown to contain bacterial components and promote antigen presentation and macrophage activation (bhatnagar and schorey, a; bhatnagar et al., b) . moreover, exosome may also promote intercellular spreading of infectious cargo, such as the observed cell-to-cell transmission of hiv (gould et al., ; izquierdo-useros et al., ). on the other hand, despite these advances in our understanding of the functions of exosome, the physiological significance of exosome in shuttling bioactive molecules key to the host defence system remains enigmatic. more recently, atanu et al. demonstrated that apobec g, which belongs to an isg family of cellular cytidine deaminases that effect to restrict replication of a variety of exogenous retroviruses, was exported by exosome and conferred antiviral phenotype to recipient cells (khatua et al., ). li et al. propose an antiviral mechanism of ifn-α activity that involves the induction and intercellular transfer of antiviral molecules (such as lamp- protein, apobec g proteins, ifi mrna, ddit mrna, hsa-mir- , hsa-mir- and hsa-mir- etc.) from liver non-parenchymal cells to hepatocytes via exosomes, by using hbv infection as a model . they also observed similar exosome-mediated transfer of ifitm mrna from macrophages to hepg . . cells, but there is not in-depth study on whether the anti-hbv activity is dependent or independent of exosome-mediated transfer of ifitm mrna . it is therefore tempting to hypothesize that direct exchange of isgs proteins transferred by exosome among host cells might contribute to the establishment of anti-viral state in uninfected cells, in addition to the direct action of ifns stimulation. in this study, we focused our investigation on the function of the extracellular ifitm protein. in cell models of denv- infection, we found that endogenous basal protein levels of ifitms inversely correlated to denv- infection, and induction of ifitms in cells with low basal protein levels was sufficient to drive protection of host cells from denv- infection at entry. conversely, loss of ifitm in host cell pronouncedly enhanced denv- infection. most importantly, our study for the first time demonstrated the presence of ifitm-containing exosome in the extracellular environment and the function of these exosome vehicles for inter-cellular transmission of antiviral proteins. in an effort to investigate whether endogenous ifitm in host cells plays a role in the interaction between denv and the host cells, we first compared the expression of ifitm in denv-permissive cell lines with versus without denv- infection. notably, following infection with denv- at multiplicity of infection (moi) of in human cell lines permissive for denv- replication, expression of ifitm was found to be inducible by denv- infection in various cell lines as demonstrated by western blotting analysis (fig. a) . next, we analysed the correlation of ifitm level in cell lines with denv- infection. we found that while endogenous ifitm expression was varied in different cell lines, cells expressing low level of ifitm protein were more susceptible to denv- infection (fig. b) . further exponential regression analysis showed that the level of ifitm protein in host cells inversely correlated with their susceptibility to denv- infection significantly (r = . , p = . , n = , fig. c ), suggesting that ifitm might be an important cellular restriction factor for denv infection. to evaluate the role of ifitm proteins in denv infection, we generated permanent cell lines stably overexpressing human ifitm , or , or control vector, respectively, with u and hela cells ( fig. s a and c). as show in fig. s b and d, ifitm , ifitm or ifitm overexpression drastically diminished the number of denv- infected cells, as indicated by twofold to fourfold reduction of denv- viral e protein accumulation in virally infected u and hela cells. similar profound restriction was also seen when ifitm was transiently transduced in t, despite a lesser extent of ifitm overexpression than that of ifitm or ifitm ( fig. s e and f), demonstrating the potency of ifitm in conferring host cells resistance to denv infection. we next examined the effect of ifitm on the entry step of denv- , and found that entry of denv- was reduced by ifitm by approximately twofold (fig. a ), as controlled with the g monoclonal neutralizing antibody to demonstrate a successful entry blockage, suggesting that the disruption of denv replication by ifitm was associated with a targeted abrogation of viral entry. since ifn stimulated signalling plays a pivotal role in protecting cells from viral infection and damage, we investigated the functional significance of ifitm in ifnmediated anti-denv response. as shown in fig. s a and b, compared with huvec cells transfected with control scramble sirna, transfection of ifitm -sirna- and ifitm -sirna- efficiently depleted basal-level ifitm expression, and increased the permissiveness of the transfected cells to denv- infection, as indicated by a > -fold increase of infection, in huvec that constitutively express high basal level of endogenous ifitm expression. similar effect of ifitm depletion was reproducible in another cell model, in which the basal ifitm level is low but greatly inducible by ifn-α. sirna silencing of ifitm markedly attenuated the protective effect of ifn-α against denv- infection in hela cells, leading to profoundly increased infection by denv- in the presence of ifn-α, by . -or . -fold ( fig. s c and d) , indicating that the depletion of ifitm could decrease the antiviral actions of ifn-α. we also performed denv binding/entry assay following knockdown of ifitm expression. our results showed no effect of sirna silencing of ifitm on the binding of denv- to hela cells, but the penetration of denv- into cells was significantly promoted by the depletion of ifitm (fig. s e) . thus, our results suggest that ifitm is involved in mediating ifn-induced cellular response against denv infection. in an attempt to analyse the biochemical properties of ifitm , we found that the protein was present both intracelluarly and extracellularly. specifically, cell culture supernatants were collected from parental huvec cells or hepg cells that express high basal level of endogenous ifitm , from huvec or hepg cells transfected with ifitm sirna- (huvec-siifitm or hepg -siifitm ), from huvec or hepg cells transfected with non-targeting control sirna (huvec-sinc or hepg -sinc), from t cells transfected with pcdna ( t-vector), and from t cells transfected with the pcdna-ifitm , , -flag construct ( t-ifitm , , ) for h. supernatants derived from each of the above groups with different treatments were collected and centrifuged at g for min, and then g for min at °c to remove cells and cell debris, followed by filtration with . μm filters. then the supernatants were concentrated by -fold using an amicon ultra- centrifugal filter unit with kda cut-off value, and subjected to western blotting analysis using an anti-ifitm antibody. our data showed that ifitm protein was present both in the cytosol and in the culture medium ( fig. a-d) , suggesting the possibility that ifitm could be released to extracellular space. it was also noteworthy that the expression of ifitm in the antiviral effect of ifitm mediated by exosome supernatants collected from cultured huvec cells is lost when ifitm was knocked down or exosome secretion was inhibited by gw (an exosome-release inhibitor) (fig. d ). as the amino acid sequence of ifitm does not contain a putative signal peptide for protein secretion, we sought to explore the possibility that the exportation of ifitm could be through an exosome-mediated a-d. cell culture supernatants of huvec or hepg cells transfected with ifitm sirna- (huvec-siifitm or hepg -siifitm ), from huvec or hepg transfected with non-targeting control sirna (huvec-sinc or hepg -sinc), from t cells transfected with pcdna ( t-vector), or from t cells transfected with the pcdna-ifitm , , -flag construct ( t-ifitm , , ) for h, were collected and concentrated for sds-page and immunoblotting, using an anti-ifitm antibody, with cell lysates as positive control. e. electron micrographs of crude exosomes negatively stained with uranyl acetate and examined at kv are shown. f. purified ifitm -containing exosomes derived from each group of cells above described were analysed by immunoblotting with anti-ifitm , anti-flotillin- , anti-cd , anti-calnexin (endoplasmic reticulum, er marker) and anti-gm (golgi marker) antibodies. ifitm is identified in the exosomes derived from each group of cells as indicated. mechanism. to achieve this, exosomes were prepared from the culture media of huvec, huvec-sinc cells, huvec-siifitm cells, ifn-α-treated t cells ( t-ifn), t-ifitm cells, t-vector cells and t cells, and subjected to sucrose gradient ultracentrifugation. the electron microscopic examination of the exosomes revealed vesicles ranging in size from nm to nm (fig. e) . interestingly, ifitm was detected in exosomes purified from the culture supernatant of huvec and huvec-sinc cells, and was also abundant in those produced by t-ifitm or t-ifn cells. in contrary, however, exosomes isolated from the t cells, t-vector cells and huvec-siifitm cells did not contain detectable ifitm (fig. f ). purified exosomes were further characterized for the presence of conventional markers for exsosomes, namely, flotillin- and cd , and non-exosomal markers calnexin (endoplasmic reticulum marker) and gm (golgi matrix marker) using western blotting analysis. as shown in fig. f , exosomes collected from each group was shown to be positive for flotillin- and cd , but negative for calnexin and gm , verifying that there is no contamination by other subcellular fractions in the purified exosome preparations. the ifitm protein contained in exosome composition was further verified by mass spectrometry (fig. s ). together, our data demonstrate that secretion of ifitm in exosome is not limited to huvec that express high level of endogenous ifitm , but also by t cells undergoing ifn stimulation or transiently expressing exogenous ifitm . the finding that ifitm is contained in exosome and exported from cells in which it is expressed prompted us to investigate whether the ifitm -containing exosomes are internalized by other cells in the system so that the anti-denv activity can be transferred to the recipient cells. in this study, we tested such a possibility by incubating hela cells, which express a low-level of ifitm , with exosomes collected from huvec, huvec-sinc, huvec-siifitm , t-ifitm or t-ifn cells. indeed, as shown in fig. a , an increased abundance of ifitm was detected in the recipient hela cells treated with huvec-, t-ifitm cell-, or t-ifn cellderived exosomes ( μg ml − ), but not with those from t-vector cells. moreover, detection of ifitm proteins in the lysates of recipient hela cells treated with ifitm -laden exosomes derived from t-ifitm and huvec cells, as determined by immunoblotting, was dose-and time-dependent ( fig. c and d) . notably, as it was reported that exosomes might be capable of delivering exogenous small rna such as sirna and microrna in vitro and in vivo (van den boorn et al., ; pan et al., ; shtam et al., ) , in this experiment we preformed the protein transfer assay in recipient hela cells transfected with ifitm -sirna (hela-siifitm ) to rule out the potential side-effect of ifitm -sirna possibly delivered by exosomes on the endogenous or exosome-delivered ifitm mrna in the recipient cells, furthermore, as shown in fig. b and d, increased abundance of ifitm was detected in the recipient hela-siifitm cells treated with huvec-sincderived exosomes in a time-dependent manner, but not in those treated with huvec-siifitm cell-derived exosomes. consistent with the western blotting data, immunofluorescence staining and confocal microscopic analysis of ifitm -flag showed that the localization in the cytoplasm in the recipient hela cells was significantly higher following treatment with exosomes derived from t-ifitm cells (fig. e ). in contrast, our results showed that t-ifitm cell-derived exosomes did not change ifitm and ifitm expression in the recipient hela cells, when compared with treatments of t cell-or t-vector cells-derived exosomes (fig. s ) . moreover, when the exosome treatments were terminated at h, further incubation with blank culture media for a total length of or h did not increase the amount of endogenous ifitm within the recipient hela cells (fig. f) , suggesting that the exosome treatment procedure per se does not change endogenous ifitm expression in the recipient hela cells. in addition, in order to verify that the increased ifitm protein level in the transfer recipient cells was due to the exosomal transfer of ifitm , rather than a stimulated expression of endogenous ifitm , we measured ifitm mrna levels in cells treated with ifitm -exosomes using quantitative real-time rt-pcr, and we found that ifitm -exosome treatment did not induce an increase of endogenous ifitm mrna in hela cells (fig. g) . furthermore, to evaluate the half-life of ifitm in cell culture supernatant, we quantified ifitm protein in the supernatant of cultured huvec at various time points ( , , , , , , and h) using western blotting. specifically, supernatants derived from huvec cells were collected and centrifuged at g for min, and then further centrifuged at g for min at °c to remove cells and cell debris, followed by filtration with . μm filters. the obtained supernatants were then concentrated by -fold using an amicon ultra- centrifugal filter unit with kda cut-off value, divided into groups, and incubated at °c in a humidified atmosphere of % co for , , , , , , and h, respectively, before being subjected to western blotting analysis using an anti-ifitm antibody. as shown in the fig. s , there was no or little change of the ifitm protein in the inter-cellular transfer of ifitm -containing exosome. hela cells were incubated with purified μg ml − exosomes derived from supernatants of cultured huvec, huvec-sinc, t-ifitm or t-ifn cells in serum-free media for h (a), incubated with increasing amounts of exosomes derived from t-ifitm cells for h (c), incubated with μg ml − ifitm -exosomes derived from t-ifitm cells or huvec for h, h, h and h (d), or incubated with μg ml − ifitm -exosomes derived from t-ifitm cells for h, followed by washing exosomes after h treatment (f). '*' indicates that hela cells were maintained in dmem after treatment with ifitm -exosomes for h. hela cells with ifitm silenced (hela-siifitm ) were incubated with μg ml − exosomes derived from huvec, huvec-sinc and huvec-siifitm (b). cell lysates were analysed by immunoblotting for ifitm or ifitm -flag proteins. to investigate whether that ifitm -laden exosome internalized into recipient cells could confer resistance to denv- infection, hela cells were cultured in serum-free conditions and exposed to increasing amounts of purified ifitm -containing exosomes for h. purified ifitm containing exosomes were used to treat hela cells that were simultaneously infected with denv- (moi = ). at h post infection when the infected cells were examined by real-time rt-pcr assay, strikingly, denv- infection was suppressed potently by the ifitm -containing exosomes in a dose-dependent manner in parental hela cells (fig. a ) or in hela cells with endogenous ifitm knocked down (fig. c) , and the copy number of denv- viral rna was reduced in the culture supernatant ( fig. b and d), indicating that the observed antiviral effect was mediated by the exosome-transferred ifitm . we also performed immunofluorescence analysis of infected hela cells treated with, or without, ifitm -exosomes, by staining for delivered ifitm -flag and a denv protein e. as shown in fig. s , our results showed that denv protein e are present only in some cells that do not contain ifitm -flag. the data from the cpe reduction assay demonstrated that ifitm -laden exosomes also attenuated cpe triggered by denv- infection (fig. e) . moreover, our results showed no effect of ifitm -exosomes on the binding of denv- to host cells or post-entry steps during denv- infection, but the penetration of denv- into cells was significantly inhibited by the ifitm -containing exosomes, with a reduction of approximately fourfold (fig. b ). in contrast, the control exosomes prepared from t cells that contained only the vector plasmid without the ifitm expression cassette did not exhibit any anti-denv activity, suggesting that ifitm was the main exosomal component responsible for the detected antiviral activity of exosome. to verify that the enhanced antiviral effects caused by ifitm -exosome transfer was a result of ifitm action rather than a stimulated paracrine ifns response, the concentration of human ifn-α/β in culture supernatant of hela cells or hela-siifitm cells treated with ifitm laden exosomes was measured with elisa by using sendai virus (sev; hau ml − ) as a positive stimulus control. our results showed that ifitm -exosomes treatment did not change ifn-α and -β expression, as compared with the control-exosomes ( fig. f and g) , implicating that reduction of virus replication was not due to a non-specific effect of ifitm -containing exosomes mediated by interferon. ifns trigger a potent antiviral response that activates host innate immunity and leads to establishment of a cellular antiviral state, which is mediated by inducing expression of isgs. accordingly, isgs represent a key line of defence against viral infection, and ifitms are among the most potent isg products with broad anti-viral spectra. a key finding of our current study, namely, that ifitm can be transferred intercellularly via extracellular exosome, provides new insights in the functional significance of ifitm as an ifn-induced anti-viral protein. the discovery of the roles of free ifitm proteins and ifitm-laden exosome as innate cellular defenders present an opportunity developing exosome-based tools to actively combat existing or emerging pathogens. conceivably, variations in the basal and inducible levels of ifitms, as well as in intracellular and extracellular levels of ifitms, might predict the severity of dengue infections among individuals or across species. exosome is secreted from many types of cells and remain stable in various body environments. by transferring proteins, mrnas and micrornas to neighbouring or distant cells, exosome contribute to modulation of important physiological or pathological processes such as immunity, angiogenesis, cell proliferation, cell migration and invasion, and cell-to-cell signalling (schorey and bhatnagar, ) . here, we propose a novel model for ifn-inducible innate immune response against denv- infection mediated by ifitm-containing exosome. noteworthy in this context is that exosome released from ifntreated cells may remotely communicate with other cells through such a mechanism that the anti-viral phenotype can be transferred to the exosome recipient cell that might not have the chance to be directly stimulated by ifn. interestingly, this inter-cellular communication model might also be applicable to explain a pioneering observation on the antiproliferative activity transmitted from ifnstimulated cells to non-stimulated cells (lloyd et al., ) . at the subcellular level, following ifn induction of ifitm expression, ifitm is transferred to the multivesicular complexes and assembled into exosomal vesicles. upon transport to the surface of a recipient cell, fusion of mvcs with the plasma membrane allows assembly of ifitm with a pre-existing exosome receptor complex that is unknown thus far. potentially, the released exosome particles containing ifitm protein can travel to non-stimulated cells where receptor assembly also occurs. such a model is consistent with the finding that the antiviral state can be transferred from ifns induced cells to non-treated cells in culture. hypothetically, this process might require ifitm proteins to be processed or relocalized in the host cells by an unknown mechanism so that it can be targeted for fig. . the antiviral activity of ifitm -containing exosome. after h incubation with ifitm -containing exosomes, hela cells (parental) or hela cells with ifitm silenced were incubated with denv- virus for h. intracellular (a and c) and extracellular (in supernatant) (b and d) viral rna was measured by real-time rt-pcr assay, and the results are presented as relative ratios of total cellular rna. (e) ifitm -containing exosome suppressed cpe triggered by denv- infection. intracellular viral rna was measured in a real-time rt-pcr assay, and the results are expressed as relative fold of total cellular rna. conditioned media obtained from exosomes derived from each group of cells treated as indicated were quantitatively measured for contained ifn-α (e) and ifn-β (f) proteins by elisa, using sendai virus (sev; hau ml − ) as a positive stimulus control. data points are presented as means ± sd of triplicated experiments. student's two-tailed t test was performed, and statistical significance is shown with asterisks (*p < . , **p < . ). exosome secretion. interestingly, it has been reported that cytosolic proteins present with exosome including members of the rab family, are involved in the formation and secretion of exosome. rab b, , and gdi are found to promote exosome docking and the membrane fusion events (thery et al., ) . interesting, feeley et al. reported that ifitm primarily resides in the late endosomal compartment and partly colocalizes with rab and lamp (feeley et al., ) . in the context, therefore, it would be of great interest to further investigate the biogenesis of ifitm exosome as well as its regulation, which might provide important clues for a rationale development of optimal anti-viral strategies. while application of ifns has been highly appreciated in the clinic, problems associated with the use of ifns, such as suboptimal efficacies against many viruses and varied degrees of adverse effects caused by ifn treatment, remain unsolved (borden et al., ) . in the intensive efforts of identifying and developing new endogenous anti-viral factors with potential clinical applicability, isgs have been attractive candidate agents for therapeutic purposes. in such a context, exosome-carried ifitm , as discovered by our present study, appears to represent a novel, attractive potential anti-viral strategy, as both natural and specially engineered exosome can serve as nano-shuttle vehicles for drug delivery. animal studies to test the feasibility and efficacy of applying exosomebased delivery of ifitm for anti-denv purposes in vivo are therefore warranted. our findings provided the first set of evidence for the existence of an extracellular, exosome-packaged form of ifitm , which enables anti-viral activities to transmit from one cell to another, leading to efficient establishment of an anti-viral state. further studies to decipher the structural and cell biological basis for the antiviral activities of ifitmexosome will be of theoretical as well as practical importance in this area of research. thp- , a , huh , t, u , hepg , hela and bhk- cells were maintained in dulbecco's modified eagle's medium (dmem; invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs; hyclone, logan, ut), mm lglutamine, μg ml − streptomycin and units ml − penicillin (invitrogen). human umbilical endothelial cells (huvec) were grown in human umbilical endothelial cell serum-free medium (invitrogen), supplemented with g ml − endothelial cell growth supplements (upstate, billerica, ma, usa) . the cultures were maintained at °c in a humidified atmosphere of % co . c / aedes albopictus cells were cultured at °c and % co in dmem supplemented with % fbs, u ml − penicillin and mg ml − streptomycin. the denv- strain ngc (new guinea c, genbank accession number m ) was kindly provided by guangzhou centers for disease control and prevention (cdc) (tian et al., ; wu et al., ) and was propagated in the c / cell line. briefly, a monolayer of c / cells was infected with denv- at an moi of , and incubated at °c and % co for days. the supernatant was harvested and centrifuged for min at g to remove pelleted cellular debris. denv- titres were determined by facs assays in c / cells, as previously described (lambeth et al., ) . cells were harvested and lysed in × sampling buffer containing mm tris-hcl (ph . ), mm pmsf, % glycerol, % sds, % mercaptoethanol and . % bromophenol blue before sonication. the protein concentration of the lysate was determined using the bicinchoninic acid protein assay kit (thermo fisher scientific, rockford, il) according to the manufacturer's instructions with bsa as the standard. protein molecular weight standards (genstar biosolutions co. ltd, beijing, china) were used to determine molecular weights of sample proteins. an aliquot of the cell lysates containing μg of protein was subjected to sds-page, and then transferred to pvdf membranes. following a blocking step using the blocking buffer (tris-buffered saline, tbs, containing % non-fat milk) for h at room temperature, the membranes were incubated overnight at °c with the following specific primary antibodies: monoclonal anti-ifitm , monoclonal anti-ifitm , polyclonal anti-ifitm (proteintech group, chicago, il), monoclonal anti-flag (sigma-aldrich, st louis, mo), d - (anti-denv- e mouse monoclonal antibody; santa cruz biotechnology, santa cruz, ca), monoclonal anti-actin (sigma-aldrich), polyclonal anti-cd (h- ; santa cruz biotechnology), anti-flotillin (c-terminal; sigma-aldrich), anticalnexin (proteintech group), and monoclonal anti-gm (cell signaling, danvers, ma) antibodies. further incubation with appropriate horseradish peroxidase (hrp)-conjugated secondary antibodies, depending on the primary antibody used, was performed for h at room temperature. membranes were washed three times in tris-buffered saline containing . % tween- for min after each incubation step. the bands were detected using enhanced chemiluminescence kit (thermo fisher scientific) with a kodak film. actin was used as a loading control for quantification normalization. intensities of the bands of interest on the pvdf membranes were quantitatively calculated with the quantity one . . measurement software (bio-rad, hercules, ca). human ifitm , , cdna with a c-terminal ha epitope tag were amplified by rt-pcr using primers shown in table s . the resultant pcr fragments were cloned into the pmscv-puro vector (clontech laboratories, inc., mountain view, ca) at bglii and ecori sites to generate plasmids pmscv-ifitm -ha, pmscv-ifitm -ha and pmscv-ifitm -ha respectively. the cloned ifitm , , cdnas were sequencing verified. a blank pmscv-puro vector was generally used as a control. these constructs were used to establish stably transduced cell lines with u and hela cells to permanently express ifitms. briefly, retroviral particles were prepared by transfecting μg of each of the pmscv-puro-ifitm -ha, pmscv-puro-ifitm -ha and pmscv-puro-ifitm -ha plasmid dna into the packaging cells t, together with μg of the pik plasmid. recombinant retroviral particles were used to infect u or hela cells, and stably transduced cell lines were selected in dmem medium supplemented with puromycin ( μg ml − ). to transiently express ifitms protein, ifitms genes were separately subcloned into the pcdna vector (invitrogen) at kpni and bamhi sites and a flagtag sequence was introduced into the coding sequence as the c-terminus. t cells were transiently transfected with the resultant plasmid pcdna-ifitms-flag by a standard calcium phosphate co-precipitation method. hela cells were transiently transfected using lipofectamine (invitrogen) by following the manufacturer's instruction. the small interfering rna (sirna) duplexes against ifitm , whose sequences are provided in table s ) were published previously (brass et al., ) , and were purchased from ribobio inc. (guangzhou, guangdong, china). huvec and hela cells were transfected with sirnas at indicated concentrations, using lipofectamine reagent (invitrogen) following the manufacturer's instructions, followed by treatment with ifn-α ( , , u ml − ) and infection with denv- at an moi of . to assess the expression of ifitm proteins, cell lysates were harvested at h after transfection and examined by western blotting analysis with corresponding specific antibodies. dengue e protein was detected in cells infected with denv- by intracellular staining and flow cytometric (fcm) analysis, as previously described (brass et al., ; chan et al., ) . briefly, cells in growth dmem medium were seeded and subsequently cultured for h, followed by infection with denv- at an moi of . cells harvested at indicated time points were washed with cold × phosphate-buffered saline (pbs) and fixed with % paraformaldehyde at °c for min. fixed cells were scraped and then permeabilized in % methanol before being incubated with nyrdeng (anti-denv- e mouse monoclonal antibody; santa cruz biotechnology), and further with appropriate fitcconjugated goat anti-mouse igg secondary antibody (santa cruz biotechnology). the fitc-positive cells were acquired and scored by flow cytometry (becton-dickinson counter, san jose, ca). the percentage of denv-infected cells was defined by fitc-positive cells distribution in a fluorescence dot plot using the cytometry list mode data acquisition & analysis software (becton-dickinson counter) . denv in cell supernatants were quantified by determining the copy number of viral rna using quantitative real-time rt-pcr as previously described (mota and rico-hesse, ). cell supernatants were harvested at indicated time points, and total rna was extracted using qiaamp minelute virus spin kit by following the instruction of the manufacturer (qiagen, chatsworth, ca). cdna was synthesized, and quantitative real-time pcr was performed with standard curve analysis using a cfx real-time pcr detection system (bio-rad). for relative quantification, results are presented as ratios relative to the quantification for gapdh. primer pairs used are shown in table s . to estimate denv rna copy number, a standard curve was generated using in vitro-transcribed rna standards, which were produced as follows. a bp fragment of the denv- ngc strain was amplified by rt-pcr and cloned into the pmd -t simple vector (takara, dalian, china). the cloning plasmid was linearized with ecorv, and rna transcripts were generated with a t megascript kit (ambion) according to the manufacturer's instructions. concentration of transcribed rna was determined with the nanodrop c spectrophotometer (thermo scientific, rockford, il), and -fold serial dilutions were prepared and used to construct a standard curve. the binding and entry experiments were performed as described previously (kanlaya et al., ; weidner et al., ) . briefly, for virus binding experiment, cells were seeded in -well plates at a density of × cells per well and cultured for h, followed by inoculation with denv- at moi of and incubation on ice for h to allow binding but impede cell entry. such a multiplicity of infection ensures that at least % of cells were infected with a minimum of one infectious viral particle. unbound virus was removed and cells were harvested to determine the amount of viral rna accumulation by quantitative rt-pcr min later. to assess denv- virus entry into cells via endocytosis, after binding on ice for h, virus inocula were removed after h of binding on ice, and infected cells were washed with × pbs followed by incubation with pre-warmed dmem for min at °c to initiate virus cell entry. subsequently, cells were rinsed three times with × pbs and then treated with . % trypsin for min, and again washed three times with × pbs to remove any cell-associated virus that did not enter the cytoplasm. for assessments of post-entry infection steps, after virus binding and entry procedures were finished, hela cells were subsequently treated with ifitm -containing or control exosomes. total cellular rna was extracted to measure the quantity of viral genomes that had entered cells by using a real-time rt-pcr assay. seven groups of cells, including huvec (with high basal-level ifitm protein), non-targeting control sirna-transfected huvec (huvec-sinc), ifitm sirna- -transfected huvec (huvec-siifitm ), ifitm -transfected t cells ( t-ifitm ), control vector-transfected t cells ( t-vector), ifn-α-treated t cells ( u ml − ; t-ifn) and t cells, were cultured in medium supplemented with % exosome-free fbs (overnight centrifugation, g), and grown to monolayer with - % confluence. exosomes were isolated from cell supernatants by filtration steps and differential ultracentrifugation as previously described (khatua et al., ) . briefly, supernatants derived from huvec and t cell culture with different treatments were collected and centrifuged at g for min, and then g for min at °c to remove cells and cell debris, followed by filtration with . μm filters (pall life sciences, port washington, ny) and ultracentrifugation at g for h at °c to pellet crude exosomes. for further purification, the crude exosomes were mixed with ml of . m sucrose in pbs and placed on the bottom of a optiseal centrifuge tube (beckman coulter inc., munich, germany), overlaid with ml m sucrose and ml . m sucrose, and ultracentrifuged for h at g. the purified exosomes accumulating at the m/ . m interface were collected, washed twice by diluted in pbs, and pelleted by ultracentrifugation at g for min. pelleted exosomes were re-suspended in pbs and used immediately or kept at − °c. gw (sigma-aldrich) was employed as an inhibitor of exosome secretion. protein concentrations of exosome preparations were determined with the bicinchoninic acid protein assay kit (thermo fisher scientific) according to the manufacturer's instructions, using bsa as the standard. finally, the ifitm -laden exosome samples were characterized by western blotting analysis, and the protein band was excised from the gel and subjected to mass spectrometry (ms) analysis according to a standard procedure (khatua et al., ). purified exosomes were processed by negative staining and analysed by transmission electron microscopy (tem) as previously described (khatua et al., ) . briefly, exosomes resuspended in μl pbs were dropped onto a sheet of parafilm, and a formvar-carbon coated nickel grid were floated for min at room temperature to absorb exosomes, followed by wash with pbs twice and fixed with % paraformaldehyde after rinsing with pbs twice, the grids were negatively stained with % phosphotungstic acid for min. finally, dry samples were viewed using a tecnai transmission electron microscope at kv or stored in a grid box for future work. cells were seeded in -well plates at × cells per well h prior to viral infection or treatments described below. to evaluate the anti-denv ability of purified ifitm -laden exosomes, and μg ml − of ifitm -exosomes, respectively, was added to cultured cells and incubated for h. subsequently, mixture of ngc denv- (moi = ) and or μg ml − of ifitm -exosomes, respectively, which had been pre-incubated for h at °c, was added to cells. after three washes with pbs to remove the unbound virus, ifitm -exosomes was added to cells and incubated for the indicated lengths of time ( , , or h). the control-exosomes was prepared from t cells that contained only the vector plasmid without the ifitm expression cassette. ifn-α and ifn-β was quantified in the supernatants derived from cells treated with ifitm -exosomes, by using enzyme linked immuno-sorbent assay (elisa) kits, according to the manufacturer's instruction (keygen biotech, china; pbl interferon source, piscataway, nj) respectively. following a pre-lysis treatment with trypsin to remove adherent exosomes, infected cells and culture medium were harvested at various times after infection and subjected to rna extraction. the amounts of dengue viral rna were measured by using a quantitative or relative real-time rt-pcr assay. for cpe reduction assay, at h post infection, microscopic examination was performed to determine the antiviral effect, and the data were confirmed using a cell viability assay. the viability of bhk- cells was measured by using the mts [ -( , -dimethylthiazol- -yl)- -( -carboxymethoxyphenyl)- -( -sulfophenyl)- h-tetrazolium] assay according to the manufacturer's instructions. briefly, μl mts solution (celltiter aqueous one solution reagent, promega, madison, wi) was added to each well and incubated for an additional h at °c, and the absorbance was subsequently measured at nm using a microplate reader (bio-tek, winooski, vt). cells treated with control-exosome were used as the control group. cell growth inhibition rates were determined using the following formula according to a previously published method (muhamad et al., ) : cell viability (%) = (od of treated cells/od of control cells) × %. the experiment was repeated at least three times from which mean and standard deviation (sd) values were calculated. the data given in the text were presented as means ± sd. comparison between two groups was evaluated by the -tailed student's t test, using . as the cut-off p-value. correlations were determined with the exponential regression analysis using the ibm spss statistics software. additional supporting information may be found in the online version of this article at the publisher's web-site: fig. s . the ifitm protein family restricts denv- infection at entry. u (a) or hela (c) cells were transduced to express ifitm , or protein or control vector alone, as verified by western blotting analysis. ifitm protein expression in t cells was measured by western blotting analysis, using aliquots of the same cells assayed in (e) respectively. actin was included as a loading control. denv- -infected cells were detected using a monoclonal antibody (nyrdeng ) directed to the envelope (e) glycoprotein of the virus, and the numbers of denv- -infected u (b), hela (d) or t (f) cells were determined by flow cytometry. fig. s . ifitm is required for the antiviral effects of interferons and silencing ifitm enhances denv- infection. (a) huvec cells transfected with indicated sirnas, or a nontargeting control sirna (sinc), were assessed for ifitm levels by western blotting analysis. at h post transfection with indicated sirna, the percentage of infected cells was assessed h after virus addition by flow cytometry for e protein (b). hela cells were transfected with the indicated sirnas, or a control, non-targeting sirna. at h post transfection, cells were incubated with , or iu ml − ifn-α for h, and the levels of ifitm were examined by western blotting (c). (d) hela cells transfected with indicated ifitm -trageting sirnas were incubated with the indicated concentrations of ifn-α for h at h post transfection, followed by infected with denv- at an moi of . the percentages of infected cells were assessed h after virus addition by flow cytometry for the denv e protein. e, effect of sirna silencing of ifitm on the binding and entry of denv- to hela cells. data points are presented as means ± sd of triplicated experiments. student's two-tailed t test was performed, and statistical differences are shown with asterisks (**p < . ). fig. s . mass spectrometric analysis of ifitm peptides. results automatically generated by the mass spectrometer. briefly, purified ifitm -containing exosomes derived from t cells overexpressing ifitm were assayed by massspectrometric analysis of proteolytic peptides. the red vertical lines represent peptide ion intensities, and the horizontal axis shows the mass-to-charge (m/z) values around the peptide ions of interest. the green dash lines were automatically given by the spectrometer for pointing the labels to the red peaks. fig. s . t-ifitm cell-derived exosomes do not change the quantities of ifitm and ifitm in the recipient hela cells, as compared with the t cell-and t-vector cell-derived exosomes. fig. s . ifitm half-life in the cell culture supernatant. the levels of ifitm protein in cell culture supernatant of huvec at various times ( , , , , , , and h) was assayed with western blotting. briefly, supernatants derived from huvec cells were collected and centrifuged at g for min, and then further centrifuged at g for min at °c to remove cells and cell debris, followed by filtration with . μm filters. the obtained supernatants were then concentrated by -fold using an amicon ultra- centrifugal filter unit with kda cut-off value, divided into groups, and incubated at °c in a humidified atmosphere of % co for , , , , , , and h, respectively, before being subjected to western blotting analysis using an anti-ifitm antibody. intensities of the bands of interest on the pvdf membranes were quantitatively calculated with the quantity one . . measurement software. the results are presented as relative ratios of intensity of the band for the h treatment group. fig. s . immunofluorescence staining and confocal microscopic analysis of ifitm -flag and denv- viral e protein in hela cells treated with t-ifitm -exosome followed by infection with denv- . table s . primer sequences and sirna oligonucleatides. exosomes released from infected macrophages contain mycobacterium avium glycopeptidolipids and are proinflammatory exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo interferons at age : past, current and future impact on biomedicine the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus ifitm proteins restrict antibody-dependent enhancement of dengue virus infection ifitm restricts the morbidity and mortality associated with influenza ifitm inhibits influenza a virus infection by preventing cytosolic entry the trojan exosome hypothesis dengue: a continuing global threat more dengue, more questions the burden of dengue infection distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus capture and transfer of hiv- particles by mature dendritic cells converges with the exosome-dissemination pathway identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections vimentin interacts with heterogeneous nuclear ribonucleoproteins and dengue nonstructural protein and is important for viral replication and release exosomes packaging apobec g confer human immunodeficiency virus resistance to recipient cells flow cytometry-based assay for titrating dengue virus exosomes mediate the cell-to-cell transmission of ifn-alpha-induced antiviral activity cell-tocell transfer of interferon-induced antiproliferative activity the ifitm proteins inhibit hiv- infection humanized mice show clinical signs of dengue fever according to infecting virus genotype antiviral actions of flavanoid-derived compounds on dengue virus type- hepatic cell-tocell transmission of small silencing rna can extend the therapeutic reach of rna interference (rnai) interferon-inducible antiviral effectors exosome function: from tumor immunology to pathogen biology exosomes are natural carriers of exogenous sirna to human cells in vitro exosomes: composition, biogenesis and function identification and immunogenicity of two new hla-a* -restricted cd + t-cell epitopes on dengue ns protein interferon-induced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms ) mir- a facilitates replication of dengue virus by dampening interferon induction by targeting traf identification of the ifitm gene as an inhibitor of hepatitis c viral translation in a stable stat cell line we owe our special thanks to professor xi huang of sun yat-sen university for providing materials essential for the study. key: cord- -qchrw authors: pu, jieying; he, li; xie, heping; wu, siyu; li, yuye; zhang, ping; yang, zhicong; huang, xi title: antiviral activity of carbenoxolone disodium against dengue virus infection date: - - journal: j med virol doi: . /jmv. sha: doc_id: cord_uid: qchrw as one of the most important mosquito‐borne viral diseases, dengue infection is now becoming a global concern due to its rapid spread and rise in incidence. currently, there is no approved vaccine or effective antiviral drug for dengue virus (denv) infection. glycyrrhetinic acid (gna) and its related derivatives have been reported to inhibit a broad spectrum of viruses. however, it is unknown whether carbenoxolone disodium (cbx), one of the gna derivatives, affects denv infection. here, we found that the production of infectious denv particles was significantly decreased by cbx treatment in denv‐permissive cells, while the viral rna and viral protein synthesis were not affected. moreover, results from time‐of‐addition study showed that the inhibitory effect of cbx on denv was exhibited by targeting the virus itself, not the host cells. directly incubating denv with cbx resulted in a remarkable reduction of virus titer and virus infectivity. furthermore, denv rna from progeny virions in the supernatants was significantly decreased by cbx treatment in a dose‐dependent manner. taken together, these data indicate that the antiviral activity of cbx against denv may be mainly due to a virucidal effect exerted by the compound itself. our work, for the first time, demonstrates that cbx has antiviral activity against denv infection, providing useful information for development of potential therapeutic interventions against dengue. j. med. virol. : – , . © wiley periodicals, inc. as one of the most important mosquito-borne viral diseases, dengue infection is now becoming a global concern due to its rapid spread and rise in incidence. currently, there is no approved vaccine or effective antiviral drug for dengue virus (denv) infection. glycyrrhetinic acid (gna) and its related derivatives have been reported to inhibit a broad spectrum of viruses. however, it is unknown whether carbenoxolone disodium (cbx), one of the gna derivatives, affects denv infection. here, we found that the production of infectious denv particles was significantly decreased by cbx treatment in denv-permissive cells, while the viral rna and viral protein synthesis were not affected. moreover, results from time-of-addition study showed that the inhibitory effect of cbx on denv was exhibited by targeting the virus itself, not the host cells. directly incubating denv with cbx resulted in a remarkable reduction of virus titer and virus infectivity. furthermore, denv rna from progeny virions in the supernatants was significantly decreased by cbx treatment in a dose-dependent manner. taken together, these data indicate that the antiviral activity of cbx against denv may be mainly due to a virucidal effect exerted by the compound itself. our work, for the first time, demonstrates that cbx has antiviral activity against denv infection, providing useful information for development of potential therapeutic interventions against dengue. j. med. virol. : - , . dengue virus (denv), a member of the genus flavivirus in the family flaviviridae, causes one of the most widespread mosquito-borne diseases in human. there are four serotypes of dengue virus, types (denv- ) to (denv- ), which have similar clinical manifestations and epidemiology in tropical and subtropical regions of the world [wang et al., ] . denv attacks a set of target cells, including dendritic cells, monocytes/macrophages, endothelial cells, and disseminates in other non-immune cells [pagni and fernandez-sesma, ] . once denv enters cytoplasm, its single-stranded, positive-sense rna genome is directly translated into a single polyprotein which is eventually cleaved into the individual viral proteins, including three structural proteins (c, prm, and e) and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) [chambers et al., ; filomatori et al., ; zybert et al., ; idrees and ashfaq, ] . dengue infection either results in a mild illness known as dengue fever (df) or the more severe and even life-threatening diseases, dengue hemorrhagic fever (dhf), and dengue shock syndrome (dss) [guzman and kouri, ] . currently, it is estimated that over million cases of dengue infection occur annually, which have swept through more than countries, with potential for further spread [simmons et al., ] . from september to december , an unexpected large outbreak of dengue infection with a significant proportion of severe cases was reported in pearl river delta of guangdong province, china. it has been the most severe dengue epidemic over the years, with more than , cases was diagnosed, including five deaths (http://www.cdcp.org.cn). the emergence of the unprecedented magnitude of dengue outbreak has aroused widespread social concern. in this situation, the who considers the development of an effective dengue vaccine to be a high priority. due to great endeavor of scientific researchers from around the world, dengvaxia (sanofi pasteur, lyon, rh) has become the first licensed vaccine available to prevent df. however, for now there is no available antiviral agent approving for combating denv [idrees and ashfaq, ] . therefore, under current circumstance, the pressing task is to develop safe and effective antiviral drugs against dengue. currently, a series of natural compounds and their derivatives are reported as important source of antiviral drugs [pompei et al., [pompei et al., , chen et al., ; fiore et al., ] . for example, triterpenic compounds, such as glycyrrhizic acid (gra), glycyrrhetinic acid (gna), and some of their derivatives have been demonstrated to block the replication of a broad spectrum of viruses, including human immunodeficiency virus (hiv), sars related coronavirus, epstein-barr virus, flaviviruses, vesicular stomatitis virus (vsv), hepatitis b and c virus, influenza virus, rotavirus, adenovirus, etc. [dargan et al., a,b; takahara et al., ; matsuo et al., ; cinatl et al., ; crance et al., ; lin, ; orlent et al., ; cos et al., ; ashfaq et al., ; hardy et al., ; smirnov et al., ] . carbenoxolone disodium (cbx), one of the gna derivatives, is similar to substances found in the root of licorice plant. with a steroid-like structure, cbx has been clinically applied in the treatment of gastric ulcers and other types of inflammation [connors, ] . besides, cbx is best known in cellular physiology as an effective, water-soluble blocker of gap junctions [rozental et al., ] . in earlier studies, cbx was reported to exhibit complex activities against herpes simplex virus (hsv), including synthesis, processing, and transportation of viral gene products subak-sharpe, , ; dargan et al., ] . nonetheless, whether cbx has an inhibitory effect on denv remains unknown. in this study, we investigated whether cbx treatment inhibits dengue infection by measuring the production of progeny virions and viral rna as well as viral protein expression. our data showed a dose-dependent inhibition of denv- and denv- progeny virions following treatment with indicated concentration of cbx. moreover, pre-incubating denv- with cbx remarkably abolished virus infectivity and decreased virus titer. furthermore, treatment with cbx significantly decreased denv rna from progeny virions in the supernatants. our results for the first time identify the old drug cbx as a novel anti-denv compound, and it might be developed as a potential treatment for dengue infection. - ) were cultured in rpmi- with % fbs at ˚c. all media were supplemented with % sodium pyruvate, mg/ml of penicillin and u/ml streptomycin (invitrogen). the denv- hawaii strain and denv- new guinea c strain were provided by guangzhou centers for disease control, and propagated in c / cells. c / cells were inoculated with denv at a multiplicity of infection (moi) of . , and incubated at ˚c for - days (denv- ) and - days (denv- ). the supernatants were collected and clarified by centrifugation. virus titer was measured by tcid assay in c / cells and viral stocks were stored at À ˚c. vesicular stomatitis virus (vsv) was kindly provided by dongyan jin [kok et al., ] , and propagated in vero cells. mtt ( -( , -dimethyl- -thiazolyl)- , -diphenyl- h-tetrazolium bromide) was purchased from sigma-aldrich (st louis, mo). a cells were cultured in -well plates with different concentrations of cbx for hr. then ml of pbs containing mtt ( . mg/ml) was added to each well. after hr of incubation at ˚c, the supernatant was removed and ml of dmso was added to each well to solubilize the formazan crystals. absorbance was measured in a microplate reader at nm after vigorous shaking. adherent a and huvec cells were seeded on cell culture plates day prior to infection. for the suspension cell line, thp- cells were seeded on the day of infection before being infected by denv. virions were diluted in dmem (a and huvec) or rpmi- (thp- ) without fbs, and inoculated to cell monolayers or suspensions at moi of (denv- ) or moi of (denv- ). after hr incubation at ˚c to allow virus attachment, supernatants were removed and cells were washed three times with pbs to remove residual virus. infected cells were then incubated at ˚c in culture medium containing % fbs for indicated time. cbx ( b-hydroxy- -oxoolean- -en- -oic acid -hemisuccinate) was purchased from sigma-aldrich and dissolved according to product instruction. appropriate volumes of medium containing specific concentration of cbx were added to cells at different time frames: starting at hr prior to denv infection (pre-treatment); hr concurrently with viral infection till hr post-infection (post-treatment); from hr prior to viral infection till hr post-infection (continuous-treatment); from hr prior to viral infection till hr post-infection (short-treatment). the cells were then incubated at ˚c for indicated time of infection before they were harvested. total rnas were extracted and purified from cells using trizol reagent (invitrogen) according to the manufacturer's instructions. reverse transcription was carried out by using mg of rna. for quantitative pcr, the reactions were performed in triplicate with each cdna template by using iq sybr green supermix (bio-rad, hercules, ca) and performed on bio-rad cfx real-time detection system (bio-rad). the mrna expression levels were normalized to gapdh expression. primer pairs used were as follows: gapdh (glyceraldehyde- -phosphate dehydrogenase), forward primer gccttccgtgtcccca ctg, reverse primer cgcctgcttcaccaccttc; d env-c gene, forward primer tcctaacaatcccacc aacagca, reverse primer agttctgcgtctcctgt tcaaga. cells were seeded onto coverslips ( - % confluency) and infected with denv- (moi of ) or denv- (moi of ) for hr, or left uninfected. then the cells were fixed with % paraformaldehyde at ˚c for min, and permeabilized with pbs containing . % triton x- . the virus protein was detected by using a monoclonal antibody against denv prm glycoprotein (abcam, cambridge, ma) and a secondary anti-mouse antibody (invitrogen). cell nuclei were stained with dapi. stained samples were visualized using zeiss fluorescence microscope. denv titer in harvested supernatants was determined by tcid assay. samples were serially diluted and inoculated into c / cells in -well plates. after -day (denv- ) or -day (denv- ) incubation at ˚c with % co , cells were examined for cytopathic effects (cpe) under a light microscope. the virus titer (tcid /ml) was calculated using the reed-muench method [reed and muench, ] . one tcid /ml was equivalent to . pfu/ml. the titer of vsv was quantified using plaque assay. ninety percent confluent vero cells in -well plates were infected with optimally diluted vsv and then covered with low melting temperature agarose (sangon biotech, shanghai, china) after rinsing with phosphate buffered saline (pbs). at hr post-infection, % crystal violet was used to stain the vero cells and plaques were quantified. denv- suspension containing  pfu/ml or denv- suspension containing  pfu/ml was incubated with an equal volume of dmem with or without indicated concentrations of cbx for indicated time at ˚c. the mixtures were then diluted in dmem, and were used for infection in c / cells to test viral titer or in a cells to test residual infectivity by tcid assay, immunofluorescence assay, or quantitative rt-pcr. for each separate set of assays, at least three independent experiments were evaluated. data are represented as mean ae sd. statistical significance was determined by two-tailed, unpaired student's t-test; p < . was considered statistically significant. chemical structure of cbx is shown in figure a . cytotoxicity of cbx was first determined by the -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide (mtt) method in a cells, one of the dengue-permissive cells, to establish suitable dosages for further studies. as shown in figure b , cell viability was close to control in treatments with , , , and mm cbx, whereas only % cells survived in the presence of mm cbx. therefore, cbx concentrations ranging from to mm were considered for the following assay. to examine the effect of cbx on denv infection, a cells were infected with denv- hawaii strain and denv- new guinea c strain, respectively, and incubated for hr in the presence or absence of cbx. as shown in figure c and d, the production of infectious denv particles was significantly decreased by cbx treatment in a dose-dependent manner. at concentrations of and mm, cbx treatment led to -and -fold reduction of denv- titer, respectively (fig. c) . likewise, at , , and mm cbx, around -, -, and -fold lower levels of denv- titer were observed than control, respectively (fig. d) . taken together, these results suggest that cbx has a dose-dependent inhibition effect on progeny denv production in a cells, and the anti-denv effect of cbx is not limited to a specific denv serotype. to determine whether cbx inhibits the production of infectious progeny denv in other dengue-permissive cells, tcid assay was performed to measure the virus titer in cbx-treated thp- and huvec cells, both of which are widely used in denv studies [halstead, ; wu et al., ] . as shown in figure a and b, both thp- and huvec cells were well infected with denv- , with about % of cells being infected. in thp- cells, treated with , , and mm cbx resulted in -, -, and -fold reduction of virus titer, respectively (fig. c) . in huvec cells, treated with , , and mm cbx led to -, -, and fold reduction of virus titer, respectively (fig. d) . these results indicate that the anti-denv activity of cbx is not cell-specific. to examine the effect of cbx on the synthesis of denv viral rna, quantitative rt-pcr was performed. as shown in figure a and b, both denv- and denv- viral rna (c protein) synthesis were not affected by cbx treatment even at the highest concentration ( mm). meanwhile, immunofluorescence assay was performed to analyze the effect of cbx on denv viral protein expression upon infection. antibody specific to the denv structural prm protein was used in this part of the study. the percentages of prm containing cells were calculated to determine the expression of viral protein ( fig. d and f) . as shown in figure c and e, cbx treatment at the highest concentration ( mm) did not reduce denv- or denv- viral protein expression. these results suggest that both denv viral rna synthesis and protein expression are not regulated by cbx treatment. to further identify the step(s) of progeny denv production inhibited by cbx, time-course of the inhibitory effect was analyzed by a time-of-addition experiment in a cells. mm cbx was added into the media at different time frames (fig. a) . starting at hr prior to denv infection (pre-treatment); hr concurrently with viral infection till hr post-infection (post-treatment); from hr prior to viral infection till hr post-infection (continuous-treatment), or till hr post-infection (short-treatment). cell supernatants were harvested for tcid assay. as shown in figure b , both posttreatment and continuous-treatment of cells by cbx led to approximately -fold reduction of progeny virus titer. by contrast, pre-treatment with cbx did not have a significant impact on denv- yields (fig. b) . as cbx can freely shuttle into cytoplasm by all three treatments mentioned above [parke, ; connors, ] , this result indicates that the inhibitory effect of cbx on denv is not exhibited by targeting the host cells. when eliminating cbx from the media at hr post-infection (short-treatment), the titer of progeny denv- particles also was not affected (fig. b) . in our denv- -infected system, newly formed virions began to secret from a cells at hr post-infection (data not shown). so at this point, short-treatment as mentioned can prevent progeny virions from contacting with cbx. therefore, these results from time-of-addition study suggest that cbx inhibits denv by directly targeting the infectious virions extracellularly. to further study the direct effects of cbx on denv particles, denv- and denv- suspensions were preincubated with different concentrations of cbx, respectively for indicated time, and then the viral titer was determined by tcid assay. as shown in figure a , denv- titer was significantly decreased by -and -fold when incubated with or mm cbx, respectively, but not with mm cbx. when denv- suspensions were incubated with or mm cbx for , , and hr, respectively, virus titer at each time point was significantly decreased (fig. b) . likewise, cbx exhibited a similar behavior on denv- . as shown in figure c , treating denv- suspensions with , , and mm cbx resulted in -, -, and -fold reduction of virus titer, respectively. moreover, longer incubation of cbx correspondingly led to a lower titer of residue denv- , peaking at hr incubation (fig. d) . surprisingly, the titer of denv- was almost undetectable when incubating with mm cbx for hr (fig. d) . taken together, these data indicate that incubation with cbx effectively decreases denv titer in a dose-and timedependent manner. furthermore, we directly incubated denv- with mm cbx at ˚c for hr, and then followed by an infection in a cells for indicated time. the viral rna synthesis, viral protein expression and progeny virus production were compared as indicated above. as shown in figure a , the levels of viral rna at and hr were barely detected in cells infected with cbx-incubated denv- . while in control cells, viral rna synthesis was gradually increased post infection (fig. a) . immunofluorescence microscopy images showed that no or just few cells were positively stained with denv prm glycoprotein antibody in cbx-incubating group, compared to approximate % of control cells were positively stained at hr post denv- infection (fig. b and c) . likewise, the production of progeny virions from - (a and b) or denv- (c and d) was directly incubated with indicated concentrations of cbx for hr (a and c) or for indicated time (b and d) at ˚c. the titer of residual virus was determined by tcid assay. data are shown as mean ae sem of at least three independent experiments. Ãà p < . ; ÃÃà p < . . cbx-incubating group was dramatically decreased (fig. d) . therefore, denv infectivity is significantly inhibited by cbx pre-incubation. data from figures and suggested that the antiviral activity of cbx against denv may be mainly due to a virucidal effect exerted by the compound itself. to test this assumption, a cells were infected with denv- and then incubated for hr with different concentrations of cbx. the supernatants were collected for virus rna detection. as shown in figure a , denv- rna from progeny virions in the supernatants was significantly decreased by cbx treatment in a dose-dependent manner. at concentrations of and mm, cbx treatment led to -and -fold reduction of denv- mrna expression, respectively (fig. a) . it suggests that cbx has a virucidal activity to inactivate the progeny denv particles. to further corroborate the virucidal effect of cbx, a cells were infected with a low moi of denv- for hr to complete at least two replication cycles, and incubated with mm cbx till the end of infection. quantitative rt-pcr result showed that denv- viral rna synthesis was significantly decreased by % in cbx-treated group (fig. b) . similarly, result from immunofluorescence assay showed that cbx treatment led to effective reduction of viral protein expression (fig. c) . the percentage of prm containing cells was half less than control group (fig. d) . taken together, these results indicate that cbx inhibits the replication of progeny denv particles through its virucidal activity. to confirm the specificity of the antiviral activity of cbx, vsv, another enveloped virus was evaluated. vsv suspensions were pre-incubated with different concentrations of cbxm, respectively for indicated time, and then the viral titer was determined by plaque assay. as shown in figure e , vsv titer was significantly decreased by -and -fold when incubated with or mm cbx, respectively, but not with mm cbx. when vsv suspensions were incubated with or mm cbx for , , and hr, respectively, virus titer was significantly decreased in fig. . infectivity of denv- in a cells is inhibited by cbxincubation. denv- particles were pre-incubated with mm cbx at ˚c for hr, and then infected a cells for indicated time. a: the total rna was isolated from the infected cells and analyzed by quantitative rt-pcr using primers specific for the viral c protein (cp) mrna. b: after hr of infection, the expression of viral protein was determined by immunofluorescence assay using denv prm glycoprotein antibody; c: the percentages of prm containing cells were determined by piccnt Â; d: the titer of progeny denv- particles was measured by tcid assay. data are shown as mean ae sem of at least three independent experiments. ÃÃà p < . . a time-dependent manner (fig. f) . potent inhibition against another enveloped virus (vsv) indicates that the antiviral effect of cbx is not specific to denv. the current epidemic of dengue infection poses a threat to public health worldwide, making it clear that safe and effective countermeasures are urgently needed against this infection. unfortunately, there is almost no specific antiviral treatment currently available for dengue diseases. given the complex pathology of the illness and the need to control four virus serotypes simultaneously, dengue vaccine development has been challenging too. in response to the urgent need for effective anti-denv drugs, our work for the first time identifies cbx, one of the glycyrrhetinic acid (gna) derivatives, as a potential natural compound against different serotypes of denv. cbx treatment did not inhibit denv- or denv- rna synthesis and protein expression in one replication cycle, but markedly reduced progeny virus the total rna was isolated from the infected cells and analyzed by quantitative rt-pcr. c: the expression of viral protein was determined by immunofluorescence assay using denv prm glycoprotein antibody. d: the percentages of prm containing cells were determined by piccnt Â. e and f: vsv was directly incubated with indicated concentrations of cbx for hr (e) or for indicated time (f) at ˚c. the titer of residual virus was determined by plaque assay. data are shown as mean ae sem of at least three independent experiments. à p < . ; Ãà p < . ; ÃÃà p < . . production, suggesting that post-translation stages of denv lifecycle as possible targets. time-of-addition experiment was also performed to narrow down the effective period of this compound. it is reported that cbx can freely shuttle into cytoplasm [parke, ; connors, ] . however, progeny denv- was inhibited only when cbx was added after virus attachment till hr post-infection (post-treatment) or throughout the course of infection (continuoustreatment), but not with pre-treatment of cells (pre-treatment) or till hr post-infection (shorttreatment), indicating that the inhibition of cbx is not exhibited by targeting the host cells. from these results, it was deduced that denv itself is the target of cbx. to test this assumption, virucidal activity assay was performed. both the titer and residual infectivity of denv were affected as assumed by preincubating with cbx, which manifested in the reduction of viral rna and viral protein, and the drop in viral titer. further study found that the level of viral rna in the supernatants was significantly downregulated in cbx-treated group, demonstrating cbx directly inactivates the progeny denv particles via its virucidal activity. the virucidal effect of cbx on denv might be exerted by irreversibly disrupting viral envelope intactness so that virus loses its protection and results in degradation. in this study, we used three different cell lines (a , thp- , and huvec) to investigate the effect of cbx on denv. given their high susceptibility to denv, a cells were the main cell line used. thp- cell line substituted peripheral blood mononuclear cells (pbmc) to mimic in vivo infection [clyde et al., ] . huvec cells, a cell line of human umbilical vein endotheliocyte, were also tested as they are a representative of non-cancerous, primary cell type. as a result, denv- was inhibited dose-dependently with increasing cbx in all three cell lines, demonstrating that cbx effectively inhibits denv- in the various cell types targeted by the virus. beside denv- , denv- viruses were also used to verify the inhibitory effect of cbx. all the results have demonstrated that the antiviral activity of cbx has no specificity on cell type or denv serotype, which might predict cbx as a new anti-denv compound to exhibit favorable application perspectives. previous study has showed that, glycyrrhizin, another effective compound derived from glycyrrhiza, inhibited denv infection in vero cells [crance et al., ] . this promoted us to suppose that cbx, one of the derivatives of glycyrrhetinic acid (gna), may play an effective role in anti-denv infections. as observed in our study, cbx treatment significantly reduced the progeny virus yield of different serotype of denv ( and ). further study found that cbx inhibits denv infection through its virucidal activity. beside denv, cbx has been previously found to inhibit another virus, hsv [partridge and poswillo, ; dargan and subak-sharpe, ]. however, the antiviral mechanisms of cbx on these two viruses are quite distinct. in regarding to hsv, cbx appeared to be active throughout the replication cycle: by reducing the synthesis of some polypeptides, affecting the transportation of certain proteins, and inhibiting the post-translational processing. as a result, cbx treatment greatly diminished progeny hsv quality, and it is thought to inhibit hsv infection in a non-specific manner, by altering cellular activities that can affect virus replication or activate host antiviral mechanisms. while in our model, cbx directly inhibited the infectivity of denv particles without targeting the host cells. likewise, further study found that pre-incubation with cbx significantly decreased vsv titer. taken together, it suggests that the antiviral activity of cbx was not limited to a specific type of virus, and the mechanisms might be something different. to be pointed out, cbx is a widely used compound as an inhibitor of gap junctions [connors, ] . gap junctions are fast-track intercellular communication routes. genetic studies have revealed that connexin (cx) protein is the molecular component of vertebrate gap junction channels [paul, ] . gap junctions comprise clusters of transcellular channels that allow small signaling molecules and inorganic ions to transfer rapidly and directly from one cell to another. recent studies have reported that gap junctions play key roles in the physiological processes and pathological conditions of several viruses, such as hiv, cytomegalovirus (cmv), borna disease virus, and rous sarcoma virus [eugenin, ] . importantly, in order to increase virus production, infection with the latter two viruses above results in down-regulation of cx expression. as a non-specific gap junction blocker, cbx inhibits various cx proteins including cx , cx , cx , and cx [tonkin et al., ] . therefore, the possibility that cbx inhibits denv infection through blocking cx proteins could not be ruled out. to this end, much more work would be needed to elucidate exactly how cbx inactivates denv through its virucidal activity. further studies in suitable animal models would also be required to evaluate the reproducibility of the inhibitory effects of cbx in vivo. in conclusion, our work provides compelling data that cbx has inhibitory effect on denv infection. importantly, the antiviral activity of cbx is not limited to a specific cell type or a specific denv serotype. combined 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carbenoxolone prevents allergic airway inflammation and airway hyperreactivity in a mouse model of asthma a simple method of estimating fifty percent endpoints how to close a gap junction channel: efficies and potencies of uncoupling agents effect of a combination of glutamyl-tryptophan and glycyrrhizic acid on the course of acute infection caused by influenza (h h ) virus in mice effects of glycyrrhizin on hepatitis b surface antigen: a biochemical and morphological study gap junction proteins and their role in spinal cord injury carbenoxolone sodium in the treatment of gastric ulcer with special reference to side-effects evolutionary relationships of endemic/epidemic and sylvatic dengue viruses human skin langerhans cells are targets of dengue virus infection functional importance of dengue virus maturation: infectious properties of immature virions key: cord- -giku r authors: manrique-saide, pablo; dean, natalie e.; halloran, m. elizabeth; longini, ira m.; collins, matthew h.; waller, lance a.; gomez-dantes, hector; lenhart, audrey; hladish, thomas j.; che-mendoza, azael; kirstein, oscar d.; romer, yamila; correa-morales, fabian; palacio-vargas, jorge; mendez-vales, rosa; pérez, pilar granja; pavia-ruz, norma; ayora-talavera, guadalupe; vazquez-prokopec, gonzalo m. title: the tirs trial: protocol for a cluster randomized controlled trial assessing the efficacy of preventive targeted indoor residual spraying to reduce aedes-borne viral illnesses in merida, mexico date: - - journal: trials doi: . /s - - - sha: doc_id: cord_uid: giku r background: current urban vector control strategies have failed to contain dengue epidemics and to prevent the global expansion of aedes-borne viruses (abvs: dengue, chikungunya, zika). part of the challenge in sustaining effective abv control emerges from the paucity of evidence regarding the epidemiological impact of any aedes control method. a strategy for which there is limited epidemiological evidence is targeted indoor residual spraying (tirs). tirs is a modification of classic malaria indoor residual spraying that accounts for aedes aegypti resting behavior by applying residual insecticides on exposed lower sections of walls (< . m), under furniture, and on dark surfaces. methods/design: we are pursuing a two-arm, parallel, unblinded, cluster randomized controlled trial to quantify the overall efficacy of tirs in reducing the burden of laboratory-confirmed abv clinical disease (primary endpoint). the trial will be conducted in the city of merida, yucatan state, mexico (population ~ million), where we will prospectively follow children aged – years at enrollment, distributed in clusters of × city blocks each. clusters will be randomly allocated (n = per arm) using covariate-constrained randomization. a “fried egg” design will be followed, in which all blocks of the × cluster receive the intervention, but all sampling to evaluate the epidemiological and entomological endpoints will occur in the “yolk,” the center × city blocks of each cluster. tirs will be implemented as a preventive application (~ – months prior to the beginning of the abv season). active monitoring for symptomatic abv illness will occur through weekly household visits and enhanced surveillance. annual sero-surveys will be performed after each transmission season and entomological evaluations of ae. aegypti indoor abundance and abv infection rates monthly during the period of active surveillance. epidemiological and entomological evaluation will continue for up to three transmission seasons. discussion: the findings from this study will provide robust epidemiological evidence of the efficacy of tirs in reducing abv illness and infection. if efficacious, tirs could drive a paradigm shift in aedes control by considering ae. aegypti behavior to guide residual insecticide applications and changing deployment to preemptive control (rather than in response to symptomatic cases), two major enhancements to existing practice. trial registration: clinicaltrials.gov nct . registered on april . the protocol also complies with the who international clinical trials registry platform (ictrp) (additional file ). primary sponsor: national institutes of health, national institute of allergy and infectious diseases (nih/niaid). aedes-borne viruses (abvs; e.g., dengue [denv] , chikungunya [chikv] , zika [zikv]) pose a major public health burden worldwide [ ] [ ] [ ] . transmitted primarily by the highly anthropophilic mosquito aedes aegypti, abvs propagate epidemically, inflicting substantial healthcare and development costs on urban tropical populations. model projections estimate that an average of million denv infections occur per year, of which million manifest clinically [ , ] . explosive denv outbreaks saturate healthcare systems [ ] , with worldwide estimates as high as $ billion ( us$) per year spent on costs related to medical care, surveillance, vector control, and lost productivity [ ] . the emergence and rapid epidemic propagation of chikv and zikv (and particularly congenital zika) have added significant burden and costs to healthcare systems [ , ] . given the heavy global burden of abv illness, and in the absence of efficacious vaccines or other therapeutic options, implementation of highly effective and currently available vector control strategies represents the most viable approach for abv prevention [ , ] . vector control methods such as larval control, source reduction, and space spraying are widely used against abvs [ , ] . unfortunately, there is limited epidemiological evidence that these methods are adequate to prevent or reduce human abv transmission in a sustainable manner [ , ] . poorly designed evaluations, a historical lack of focus on quantifying intervention impact using epidemiological endpoints, and limited funding for large-scale randomized controlled trials with epidemiological endpoints have all contributed to the lack of rigorous, evidence-based, assessments of abv vector control interventions [ , ] . furthermore, the classic deployment of house-based interventions in response to reported clinical abv cases has failed to account for the important contribution of out-of-home human exposure to ae. aegypti [ ] and the silent contribution of asymptomatic infections in sustaining infectious virus in local mosquitoes [ ] . novel vector control approaches and intervention delivery strategies with proven and robust epidemiological evidence of their impact on abv transmission are urgently needed. indoor residual spraying (irs) is the use of longlasting residual insecticides applied to the walls, eaves, and ceilings of houses or structures targeting vectors that land or rest on these surfaces [ ] [ ] [ ] . the residual component of the application means that, for several weeks or months, the insecticide will kill mosquitoes and other insects that come into contact with treated surfaces. historical evidence has shown that, when expeditiously implemented, residual insecticide applications can significantly reduce abv transmission [ ] [ ] [ ] . despite this evidence, the fact that it is time consuming and dependent on specialized human resources has limited widespread adoption of irs by abv control programs due to the perceived challenge of scaling-up the intervention over large urban areas. in urban settings, adult ae. aegypti typically rest indoors, where they feed frequently and almost exclusively on human blood [ ] . studies performed in panama, peru, and mexico have shown that ae. aegypti rest predominantly below heights of . m, mainly inside bedrooms and on surfaces made of cement, wood, and cloth [ ] [ ] [ ] . selectively applying residual insecticides below . m and on common mosquito resting surfaces provides an entomological impact similar to spraying entire walls (as performed in classic irs), but in a fraction of the time (< %) and insecticide volume (< %) compared to classic irs [ ] . this selective insecticide application mode is called "targeted indoor residual spraying" (tirs), and it involves the application of residual insecticides on exposed lower sections of walls [< . m], under furniture, and on dark surfaces throughout houses with the exception of the kitchen (fig. ). as such, tirs is a rational vector control approach whereby ae. aegypti resting behavior guides targeted insecticide applications, thus reducing unnecessary exposure to chemicals for both applicators and household residents (fig. ) , and also reducing the time it takes to spray a premise with no apparent loss in insecticidal efficacy [ ] . in cairns, australia, an observational study found that tirs can reduce the probability of future denv transmission by - % as compared to unsprayed premises [ ] . concurrent trap collections of ae. aegypti in the heart of the outbreak showed that tirs was associated with a~ % reduction in gravid ae. aegypti female abundance [ ] . in merida, mexico, a phase ii cluster randomized controlled trial (crct) evaluated the entomological impact of irs with bendiocarb (ficam®, bayer, a carbamate insecticide to which local ae. aegypti are fully susceptible) and reported reductions in indoor adult ae. aegypti abundance up to % over a -month period, compared to no reduction when the pyrethroid deltamethrin was used [ ] . fitting such entomological information to an agent-based model of yucatan state, mexico, showed that high levels of tirs coverage ( % of houses treated once per year) applied preemptively before the typical dengue season (before july) could reduce denv infections by . % in year and . % cumulatively over the first years of an annual program [ ] . such findings were confirmed with another modeling study comparing tirs with indoor space spraying in iquitos, peru [ ] . these findings suggest that preemptive tirs may provide high short-term and long-term effectiveness in preventing abvs in endemic areas where transmission is seasonal. a systematic review has identified tirs as a highly promising approach for abv prevention [ ] , but highlighted the limited evidence for tirs due to the absence of impact estimates from randomized controlled trials with epidemiological endpoints performed in endemic settings. the study protocol presented here introduces the design for a crct to test whether tirs, applied preventively, reduces laboratory-confirmed cases of abv illness and infection in the city of merida, yucatan state, mexico. trial endpoints are listed in table and the approaches followed to quantify them will be described in subsequent sections. merida, the capital city of yucatan state, is the largest urban center in the region with , inhabitants [ ] . the city has a tropical climate characterized by a mean annual temperature of . °c and an annual fig. targeted indoor residual spraying (tirs) to control ae. aegypti. in urban environments, houses are primarily built of brick and cement, and ae. aegypti rests preferentially below . m of height. spraying residual insecticides in walls below . m and in key resting sites such as under furniture (# in figure, represented in green) will eventually kill ae. aegypti that may be emerging from immature larval habitats outdoors ( ) and rest indoors on treated surfaces ( ) . after exposure to the residual insecticide, mortality can occur immediately ( ) or after several hours/days ( ) precipitation of mm. merida is endemic for abvs, with denv being persistently transmitted since and, more recently, co-circulating with chikv (since ) and zikv (since ) [ , ] . abv transmission in merida is seasonal, beginning in july and peaking in october-november. baseline serological information (captured by elisa methods) on natural abv infection rates has been collected from merida in - through a school-based cohort that followed all family members living in the same household as the enrolled children [ ] [ ] [ ] . in , denv seroprevalence in the cohort was . %, which increased with age from % in - -year-olds to % in adults ≥ years. in - , the incidence of lab-confirmed abv illness in the cohort was . per person-years ( % ci . , . ) [ ] . the incidence of symptomatic dengue infections observed during the same period was . cases per person-years ( % ci . , . ). the majority of seroconversions occurred in the younger age groups (≤ years old) [ ] [ ] [ ] . the incidence of symptomatic chikungunya illness was . per person-years ( % ci . , . ) and the incidence rate of symptomatic zika illness was . per person-years ( % ci . , . ) [ ] . zika virus symptomatic attack rate in pregnant women from the cohort was % [ ] . data from~ , geocoded denv, zikv and chikv symptomatic cases captured by mexico's national passive surveillance system from to identified denv transmission "hot-spots" in merida (areas with higher-than-average numbers of cases), which overlapped with chikv and zikv hot-spots [ ] . combining these data with information from the cohort, we found that denv seroprevalence rates are~ × higher in hot-spot areas compared to other areas [ ] . merida also has entomological laboratory infrastructure and trained personnel to conduct and evaluate tirs [ , ] . the collaborative unit for entomological bioassays (ucbe) is a reference laboratory within the autonomous university of yucatan (uady) and is currently a world health organization good laboratory practice (glp) site for evaluating insecticide products for vector control [ ] . the two-arm crct will include a total of clusters of × city blocks each, with clusters randomly allocated to the intervention (tirs) arm and clusters allocated to the control arm (fig. ) . routine ministry of health (moh) vector control actions performed in response to symptomatic abv cases reported to the healthcare system will not be interrupted and could occur across both study arms. upon detection of a suspected abv case in the national epidemiological database, yucatan moh mobilizes its staff aiming at containing local transmission by focusing efforts on adult mosquito control. truck-mounted ulv spraying with the organophosphate insecticides chlorpyrifos and malathion is widely implemented in merida, despite scientific evidence of its poor efficacy [ ] . moh response also involves indoor space spraying (iss) with pyrethroids (mainly deltamethrin) and organophosphates (malathion) in houses that allow entry. limitations in personnel, geographic extent of outbreaks, and availability of resources (e.g., insecticides) commonly challenge moh operations, reducing the coverage and effectiveness of their actions [ ] . all moh actions will be mapped and included in secondary analyses evaluating the impact of tirs in addition to routine vector control. participants in both arms will have access to any concomitant care they may choose to pursue, including cleaning their own yard and eliminating mosquito breeding habitats or using commercially available insecticide sprays or repellents (e.g., transfluthrin coils). clusters will be located within the areas previously identified as hot-spots of abv transmission [ ] (fig. ) . placing all clusters within areas of high abv incidence will increase power because of higher event rates and decrease the potential for imbalance across trial arms. to reduce contamination and edge effects, while all households in tirs clusters will be offered the intervention, epidemiological and entomological evaluations will occur in the center of each cluster, following a "fried egg" design ( fig. ). entomological interventions that are constrained to a given area suffer from immigration of mosquitoes from untreated neighboring areas, as observed in a recent study that released wolbachia-infected mosquitoes in fresno, ca, and quantified mosquito dispersal up to m from their release point [ ] . by focusing participant enrollment on the central × blocks of the × clusters, we will minimize any contamination in our primary and secondary endpoints emerging from mosquitoes flying into treatment areas (fig. ). this "fried egg" design is novel for vector-borne diseases and has been proposed as a rational approach to quantify the epidemiological impact of vector control [ ] . to prevent selection bias, enrollment into the trial will occur in all clusters before tirs allocation has been determined. to assess power and sample size requirements, we analyzed historical passive surveillance data from the hot-spot census tracts with population size of at least (from our previous work characterizing the abv hot-spot area [ ] ). we used yearly data from to on the number of dengue, chikungunya, and zika cases recorded in children - years each year by census tract [ ] . data were combined into pairs of adjacent years to mimic a -year trial period, and table summarizes the mean incidence (number of cases over -year period/number of children) and intracluster correlation coefficient (icc) for a given -year period [ ] . assuming % incidence over a -year period, % tirs efficacy, an icc of . , and % loss to follow-up, we will require age-eligible children enrolled per cluster for an overall sample size of clusters and children to have % power to detect a significant reduction in abv incidence between arms (table ) . clusters will be selected from the set of census tracts within the abv hot-spot area [ ] that have a total population size of at least and at least children aged - years, per the census (fig. ) . clusters are also selected to maximize the distance between the centroid of each cluster to the centroid of its nearest neighbor also in the trial. given a set of clusters, covariate-constrained randomization [ ] will be used to limit imbalance across trial arms with respect to the following census tract-level variables: population size, per census; population density, per census; percent employed population, per census; and cumulative number of abv cases between and , per passive surveillance. these variables were selected because of their association with abv transmission risk. for each balancing factor, only allocation patterns where the mean value of clusters in group a divided by the mean value of clusters in group b is within / . to . are retained. furthermore, we eliminate any allocation pattern with imbalance in the number of clusters per arm per sector greater than ± . to ensure randomization is not overly constrained, we only consider sets of clusters that have many acceptable allocations into two groups of , satisfying validity criteria proposed by moulton [ ] (e.g., pairs of clusters always or never appearing in the same arm). given the set of allocation patterns that meet the above balancing criteria, the biostatistics team at uf will use equal probability sampling to randomly select one allocation. a sample allocation pattern is plotted in fig. . for participant enrollment, the study teams will be provided with a list of census tracts for inclusion in the study, without a record of which census tracts are in group a or b. a random number generator produced by biostatisticians from uf will assign one group to tirs and one group to control. the trial will focus on the pediatric population, enrolling children aged - years in a longitudinal cohort to track their abv illness and lab-confirmed seroconversion over two (and potentially three) transmission seasons (fig. ) . the previously conducted cohort study in merida indicated that the majority of dengue-naïve infections and seroconversions occurred in children ≤ years old [ ] [ ] [ ] . by following children aged - years at enrollment, we will capture the segment of the population with the highest probability of abv illness. we excluded younger children (< years) because of the difficulties in obtaining blood specimens and potential for cross-reactivity with maternal antibodies [ ] . there will be two levels of participation: at the household level and at the individual child level. table shows the inclusion/exclusion criteria for each level. for each participation level, consent (and assent) will be obtained, as follows. on august , after being given time to review information about the intervention, one adult household decision-maker will be asked for written consent to have their house included in the trial (at the time of consent, neither study personnel nor householders will know to which arm of the trial the house will be allocated). in consenting houses with children meeting the inclusion criteria (table ) , individual consent/assent will be obtained during december -january . parental informed consent will be obtained for children aged - years, and both assent to participate from children and a parental informed consent will be obtained for - -year-olds (additional file ). enrollment of children will be focused in the central × city blocks of each cluster and will extend beyond if not enough children are enrolled in the core. consent will be obtained in participants' homes. study explanations will be provided to small groups of adults present in the household, whereas written consent and assent will be obtained from each individual participant. engaging communities early in the trial will be essential for maximizing participant acceptance and retention [ , ] . an experienced team of social workers, who will interact directly with study participants (through informal conversations, games, and other educational activities with children), will ensure they remain engaged throughout the duration of the study [ ] . several factors may lead one household to withdraw from the intervention. householders may sell their home and move to a different location, and we will consider them lost to follow-up. householders may refuse to receive the intervention on a second or third opportunity, meaning they will not be subject to treatment (and therefore excluded from any future analysis). our team will document voluntary withdrawals and communicate them as part of the trial reporting. trial performance milestones table shows our proposed milestones for the trial, following the spirit checklist, and sections below provide information on each step (see additional file ). they can be divided into (a) trial planning, (b) tirs evaluation, and (c) trial analysis and reporting. trial design will be finished during the first year. enrollment is expected to last up to months, when all children will enter follow-up. trial evaluation will occur for two transmission seasons, with the possibility of adding a third season should incidence of the primary endpoint be lower than assumed. trial analysis will include a projection of tirs impact, based on results from the crct, using our stochastic simulation model fitted to our study population. a baseline assessment of household characteristics (size, building materials, number of rooms, number of inhabitants) and ae. aegypti infestation and susceptibility to insecticides will occur july-december (fig. ) . entomological collections will be conducted monthly in % of all houses located in the centers of the clusters (blue blocks in fig. , equal to houses across clusters). standard ovitraps will be placed to collect eggs that will be reared for assays to characterize insecticide susceptibility in mosquito populations. after the transmission season (january-april ) and during individual child enrollment, a baseline sero-survey will quantify levels of abv seroprevalence. all enrolled children will provide a blood sample by venipuncture, which will be tested for the presence of neutralizing antibodies against denv, chikv, or zikv (see laboratory methods below). personnel from the servicios de salud de yucatan (ssy; yucatan's ministry of health) will conduct the tirs after proper training [ ] . based on our model [ ] . we will prioritize the use of the organophosphate pirimiphosmethyl (actellic cs®), given its longer residual power in comparison to the carbamate bendiocarb (ficam®) [ ] . however, if insecticide resistance profiles of mosquitoes after the first year of spraying show decreases in susceptibility to the active ingredient in actellic cs®, we will switch to ficam®. insecticide application will follow strict procedures developed by project team [ ] . residents will be asked to temporarily leave the house during treatment and wait h for the product to dry before re-entering. staff will wear branded uniforms with identification and use appropriate personal protective equipment. the epidemiological impact of tirs on the primary endpoint will be evaluated by active surveillance to detect and lab-confirm symptomatic denv, chikv, or zikv from july to december of each season (fig. ) . enhanced symptomatic abv case detection will rely on three sources (fig. ). ten field teams consisting of a nurse and a social scientist will conduct wellness visits to all enrolled children once per week, with the goal of identifying any probable case of abv illness. in addition to wellness visits, nurses will call parents/guardians of enrolled children regularly (twice per week) to check for the occurrence of any abv symptoms. when interacting with parents/guardians, nurses will also remind them that they can call our toll-free - number in case of any illness compatible with an abv infection. widely used by the previous cohort, the - number enhanced the detection of symptomatic individuals by providing study participants - access to a toll-free phone number to consult an "on call" project physician about any symptom in their children [ ] . additionally, our project will access the online abv database managed by mexico's national center of preventive programs and diseases control (cenaprece) [ ] to identify all reported symptomatic cases (including all ages, not only children) residing within study clusters in real time, and to map routine vector control actions performed by ssy. for ascertaining the primary endpoint, a suspected symptomatic abv case is defined as a participant with acute onset of fever (axillary temperature ≥ °c) or a non-focal rash plus any additional symptom such as headache, conjunctivitis, arthralgia, or myalgia. when a suspected abv case is identified through active surveillance, they will be visited preferably on the same day by one project physician to perform a physical examination (physical exam, temperature, vital signs). the doctor will be joined by one field team member, who will obtain demographic and behavioral data, and collect blood specimens. acute and convalescent (obtained [range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days after symptom onset) blood specimens will be collected from each suspected case to confirm abv infection. additionally, history of movement (by a retrospective movement survey) [ , ] will provide information on potential exposure locations for each case. after laboratory confirmation, participants will meet with study physicians, who will explain the diagnosis and potential steps if symptoms worsen. epidemiological impact will be further assessed via a secondary endpoint capturing serological evidence of abv infection (table ) . yearly blood samples from all enrolled participants will be collected after the regular transmission season (from january to april) to test for serologic evidence of interval infection by denv, chikv, or zikv, as in [ ] [ ] [ ] . in addition to collecting blood specimens, project team will also conduct annual prospective movement surveys to characterize the routine mobility patterns of participants. entomological impact will be measured by standardized monthly collections of indoor adult ae. aegypti (table ) . a random sample of % of the houses located in the center ("yolk" of our fried egg design) of each cluster (~ houses in total) will be visited and surveyed for the presence of adult ae. aegypti mosquitoes indoors using prokopack® aspirator collections performed for min per house, as described in [ , ] . female ae. aegypti collected indoors will be pooled by city block and tested for abv infection. entomological surveys will begin immediately following tirs implementation (july ) and will be performed monthly for months (until dec ). monthly who cone bioassays [ , ] will be done in a random sample of treated houses to monitor the residual efficacy of the insecticide used. venipuncture procedures will be performed using standard aseptic techniques. an experienced phlebotomist will take the blood sample from an antecubital vein. blood will be collected into vacutainer® collection tubes or by a needle and syringe. a -gauge needle will be used for - -year-olds, and a -gauge needle for children < years. blood specimens will be immediately taken to yucatan state diagnostics laboratory, dependent of the ministry of health for immediate molecular diagnostics (acute samples) or serum separation, followed by elisa tests (convalescent samples and annual blood draws). aliquots of all specimens will be stored at − °c in labeled polypropylene cryogenic vials at uady, and then transported to emory university for advanced diagnostics. long-term specimen storage will occur at emory university. specimens from individuals who did not sign the "future use" clause of the consent will be discarded after diagnostics, following sample processing procedures established by yucatan state laboratory. figure shows all lab testing components of the trial, which will occur at ssy, uady and emory university. acute samples from active surveillance will be tested at the yucatan state laboratory using a multiplex reverse transcriptase-polymerase chain reaction (rt-pcr) [ ] and virus-specific igm elisas. annual serologic samples will be tested at yucatan state laboratory by antigen capture elisa for human igg [ ] , and positive samples will be taken to emory university for focus reduction neutralization testing (frnt) [ ] [ ] [ ] . natural abv infection rates in ae. aegypti will be detected by rt-pcr [ ] at uady. standard cdc bottle bioassays [ ] will assess phenotypic resistance of adult ae. aegypti from treatment and control clusters pre-intervention and at and months post-intervention every year. f , or f progeny, from field-collected eggs will be screened for susceptibility to pirimiphos-methyl [ ] . if resistance is detected, both dna and rna will be analyzed from a subset of the phenotyped mosquitoes to calculate the frequencies of known resistance alleles as well as expression of resistance-associated genes. given cross-reactivity and variable sensitivity of assay methods, we will use a composite approach to diagnose abv infections (fig. ) . for active surveillance, two diagnoses are used: preliminary diagnosis-suspected cases are confirmed if rt-pcr is positive for any abv. if rt-pcr is negative, the acute specimen igm result is considered and any positive igm result indicates a preliminary diagnosis of abv infection. if both zikv and denv igm assays are positive, the case is designated as a case of flavivirus infection. final diagnosis-paired acute and convalescent specimens will be tested for igm and igg seroconversion. these results will refine the case designation. a case with laboratory evidence of abv infection in the acute testing must also demonstrate seroconversion or increasing levels of igg or igm in the convalescent specimen. rt-pcr+ suspected cases that do not exhibit seroconversion or increase in igg or igm levels will be designated recent infections, but not abv cases (that is, this result is most consistent with an etiology other than abv infection as the cause of the symptomatic illness). additionally, igg or igm seroconversion or increasing igg that is observed when rt-pcr and acute specimen igm are negative will be considered confirmation of an abv case. this approach may increase the sensitivity to detect abv cases that have false negative pcr testing and have not yet mounted an igm response at the time of presentation. finally, if convalescent serology does not distinguish between denv and zikv infection, the annual surveillance sample for that subject will be considered. if it is clear from neutralization testing on the annual surveillance specimen what the intervening viral infection was, that will become the designation of the abv case captured during active surveillance. the annual serologic surveillance takes into account that the majority of abv infections are inapparent. it will also account for the known cross-reactivity among abvs. chikv is an alphavirus, and serologic assays for chikv perform with high sensitivity and specificity. elisa is likely sufficient for annual chikv serosurveillance. denv and zikv are related flaviviruses, and conventional approaches to serologic diagnosis of flavivirus cases can exhibit reduced specificity. however, the antibody response to denv and zikv is dynamic, and cross-reactive antibody levels are greatest in the first few months after infection. thus, cross-reactivity is present but less intense in late convalescence, which is one reason for performing serosurveillance in the lowtransmission season. for flavivirus surveillance, neutralizing antibody titers will be compared using the frnt (inverse of serum dilution that exhibits % of maximum neutralization). conversion of neutralization assays from negative to positive in subsequent years is strongly supportive of interval infection. the precise infecting virus (denv - serotype or zikv) can often be identified by comparing relative frnt values for each virus. a ≥ -fold difference in the frnt is considered a significant difference. once an individual has high titers to multiple denv serotypes, detection of additional denv infection is challenging by serosurveillance alone. the details of interpreting all possible flavivirus neutralizing antibody profiles are beyond the scope of the article. we have reviewed the key concepts recently [ ] . the primary analysis will estimate the overall efficacy of tirs in reducing the rate of laboratory-confirmed abv illness, where the overall efficacy is estimated as one minus the hazard ratio from a cox proportional hazards model [ ] . the hypothesis test for the primary outcome will be a score test of the null hypothesis that tirs efficacy is ; the two-sided test will be conducted at the α = . level. the cox proportional hazards model will be fit using individual-level data for eligible and consenting children. the primary endpoint will be time to symptom onset of first laboratory-confirmed abd. the time origin will be july prior to the first season, by which time spraying will have been completed. the analysis will consider events occurring between july and december of each year of the study, as this corresponds to the time when the residual effect of the insecticides used in tirs is expected to be active and while active surveillance is ongoing. to account for clustering, the model will include a robust variance estimator with two parameters; one characterizes the level of correlation in outcomes between children within the same household, and one characterizes the level of correlation in outcomes between children in different households but within the same cluster. we will use schoenfeld residuals to assess departures from proportionality, as would occur if the effect of tirs varies over time [ ] . we will use timedependent (piecewise) models where significant nonproportionality occurs [ ] . planned secondary analyses of clinical and human serological data include: cox proportional hazards model with time to first laboratory-confirmed symptomatic abv disease as the endpoint, adjusting for additional cluster-and household-level covariates (e.g., population density, household size, socio-economic status). cox proportional hazards model with time to first laboratory-confirmed symptomatic abv disease as the endpoint, adjusting for routine human movement as measured by the prospective movement survey (measured in all enrolled participants). the proportion of time in treated areas will be included as a further covariate, as described in [ ] . disease-specific versions of the primary analysis (e.g., time to first laboratory-confirmed symptomatic dengue disease as the endpoint), if data permit. analysis of recent human movement measured by a retrospective movement survey in enrolled participants presenting with symptoms for laboratory confirmation. the data will be analyzed using a test negative design-type structure, where individuals testing negative for any abv will serve as a comparator group for individuals testing positive for abv. the analysis will adopt recently developed methods for cluster randomized vector control trials [ , ] . binomial generalized linear mixed effects model to assess the efficacy of tirs for reducing laboratoryconfirmed denv, chikv, or zikv infection will be analyzed as cumulative incidence over the two (or potentially three) transmission seasons, as measured from annual serological samples. given the larger number of sub-clinical and undetected abv infections compared to symptomatic abv illness, the study will be amply powered to detect a statistical difference in abv infections (measured by annual serology). using the passive surveillance data, we will quantify the community impact of tirs on symptomatic abv cases reported to the public health system, beyond our pediatric cohort. poisson regression will be used to compare cluster-level incidence rates across trial arms. acceptability of tirs intervention will be assessed by calculating summary statistics from the postintervention data. acceptability measures will be paired with any adverse reactions experienced or reported by study participants and assessed by our team of physicians. for mosquito data, planned secondary analyses include: the following ae. aegypti adult indices will be calculated for each sampling date and compared between treatments and over time: presence (binomial variable) and abundance (count variable) of adults, females, and blood-fed females per house. generalized linear mixed effects models (glmm) nested at the cluster (level ) and city block (level ) levels will be used to compare each entomological index between treatment and control arms, as in [ ] . link functions for glmms will be binomial for presence indices and negative binomial for abundance indices. the best fit models (after comparing aic values for models including all levels or only level ) will be used to calculate odds ratios (or; for mosquito presence/absence) and incidence rate ratios (irr; for mosquito abundance) using control houses as the unit of comparison. we will calculate the operational efficacy of the intervention as e = ( − irr) × . this measure, ranging between and , describes the percent reduction of mosquito abundance in treated houses with respect to the control. similarly, a negative binomial glmm will test for differences in treatment and control arms for infection rates with denv, chikv, or zikv, calculated as minimum infection rate, following similar statistical methods as for ae. aegypti abundance. epidemiological and entomological information will be combined to quantify the relative reduction in the incidence of symptomatic abv illness at the cluster level observed from a measured entomological reduction due to tirs (measured as number of adult or female ae. aegypti). binomial glmms, with random intercepts at the cluster and year levels, will quantify the association between both variables for the duration of the trial and provide values of threshold vector densities associated with a significant reduction in the odds of human symptomatic infection. transmission modeling our existing mathematical model for yucatan [ , , ] will simulate the effectiveness of tirs for different scenarios of intervention coverage and insecticide residual power, using the observed trial data as a critical model input. this agent-based model of individual people and mosquitoes incorporates household demography, a spatially heterogeneous population structure based on census and remote sensing data, movement of workers and students, and seasonal fluctuations in mosquito population and incubation period. different movement (e.g., mosquito vs. human) and transmission (e.g., pathogen introduction and elimination) dynamics become relevant at different spatial scales; thus, we will predict the impact of scaling up tirs to the entire state rather than treating just merida. simulating epidemiological trends of scaled-up tirs for periods longer than the duration of this trial (e.g., a decade) will evaluate the effect of changing population-level immunity and generate measures of effectiveness that are more informative for programmatic decision making. emory university will coordinate all aspects related to data storage, management, and sharing. a data management core (dmc) provides timely and efficient curation and dissemination of study data from multiple sources (e.g., clinical, laboratory, passive surveillance, entomology, demographic, ministry of health interventions), all essential to the success of the trial (fig. ) . information from the trial including consent forms, surveys, active surveillance forms, laboratory diagnostics, entomological surveys, mobility surveys, withdrawal forms, intervention acceptability, and annual blood draws will be collected in paper form and digitally recorded into our redcap database (see below) by the data entry staff at uady. staff will enter information in a private dedicated space at uady-ucbe. laboratory results at emory university will be entered directly into the redcap database by laboratory staff using an online form. all forms were developed by our team specifically for this study. all data will be stored on secure data servers and kept strictly confidential (with participant identifiers blinded by using non-identifiable ids). households are assigned codes unique to the project database, which are then used to identify all subsequent data we will collect. outside of the database, these codes will not be interpretable, rendering the data effectively unidentifiable without access to our servers. blinding of identifiable data will occur in the analysis stage also. all diagnostics of specimens will be conducted using the sample id, blinding laboratory personnel from any identifiable information or membership of samples to a given study arm. access to the database will be primarily administered through a custom, web-based interface with restricted access privileges and encrypted data transfer (redcap, https://www.project-redcap.org/). different data entry interfaces will be generated for each component. access will be limited to certified project personnel and certified associates, who will be provided unique login and password combinations. database servers will be protected by multiple layers of security. databases will be shared electronically through secure servers among key project personnel for analyses, publications, oral presentations, and project development. regular checks of the database for completeness and accuracy will be performed. the heterogeneous nature of abv transmission may dictate the need for a third transmission season to evaluate the epidemiological impact of tirs. the decision to continue into a third season will follow an event-driven decision process. after the second season evaluating tirs, the statistical team will quantify the number of total primary endpoints. we will pursue the following ranking in order to evaluate whether to stop or continue into a third season: the choice of < endpoints represents the target number of events needed for a power of % when tirs efficacy is %. overall, the risks to study participants are minimal in all of our study procedures ( table ). the most serious risk is related to potential intoxication with the insecticides used in tirs (table ). both actellic cs® and ficam® have been approved by the world health organization (who) for indoor control of mosquitoes [ , ] . the who's hazard assessments concluded that, when used for indoor residual spraying as instructed and at the recommended doses, both products do not pose undue hazards to the spray operators or residents of the treated dwellings [ ] [ ] [ ] . provided that operational guidelines are followed, routine cholinesterase monitoring of spraying personnel during indoor residual spraying programs is not required [ ] [ ] [ ] . during the period of active surveillance, immediately after tirs application, study participants will be contacted regularly ( ×/week in-house or ×/week by phone calls) by our team, who will ask for the presence of any sign of intoxication in any of the members of the house. such contacts will coincide with the epidemiological evaluation of the intervention. in addition to our team's direct contact, households receiving tirs will receive a pamphlet with a - toll-free number for them to self-report any signs of intoxication. once in the presence of a probable case of intoxication, a physician will medically assess the patients to diagnose the extent of their condition. vital signs, together with respiratory distress (i.e., bronchorrhea, bronchospasm) and clinical evidence of cholinergic excess (i.e., salivation, vomiting, urination, defecation, miosis), will be followed until they resume. in cases of severe intoxication, plasma cholinesterase activities will be assessed, together with electrolytes and serum lipase (both tests can be performed at uady's school of medicine public health laboratory, which routinely performs such tests for pesticide occupational exposure assessments). given the insecticide dose and mode of application used in tirs, we expect most intoxications to be mild and resume after exposure ends (i.e., after individuals are exit their home). our preliminary results from our phase ii entomological trial utilizing actellic cs showed that in houses (including individuals) a total of cases ( %) of symptoms compatible with a reaction to the insecticide were detected (vazquez-prokopec et al. unpublished). the most common signs (accounting for % of symptoms) were headache, nausea, and mild skin irritation. however, if the physician considers that a moderate to severe intoxication occurred, serological tests will be performed to confirm the cause of their condition. all probable aes will be noted in the adverse event log (ael), which will be the primary form of communication between physicians and the pi. aels will be filed immediately (one record per event) after the detection of a probable ae (the form will include links to any specific medical record or laboratory record associated with each case). once an ael is filed in the database, the pi will receive an alert requiring his attention. upon conversation with the study doctors, the pi will make an informed decision as to whether the condition represents a reportable ae or not. any ae or unanticipated problems (up; serious, life threatening, or result in death and unexpected and caused by the intervention) involving risk to participants will be notified to the irb within calendar days of their occurrence. emory irb will generate specific forms within their eirb platform to report any aes or ups associated with this study. the irb reports on aes or ups will be received by the nih program officer assigned to this study. in the unlikely situation that ups emerged due to tirs implementation, emory irb and the nih program officer will coordinate with the pi about the temporary or permanent suspension of this study. this project will strengthen a unique us-mexico partnership involving universities and research centers (emory, uady, fred hutch, uf) and federal agencies (cenaprece, mexico's national institute of public health, cdc) together with state agencies (ssy). emory university will lead the project and will be in charge of overall coordination, procurement of commodities (e.g., insecticides, diagnostic reagents), and data coordination, advanced diagnostics, and irb approval. the autonomous university of yucatan will coordinate all aspects of the field implementation of the trial as well as the integration of field and laboratory data streams. trial design will be led by fred hutchinson cancer research center. analyses for the primary and secondary endpoints as well as for evaluation of trial continuation will be conducted by uf (ira longini, natalie dean), with input from biostatisticians from fred hutchinson cancer research center. uf will also lead the mathematical modeling component. technical support will be provided by the us cdc to evaluate patterns of insecticide resistance in space and time. mexico's cenaprece will provide access to the online abv database. the ssy will contribute spraying personnel and access to samples for laboratory testing in support of the trial's active surveillance procedures, as well as help with communication about tirs and the trial's goals. dr. silvina contreras-capetillo, md (hospital o'horan, merida, mexico), expert in clinical aspects of aedes viruses, particularly genetic malformations in zika, will act as an independent trial monitor. the funder (nih) considered the data gathered in this project will be identifiable and certain data types, such as movement interview, are sensitive. the primary risks lie with identifying the individuals who provided information they consider confidential (e.g., movement to private locations). there is a small risk that the repeated blood collections will cause or exacerbate anemia. in-depth interviews (prospective and retrospective movement interviews) risks to study participants are minimal. participants may feel that in-depth interviews take up too much time-but they have the option of ending their participation at any time. there are no sensitive topics covered, but if any participant feels that there is something he/she does not want to talk about, he/she does not need to answer all questions. the low risks associated with the intervention not to merit the establishment of a dsmb. as such, the study team and the nih program officer(s) will communicate directly about study findings, reports from independent trial monitor, continuation rules, and adverse events. any deviation from protocol will require prior approval by the nih program officer. the study protocol and associated documents including informed consent forms are approved by the respective institutional review boards (irb) of all collaborating institutions as well the national institutes of health. the trial protocol was registered on clinicaltrials.gov (nct ) on april , . it will be made clear during the consent process that no information can be shared with anyone other than designated study personnel, the paper and computer files will be well protected, and we will ask that interviews be carried out one-on-one to prevent other family members listening in. consent and assent forms include a separate section where participants give permission to the pi to keep their specimens for future tests or studies. we will take all necessary measures to ensure confidentiality. it will also be made clear to study personnel that any violation of confidentiality would be a fireable offense. all paper data forms will be stored in locked files or cabinets in uady in a specified storage facility with limited access. access to computer data files will be password protected to allow exclusive access to appropriate study personnel. the paper data forms associated with the project (e.g., consent forms, questionnaires, census) will be stored in accordance with irb regulations. should consent be given for future use, then serological samples will be stored indefinitely. the samples will not have any participant identifiers, beyond the participant's code. if, however, consent for future use is not given, the blood samples will be destroyed immediately (using strict protocols at uady for disposal of biological samples) following completion of the project. monitor evaluations will occur once a year and will be timed to occur right after the epidemiological evaluation of tirs (january-march). on every visit, dr. contreras-capetillo will file a monitoring log and a self-monitoring tool form. selfmonitoring will be performed on a random selection of % of study participants. the monitor will also review records of all adverse events as well as the information of any dropouts that occurred between monitoring periods. after the visit, the monitor will submit the self-monitoring tool to the pi, together with any recommendations based on the visit. a phone call between the monitor and the pi will be scheduled, should corrective actions be required. novel tools and strategies that are operationally feasible and widely scalable are desperately needed to prevent and control abvs. this phase iii crct trial will quantify the epidemiological impact of tirs in preventing abvs and generate a definitive evidence base for assessing the public health value of this approach. the heavy reliance on pyrethroid insecticides for mosquito control has led to widespread pyrethroid resistance on a global scale [ ] . the high levels of resistance to pyrethroids found in mexico [ ] , including the yucatan [ ] , prompted cenaprece to expand the chemical groups used for aedes control to other insecticide classes such as carbamates and organophosphates, to which local ae. aegypti are susceptible [ , ] . a recent entomological crct performed in merida, yucatan, demonstrated that utilizing an insecticide to which ae. aegypti were susceptible had a significant impact on indoor mosquito density, as compared to the use of a pyrethroid to which the local population was resistant [ ] . the selection of new insecticide formulations (e.g., microencapsulated insecticides) with longer residual power (ca. - months) can further increase the effectiveness of tirs. fortunately, r&d for new insecticide formulations as well as novel chemistries for vector control has expanded, and new products are at various stages in product development pipelines [ ] . findings from this trial will not only aid in understanding how residual insecticides can function effectively for abv control but also help catalyze r&d for residual insecticide formulations better suited for the surfaces and materials found in urban areas. responding only to symptomatic abv cases likely misses a significant number of cases as a large proportion of abv infections are asymptomatic, which can still successfully infect mosquitoes [ ] and in turn significantly contribute to abv transmission [ ] . findings from a spatially explicit agent-based model of dengue dynamics in yucatan, mexico [ , , ] , suggested that tirs maximal effectiveness occurs when it is deployed preemptively (before the seasonal peak of abv transmission) rather than reactively. our trial will evaluate the preemptive implementation of tirs (spraying - months prior to the beginning of the peak abv transmission season). if found efficacious, the trial will make a strong case for the public health value of preemptive, long-lasting vector control measures against abvs. this finding would contribute to a paradigm shift in aedes control and abv prevention, leading to innovations in the way that interventions are conceptualized and brought to scale in operational settings. while the crct approach itself is largely standard, focusing on adherence to core epidemiological principles [ ] , our trial will incorporate several innovative features into the randomization and analysis. we have modified the covariate-constrained randomization procedure [ ] to include a selection step to maximize the geographical spread of the clusters. this strategy may be useful in future vector control trials. through the use of highly spatially resolved prospective and retrospective movement surveys, we will be able to refine our estimates of tirs efficacy to account for participant time spent in treated and untreated areas [ ] . finally, we are able to directly integrate trial data on mosquito abundance, human movement, and clinical outcomes into an existing mathematical model to better understand the potential population-level impacts of tirs. using statistical simulations to help interpret and contextualize the results of an infectious disease trial is an emerging area of research [ ] . to fulfill the critical need for carefully designed trials for vector control [ ] , this study will provide key data on the epidemiological impact of tirs on abvs and contribute methodologies and approaches for the design of future crcts. at the time of submission, the project is on its second trimester (table ) and main administrative activities have been activated. initial community contacts are expected to occur on mid-october , with concurrent participant enrollment (level ) and baseline serology occurring january-march . such timeline 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control programmes asymptomatic humans transmit dengue virus to mosquitoes cluster randomised trials simulations for designing and interpreting intervention trials in infectious diseases springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors acknowledge scott ritchie for his inspiring contribution to the development of the tirs methodology. drs. michael dunbar, gregor devine, richard reithinger, gabriela gonzalez-olvera, wilbert bibiano-marin, and amy crisp provided feedback for the design or conceptualization of the trial. the findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention or the national institutes of health. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the full trial protocol will be made publicly available within year of the conclusion of data collection. the datasets generated in this study will be made available by the corresponding author on reasonable request, within year of the conclusion of data collection. this trial protocol has been approved by emory university (irb ) and the autonomous university of yucatan (cei- - ) and endorsed by the secretarias de salud de yucatan. written consent/assent will be obtained from participants and kept in a secure place for record-keeping and trial monitor evaluation. the authors declare that they have no competing interests.author details unidad colaborativa de bioensayos entomológicos, campus de ciencias biológicas y agropecuarias, universidad autónoma de yucatán, merida, key: cord- - o v a authors: halstead, scott b; katzelnick, leah title: covid vaccines: should we fear ade? date: - - journal: j infect dis doi: . /infdis/jiaa sha: doc_id: cord_uid: o v a might covid vaccines sensitize humans to antibody dependent enhanced (ade) breakthrough infections? this outcome is unlikely because coronavirus diseases in humans lack the clinical, epidemiological, biological or pathological attributes of ade disease exemplified by the dengue viruses (denv). in contrast to denv, sars and mers covs predominantly infect respiratory epithelium, not macrophages. severe disease centers on older persons with pre-existing conditions and not young infants or individuals with previous coronavirus infections. live virus challenge of animals given sars or mers vaccines has resulted in vaccine hypersensitivity reactions (vah), similar to those in humans given inactivated measles or respiratory syncytial virus vaccines. safe and effective covid vaccines must avoid vah. a c c e p t e d m a n u s c r i p t introduction. not since pandemic smallpox or the influenza have humans confronted a epidemic viral pathogen as successful as sars cov- , a member of a family of viruses that cause serious diseases in many vertebrates. [ ] it has proved difficult to achieve robust vaccine protection against avian, bovine, porcine, canine and feline coronaviruses, failures sometimes attributed to "antibody dependent enhancement (ade)." [ ] the possibility that a sars cov- vaccine may sensitize recipients to ade has received considerable scrutiny. [ ] on inspection, ade is not one but two vaccine-related immunopathological phenomena: intrinsic ade (iade) and vaccine hypersensitivity (vah). iade describes interactions between igg antibody and microbial pathogen immune complexes that attach to fc receptors to initiate infection but also enhance replication of the microbe by suppressing innate cellular immune defenses. [ , ] vah was first described in humans in the early s, after formalin-inactivated measles vaccines were introduced in the us and europe. within months large numbers of vaccinated children developed a severe breakthrough disease, called "atypical measles." [ ] a similar outcome, "vaccine associated enhanced respiratory disease (vaerd)," was observed in infants, - months of age, who were given formalininactivated respiratory syncytial virus (rsv) and a few months later infected by rsv. [ ] the outcomes observed were attributed to delayed type hypersensitivity and/or an arthus reaction. [ ] lung lesions revealed damage to parenchymal tissue, a pulmonary neutrophilia with abundant macrophages and lymphocytes and excess eosinophils. from studies in laboratory animals, it is thought that formalin de-conformed viral antigens raised nonprotective antibodies that led to a th polarization of the immune response and a deficit of cytotoxic t cells. it was also the case that mice immunized with rsv inactivated with uv radiation, a purified fusion (f) protein, or a vaccinia-rsv replicative construct experienced similar pathology following challenge with wild-type virus. a similar pathological response has repeatedly accompanied live virus challenge in several species of laboratory animals a c c e p t e d m a n u s c r i p t vaccinated with sars and mers cov constructs, with and without adjuvants. [ , ] vah may best be defined as a coombs type iii antigen hypersensitivity. it should be emphasized there is no formal proof that vaerd is antibody mediated. the mechanism(s) of the postmeasles vaccine disease enhancement and its similarity to vaerd are not known. the biological behavior of some coronaviruses in non-human species together with evidence that human coronavirus antibodies enhanced infection of sars or mers covs in fc receptor-bearing cells, in vitro, have led to speculations that ade contributes to disease severity in humans. [ ] it has been reported that high levels of sars cov- igg antibodies circulated in severe sars cases and that anti-s igg neutralizing antibody (nab) responses developed significantly faster after the onset of clinical symptoms in fatal compared with recovered cases leading some to attribute enhanced tissue damage to ade. [ ] because sera from sars or mers vaccinated animals sera enhanced cov infections, in vitro, it was assumed that post-vaccination pathologies, too, were ade responses. [ ] dengue ade if sars or mers infection outcomes are affected by iade they should have epidemiological and disease features in common with denv. these are compared in table . in vivo, iade requires an initial immunological event, termed "sensitization." in dengue, this occurs in three settings: ) first infections, [ ] ) multitypic dengue antibodies passively transferred to infants (high antibody levels protect, low levels enhance), [ ] and ) vaccination resulting in incomplete protective immunity. [ , ] crucial to the occurrence of iade is the circulation of four antigenically related denvs. after a first infection, there is a - year period of relative cross protection after which heterotypic denv infection may cause severe disease. [ ] third or fourth sequential infections are not pathogenic. pre-outbreak age-specific distributions of dengue antibodies control age-specific ade disease attack rates. during heterotypic infections, viremias may be enhanced early but as illness progresses the titers and duration of viremias are shortened. [ ] a c c e p t e d m a n u s c r i p t dengue disease is a serious and widespread global health problem. in many dengue endemic countries there is an estimated % lifetime risk of hospitalization for enhanced dengue disease. [ ] severe denv iade infections are short duration illnesses that elicit a stereotypical clinical course: an abrupt onset of fever and generalized symptoms followed around the time of defervescence by a rapid loss of fluid from the vascular compartment and, in turn, anoxia, shock and gastrointestinal hemorrhage. [ ] the first suggestion of an immunopathology was finding that denv infected peripheral blood leukocytes (pbl) from dengue-immune monkeys and humans but not pbls from non-immune donors. [ ] severe denv infections in infants implied that antibodies were etiological factors, a hypothesis confirmed when antibody mediated enhanced denv infection was produced in monkeys. [ ] peak viremia titers observed early in illness are predictive of severe dengue in humans. [ ] careful pathologic studies identified splenic and lymph node monocytes, macrophages and dendritic cells as major targets of denv infection. [ ] fluid loss from the vascular compartment is attributed to capillary damage caused by circulating toxic viral protein (nonstructural protein -ns ). [ ] in humans, disruption of endothelial glycocalyx components by ns correlates with plasma leakage during severe denv infection. [ ] denv ns -induced endothelial cell intrinsic pathway vascular leakage is related to loss of integrity of endothelial glycocalyx components both in vitro and in vivo and is independent of inflammatory cytokines. [ ] two corollary iade phenomena have been described: ) passively acquired dengue antibodies efficiently enhance infection/disease and ) disease severity rates may increase rapidly during epidemics. severe dengue accompanies first infections in infants circulating dengue antibodies acquired from multi-immune mothers. [ ] during the course of secondary denv epidemics in cuba in and , disease severity increased month to month. it has been suggested that a single amino acid mutation in non-structural protein (ns ) may observed as early as days after onset of illness. it is thought that competent t cell immunity is essential for recovery. [ ] while many clinical and pathological features are shared by sars, mers and covid , lungs from patients with covid- show distinctive severe endothelial injury associated with the presence of intracellular virus and disrupted cell membranes. histologic analysis of pulmonary vessels in patients with covid- showed widespread thrombosis, microangiopathy and a reactive angiogenesis. [ ] indeed, there is growing evidence of thromboembolic phenomena in covid . [ ] in severe and fatal sars and mers the dominance of the inflammatory response gave rise to the concept that cellular damage was due to a "cytokine storm." [ ] because cytokines are stimulated by viral infection itself, it is difficult to distinguish between cytokines as cause or effect of infection. "cytokine storm" has also been invoked as a pathogenic mechanism in dengue, instead, capillary damage results from a circulating viral toxin. [ ] concluding remarks: with others, we conclude that the differences in clinical, epidemiological and pathological features of sars and denv diseases suggest that iade does not contribute to the severity of natural human coronavirus infections. [ ] a question asked frequently is whether sars or mers cov infections convey solid protective immunity. viral respiratory infections often fail to protect the respiratory tract from reinfection by the same organism. among immune individuals, respiratory tract superinfections occur frequently, but, usually without systemic disease. [ ] for example, natural and vaccine immunes were re-infected with measles or rubella viruses and these infections may contribute to the spread of virus. [ ] vah is a post-vaccination outcome that may be associated with non-protective antibodies. vah is a complex and poorly defined immunopathology. several different sars and mers vaccines have been shown to elicit a post-challenge vah in laboratory animals. ominously, when sars-cov- -immune monkeys were challenged with homologous virus most animals had evidence of lung inflammation. [ ] it is important to note that inactivated measles vaccine and dengvaxia exhibited short-term protection. [ , ] a central challenge to sars cov- vaccine development will be differentiating early from sustained protection and will be greatly aided by a sars cov- model of vah in laboratory animal models. recognition of vaccine constructs that achieve solid protection in humans might be controlled by prevalence of st denv infection antibodies. [ ] no antibody effect observed ade with passive antibody - month-old infants born to denvimmune mothers [ ] not observed viral pathogenicity increases month to month; single mutation controls [ ] not observed vaccine ade dengvaxia raises non-protective (ade) antibodies, sensitizing non-immunes [ , ] challenge virus produces vah in vaccinated animals [ , , ] animal coronavirus vaccines: lessons for sars an update on feline infectious peritonitis: virology and immunopathogenesis a perspective on potential antibody-dependent enhancement of sars-cov- intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes how innate immune mechanisms contribute to antibody-enhanced viral infections altered reactivity to measles virus. atypical measles in children previously immunized with inactivated measles virus vaccines field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population production of atypical measles in rhesus macaques: evidence for disease mediated by immune complex formation and eosinophils in the presence of fusion-inhibiting antibody immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins anti-sars-cov igg response in relation to disease severity of severe acute respiratory syndrome anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcgammar pathway risk factors in dengue shock syndrome: a prospective epidemiologic study in rayong, thailand. i. the outbreak maternal antibody and viral factors in the pathogenesis of dengue virus in infants effect of dengue serostatus on dengue vaccine safety and efficacy detection of post-vaccination enhanced dengue virus infection in macaques: an improved model for early assessment of dengue vaccines a shorter time interval between first and second dengue infections is associated with protection from clinical illness in a school-based cohort in thailand dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity neutralization and antibody dependent enhancement of dengue viruses shock associated with dengue infection. i. clinical and physiologic manifestations of dengue hemorrhagic fever in thailand immunologic enhancement of dengue virus replication in vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody pathologic highlights of dengue hemorrhagic fever in autopsy cases from myanmar the good, the bad, and the shocking: the multiple roles of dengue virus nonstructural protein in protection and pathogenesis association of endothelial glycocalyx and tight and adherens junctions with severity of plasma leakage in dengue infection dengue virus ns cytokine-independent vascular leak is dependent on endothelial glycocalyx components dengue in vietnamese infants--results of infectionenhancement assays correlate with age-related disease epidemiology, and cellular immune responses correlate with disease severity a t s mutation in the dengue virus ns protein is associated with greater disease severity in mice pathogenesis of feline infectious peritonitis: nature and development of viremia the severe acute respiratory syndrome time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars pathogenesis of severe acute respiratory syndrome t-cell immunity of sars-cov: implications for vaccine development against mers-cov pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- covid- and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology the macrophage in the pathogenesis of severe acute respiratory syndrome coronavirus infection dengue virus and dengue hemorrhagic fever primary severe acute respiratory syndrome coronavirus infection limits replication but not lung inflammation upon homologous rechallenge rubella: reinfection of vaccinated and naturally immune persons exposed in an epidemic human challenge studies to accelerate coronavirus vaccine licensure an urgent need for "common cold units" to study novel coronavirus disease (covid- ) tropism of dengue virus in mice and humans defined by viral nonstructural protein -specific immunostaining observations related to pathogenesis of dengue hemorrhagic fever. vi. hypotheses and discussion reduced risk of disease during postsecondary dengue virus infections observations related to pathogenesis of dengue hemorrhagic fever. iv. relation of disease severity to antibody response and virus recovered observations related to pathogenesis of dengue hemorrhagic fever. iii. virologic studies of fatal disease virus role during intraepidemic increase in dengue disease severity anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection a c c e p t e d m a n u s c r i p t key: cord- -v lpw r authors: viktorovskaya, olga v.; greco, todd m.; cristea, ileana m.; thompson, sunnie r. title: identification of rna binding proteins associated with dengue virus rna in infected cells reveals temporally distinct host factor requirements date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: v lpw r background: there are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. a better understanding of the host pathogen interaction is required to develop effective therapies to treat denv. in particular, very little is known about how cellular rna binding proteins interact with viral rnas. rnas within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses) replication. methodology/principal findings: seventy-nine novel rna binding proteins for dengue virus (denv) were identified by cross-linking proteins to dengue viral rna during a live infection in human cells. these cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in denv amplification. knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. their requirement by denv for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. the protein abundances of these host factors were not significantly altered during denv infection, suggesting their interaction with denv rna was due to specific recruitment mechanisms. however, at the global proteome level, denv altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated. conclusions/significance: the method for identification of host factors described here is robust and broadly applicable to all rna viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral rna within cells. this study significantly increases the number of cellular factors known to interact with denv and reveals how denv modulates and usurps cellular proteins for efficient amplification. dengue is a mosquito-borne viral disease that infects - million people annually, resulting in dengue fever that is either asymptomatic or flu-like. however, tens-of-thousands of people develop the more severe, and sometimes fatal, dengue hemorrhagic fever/shock syndrome (dhf/dss) [ ] . denv is found in most tropical and many subtropical areas with more than countries being endemic for denv [ ] . there is no approved vaccine or antiviral therapeutic available for this life-threatening disease. given the seriousness of infection, the expanding geographical range of the denv, and the limitations in the existing measures of control and prevention, there is a pressing need to better understand the biology and pathogenesis of denv. denv is a single-stranded positive-sense rna virus that belongs to the flaviviridae family. it has a ' cap, no poly(a) tail, highly structured '-and '-untranslated regions (utr), and a single open reading frame (orf) [reviewed in [ ] ]. following virus entry, the viral rna is released into the cytoplasm. viral translation and replication occur in membranous assembly "factories" localized in the perinuclear region of endoplasmic reticulum (er) [ ] . the positivestranded rna molecules are encapsidated; virions are further processed as they are transported through the secretory pathway to the cell surface and released extracellularly [reviewed in [ ] ]. in addition to the viral proteins, cellular proteins, termed host factors, participate in most, if not all, steps of the denv life cycle, including entry, translation, replication, virion assembly, and release [ ] . since viruses require host factors for efficient amplification, targeting host factors can provide an effective antiviral target for which the virus has no genetic control over. therefore, it may be more difficult for viruses to evolve escape mutants that can replicate efficiently in the absence of the host factor [ , ] . several cellular proteins are known to impact denv infection. for example, the polypyrimidine-tract-binding protein (ptbp ) is relocalized from the nucleus to the cytoplasm following denv infection where it enhances denv amplification by binding to the denv 'utr and to ns a, a viral protein required for the formation of the viral replication complex [ ] [ ] [ ] [ ] . ptbp may also stimulate denv translation [ ] , although this is still controversial [ , ] . while most of the known denv rna binding proteins enhance viral amplification, several reduce denv titers [ ] [ ] [ ] . one such factor, ybx , inhibits viral translation [ ] . although previous studies have laid a foundation for establishing critical interactions between viral rna and cellular proteins [ [ ] and reviewed in [ ] ], the host factors identified thus far likely represent only a fraction of the total network of denv host factors. previously, we have described a high-throughput mass spectrometry method termed tux-ms (thiouracil cross-linking mass spectrometry) to identify host factors that interact with viral rna during a live infection in cell culture [ ] . importantly, tux-ms allows for identification of proteins that are bound directly to the viral rna in living cells. briefly, during a viral infection in cell culture, thiouridine is biosynthetically incorporated into the viral rna to serve as a zero-distance cross-linker upon exposure to ultraviolet (uv) light. thus, proteins that are bound directly to the viral rna during a live infection are cross-linked to the rna prior to disruption of cellular compartmentalization. this is particularly valuable for the identification of denv host proteins, since denv amplification is tightly associated with cellular membrane structures [ ] . following cross-linking, the viral rna together with cross-linked proteins are isolated under denaturing conditions and identified by mass spectrometry-based proteomics. using tux-ms, we reported previously the successful identification and validation of host factors for poliovirus, pointing to a low false discovery rate of < % [ ] . here, we expanded the tux-ms methodology for use with other types of rna viruses, and investigated rna-protein interactions during denv infection. we modified the method to use virus-specific dna oligos to capture the viral rna and cross-linked proteins. furthermore, we used metabolic labelling with stable isotopes to accurately quantify relative protein levels. this quantitative thiouridine cross-linking mass spectrometry (qtux-ms) analysis identified novel host proteins, which were not previously shown to be involved in denv infection. we placed these findings in the context of whole proteome changes upon denv infection, and further validated and functionally analysed a subset of the novel denv host factor candidates. overall, validation of the qtux-ms identified factors using secondary assays indicates a low rate of false positives ( %), suggesting that the majority of the other identified qtux-ms factors may also play significant roles in denv viral amplification. hela uprt cells expressing uracil phosphoribosyltransferase (uprt) were generated previously by transduction of hela cells (atcc, ccl- ) with uprt-gene containing lentivirus [ ] . huh . uprt cells were generated by transduction of huh . cells (a kind gift from charles m. rice, rockefeller university) with the same lentiviral construct as in [ ] . hela uprt and huh . uprt cells were cultured at °c and % co in complete dulbecco's modified minimum essential medium (dmem) supplemented with % fbs (fetal bovine serum; atlanta biologicals) and penicillin-streptomycin and grown with mg/ml g (sigma) to select for the uprt gene; huh . uprt cells were additionally supplemented with non-essential amino acids (cellgro). for silac labelling huh . uprt cells were passed : at least twice in silac dmem (thermo scientific) with % dialyzed heat-inactivated fbs (thermo), l-proline ( mg/l) and penicillin-streptomycin, and either mg/l 'heavy' ( c l-lysine and c - n l-arginine; cambridge isotope laboratories, inc.) or mg/l 'light' l-lysine and l-arginine amino acids [ ] . dengue virus serotype (denv ), strain (genebank accession number u ) was kindly provided by dr. robert striker (university of wisconsin-madison). denv was propagated in the mosquito c / cells at °c and % co in advanced dmem supplemented with % fbs, penicillin-streptomycin (cellgro), l-glutamate and % tryptose phosphate broth ( g/l tryptose; g/l glucose; g/l sodium chloride and . g/l disodium hydrogen phosphate). titers for denv were determined using plaque assays in bhk cells. for infections, cells were incubated with virus containing media for hours, washed twice with the dmem media after removal of the virus and incubated in the serum-free dmem for the indicated time. infections and titer determination of adenovirus (ad ) were performed exactly as in [ ] . the denv antisense biotin-labelled dna fragments were generated using pcr and primers listed in (s table) from the denv complementary dna (cdna) and correspond to positions - and - of denv genome. pcr was followed by removal of the unlabelled dna strand according to the nanolink streptavidin magnetic beads (solulink) manual. the mixture of two biotinylated single-stranded dna fragments of base pairs (bp) and bp long were bound to nanolink streptavidin magnetic beads magnetic beads according to the manufacturer's protocol. - x human hepatoma huh . uprt cells labelled with either 'light' or 'heavy' silac media were infected with denv (moi = ) or mock-treated, respectively. then, virus was replaced with silac media with mm -thiouracil and % fbs. at hours post-infection (hpi) the cells were washed with pbs and irradiated at nm uv light for min, collected, cell pellets were frozen on dry ice and stored at - °c. cell pellets were lysed in the lysis buffer ( mm tris-hcl ph , mm magnesium chloride, mm sodium chloride, . % tween- , mm dithiothreitol, recombinant rnasin ribonuclease inhibitor [ units/ml; promega], × complete protease inhibitor cocktail [roche]), with a half volume of - μm glass beads (sigma) by shaking at frequency of hz for min using a retsch mm mixer mill. an aliquot of 'light' and 'heavy' cell lysates were removed and the remaining lysates were incubated with the streptavidin magnetic beads labelled with denv antisense dna fragments for to min allowing for viral rna to bind. the beads were washed twice with wash buffer i ( mm tris-hcl ph , mm potassium chloride, . % tween- ), once with wash buffer ii ( mm tris-hcl ph , mm sodium chloride, . % sodium deoxycholate) and once with mm tris ph . . the samples were eluted at °c for min in μl of mm tris ph . . the eluted 'light' and 'heavy' samples were mixed at a : ratio, rna was degraded with . ng/μl bovine rnase a (fisher scientific) at °c for hrs and subjected to quantitative mass spectrometry-based proteomic analysis. quantitative mass spectrometry-based proteomic analysis of rnainteracting proteins (qtux-ms) protein eluates and their respective mixed light/heavy input lysates were subjected to in-solution enzymatic digestion using a filter-aided sample preparation approach [ ] , then analyzed by nlc-ms/ms, as described in s methods and in [ ] . light (denv ) and heavy-labelled (mock) cell pellets were lysed in mm abc containing % sodium deoxycholate at °c to ensure denaturation and virus inactivation. the protein concentrations were determined by the bca assay and mixed in equal protein amounts ( μg total). proteins were subjected to in-solution digestion with trypsin, then fractionated and analyzed by nlc-ms/ms as described in the s methods. proteomic data analysis qtux-ms, its respective mixed input lysate, and the whole cell silac instrument raw data were separately processed using the maxquant software (ver. . . . ), configured with default settings, except for experiment-specific parameters, which are described in the s methods. the mass spectrometry proteomics data have been deposited to the proteomexchange consortium via the pride [ ] partner repository with the dataset identifier pxd . using the filtered list of protein identifications, unique gene symbols were used for downstream functional ontology analyses. the gene ontology annotations from uniprot were used to generate and assign the denv rna interacting candidates into broader functional categories. for the whole cell silac protein expression study, genes and their associated ratios were analyzed by panther gene enrichment (panther database ver . , - - ) [ ] using the panther pathway and protein class ontologies. significant differential protein abundance was determined as a function of ontological classification versus the overall population (bonferroni-corrected p-value < . ). for specific functional protein ontologies that were differentially regulated, a subset were selected for analysis by the reactome functional interaction (fi) network cytoscape plugin [ ] . the reactome fi plugin was used to visualize candidate host factors identified by qtux-ms. sirna transfections x hela uprt cells were transfected with pmol of silencer select negative control (ambion) or the specified sirnas (s table) using the xtreamgene sirna transfection reagent system (roche). hrs later, x cells/well were plated for infection. hours post transfection cells were infected (moi = . ) with denv or ad (n = ). at either (ad ) or (denv ) hpi, virus titers were determined by plaque assays using or bhk cells, respectively. knockdown efficiency was measured hrs post transfection by rt-qpcr. experiments were performed in two biological replicates for each host factor. statistical analysis was performed using student's t-test. cell viability and proliferation assay hrs post sirna transfection equal numbers of cells (either x or x ) were seeded in -well plates. cell viability was measured hours after sirna-mediated knockdown of individual host factors using the vybrant mtt [ -( , -dimethyl- -thiazolyl)- , -diphenyl- h-tetrazolium bromide] assay kit (invitrogen) according to the manufacturer's protocol and reported relative to the negative-control sirna (set to %; n = ). statistical analysis was performed using student's t-test. cdna was generated from μg trizol (ambion) purified total rna using moloney murine leukemia virus (mmlv) reverse transcriptase (promega) as described by the manufacturer using random primers (invitrogen). qpcr was performed using iq sybr green supermix (bio-rad) with the primers listed in the s table. the amplification efficiency for each primer set was ± % as determined using a standard curve. development of a quantitative thiouridine cross-linking mass spectrometry (qtux-ms) method for identification of proteins associated with the denv rna tux-ms can be used to identify host factors by incorporating -thiouridine ( su), a zero-distance cross-linker, into the viral rna (vrna) to enable cross-linking of proteins bound to vrna during a live infection in cell culture [ ] . cross-linking is carried out under physiological conditions prior to cell lysis to ensure specificity and reduce false-positives from non-specific rna protein interactions that occur upon loss of compartmentalization. vrna is isolated under denaturing conditions and cross-linked proteins are identified using liquid chromatography-tandem mass spectrometry (lc-ms/ms). to improve quantification of the tux-ms identified host proteins (qtux-ms), a silac (stable isotope-labelled amino acids in cell culture) approach [ ] was used to label the uninfected (mock) and infected cells with either 'heavy' or 'light' amino acids, respectively ( fig a) . when -thiouracil ( tu) is present in the medium, huh . uprt human hepatoma cell lines stably expressing uprt (uracil phosphoribosyltransferase) convert tu to ump. then, the ump is converted to thiouridine triphosphate ( sutp) by cellular kinases [ ] . both cellular and viral rna polymerases use sutp as a substrate during rna synthesis, which serves as a zero-distance cross-linker, covalently binding proteins to rnas upon exposure to long wave uv-light. importantly, protein-protein crosslinking is very inefficient at long uv wavelengths, ensuring that only proteins in direct contact with the reactive thiol group of the su-containing rna will be cross-linked [ ] . we have shown previously that immunoisolation of candidate vrna-binding proteins identified by tux-ms (and confirmed by western) could be specifically co-isolated with viral rna [ ] . together, this study established that tux-ms can identify bona fide interactions between host proteins and viral rna. the tux-ms method was originally developed to capture polyadenylated rna using oligo(dt) beads [ ] . however, as denv rna is not polyadenylated, we modified the method to use sequence specific capture of the vrna using magnetic beads. following crosslinking in huh . uprt cells infected with denv at hpi and affinity capture of the vrna, the ribonucleoprotein complexes were eluted from the beads, 'heavy' and 'light' eluates were mixed, and rnase a was used to degrade the rna. the proteins were digested insolution with trypsin and subjected to quantitative ms-based proteomics ( fig b) . the median 'light' to 'heavy' peptide and protein ratios were calculated, reflecting the specificity of vrnaprotein capture. we identified several classes of proteins, including denv proteins, known denv host factors, and putative rna-interacting host proteins, but most of the qtux-ms identified factors have not been previously identified through interactions with denv (s table) . consistent with previous knowledge of flaviviral rna [ ] [ ] [ ] [ ] , our qtux-ms analysis identified several viral proteins-c, e, ns , ns a and ns -as associated with vrna. in addition, qtux-ms identified six known denv host factors: polypyrimidine tract-binding protein (ptbp ), interleukin enhancer-binding factor (ilf ), calreticulin (calr), calnexin and heterogeneous nuclear ribonucleoproteins hnrnp h and hnrnp k, as well as a known denv anti-viral protein-eukaryotic initiation factor a (eif a)-and other proteins previously shown to associate with denv rna or proteins (s table) . altogether, since several known host factors were identified using qtux-ms, this suggests that the adapted method is effective at identifying host factors for denv. for identification of novel host factors, cellular proteins with a denv/mock silac ratio of . -fold (n quantified peptides) were considered putative denv vrna interactions. this threshold was selected when considering the median variance in the silac ratio (for (blue) amino acids are infected with denv or treated with mock, respectively, in the presence of tu. su is incorporated into cellular and denv rnas and proteins are uv cross-linked to the contacting thio-containing rna (represented as either balls to indicate native conformation or curved lines to indicate denatured proteins) in living cells at hpi prior to cell lysis under denaturing conditions. viral ribonucleoprotein complexes were isolated using dna molecules complementary to denv rna bound to magnetic beads, the rna was degraded with rnase a and the proteins were identified by mass spectrometry. (b) workflow for quantitative proteomic analysis of rna-bound host factors isolated in (a). isolated proteins were mixed between mock and virus-infected samples, digested into peptides, and analysed by mass spectrometry. relative 'light' and 'heavy' peptide abundances were quantified to determine the specificity of interaction. host factor candidates were identified and subjected to functional validation. doi: . /journal.pntd. .g proteins with > quantified peptides), which was approximately %. therefore, we opted for a conservative cut-off at %, representing twice this median value or . -fold. common environmental and non-human cell culture contaminants were excluded, since they existed in only the 'light' silac state. in addition, our qtux-ms samples also contained histones: h , h , h a, h b, h . and macroh a. . in a previous study, histones were shown to play roles in dengue infection [ ] . however, their functions were mediated through an interaction with a viral capsid protein and were shown to be independent of rna. in addition, histones are primarily nuclear and highly abundant proteins commonly detected (> %) in control affinity purifications compiled across diverse protein-protein interaction studies [ ] . for these reasons, histones likely represent non-specific associations rather than denv rna binding factors, and thus were excluded from further analysis. in total, cellular proteins passed these selection criteria, of which have not been previously shown to associate with denv (s table, s dataset). several of the known denv host factors were enriched but did not meet the stringent inclusion criteria (s table) , suggesting that there may be additional host factors below our enrichment threshold (see s dataset). importantly, the subset of host factors represents a significant potential expansion in the number of known denv host factors, providing a valuable resource to test for pro-viral or antiviral activities during denv infection. it is well recognized that viral infections can induce significant changes in cellular proteomes [ ] [ ] [ ] and an increase in protein levels during denv infection may contribute to the increased protein capture measured by qtux-ms. to address this, we used silac-ms to quantify proteome (i.e., total protein abundance) changes following denv infection. comparison of qtux-ms and proteome silac ratios showed that the protein abundances for the qtux-ms-identified vrna-binding factors remained largely unchanged (fig a, s fig and s dataset). on average, for these proteins, the denv-induced changes in whole cell abundance were ± . -fold, suggesting that their identification by qtux-ms was not due to an increase in their abundance in the cell following denv infection. noteworthy, a retrospective qualitative comparison of the qtux-ms identified factors for denv with those identified in the tux-ms analysis on poliovirus revealed less than % were identified for both viruses [ ] ( fig b) . since the identified proteins are largely denv specific, qtux-ms is not biased towards identifying a sub-set of cellular rna binding proteins. taken together, our results indicate that the enrichment ratios measured by qtux-ms is predominantly due to their specific association with the denv rna. while proteins that bound denv rna did not show significant changes in abundance upon infection, we performed bioinformatics analysis on the complete proteome dataset of whole cell abundance to determine the global proteome effects of denv infection under these conditions. in total, , host proteins were quantified by silac ms in biological duplicates (s dataset). the abundance ratios between biological duplicates were reproducible, with only~ % of the ratios varying by > % (fig a) . from these duplicates, an average abundance ratio was calculated and the respective proteins were analyzed by panther gene enrichment analysis (s fig) [ ] . this analysis found systematic up regulation of proteins in the ubiquitin proteasome pathway (upp), comprising members of the s proteasome as well as various ubiquitinconjugating enzymes (s fig). many of these enzymes are linked to ubiquitin-dependent proteasome degradation, consistent with the current knowledge that the upp is important for production of infectious virions [ , , ]. yet, other enzymes, such ube n and ube v , catalyze polyubiquitination at lys- , which does not lead to proteasome degradation but rather participates in transcriptional activation of target genes and may promote innate immune signaling [ , ] . in contrast, proteins in the transporter protein class were on average down regulated (fig b and s fig). assembly of the annotated proteins into reactome protein networks identified several subnetworks with various transporter activities (s fig). while the abundances of mitochondrial transporters and nucleoporins were the most consistently decreased, not all transporters were down regulated; for example, the lipoprotein (apo) transporters were increased in expression (fig c) . interestingly, the rna binding protein class was significantly down regulated (fig b and s fig) , though the effect was not as pronounced as the transporter class (fig b) . the overall down-regulation of rna binding proteins appears to be driven by changes in cytoplasmic and mitochondrial ribosomal subunits, and proteins involved in rna degradation and processing (s fig). nevertheless, the relative protein abundance for the set of (known and putative) denv binding factors identified by qtux-ms was largely unchanged, despite being enriched in rna processing and translation factors (fig a) . overall, the quantitative proteome analysis suggests that denv selectively alters the abundance of proteins, and reveals several pathways that could be directly or indirectly modulated in the host response to denv infection. to gain insight into potential molecular mechanisms and biological processes of the qtux-ms identified factors, we performed a functional network-based analysis using the curated human pathway relationships from the reactome database. this analysis revealed a high degree of connectivity, with proteins forming a large interconnected network (fig ) . the densest network connectivity included proteins involved in rna processing/translation (orange nodes) and dna binding/transcription (blue nodes). several additional proteins with rna and/or translational activities were also identified, but lacked annotation in reactome (orange single nodes). overall, our bioinformatic evaluation further supports the ability of qtux-ms to capture vrna-bound host factors and points to their possible function in denv amplification. since most of the factors were associated with rna processing in the reactome analysis (fig ) , we focused on these factors for functional analysis of their roles in dengue infection. we have randomly selected six qtux-ms identified host proteins with functions in rna processing/translation, which were enhanced in the denv sample ranging from . -to . -fold (denv/mock). thus, by validating factors that are only modestly enhanced in the qtux-ms analysis this will indicate if the qtux-ms identified factors that are near the cut-off of . -fold are bona fide host factors or not. we assessed the effect of sirna knockdown of these factors (fig a) on viral production. hela uprt cells were used for sirna silencing experiments due to higher sirna transfection efficiency compared with huh . uprt cells. knockdown of five out of six qtux-ms identified candidates: non-pou domain-containing octamer-binding protein (nono), embryonic stem cell-specific -hydroxymethylcytosine-binding protein (hmces), rbmx (rna-binding motif protein, x chromosome), hnrnp m and hnrnp f significantly decreased denv production, while knockdown of hnrnp l had no effect on denv titer ( fig b; s fig) . knockdown of ptbp , a positive control [ , ] , also resulted in decreased viral titers (fig ) . for negative controls, we selected two rna binding proteins (ddx and hnrnp a ) that were not identified by qtux-ms. denv titers were not altered following silencing of these two proteins, suggesting that only specific rna binding proteins are used by denv. altogether, our data suggests that rbmx, nono, hmces, hnrnp m, hnrnp f are required for viral production. these results demonstrate that qtux-ms is a robust method with a low false discovery rate for high-throughput identification of viral host factors. reduced viral amplification could be due to compromised cell fitness rather than a specific requirement of the virus for a particular host factor. using an mtt assay, we confirmed that knockdown of these factors did not impact cell viability (fig c) . as a positive control, knockdown of g bp did reduce cell fitness, as previously shown (s fig) [ ] . to more rigorously rule out any potential effects of host factor knockdown on cell fitness that would affect viral amplification, an unrelated virus (adenovirus ), was amplified following knockdowns of the candidate factors. adenovirus replication was not significantly decreased by rbmx, nono, hnrnp m, hnrnp f or hmces sirna knockdown (fig b and s c fig) demonstrating that knockdown of these factors does not affect cell fitness for viral amplification. given that these proteins bind directly to viral rna and are required for viral amplification, rbmx, nono, hnrnp m, hnrnp f and hmces are novel denv host factors. to determine if the host factor is required for a step prior or subsequent to viral replication, vrna was quantified by qrt-pcr in the denv infected cells knocked down for rbmx, nono, hnrnp m, hnrnp f or hmces (fig ) . as a positive control, knockdown of ptbp , which is required for denv replication [ ] , reduced denv rna levels. similarly, the intracellular vrna levels were reduced in cells knocked down for either hnrnp f, rbmx or hmces. the decrease in dengue rna levels (fig ) is consistent with the decrease in viral titers ( fig b) . thus, hnrnp f, rbmx or hmces are required for the early steps in the viral replication cycle, such as translation, replication or rna stability. in contrast, knockdown of hnrnp m and nono did not change intracellular viral rna levels despite the dramatic decrease in viral titers, suggesting they may play a role downstream of replication. altogether, we have identified and validated five novel host factors for denv, demonstrating that qtux-ms can identify factors that function at different stages of the virus life cycle. an estimated % of the world's population is at risk from dengue for which vaccines or antivirals are not yet available. since diagnostic tests can detect denv infection at early stages, administration of antivirals could significantly improve survival rates as viral load is correlated with symptom severity [ ] . using antivirals that target host factors may limit the appearance of drug-resistant viruses and may be effective for all denv serotypes and possibly for multiple flaviviruses [ , , , ] . the qtux-ms analysis identified novel cellular proteins, for which the majority are distinct from those previously identified for poliovirus using a similar approach [ ] . this suggests that unrelated rna viruses have evolved to utilize distinct host rna binding proteins. interestingly, ptbp and nono, which were identified in both the poliovirus and denv tux-ms analyses, were shown to be required for production of both viruses (this study, figs and ) [ , , ] . further analysis of virus-specific and shared host factors will reveal whether unrelated viruses utilize similar or diverse mechanisms to control viral rna replication, processing and packaging within cells. the host factors that enhance amplification of both poliovirus and dengue, (fig b) could serve as attractive targets for the development of broad-spectrum antivirals. the novel denv rna interactions identified in our study reveal a large network of cellular proteins which belong to different functional classes primarily associated with the nucleic acid metabolism, including numerous components of splicing, rna processing and translation machineries. these factors likely play direct roles in denv translation, replication or packaging. in addition, we have detected multiple components of cell signaling and stress response, such as several members of - - adapter proteins, heat shock proteins and β-catenin. these factors are known to regulate diverse pathways, including host innate immune and cellular homeostasis [ ] [ ] [ ] suggesting their possible role in host antiviral response or viral strategies to subvert the innate immune response. we demonstrated that the majority of the qtux-ms factors that we selected for validation were required for efficient denv amplification (fig ) . specifically, we found that hnrnp f, hmces and rbmx are required for the early steps in the viral life cycle. in contrast, hnrnp m and nono appear to act downstream of viral rna replication (fig ) , which may be significant given that both have been shown to be in a complex together [ ] . nono and its binding partners are predominantly nuclear, bind rna, and are involved in pre-mrna processing, splicing, and rna transport, as well as in transcriptional activation and repression [ ] [ ] [ ] . interestingly, other nono binding partners: psf/sfpq (polypyrimidine tract-binding protein (ptb)-associated splicing factor) and matrin were identified by qtux-ms as well. many of the qtux-ms identified cellular proteins are hnrnps, which encompass a large class of rna binding proteins that either localize to the nucleus or shuttle between the nucleus and the cytoplasm in order to perform multiple functions in rna metabolism, from transcription to rna turnover [ ] [ ] [ ] . importantly, the vast majority of these factors have established roles in viral infections or in modulating the antiviral host response to various viruses [ ] [ ] [ ] [ ] , including denv (s table and references within). our study establishes that rbmx (hnrnp g), hnrnp f and hnrnp m are required for efficient denv amplification (fig b) . since several hnrnps, such as ptbp (hnrnp i), hnrnp a and hnrnp k, were previously shown to re-localize from the nucleus to the viral replication sites during denv infection [ , , ] , rbmx, hnrnp f, hnrnp m and possibly other nuclear qtux-ms identified factors are also likely to be either actively recruited to the viral replication sites or retained in the cytoplasm upon denv infection. interestingly, the qtux-ms identified hnrnps affect different steps in the denv life cycle (fig ) , suggesting that they have distinct functions during infection. this is consistent with studies that have suggested that denv rna structures are dynamic during the viral life cycle [ ] and may suggest that host factors play an important role in these structural changes. furthermore, we showed that knockdown of some hnrnps (hnrnp a and hnrnp l) did not affect dengue viral titers significantly demonstrating that only certain hnrnps are required for denv amplification. since some of hnrnps are known to modulate cellular gene expression in response to dengue infection [ , ] , we can not rule out that some of the observed effects on virus titers derive from their roles in regulating host mrnas. among the numerous qtux-ms identified factors of interest, our study is the first to demonstrate the involvement of hmces (or c orf ) in viral infection. while the cellular role of human hmces is currently unknown, the mouse homologue was suggested to be an rnabinding protein and predicted to contain a putative peptidase domain [ , ] . interestingly, it is possible that the nucleic acid binding domain enhances the protease activity or visa versa as has been shown for other such proteins [ ] [ ] [ ] . for example, adenovirus uses a nucleic acid binding protease to localize the protease activity to the viral substrates [ ] . it has been suggested that the protease is recruited to the empty capsid as an inactive protease, then it becomes fully activated once bound to the viral dna inside the virion. using the dna as a guide wire, it moves along the nucleic acid, searching for capsid and core proteins to cleave, which is required to render the viral particle infectious [ , , ] . however, since we observed that knockdown of hmces resulted in a decrease in viral rna, it seems more likely that it might participate in translation, replication or the switch from translation to replication as has been shown for other nucleic acid binding proteases [ , ] . only one of the qtux-ms identified factors was increased at the protein level in whole cells following denv infection, suggesting that qtux-ms identifications derived from the specific associations to vrna rather than changes in protein abundances. our analysis of the host cell proteome upon infection also revealed interesting changes, including up regulation of proteins in the ubiquitin pathway and down regulation of transporter proteins. the ubiquitin-proteasome pathway is one of two major cellular pathways used to degrade to % of proteins. previous studies on denv infected cell lines and patient samples showed that the ubiquitin pathway was upregulated [ , , ] . many groups have consistently shown that the ubiquitin proteasome pathway is critical for amplification of a number of flaviruses, including denv and west nile virus [ , , , ] . however, it remains controversial as to which step in viral amplification is affected by ubiquitination, but it appears to be early during internalization or viral genome release [ , , ] . further studies will be required to understand how denv up-regulates the pathway and the mechanism that ubiquitination has in denv amplification. altogether, our study has significantly increased the number of cellular proteins known to interact with the denv rna during a live infection in cells. we have also placed these interactions in the context of proteome abundance changes in the infected cells. a recent study by phillips and colleagues [ ] exploited a cross-linking label-free ms approach to identify denv rna associating proteins in cell culture by cross-linking the rna to the proteins using short wavelength uv light and isolating denv rna bound proteins by anti-sense dna affinity capture [ ] . while their method identified several denv host factors [ , [ ] [ ] [ ] , the qtux-ms method reported here resulted in improved identification of known dengue host factors and putative denv rna interacting proteins. there could be several reasons for these results, such as, the qtux-ms approach achieves greater cross-linking efficiency by using long wave uv light to form crosslinks to -thio-uridines compared to short-wavelength uv light, which is inherently inefficient [ ] . moreover, since thio-uridine is a zero distance cross-linker for rna-bound proteins at long uv wavelengths and protein-protein cross-links are not formed at long uv wavelengths, qtux-ms may also have achieved improved specificity, as only proteins in direct contact with the viral rna would be captured [ ] . additionally, qtux-ms used an ms-based silac approach to determine which host proteins were specifically enriched in the vrna isolations versus mock. though isotope-labelling is not applicable in all model systems, it does afford greater quantitative accuracy compared to label-free ms strategies [ ] . overall, the qtux-ms method identified cellular proteins that bind to denv rna, which include previously known or putative interactions. importantly, five out of the six qtux-ms identified novel factors that were tested were shown to be bona fide host factors. we used robust assays to show that the identified host factors were specifically required for denv amplification and did not merely result in a decrease in cell fitness for viral amplification. future studies will reveal whether the identified factors may also be required for other flavivirus infections that cause life-threatening illnesses, such as yellow fever, west nile, zika, japanese and tick-borne encephalitis. therefore, our data demonstrates that qtux-ms is an effective technique for identifying novel virus host factors that can be used for a broad spectrum of rna viruses by simply designing antisense dna oligonucleotides to allow for efficient sequence-specific isolation of the vrna. supporting information s table containing all proteins quantified by qtux-ms, including proteins enriched in denv infection (red highlighted rows) and those that did not meet the specificity threshold. for each protein group, the following are provided (from left to right), uniprot accession number, gene name, protein name, the linear and log denv/ mock qtux-ms enrichment ratios (columns d and e), the qtux-ms ratio variability, the number of qtux-ms quantified peptides, the average log denv/mock whole cell relative abundance silac ratio, whether this ratio was up (u) or down (d) regulated by more than ± . -fold, the average silac ratio variability, the average number of quantified peptides, the number of razor+unique peptides and sequence coverage for qtux-ms, the protein's molecular weight and sequence length, and the complete fasta header entry for the primary protein group member. columns h-k are cross-referenced from the respective whole cell data (b). nd = not detected. (b) table containing all proteins quantified in whole cell lysates by silac. for each protein group, the following are provided (from left to right), uniprot accession number, gene name, protein name, the log denv/mock relative abundance ratios for replicates , , and the average (columns d and e), whether this ratio was up (u) or down (d) regulated by more than ± . -fold. for each replicate, the following are provide: the silac ratio variability, the number of quantified peptides, the number of razor+unique peptides, and the number of unique peptides, total protein intensity for light (denv) and heavy (mock) conditions, and the overall sequence coverage. the complete fasta header entry is listed for the primary protein group accession number. (xlsx) s (a) hela uprt cells were transfected with either control or specific sirnas. hours post transfection cells were counted and seeded ( x cells/well) in -well plates in triplicates. hours post transfection cells were infected with denv at moi . and virus released to the media collected hours post infection. denv titers were measured using plaque assays. the bars represent average values from triplicate, a standard error is reported. the representative data from one of at least two independent experiments is shown. (b) to verify sensitivity of the mtt assay, we performed sirna knockdown of g bp , which is known to bind denv and was previously shown to affect cell viability [ , ] . relative viability of non-infected cells was measured using an mtt assay (invitrogen) and represented exactly as described in fig c. cell viability or proliferation is decreased by knockdown of g bp compared with control sirna (p< . ). sirna transfection was performed as described in the methods section using previously published sirna sequence [ ] . in cells treated with g bp -specific sirna but not control sirna, g bp protein was knocked down to the levels undetectable by western analysis using antibodies against g bp (abcam, ab ). (c). hela uprt cells were seeded . x cells in mm plates and transfected with either control or specific sirnas. hours post transfection cells were infected with ad at moi . and collected hours post infection. sirna knockdown efficiency was determined by measuring respective mrna levels in comparison to β-actin mrna abundance as described in the experimental section and reported relative to control sirna transfection (upper panel). ad titers were determined using plaque assays on cells. the bars represent average values from triplicate, a standard error is reported. the representative data from one of at least three independent experiments is shown. 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-w finxf authors: heaton, nicholas s.; randall, glenn title: dengue virus and autophagy date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: w finxf several independent groups have published that autophagy is required for optimal rna replication of dengue virus (denv). initially, it was postulated that autophagosomes might play a structural role in replication complex formation. however, cryo-em tomography of denv replication complexes showed that denv replicates on endoplasmic reticulum (er) cisternae invaginations and not on classical autophagosomes. recently, it was reported that autophagy plays an indirect role in denv replication by modulating cellular lipid metabolism. denv-induced autophagosomes deplete cellular triglycerides that are stored in lipid droplets, leading to increased β-oxidation and energy production. this is the first example of a virus triggering autophagy to modulate cellular physiology. in this review, we summarize these data and discuss new questions and implications for autophagy during denv replication. classical autophagy is a homeostatic process wherein cytoplasmic material is sequestered in double membrane vesicles and degraded [ , ] . the process of autophagy is initiated when a cell perceives a signal such as starvation or pathogen infection. these signals are integrated through mtor to initiate a pathway autophagosome formation, which requires numerous autophagy (atg) proteins, which are conserved from yeast to humans [ ] . although the process of autophagosome formation is not completely understood, many of the required components are known [ ] . a limited description of autophagosome formation follows. for more comprehensive reviews on autophagy as a process, please open access see [ , , ] . initially, an immature autophagosome, termed a phagophore, forms from a variety of cytoplasmic membrane compartments [ ] . the atg /ulk complex positively regulates phagophore formation, while atg /beclin- recruits a class iii phosphatidylinositol -kinase (vps ) and atg to generate phosphatidylinositol -phosphate and nucleate the phagophore [ ] . two ubiquitin conjugating systems are involved in the elongation of the phagophore into an isolation membrane. first, atg is conjugated to atg , which then associates with atg l to form the pre-autophagosomal structure. second, atg /lc is cleaved by atg to form lc -i and subsequently becomes conjugated to phosphatidylethanolamine to form lc -ii and specifically associate with autophagosomal membranes [ ] . once the isolation membrane recognizes its cargo, it then engulfs the cargo and fuses, generating an autophagosome. lc -ii plays critical roles in cargo identification and membrane fusion [ ] . after a mature autophagosome has enveloped its cargo, it fuses with a lysosome to form a degradative compartment, the autolysosome. once the contents of the autolysosome have been broken down, the components are released for use by the cell. the process of autophagy is critical for the maintenance of cellular homeostasis as well as providing a mechanism to avoid cell death during starvation conditions [ ] . outside of the context of viral infection, deregulation of autophagy can lead to various pathologies including heart disease, neurodegeneration and cancer [ ] . during viral infection, autophagy can play either a pro-or anti-viral role [ ] . autophagy can act as an anti-viral component of the innate immune system, presumably by sequestering and degrading viral structures in the cells to help reduce viral replication. autophagy can be induced by toll-like receptor (tlr) ligands, which further indicates that autophagosomes can have anti-viral functions [ ] [ ] [ ] . in addition, autophagy can also play a role in delivering viral antigens for presentation to tlrs as has been reported for sendai virus and vesicular stomatitis virus [ ] . autophagy can also function in the adaptive immune response. recent studies have found that autophagy in antigen presenting cells can facilitate loading of antigen onto mhc class ii molecules [ , ] ; in at least one case this occurs via the fusion of autophagosomes with multi-vesicular mhc class ii loading compartments [ ] . despite its antiviral functions, autophagy is subverted by some viruses to perform pro-viral roles [ ] . poliovirus, coxsackievirus b virus, coronaviruses, hepatitis c virus, and dengue virus are among some of the best characterized examples of viruses that activate and require some aspect of autophagy for robust viral replication [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while the exact mechanisms of how autophagy can contribute to viral replication are for the most part unclear, progress has been made in characterizing the proviral roles of autophagy. some picornaviruses appear to use autophagosomal membranes as components of the viral replication complex [ ] [ ] [ ] . other viruses such as coronaviruses can utilize specific components of the autophagy machinery; in this case a non-lipidated form lc , to help reorganize cellular membranes [ ] . hepatitis c virus also has a requirement for autophagy [ ] , likely for an early viral rna translation step [ ] and/or suppressing innate antiviral immunity [ ] . dengue virus (denv) is a positive-stranded rna virus of the family flaviviridae. it is composed of a group of four serotypes (denv - ). denv is transmitted to vertebrate hosts via the mosquito vectors aedes aegypti or aedes albopictus. infection with denv can lead to a spectrum of clinical diseases ranging from subclinical infection to dengue fever to the most severe forms, dengue hemorrhagic fever and dengue shock syndrome [ ] . globally, there are an estimated - million infections annually, making denv the most important arbovirus to human disease [ ] . due in part to the large impact on human health, basic research on denv has expanded in recent years. denv initiates infection of a permissive cell via clathrin-mediated endocytosis and then releases its genomic rna into the cytosol after fusing with the late endosome [ , ] . the viral rna is translated as one open reading frame, and is subsequently cleaved by cellular and viral proteases to release three structural proteins and seven non-structural proteins. the non-structural proteins replicate the viral rna and the structural proteins assemble with the nascent viral rna to generate new virions [ ] . during viral infection, the virus manipulates many different cellular pathways, including autophagy. denv has been published to induce and require autophagy by four independent laboratories [ ] [ ] [ ] [ ] [ ] . lee et al. performed the initial characterization of autophagy during denv infection in [ ] . the authors showed that denv infection of a hepatocyte cell line induced autophagy and that inhibiting autophagy with the drug -methyladenine ( ma) or sirnas targeting autophagy gene expression compromised viral infection. they further showed that the denv induced autophagosomes co-localized with lamp , a marker of lysosomal fusion. this work was expanded upon the next year by panyasrivanit et al. [ ] . it was again shown that denv infection of hepatocytes induced and required autophagy via immunofluorescence assays and drug inhibition. it was also shown that a proportion of denv nonstructural protein (ns ) protein co-localized with autophagosomes as well as lamp and the ribosomal protein l . the authors also showed that an endosomal marker (m p-r) co-localized with autophagosomes, indicating that some autophagosomes may fuse with endosomes to form organelles called amphisomes. since denv replicates on virally induced characteristic double membrane vesicles (dmvs), and autophagosomes are dmvs, the authors hypothesized that denv might replicate on amphisomes and thus link virus entry and replication. the authors also showed that inhibiting lysosomal fusion with autophagosomes increased viral replication, indicating a role for immature autophagosomes during denv replication. soon after this publication, work from the same lab (khakpoor et al.) examined the role of autophagy in denv infection [ ] . similar to denv , denv infection also induced and required autophagy. lamp was observed to co-localize with autophagosomes, but in contrast to the previous denv study, treatment with a lysosomal fusion inhibitor decreased denv replication. this indicated a role for mature autolysosomes in denv infection. the mechanism for how autolysosomes could contribute to viral replication remained unclear. following these initial characterizations of denv-induced autophagy, an electron tomography study was performed by welsch et al. which showed the d structure of denv replication complexes in hepatocytes [ ] . while traditional thin section em appears to show virally induced replication complexes to be a cluster of independent double membrane vesicles [ ] , the d reconstruction clearly showed that these vesicles were actually contiguous invaginations of the er. complementary immuno-em studies demonstrated that the viral replicase proteins are present on the er invaginations as well as double-stranded rna, the viral replication intermediate [ ] . interestingly, the er invaginations that contain the replication complex were physically linked to er membrane compartments that contained capsids [ ] . a model was proposed wherein denv rna is transported through a neck that links the replication complex compartment to sites of capsid assembly. following assembly, the capsid would bud into the er to acquire its envelope. thus, this study clearly ruled out the hypothesis that denv was replicating on classical autophagosomes; however, there was still a very clear requirement for autophagy during viral replication. this indicated that autophagy might be playing an indirect role to enhance viral replication. in addition to bulk macroautophagy, which is relatively non-specific, different types of selective autophagy exist that target specific organelles (reviewed in [ ] ). the hypothesized role of selective autophagy is that the cell frequently needs to initiate a physiological response that appropriately addresses a specific stress. relevant to denv infection, a type of selective autophagy termed lipophagy was described, wherein autophagosomes can target cellular stores of lipids known as lipid droplets (lds) to generate energy for the cell [ ] . heaton et al. performed a limited sirna screen to identify cellular co-factors of denv replication in hepatocytes, which identified, among others, a gene involved in the induction of autophagy [ ] . in subsequent work, the authors reproduced the published results that denv induces and requires autophagy for robust viral replication [ ] . the initial observations were expanded upon by showing that denv induced autophagosomes not only acquire lamp , but complete their maturation and become autolysosomes [ ] . these autophagosomes did not co-localize with markers of the viral replication complex, suggesting that they may play an indirect, non-structural role in denv replication. the denv-induced autophagosomes did, however, significantly co-localize with lipid droplets. lipid droplet volume decreased during denv infection in an autophagy-dependent manner, as did cellular triglycerides, a major component of lipid droplets. denv-induced autophagy stimulated the delivery of lipids to lysosomal compartments, resulting in the release of free fatty acids, which undergo -oxidation in the mitochondria to generate atp. this produces a metabolically favorable environment for viral replication. importantly, the authors showed that the defect in viral replication caused by inhibition of autophagy could be completely complemented by adding exogenous free fatty acids. this complementation of defective autophagy by free fatty acids required -oxidation. thus, despite the many roles of autophagy in regulating cellular homeostasis, its regulation of lipid metabolism is a major contributor for robust denv replication [ ] . many viruses, including cytomegalovirus, hcv, and denv alter cellular metabolism to promote their replication (reviewed in [ ] ). the induction of lipophagy is a novel mechanism by which viruses can manipulate the metabolic state of the infected cell. it is also a very different interaction with autophagy than has been proposed with other viral infections. many viruses appear to induce a bulk autophagy and then inhibit its progression at various steps to prevent anti-viral functions (reviewed in [ ] ). alternatively, denv infection induces a selective autophagy that is preferentially targeted to lipid droplets, which leads to changes in cellular metabolism. in addition to modifying cellular metabolism, it is possible that this serves a secondary function in immune evasion. the targeting of autophagosomes to lipid droplets may also divert autophagosomes from processing viral antigens for antigen presentation as an immune evasion strategy. the mechanism by which denv induces autophagy is unclear. a recent report showed that ns a expression can induce autophagosome formation during denv infection and help infected cells avoid apoptosis in renal epithelial cells and thus, contribute to prolonged viral replication [ ] . the unfolded protein response (upr)/autophagy pathways have been shown to modulate the denv pathogenassociated molecular pattern (pamp) rna-induced innate immune response [ ] , suggesting that autophagy may promote denv replication through repressing innate immunity. more work, however, is required to show whether the proposed viral triggers of autophagy reproduce all cellular signals and phenotypes that accompany autophagy induction in denv-infected cells.  denv infected cells are resistant to apoptosis by exogenous stimuli.  denv induces autophagy  ma treatment inhibits viral replication  inhibition of autophagy prevents denv mediated resistance to apoptosis  over-expression of ns a alone prevents apoptosis  ns a mediated protection from apoptosis is dependent upon autophagy given the many functions of autophagy in the cell, it is perhaps not surprising that different studies have identified multiple possible roles for autophagy in denv infection (figure ). while it is clear that denv replication does not occur on discreet, classical autophagosomes but rather the er, there are possible explanations as to why denv proteins are sometimes observed on membranes positives for autophagosomal markers. perhaps the components of the autophagosomal machinery are involved in the er membrane reorganization. this has been shown to be the case for the coronavirus mouse hepatitis virus [ ] . additionally, it is possible that denv may have different interactions with autophagy, dependent on the cell type. the majority of studies with denv and autophagy have focused on non-phagocytic cells, including hepatocytes and epithelial kidney cells. the impact of autophagy on denv replication also needs to be characterized in phagocytic cells. while the liver is an in vivo target during denv infection, it will be important to repeat these experiments in monocytes, which are thought to be a primary target during infection. further, the majority of this work has been done with denv , with only one report examining denv . it will be important to determine which of the interactions between denv and autophagy are conserved between serotypes. autophagy responds to various stress stimuli to maintain cellular homeostasis. since viral infections frequently induce stress, it was initially assumed that autophagy induction might be a byproduct of the infection. in this model, autophagy would be generically triggered and then subsequently inhibited by a viral protein prior to maturation into its degradative form. the demonstration that denv can induce at least one form of selective autophagy that goes to completion to modify cellular metabolism suggests that it triggers either the induction or the marking of lipid droplets as cargo in a very specific way. an important question is how the stimulation of atp production by denv-induced autophagy benefits replication. the answer that -energy is good‖ is not wholly satisfying. atp production might impact cellular energetics, atp-dependent enzymatic processes required for replication such as the ns atpase activity, or cellular signaling pathways that are regulated by atp levels. the importance of viral modulation of cellular metabolism is an emerging field within virology with more questions than answers at this stage. the cellular pathways that are modified and the viral functions responsible for lipophagy induction are not yet known. indeed, the cellular pathway of lipophagy induction is also poorly characterized. future studies characterizing the mechanism by which denv induces lipophagy should enlighten our understanding of how selective autophagy is triggered. it may also produce a novel antiviral therapeutic strategy. a generic interference with autophagy is a dubious antiviral approach given the importance of autophagy to cellular survival. however, it is possible that a denv-specific interaction with the autophagy machinery may be targeted for therapeutic intervention. inhibition of the denv-autophagy interaction stimulating lipophagy would limit cellular metabolic changes and inhibit denv replication. it may also enhance immune recognition of denv antigens, since autophagosomes would no longer be diverted to lipid droplets. in sum, there have been many proposals for the role of autophagy during denv infection. one role is the modulation of the cellular metabolic state, however, this is not mutually exclusive with additional pro-viral roles (such as the inhibition of apoptosis). future work will help to further characterize the role(s) and relative contributions of autophagy to the various aspects of denv replication. regulation mechanisms and signaling pathways of autophagy autophagy: basic principles and relevance to disease autophagy: process and function a comprehensive glossary of autophagyrelated molecules and processes the origin of the autophagosomal membrane the beclin -vps complex-at the crossroads of autophagy and beyond lc , a mammalian homologue of yeast apg p, is localized in autophagosome membranes after processing physiological role of autophagy as an intracellular recycling system: with an emphasis on nutrient metabolism viruses and autophagy myd and trif target beclin to trigger autophagy in macrophages toll-like receptors in control of immunological autophagy multiple regulatory and effector roles of autophagy in immunity autophagy-dependent viral recognition by plasmacytoid dendritic cells major histocompatibility complex class ii-restricted presentation of a cytosolic antigen by autophagy autophagy promotes mhc class ii presentation of peptides from intracellular source proteins antigen-loading compartments for major histocompatibility complex class ii molecules continuously receive input from autophagosomes subversion of the cellular autophagy pathway by viruses modification of cellular autophagy protein lc by poliovirus potential subversion of autophagosomal pathway by picornaviruses autophagosome supports coxsackievirus b replication in host cells coronaviruses hijack the lc -i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication induction of incomplete autophagic response by hepatitis c virus via the unfolded protein response the autophagy machinery is required to initiate hepatitis c virus replication activation of the unfolded protein response and autophagy after hepatitis c virus infection suppresses innate antiviral immunity in vitro dengue and dengue hemorrhagic fever dengue-clinical and public health ramifications dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells dengue virus ensures its fusion in late endosomes using compartment-specific lipids recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis dengue virus nonstructural protein redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis autophagic machinery activated by dengue virus enhances virus replication a role for autophagolysosomes in dengue virus production in hepg cells co-localization of constituents of the dengue virus translation and replication machinery with amphisomes flavivirus ns a-induced autophagy protects cells against death and enhances virus replication composition and three-dimensional architecture of the dengue virus replication and assembly sites modification of intracellular membrane structures for virus replication dikic, i. a role for ubiquitin in selective autophagy autophagy regulates lipid metabolism dengue virus-induced autophagy regulates lipid metabolism multifaceted roles for lipids in viral infection we would like to thank kristi berger for critical reading of the manuscript. g.r. acknowledges membership within and support from the region v great lakes rce (nih award -u -ai- ). n.s.h. is funded by nih training grant t ai - and the william rainey harper fellowship. the authors declare no conflict of interest. key: cord- - acz l authors: he, miao; wang, jingxing; chen, limin; liu, jing; zeng, peibin title: the impact of emerging infectious diseases on chinese blood safety() date: - - journal: transfus med rev doi: . /j.tmrv. . . sha: doc_id: cord_uid: acz l emerging infectious diseases (eids) have always been one of the major threats to public health. although the implementation of mandatory testing for classical transfusion-transmitted infectious—human immunodeficiency virus, hepatitis b virus, hepatitis c virus, and syphilis—has reduced the transfusion risk of these pathogens, the potential threat of various eid agents and their constantly evolving variants to blood safety in china is not fully understood. this review presents representative eid agents that are autochthonous and epidemic nationally or regionally in china. the epidemiologic status and distribution of these eid agents among donors and/or healthy populations are summarized. the potential risks of these eid agents to blood safety are discussed. the review also explores strategies to strengthen hemovigilance systems and studies to further evaluate the impact of eid agents on blood safety. emerging infectious diseases (eid) agents are considered as major threats to transfusion safety. the most notorious eid agent that sabotaged blood safety during s was human immunodeficiency virus (hiv). it raised global concerns of eids and triggered organized activity to systematically prevent eid agents from threatening transfusion safety. nowadays, blood donations are screened for various infectious agents in developed countries or regions where eid agents have been well studied. in the united states, the blood supply is routinely screened for human t-lymphocyte virus (htlv). west nile virus (wnv); trypanosoma cruzi [ ] [ ] [ ] and babesia spp. [ ] [ ] [ ] [ ] have been systematically scrutinized to evaluate the value of donor screening [ ] ; and zika virus has recently emerged as an eid threat [ ] . in china, however, many of the eid agents are rarely investigated among blood donor and evidence for their impact on blood safety is absent. in , the american association of blood banks published a catalog of pathogens relevant to blood transfusion medicine reviews j o u r n a l h o m e p a g e : w w w . t m r e v i e w s . c o m safety in which eid agents were listed as confirmed or suspected to be associated with transfusion transmissible infection (tti) [ ] . the threat of these eid agents to blood safety varies, due to different spreading patterns, transmission routes, epidemiologic characteristics, and endemic status; therefore, they need to be specifically evaluated in each country or area. in china, blood donors are routinely tested for only pathogens: hiv- / , hepatitis b virus (hbv), hepatitis c virus (hcv), and syphilis [ ] . with the broad implementation of nucleic acid testing (nat), the improvement in performance of enzyme immunoassay assays, and the more rigorous policies for donor recruitment and testing, the transfusion risks of the conventional ttis have been largely reduced in china [ , ] . however, many of the diversely distributed eid agents still pose potential threats to blood safety, yet their risks have never been fully evaluated. nevertheless, china has witnessed recurrent outbreaks of highly virulent eids including severe acute respiratory syndrome (sars), highly pathogenic avian influenza (h n ) and streptococcus suis, a zoonotic bacterium mostly carried by pigs or pork products which causes syndromes of streptococcal toxic shock, sepsis and meningitis. [ ] . for example, outbreaks of human streptococcus suis infection in china were reported in sichuan province, resulting in laboratory confirmed cases and deaths from mid-july to the end of august [ ] . some eid agents with asymptomatic distribution among chinese general population, such as malaria, hepatitis e virus, and dengue virus may emerge as greater threats to chinese blood safety and require interventions. in this review, we highlight representative, autochthonous eids agents that are nationwide or regionally epidemic. the epidemiology of these eid agents and the investigations among blood donors and/or the general population are reviewed. the geographic distribution for eid agents that are regionally epidemic is summarized, and the prevalence of these eid agents among chinese donors is summarized. human parvovirus b (b v) is a small, non-enveloped, singlestrand dna virus [ ] , causing various clinical manifestations such as chronic anemia, aplastic crises and arthropathies, and a variety of other syndromes among immune-compromised or immunosuppressed patients [ ] [ ] [ ] [ ] . b v has been confirmed to be one of the ttis transmitted through blood or blood products [ ] . the us food and drug administration (fda) and european pharmacopeia have proposed a limit of × geq/ml for pooled-plasma in order to reduce the potential risk of transmission [ , ] . there is limited data on b v prevalence among blood donors or the general population in china. according to several reports, the estimated prevalence rate could be as high as . % in the general chinese population [ ] and . % in hiv co-infected individuals [ ] . in the tibetan area, the b v dna positivity rate was . % among the general population [ ] . among chinese blood donors, the b v dna prevalence rate was . %, which is much lower than that in the general population [ ] . the same study also reported that different geographic locations demonstrated different prevalence rates for b v, and that the dna sequences in xinjiang province showed a different genetic lineage than in other places of china [ ] . in another study, b v dna was detected in . % ( / ) of plasma pools from chinese blood product manufacturers of intravenous immunoglobulin (ivig), factor viii, fibrinogen and prothrombin complex concentrates, with levels of b v-dna varying from × geq/ml to × geq/ml [ ] . the viral load in one donation sample was . × geq/ml, which was significantly higher than the threshold recommended by the us fda and european pharmacopeia ( × geq/ml). while further investigation is necessary to determine whether b v nat screening should be implemented as a routine in chinese blood centers, the current b v contamination in plasma products is a serious concern. malaria malaria is caused by infection with the parasites of plasmodia spp. and the current distribution covers the tropics and large parts of the subtropics [ ] . infection with plasmodia spp. often resembles a common viral infection which may lead to a delay in diagnosis [ ] . malaria is considered a transfusion risk since asymptomatic immigrants who reside in and travelers who visit endemic areas might import malaria to non-endemic areas [ ] . therefore, us fda has initiated a donor deferral policy that temporarily defers donors who travel to endemic areas. as a result, about % of us donors are deferred for that reason [ ] . malaria was once highly endemic in china with an estimated million cases per year [ ] . the chinese government began a tremendous effort to eliminate malaria in when the national malaria control program was launched [ ] . the main species that causes malaria in china is p vivax and has been found in many regions; however, other species of plasmodia have also been reported [ ] . although malaria has been well controlled in the last decades, sporadic outbreaks are frequently reported [ ] , and malaria remains a reportable infectious disease in china [ ] . several cases of malaria transmission by transfusion were recently reported ( table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . meanwhile, the dna of p knowlesi was also found in pooled plasma from a manufacturer in guizhou [ ] . however, currently chinese blood centers do not have a policy to defer donors who have traveled to malaria endemic areas or malaria infected countries during epidemic seasons. the actual prevalence of malaria and its residual risks among voluntary blood donors is unknown. further studies on malaria infected blood donors in china, such as a survey on the risks of infection and demographic characteristics of plasmodium infected donors, are crucial to better understand the transfusion risks of malaria in china. hepatitis e virus (hev) is an enterically transmitted, positive-sense, single-stranded non-enveloped rna icosahedral virus [ ] . it usually causes an acute and self-limiting infection. hev has a worldwide distribution and substantial morbidity and mortality in some developing countries [ ] . many cases of hev transmission by blood transfusion have been documented all over the world [ ] [ ] [ ] [ ] [ ] [ ] [ ] . routine screening of donors for hev rna was suggested by some studies [ ] . in china, the anti-hev seroprevalence is about % in the general population and increases with age by % per year [ ] . approximately . % of individuals are igm positive (indicating acute infection) and . % are asymptomatic with viremia [ ] . in the early s, due to the frequent occurrence of illegal blood donation in central china, the anti-hev igg prevalence spiked to . % and anti-hev igm prevalence was . % among illegal blood donors [ ] . in a recent study using test results from routine donations collected at urban blood centers, investigators reported a prevalence of . % for anti-hev igg, . % for anti-hev igm, and . % for hev rna among donations. in addition, they found that prevalence rates varied by blood center locations [ ] . in a comparative study [ ] , where samples from both qualified blood donors and donors deferred due to elevation of alanine aminotransferase (alt) were examined, the prevalence rates of anti-hev igm and anti-hev igg in alt-elevated donors ( . % and . %, respectively) were significantly higher than those in qualified donors ( . % and . %, respectively). meanwhile, the prevalence of hev antigen among the alt-elevated donors ( . %) was also higher than that among qualified donors ( . %), but not at a statistically significant level. these data suggest that routine pre-screening and post-donation alt tests can reduce, but not eliminate the potential risks of hev infection from otherwise qualified donations in china. dengue viruses (denv) denv causes dengue fever, dengue hemorrhagic fever (dhf), and dengue shock syndrome. denv is a life-threatening mosquito-borne disease with global spread [ ] . denv was shown to be capable of transmission by transfusion [ ] . transfusion of red blood cells which tested denv seronegative but were rna positive with high viral load (n copies/ml) can lead to denv infections in transfusion recipients [ ] . several countries and regions with a high prevalence of denv have investigated denv among blood donors and recipients to evaluate its impact on blood safety, as well as to develop a proper donor enrollment strategy to reduce the transfusion-transmission risk [ ] [ ] [ ] . in china, early outbreaks of dhf in s were reported mostly in guangdong, guangxi, yunnan, hainan, fujian, and zhejiang provinces located in the southeast coastal regions or border areas next to southeast asia [ ] . more than % of the documented dhf cases were from guangdong province, one of the most developed provinces located in the south coastal area [ ] . since dengue fever first reemerged in guangdong province in [ ] , the epidemic of denv spread to chinese provinces by [ ] . from to , the epidemic status of dhf gradually changed from sporadic imported to autochthonous endemic cases [ ] . in , there was a large dhf outbreak from july to november in foshan city, guangdong province with suspected febrile cases [ ] . among them, denv infections were confirmed by laboratory testing ( rna positive and igm positive) [ ] . the following year, the third historically largest dhf outbreak spread throughout guangdong province ( of cities with reported cases) with febrile cases ( denv infections confirmed) resulting in deaths [ ] . partly in response to the outbreaks in and , the chinese center for disease control and prevention (cdc) amended the guidelines for dhf prevention and control to strengthen the implementation of mosquito control [ ] . in , the dhf cases in guangdong province dropped sharply to cases by the end of september [ ] , reflecting the effectiveness of denv control measures in this area. denv sentinel monitoring by sero-surveillance has been in operation in dhf epidemic regions in china since s. the denv seroprevalence rate varied (igg: . %- . %; igm: . %- . %) in the denv endemic areas in guangdong [ , ] , guangxi [ ] , yunnan [ , ] , hainan [ ] , and fujian [ ] provinces. a pilot study of denv serology and viremia among asymptomatic donors in guangzhou city (guangdong province) in september and october found that the denv igm prevalence rate was . % (n = ). the study also identified one denv rna positive donor with viral load of copies/ml [ ] . another post-outbreak serological investigation among healthy populations in foshan city (next to guangzhou city) found significantly higher denv seroprevalence rates in the towns that experienced dhf outbreaks in (igg rate: . %, n = ) compared with the towns without autochthonous cases (igg rate: . %) [ ] . a blood product with denv rna viremia can potentially lead to transfusion-transmitted infection. however, to date, there are no published studies on denv transmission via transfusion in china. human brucellosis caused by brucella spp. is one of the most severe zoonotic diseases worldwide [ ] . transfusion-transmitted brucellosis cases have been reported since the s [ ] [ ] [ ] [ ] , and brucella spp. are considered a potential risk for blood safety [ ] . human brucellosis is one of the leading threats to public health in farming areas with livestock in china [ ] . more than % of brucellosis cases in china were found in north china and were concentrated in inner mongolia, shaanxi, heilongjiang, jilin, and hebei provinces [ ] . a total of brucellosis cases were confirmed in these areas. in addition, a rapidly increasing trend was observed in brucellosis incidence, from . cases per people in to . clinical cases per people in [ , ] . an epidemiological investigation in inner mongolia reported the incidence rate as high as . % among people who had close contact with livestock [ ] . in china, most of brucella surveillance has focused on risk factors for infections [ , ] , prevalence in dairy cattle [ ] and local animals [ , ] with the goal of providing information for brucellosis prevention and control. the dna prevalence rate of brucella in raw whole milk samples was found to be . % in provinces [ ] . the low-risk population in urban areas is at increasing risk of brucellosis from contaminated milk or raw meat [ ] . limited data showed the seroprevalence rate of brucella among the healthy human population to be . % in endemic areas [ ] . recently, a brucella spp. investigation based on enzyme immunoassay testing among blood donors in an endemic area (kashi, xinjiang province) showed that the reactive rate was % ( / ), in which . % ( / ) samples were further confirmed by western blot (wb) testing, and the brucella dna prevalence rate was . % ( / ) [ ] . although no infection cases transmitted by transfusion have been reported, the existence of brucella dna in donors' plasma samples indicates potential risk of transfusion-transmitted brucellosis in endemic areas and warrants future investigation. as the first retrovirus discovered in humans [ ] , htlv is one of the important ttis that may lead to various human diseases such as adult t-cell leukemia/lymphoma, myelopathy/tropical spastic paraparesis, opportunistic infections, and inflammatory disorders [ ] . the implementation of mandatory testing on donors has been in effect since mid- s in many countries to reduce the risk of transfusion transmission. [ ] in china, sero-surveillance of htlv among the general population started in s when an early study in beijing and other provinces found a . % positive rate in samples-all connected to the endemic country japan [ ] . in and , nationwide investigations reported the prevalence of htlv among blood donors to be . % (n = ) [ ] and . % (n = ) [ ] respectively. a meta-analysis of studies among donors in provinces and regions found that the pooled estimates of htlv- prevalence in fujian and guangdong were . / ( % ci, . / - . / ) and . / ( % ci: . / - . / ), respectively. most isolates belonged to the transcontinental subgroup a whereas only cases of htlv- infection were found among donors in other provinces and regions [ ] . data from these studies indicate that while htlv prevalence is low in china, the infection has expanded from concentrated coastal regions in fujian and guangdong to the neighboring provinces where seroprevalence remains low (ranging from . % in shanghai and . % in jiangxi) [ , ] . although no transfusion-transmitted htlv infections have been reported, the surveillance of htlv among blood donors and the evaluation of its impact on blood safety continue to be studied in china. severe fever with thrombocytopenia syndrome virus (sftsv), a novel tick-borne bunyavirus, was first identified in china to be the etiologic agent of severe fever with thrombocytopenia syndromes (sfts) with initial fatality rates between % and % in [ ] . sftsv is concentrated in the mountainous rural areas in central and eastern china [ ] [ ] [ ] . the epidemic seasons of sfts are mainly from spring to autumn and peak in may to july [ ] . farmers are at high risk for sfts due to more exposure to ticks [ ] . the molecular characteristics, epidemiologic distribution, risk factors and clinical symptoms have been well summarized in several reviews [ , ] . by the end of , sftsv had spread from provinces in [ ] to provinces with a growing incidence but a decreasing fatality rate [ ] . the increasing epidemic of sfts was reported and annual incidence was estimated to be cases per populations in autochthonous endemic area [ ] . meanwhile, increasing numbers of sfts cases were reported in japan [ ] [ ] [ ] and south korea [ , ] . migratory birds are suspected to have played an important role in promoting the spread of sftsv in east asia [ ] . although there is no recorded transfusion-transmitted sfts case at present, sftsv has the potential to become a transfusion-transmitted infectious agent due to several considerations: ( ) nosocomial transmissions caused by direct exposure to sfts patients' blood have demonstrated that sftsv is a blood-borne pathogen [ ] [ ] [ ] [ ] . ( ) the incubation period from infection to onset of the disease is one or weeks on average [ ] , and in some cases up to thirty days [ ] . viremic asymptomatic blood donors within the incubation period may therefore potentially transmit sftsv to recipients leading to sfts. in a study launched by the national heart, lung, and blood institute, johns hopkins university, the chinese institute of blood transfusion, and chinese blood centers (one located in an endemic and in nonendemic regions), antibody screening and follow-up sftsv rna detections were performed on blood donor plasma samples collected between april and october . the seroprevalence rates were . % ( / ), . % ( / ) and . % ( / ) in an endemic area (xinyang) and non-endemic regions (mianyang and luoyang) respectively, with no significant difference observed (p n . ). among donors screened by -sample minipool sftsv rna testing, suspected viremic samples were detected each with a viral load less than plaque-forming units (pfu)/ml [ ] . other regional sftsv among the healthy individuals reported seroprevalence rates varying between . % and . % [ ] [ ] [ ] in epidemic areas, with finding asymptomatic viremic cases. continued investigation is needed to evaluate whether sftsv presents a risk to transfusion recipients in china. leishmania infection is responsible for cutaneous and visceral leishmaniasis (kala-azar). leishmania spp. are usually transmitted to people through the phlebotomine sandfly [ ] . the infected individual may harbor a persistent infection up to years before recovery [ ] . the transmissibility of leishmania infection via blood has been demonstrated in animals [ , ] as well as human beings [ ] [ ] [ ] [ ] . leishmania in human red blood cells (rbcs) can survive for as long as days under blood bank storage conditions [ ] . it has been reported to cause cutaneous or visceral leishmaniasis in infants and immunocompromised patients [ ] [ ] [ ] [ ] . asymptomatic infections are usually found in healthy blood donors from endemic areas [ ] [ ] [ ] [ ] [ ] . us military blood banks enforce permanent deferral for individuals with any history of leishmaniasis, but there is no existing regulation or standard for leishmania testing in civilian donor screening [ ] . during the s, there were more than documented kalaazar cases in china, mainly in rural areas in the north [ ] . with the efforts of a national control program, kala-azar was almost eliminated in s [ ] . currently, more than cases of kala-azar are reported each year, mainly from xinjiang province and other provinces in western china [ ] . recently, a retrospective study of visceral leishmaniasis by the chinese cdc reported an increase in the number of visceral leishmaniasis cases in endemic areas between and [ ] , indicating that prevention and control strategies must be taken to restrain the increasing incidence and spread of leishmaniasis. otherwise, frequent population migration, tourism, and a rapidly growing public transportation may lead to further spread of the infection and push it up on the list of transfusion-transmitted infectious diseases in china. to date, there are still no survey studies of infection among donors and no documented transfusion-transmitted cases for leishmania infection in china. q fever is a zoonosis due to coxiella burnetii infection. c burnetii usually causes asymptomatic clinical manifestations such as a flu-like disease or atypical pneumonia in humans [ ] ; however, acute [ , ] and fatal chronic infections [ ] [ ] [ ] [ ] have been reported. in , there was a large outbreak of q fever in the netherlands [ ] . hogema et al initiated a surveillance of c burnetii dna and antibodies in local blood donations and found that the c burnetii dna positive rate was . %, while the igg seropositive rate was . % [ ] . furthermore, seroconversions were detected in donors with an incidence rate of . % per year during the outbreaks from to [ ] . after the great outbreaks of q fever, slot et al. started to investigate if chronically infected donors posed a threat to blood safety in the netherlands. the serological results from serum samples collected in the most affected area during august to january showed that chronic c burnetii infection was absent in the epidemic blood donors, which led to the donor re-entry policy that had already been initiated elsewhere in europe [ ] . in china, most c burnetii infections have been observed in tibet, yunnan, xinjiang and inner mongolia [ ] . the c burnetii dna positivity rate was about % in ticks and % in humans in western china [ ] . as q fever is mainly an airborne disease, the individuals with exposure to livestock bear a higher risk. some epidemiological studies show that more than % of rural farmers had antibodies to c burnetii [ , ] . although blood donor screening of c burnetii has not been initiated in china, the dna of c burnetii was found in pooled plasma from a manufacturer in guizhou [ ] . a cross-sectional study of the seroprevalence and viremia status of c burnetii among blood donors, especially in nomadic herding and livestock regions, would provide a more comprehensive assessment of its impact on blood safety in china. due to its biological and geographic diversity, china will always be at high risk for various eid agents. as a populous country, a dramatic ramp-up in the number of blood donations was reported recently, with more than million donations collected in at the donation index of . donations per people [ ] . however, monitoring of the classical ttis such as hiv, hbv, hcv and syphilis, as well as eid agents has only been implemented by the chinese cdc and mostly based on sentinel investigations and clinical case report systems. the monitoring systems have demonstrated effectiveness in establishing proper guidelines for disease prevention and control based on epidemiological analysis of transmission routes and infection risk factors. the fatality rates and clinical symptoms from the documented cases are periodically summarized and analyzed to help with diagnosis and therapy of these pathogens, as well as with the development and improvement of laboratory diagnostic assays. effort towards disease control has benefitted from the system in the face of several outbreaks of eid epidemics, such as: sars [ ] , h n [ ] , sftsv [ ] , and denv [ ] . the regional spread of infection is an important aspect to consider when evaluating the transfusion risk for eid agents. among the eid agents listed above, all of them have been proven to result in pathological and autochthonous epidemics in china. b v, hev and plasmodium spp. have nationwide distribution. three insect-borne eid agents (denv, sftsv, and leishmania spp.) and zoonoses (brucella spp. and c burnetii) display significant geographic diversity due to their patterns of transmission. htlv is clustered in one coastal region. the regional distribution of eid agents is shown in fig. . not surprisingly, the regional distribution of insect-borne pathogens matches that of disease vectors (mosquitos, sand-flies and ticks). denv, a well-described mosquito-borne virus, has a global distribution in tropical and subtropical areas, the location of recent outbreaks of dhf. the infections of sftsv, the novel tick-borne bunyavirus, were identified from sfts patients in rural mountainous areas in central and eastern china, mainly because of the higher risk of tick exposure. in a similar way, most of the infections of zoonotic diseases (q-fever and brucellosis) were found in rural livestock farming areas in northern and western china. by compiling the distribution of reported clinical cases and the prevalence of eid agents in donors and/or general population, we provide estimates of the frequency of eid agents among chinese blood donor populations. see fig. . hev and b v are estimated to have national distribution among donors and general population in china based on previous cross-sectional studies. spread of brucella spp. depends on transmission routes from contaminated milk and raw meat produced from endemic regions. considering the current dna prevalence rate ( . %) of brucella spp. among blood donors in endemic regions and its potential high frequency in the general donor population, this eid is a matter for concern. in the shadows of recent global outbreaks and the fast expanding trends of dhf, denv should also be highlighted for its potential risks in endemic areas in china. the other regionally concentrated eid agents: qfever, leishmania spp., and sftsv are estimated to have moderate or low frequency among the blood donor population. however, ongoing surveillance on these eid agents in donors is prudent in order to provide sound evidence regarding their impact on blood safety in china. in order to evaluate the impact of eids on blood safety, a practical and feasible approach is to monitor the existence of eid agents among asymptomatic blood donors and obtain direct evidence from transfusion-transmitted cases confirmed by molecular analysis. firstly, the surveillance among blood donors through sero-markers and nucleic acid of pathogens provides data for estimating transfusion risk. among the eids discussed in this review, only htlv has been continuously investigated at blood centers since s. there are a few investigations and case reports on b v, hev and plasmodium spp. among blood donors, but no systematic and continuous investigation on these eids. data on sftsv, denv and brucella spp. in china are very limited with only one documented donor surveillance study available for each agent. c burnetii (q fever) and leishmania spp. have not been investigated. in addition, well-organized and appropriately designed donorrecipient linkage studies may directly confirm transmission by transfusion, facilitate further evaluation of the post-transfusion outcomes, and yield more convincing evidence to support strategies for screening donors for eid agents. although china has not performed such linkage studies, brazil examined donor-recipient linkage for dengue virus using a large, nationally representative database [ ] . this study may serve as an example to follow in order to evaluate transfusion risk of eids in china. collaborative efforts from global surveillance programs may prove useful to mitigate the transfusion risk of eids. although most eid agents in china were historically imported, some agents, such as sars and sftsv, originated in china and are expanding to other adjacent countries. also, non-endemic eid agents such as west nile virus (wnv), chikungunya virus, variant creutzfeldt-jacob disease (vcjd) and zika virus (zikv) should be immediately addressed due to their worldwide distribution and severity of clinical outcomes. recently, transfusiontransmitted zikv has been documented [ ] [ ] . several cases of zikv infection among travelers from epidemic areas have been reported in china [ ] [ ] . currently immigration authorities recommend oral fig. . the distribution of major regional eid agents in china. declaration of infection at entry-exit inspection and quarantine of symptomatic travelers from zikv epidemic areas upon entry into china. travelers from countries epidemic for vcjd are deferred from donation by health questionnaire in chinese blood centers. however, whether wnv, chikungunya virus and zikv should also be added to the deferral list remains a matter of controversy that needs evidence from further investigation. pathogen reduction of blood products is an alternative defense against eid agents. several studies described effective inactivation of eid agents such as denv [ ] and wnv [ ] . currently the only pathogen reduction technology in use in china is the methylene bluephotochemical technology applied to plasma products [ ] . the technology is not mandatory and only performed at a few blood centers. more efforts should be made to evaluate whether and how pathogen reduction can be used to safeguard the 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multiple arboviral infections during a denv- outbreak in solomon islands date: - - journal: trop med health doi: . /s - - - sha: doc_id: cord_uid: xyie s background: solomon islands, a country made up of tropical islands, has suffered cyclic dengue fever (df) outbreaks in the past three decades. an outbreak of dengue-like illness (dli) that occurred in april prompted this study, which aimed to determine the population’s immunity status and identify the arboviruses circulating in the country. methods: a household survey, involving participants in two urban areas (honiara and gizo), and a parallel hospital-based clinical survey were conducted in april . the latter was repeated in december after a surge in dli cases. arbovirus igg elisa were performed on the household blood samples to determine the prevalence of arboviruses in the community, while qpcr testing of the clinical samples was used to identify the circulating arboviruses. dengue virus (denv)-positive samples were further characterized by amplifying and sequencing the envelope gene. results: the overall prevalence rates of denv, zika virus, and chikungunya virus were . %, . %, and . %, respectively. the qpcr positivity rates of the clinical samples collected in april were as follows: denv . %, zika virus . %, and chikungunya virus . %, which increased to %, %, and % respectively in december . the displacement of the circulating serotype- , genotype- , with denv serotype , genotype cosmopolitan was responsible for the outbreak in . conclusions: a denv outbreak in solomon islands was caused by the introduction of a single serotype. the high prevalence of denv provided transient cross-protection, which prevented the introduction of a new serotype from the hyperendemic region for at least years. the severe outcomes seen in the recent outbreak probably resulted from changes in the causative viruses and the effects of population immunity and changes in the outbreak pattern. solomon islands needs to step up surveillance to include molecular tools, increase regional communication, and perform timely interventions. dengue fever (df) is the most prevalent mosquito-borne viral infection in tropical and subtropical regions. it is estimated to cause - million symptomatic infections annually [ ] . the complex interactions between the virus and the human population have resulted in an unpredictable disease evolution and variable clinical outcomes associated with the infection. the majority of infections are mild and self-limiting, but a small number progress to severe clinical manifestations, which can lead to severe organ complications and even death [ ] [ ] [ ] . dengue virus (denv) is a flavivirus from the flaviviridae family. its rna genome is approximately -kb long and contains an open reading frame, which codes for three structural and five non-structural proteins. the virus has four distinct antigenic serotypes and several genotypes, which produce different outcomes during outbreaks [ ] [ ] [ ] . most global df cases occur in the asia-pacific region [ ] . countries in southeast asia (se asia) are generally more urban and have high population densities, while the pacific island countries (picts) are rural, and their populations are scattered across many islands. previous df outbreaks in the picts were caused by a single serotype, which occurred every to years [ ] . on the other hand, in se asia all four denv serotypes are circulating, and a -year wave-like outbreak pattern, in which the disease emanates from a central metropolitan city, is seen. there have been very few quality descriptive studies of df conducted in the picts, resulting in a paucity of information about the magnitudes and drivers of df epidemics in this region. the available studies were mostly based in a clinic setting or involved specific groups like a blood donor population [ , ] . the principal vectors of df, aedes aegypti (linnaeus, ) and aedes albopictus (skuse, ), are present in both se asia and the picts. solomon islands is an archipelago, consisting of a double chain of islands, located in the southwest pacific region. it has a population of more than half a million people. it shares a border with papua new guinea, and the densely populated region of se asia is located to the west (fig. ) . while the majority of the population live in rural areas and are involved in subsistence agriculture, fast-growing unregulated urban settlements are also common. the first reported df outbreak in solomon islands occurred in honiara, the capital city of solomon islands, in , but it was only confirmed serologically in [ ] . a -year inter-epidemic cyclic pattern was observed until , when denv- first started to circulate the region [ ] . a subsequent outbreak in was more severe, resulting in the first reported deaths [ ] . this outbreak was unique, as it lasted longer than usual, i.e., previous outbreaks lasted years, but the outbreak lasted until early . this, in addition to the global expansion and reported occurrence of zika virus (zikv) and chikungunya virus (chkv) infections in the region [ , ] , prompted us to carry out this study. the study objectives were to estimate the seroprevalence of arboviruses in the urban population of solomon islands while using qpcr to determine the circulating viruses from the clinical samples; furthermore, to characterize and compare the dengue virus isolated from our study to previous published isolates from the region using the phylogenetic tree analysis. suspected cases of df were reported syndromically as dengue-like illness (dli), which was defined as the sudden onset of fever, testing negative for malaria, and exhibiting one or two clinical manifestations of df, as described previously [ ] . in order to obtain a complete picture, we only included the clinics in honiara, including gizo, that started reporting from to . this ensured that we would be able to detect trends using consistent and complete datasets. the virological investigations involved -weeklong blood sample collection periods (in april or december ), during which blood samples were collected from dli patients that visited the hospitals in honiara and gizo. the household seroprevalence study was performed in april in gizo (western province) and a community settlement (tuvaruhu) that was representative of honiara. each of the subjects that gave their consent filled out a questionnaire, before approximately μl of blood was collected in a pediatric tube, containing ethylenediaminetetraacetic acid (edta), using the finger prick method (becton dickinson collection system). the blood samples were brought back to the hospital laboratory and processed by centrifugation at rpm for min to separate the plasma. the samples were then transported to our laboratory overseas, where they were stored at − °c, before being used for various enzymelinked immunosorbent assays tests. to determine the seroprevalence of arboviruses in the household study, tests for igg antibodies against various arboviruses were performed using commercially available elisa. these included tests for anti-denv igg antibodies (vircell, granada, spain), anti-zikv igg antibodies (mybiosource inc., ca, usa), and anti-chikv igg antibodies (abcam, cambridge, uk). the controls provided with the kits and the cutoff values listed in the associated information were used to determine the results, as recommended by the manufacturers. the clinical samples from recent dli cases were tested using non-structural protein- (ns )/igm/igg combo rapid diagnostic test (rdt) kits for denv. tests for the other arboviruses were performed on selected december samples using commercial igm elisa kits (zikv igm elisa, mybiosource inc., and chikv igm elisa, abcam). the qpcr screening was performed using cdna extracted from μl of the serum samples from the patients with suspected df. the cdna was synthesized using the random primer protocol in the revertra ace-α-® system (toyobo, osaka, japan), as recommended by the manufacturer. the cdna samples were stored at − °c, before being used for various molecular tests. the qpcr screening used previously described denv serotype-specific and zikv-specific primers ( table ) with some modifications [ ] [ ] [ ] . a -μl reaction mixture, containing . μm of each forward and reverse primer, μl of × ssoadvanced universal sybr green supermix taq (bio-rad laboratories, hercules, ca), and μl template, was used together with the rotor-gene qpcr system (qiagen, hilden, germany). the qpcr setup involved an initial temperature of °c for s, followed by cycles of s at °c, and s at °c. to detect chkv, the primers listed in table were designed based on the conserved region of the envelope gene, which was based on an alignment that included the papua new guinea (png) sequence (mf ). the primer was first characterized by making -fold serial dilutions of . × copies of the e gene using the qpcr conditions described above. the amplification curve and t m peak value were analyzed to calculate the correlation coefficient and consensus peak values of the primer. sequence confirmation of % of the qpcr products was then performed to further validate the primer. the denv envelope genes were investigated using cdna templates from patients that were found to be positive for denv in the clinical surveys conducted in gizo and honiara. the templates were amplified using tksgflex tm dna polymerase (takara bio, shiga, japan) and previously described overlapping primers for denv- and denv- [ ] . the pcr products were then cycle-sequenced using the bigdye terminator cycle sequencing kit (version . ), before being sequenced on the abi xl automated sequence analyzer (thermo table the primers used in the qpcr screening of the arbo-viruses fisher scientific, inc.). the final -bp sequence for denv- and the final -bp sequence for denv- were obtained by manually assembling the overlapping sequences in bioedit (version . ) [ ] . our study sequences were uploaded to the dna data bank of japan (ddbj) under accession numbers lc to lc . these sequences were combined with those circulating se asia, australia, and picts (from genbank) to construct a phylogenetic tree using the maximum likelihood (ml) method in the software mega (version ) [ ] . multiple full-envelope nucleotide sequences were aligned using clustal w, which is integrated into the mega software, while evolutionary distances were computed using the recommended tamura-nei + g model. the reliability of the ml analysis was evaluated via a bootstrap test of replications. a different genotype from that of the interest group was set as an outgroup in each of the dengue trees. descriptive statistical analyses were performed using the spss statistical software (version . ; spss inc., il, usa), and individual subjects were used as statistical units. multinomial logistic regression analysis was performed to analyze the associations between selected variables and dengue infections. the prevalence rates for each variable were also determined, together with % confidence intervals ( % ci). a catalytic model with a constant force was used to calculate the force of infection (foi), which was determined using the ml method, as described previously [ , ] . the sentinel clinic data for dli reported for honiara, which covered the period from to , are shown in fig. . the graph shows that two outbreaks with very similar outbreak patterns occurred within years, i.e., in and . the outbreaks involved a sharp increase in the number of dli cases in the first month and peaked in the subsequent months. the sentinel data represented % of all dli syndromic cases, as reported previously [ ] . the outbreak differed from the previous outbreak in that it started in august rather than in january. furthermore, the continuous persistent occurrence of dli was seen before the spike in august . the household survey involved participants, who were recruited from honiara (n = ) or gizo (n = ). no refusals to participate were recorded in this study. there were slightly more females than males (male:female = : . ). the subjects' mean age was . years ( % ci . - . ; range: from to years old). the most common main source of income for the surveyed households was being employed in the private sector ( %), followed by being employed in the public sector ( %). the overall seroprevalence of the denv, as measured by the rate of positive anti-denv igg antibody elisa results, was . % ( % ci . - . %) ( table ). the prevalence of the denv was slightly higher in honiara ( . %) than in gizo ( . %), but the difference was not significant (p = . ). the prevalence of the denv differed significantly between females ( . %) and males ( . %) (p value = . ). neither the main household source of income nor recalled illnesses in the last months significantly affected the prevalence of the denv. the prevalence of the denv increased with age, with the lowest prevalence rate ( . %) seen in the - years age group and plateaued in the - years age group (fig. ) . the mean ages of the subjects that tested positive and negative for anti-denv igg antibodies were . years and . years, respectively. the difference between the two groups ( . years) was significant. the foi, which was defined as the annual risk of infection by any denv serotype among denv-naïve individuals, was . % in the - years age group. the prevalence results for the other arboviruses were . % for the zikv and . % for the chkv (table ) . the high positivity rate for anti-denv igg antibodies ( . %) and the high denv positivity rate detected in the qpcr analysis of the clinical samples ( . %) were indicative of a high number of secondary infections in the outbreak. our high prevalence results appeared to support the idea that igg is a useful serological marker of past exposure to the denv. among the samples tested in april , all of the detected denv belonged to the denv- serotype. in december, however, the dominant serotype detected in tested samples was denv- ( %), although samples were co-infected with the denv- serotype. the qpcr-based zikv detection rate was . % in april, and it increased to % in december, while the qpcr-based chkv detection rate increased from . % in april to . % in december. further analysis of the qpcr results showed that triple infections with denv, zikv, and chkv were detected in . % of the december samples. analysis of the december samples also revealed that there were only chkv-positive and zikv-positive samples among the denv-negative samples. among the clinical samples collected in december, a subset of samples was used to calculate the sensitivity and specificity of the denv rdt compared with the qpcr reference test ( table ) . the denv rdt combo kit included tests for ns , igm, and igg. the sensitivity and specificity of the ns based rdt were found to be % and %, respectively. when the ns -based test was combined with the igmbased test, the sensitivity of the rdt increased to %, but its specificity was reduced to %. the sensitivity and specificity of the igg-based rdt were % and %, respectively, which the kit manufacturer's information suggested was indicative of a high rate of secondary infections. the phylogenetic tree for denv- shown in fig. was constructed using sequences from se asia, australia, and the pacific region. the solomon island sequences were isolated from to and included isolates from our study in april. the sequences in the tree were classed under denv serotype , genotype- . the pict lineage, which included isolates from solomon islands, fiji, vanuatu, samoa, and nauru, shared a bootstrap value of and circulated the region. they shared a common linkage with isolates from png (kt / and kt / ). an ancestral link with a bootstrap value of to an isolate dating back to (ky / ) was shared by all isolates found in png, australia, and the picts. they also shared ancestral links with the isolates that had been circulating from to . the ancestral links also showed that se asia played an important role in the evolution of df and its introduction to the picts. the denv- phylogenetic tree shown in fig. was constructed using the sequences we isolated in december and sequences from se asia, australia, and the picts, which were obtained from genbank. they were all classified as denv- , genotypecosmopolitan. there were two distinct denv- lineages circulating the pacific region in . the solomon island samples were all very similar and had a very strong bootstrap value of , which indicated that the virus was introduced at a single point and was circulating for a very short period of time. the the prevalence of denv in our study was comparable to those seen in other studies conducted in the region. for example, the prevalence of denv was reported to be . % in samoa, % in png, and - % in french polynesia [ , , ] . these values were similar to those reported in asian countries: bangladesh - . % [ ] , the philippines . % [ ] , and thailand . % [ ] . based on the syndromic data obtained in , the high anti-denv igg antibody prevalence rate found in our study before the surge must have arisen during the first months of an outbreak. this would have resulted in at least two-thirds of the population being exposed and becoming immune to the circulating serotype. a study conducted in bangkok suggested that such exposure induces years transient cross-protection against other denv serotypes [ ] . this would have provided herd immunity, which would have helped control the outbreak and could have been responsible for the alternative serotype outbreak pattern seen in the country [ ] . it was also suggested that denv-immune individuals are cross-protected against zikv [ ] , which would probably explain the low prevalence of zikv in april despite its circulation in the country in (genbank kx ) [ ] . the high qpcr-based denv detection rate seen in our denv-immune study population pointed to a high secondary denv infection rate during the outbreak. secondary infections involve heterologous denv infections and are associated with an increased risk of severe df [ , ] . the denv responsible for the new infection will trigger antibody production from preexisting plasma cells, but these antibodies will not be able to neutralize the newly introduced serotype. the non-neutralizing binding of the antibodies to the virus results in the formation of a complex that binds to the fcγ receptor, which is expressed on monocytes, macrophages, and dendritic cells, to facilitate antibodydependent enhancement [ ] . this process promotes virus production and an altered immune response, which is linked with severe df. the use of ns -based denv rdt in a resource-poor setting was very useful for diagnosis and management. however, the sensitivity value of the ns -based rdt ( %) was low compared to the % sensitivity value reported in the previous denv- outbreak in [ ] . secondary infections and the denv serotype are important factors affecting the diagnostic sensitivity of ns -based rdt [ , ] . in our study, the ns -based rdt was found to have lower sensitivity for the denv- serotype than for the previously reported denv- . the phylogenetic tree analysis and the serotypespecific qpcr results indicated that there was a single denv- serotype in circulation from to in solomon islands. the newly introduced denv- serotype in was responsible for the spike in cases seen in the latter part of . this successful introduction was unique, as it was the first time that two serotypes were found to be co-circulating in the country. the cocirculation of multiple serotypes is a feature of endemic settings and was also recently reported in neighboring png [ ] . the co-circulation of denv- and denv- has also been reported in new caledonia [ ] , suggesting that such co-circulation is a potential threat in the region. the presence of two lineages of the denv- cosmopolitan serotype circulating the pacific region in confirmed the heterogeneous nature of the virus [ ] and the importance of monitoring the outbreak potential of different lineages. it also highlighted the importance of performing molecular sequencing analysis to identify genotype and subgenotype lineages. the denv has probably been introduced into the picts multiple times from multiple sources. our study, however, seemed to highlight ancestral linkages to png where the circulation of multiple serotypes and the formation of local lineages had been described [ ] . better molecular surveillance in png will enable more accurate prediction of denv outbreaks in the pacific region. recent improvements in airline travel links to the picts will make it easier to introduce new viruses into the region as airline travel was reported to increase the risk of df spreading in asia [ ] . it is important for countries to have good surveillance mechanisms for both diseases and their vectors to enable timely interventions in future outbreaks. the seroprevalence study was initially designed to explore the population's immunity to denv in order to predict its outbreak potential. the sample size was small, as it was based on the high denv positivity rate (approximately %) seen during the last outbreak. it was therefore not ideal for investigating the zikv or chkv, which have low prevalence rates. as the clinical study was initially only planned to involve molecular virological surveillance, it lacks demographic and clinical information. this also explains why the april samples were not subjected to serological testing. however, this study provided important information that will aid understanding of the unique outbreak pattern seen in this small population. this study found that the dli outbreak in solomon islands was caused by multiple circulating arboviruses. the displacement of a single serotype (denv- ) by a newly introduced one (denv- ) was the predominant cause of the dli outbreak, but there was evidence of zikv and chkv co-circulating. the outbreak pattern suggests that natural population immunity is still the main control mechanism and that the population will always be vulnerable to vector-borne diseases. there is a need to step up vector-control activities and community mobilization to reduce the threat of such diseases in solomon islands. we recommend that solomon islands needs to increase its capacity for disease surveillance to include molecular screening of different arboviruses and some capacity to perform regular sequencing of the envelope genes of viral isolates. we also recommend the surveillance of disease vectors and their insecticide-resistance status. the communication of this information within the region will enable each country/ territory to prepare as best as they can for the next outbreak. global strategy for dengue prevention and control clinical and hematological profile of patients with dengue fever at a tertiary care hospital -an observational study characteristics and risk factors for fatality in 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detection tests with acute sera from patients infected with dengue virus in venezuela dengue viruses in papua new guinea: evidence of endemicity and phylogenetic variation, including the evolution of new genetic lineages analysis of genotype diversity and evolution of dengue virus serotype using complete genomes increasing airline travel may facilitate co-circulation of multiple dengue virus serotypes in asia publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank miss kaoru miyata, central research laboratory of kansai medical university, for her help with the dna sequencing. we also thank our partners in the solomon islands ministry of health and medical services (mhms), including the surveillance unit, the national vector borne disease control program (nvbcp), and participating hospitals and laboratories, for their input during the sample collection process. authors' contributions nt conceptualized the project, while the rest of the research team supported the planning and implementation of the study. dt, jc, and daw mobilized the field team and supported the sampling fieldwork and blood collection. ks, st, mn, and lp supervised the clinical and laboratory work and analyzed the results. daw drafted the manuscript, while all of the authors have reviewed the final draft and agreed to its publication. this research was partially funded by grant d from the kansai medical university. the funders had no role in the study design, data collection or analysis, the decision to publish, or the preparation of the manuscript. ethics approval and consent to participate the study protocol was reviewed, and ethical approval was obtained from the solomon island national health research committee (hrc / ) and kansai medical university research ethics committee, japan (no ). not applicable. the authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcomes.author details key: cord- - u mtp authors: lokida, dewi; lukman, nurhayati; salim, gustiani; butar-butar, deni pepy; kosasih, herman; wulan, wahyu nawang; naysilla, adhella menur; djajady, yuanita; sari, rizki amalia; arlinda, dona; lau, chuen-yen; karyana, muhammad title: diagnosis of covid- in a dengue-endemic area date: - - journal: am j trop med hyg doi: . /ajtmh. - sha: doc_id: cord_uid: u mtp emergence of sars-cov- in dengue virus (denv)–endemic areas complicates the diagnosis of both infections. covid- cases may be misdiagnosed as dengue, particularly when relying on denv igm, which can remain positive months after infection. to estimate the extent of this problem, we evaluated sera from confirmed covid- patients for evidence of denv infection. no cases of sars-cov- and denv coinfection were identified. however, recent denv infection, indicated by the presence of denv igm and/or high level of igg antibodies, was found in seven patients. dengue virus igm and/or high igg titer should not exclude covid- . sars-cov- reverse transcription polymerase chain reaction (rt-pcr) testing is appropriate when dengue nonstructural protein (ns ) or rt-pcr is negative. given the possibility of coinfection, testing for both denv and sars-cov- is merited in the setting of the current pandemic. emergence of sars-cov- in dengue virus (denv)endemic areas has raised concern regarding coinfection with the two viruses. , difficulty in distinguishing dengue and covid- , particularly during the acute stage, can engender inaccurate diagnoses. during the covid- pandemic, patients who screen positive on the sars-cov- questionnaire (supplemental table ) are tested for sars-cov- by rt-pcr. when confirmed, no further investigation for other etiologies is commonly performed. when sars-cov- is negative and clinical indication is present (at least fever and thrombocytopenia), denv ns antigen and/or igm/igg antibody testing may be performed. clinicians from singapore reported two covid- cases that were misdiagnosed as dengue among patients who presented with clinical manifestations and hematology profiles, suggesting dengue infection and false-positive denv igm antibody using a rapid diagnostic test (rdt). this may have occurred because of persistence of denv igm from a prior denv infection. indonesia has experienced a surge in covid- cases against the backdrop of dengue endemicity. because the prevalence of denv igg antibodies in singapore is significantly lower than that in indonesia, , we expect indonesia to face greater challenges with diagnosing sars-cov- , typically performed by rt-pcr, while denv is co-circulating. to estimate the extent of this problem, we evaluated sera from confirmed covid- patients for evidence of denv infection. covid- cases were defined as inpatients who met the covid- criteria based on a predetermined combination of symptoms, laboratory testing, imaging, and risk exposure at tangerang district hospital, indonesia (see supplemental table ), and had a positive nasopharyngeal or oropharyngeal real-time rt-pcr for sars-cov- . blood and sera were collected from all suspected covid- patients for clinical and research testing. for this study, admission sera for all cases were evaluated for denv ns using rdt (panbio ® dengue early rapid, abbot, brisbane, australia) and elisa (dengue ns antigen dxselect™, focus diagnostics, cypress, ca) assays, and for denv igm and igg using rdt (panbio ® dengue duo cassette, abbot, sinnamon park, australia) and elisa (focus diagnostics) assays. if available, follow-up sera from . ± . days later were evaluated for denv igm and igg by the same rdt and elisa methods. admission sera from cases with positive denv igm were evaluated using rt-pcr. results were not returned in real time for patient care purposes. clinical and laboratory information on admission was obtained by chart review. descriptive statistics were performed to characterize the presentation of covid- among these cases and to assess denv infection status. this research was approved by the tangerang district hospital ethics committee. admission sera were available for covid- cases. follow-up sera were available for of these patients. the mean age was . (sd ± . ) years, with a male predominance ( . %). time from the onset of illness to serum collection was . days (sd ± . ) days. the most common signs and symptoms were fever ( . %); cough ( . %); fatigue, dyspnea, and dysgeusia ( . % each); sore throat ( . %); headache ( %); and anosmia and diarrhea ( . % each). lymphopenia (< , /mm ), leukopenia (< , / mm ), and thrombocytopenia (< , /mm ) during admission were found in . %, . %, and . %, respectively. a comparison of signs and symptoms for our patients with covid- and dengue patients from a recent fever study is shown in supplemental table . none of the subjects was positive for dengue ns or showed seroconversion or increasing denv igm and igg index values, suggesting no acute denv infection among these covid- cases. however, both denv igm and high igg titer, as indicated by positive igg rdt, were identified in three patients. dengue virus rt-pcr was negative in all cases. the detection cutoff of igg panbio rdt is set at a high titer (equivalent to a hemagglutination inhibition titer ³ , ), which is commonly found in recent secondary denv infection. thus, these cases were probably acute covid- cases with recent secondary denv infection. retrospective assessment of exposure history revealed that one patient could have been infected by sars-cov- during hospitalization for dengue a week prior at a different hospital. another patient reported that a clinician he visited before hospitalization suspected denv infection clinically, but the ns result was negative. the third patient did not recall having a fever before acute covid- illness, suggesting asymptomatic or mild dengue, the most common presentation of denv infection. in four patients, denv igm was detected but did not increase in follow-up samples, denv igg was only detected by elisa, and denv rt-pcr was negative, suggesting that denv infection occurred less recently than in the three patients described earlier. detection of igm is plausible as it may be detected until year postinfection. most patients ( , . %) only had denv igg antibodies, implying past denv infection. this is consistent with previous studies conducted in indonesia, which demonstrated that more than % of adults aged > years had been infected by denv. no evidence of denv infection was identified in two ( . %) patients. the distribution of dengue diagnostic results is shown in table . despite concurrent high incidence of covid- and dengue in indonesia, acute coinfection with denv was not detected in this cohort of patients identified to have covid- . this may be because of sars-cov- and denv testing practices, which focus on symptomatic cases. it is likely that coinfection is occurring but is often asymptomatic. it is also possible that some patients had already been infected with current circulating denv serotypes and thus had immunity, as indicated by high prevalence of patients with igg antibodies. identification of seven ( . %) covid- cases with denv igm in our cohort may be related to the occurrence of covid- during the yearly dengue season. this finding is concerning, particularly because clinicians frequently diagnose dengue based only on denv igm, which may persist for months after resolution of infection. our study demonstrates that adding ns to the diagnostic algorithm may reduce dengue overdiagnosis attributable to reliance on igm. missed diagnosis of acute covid- due to presumption of dengue can result in inadvertent omission of targeted precautions, which could lead to transmission to contacts, including family, colocated patients, and healthcare workers. a missed diagnosis could also delay the receipt of standard of care covid- treatment. missed diagnoses have been reported in singapore due to false-positive denv igm rdt results versus persistence of denv igm. in the setting of the current pandemic and in light of overlapping symptomatology, clinicians should test for both denv and sars-cov- . it is notable that one of the patients may have contracted sars-cov- during hospitalization a week prior. in resourcelimited settings, adequate infection control practices are difficult to implement. hence, nosocomial infection should be considered in the setting of recent contact with the healthcare system, including due to dengue. sars-cov- infection control strategies for resource-limited settings are needed. findings from our study should be interpreted with caution. the study population was small and from only one hospital at tangerang district, during march and april . therefore, results have limited generalizability as they reflect the epidemiology of covid- and dengue in that area during the study period. furthermore, as the assays were qualitative (rdt) or semi-quantitative (elisa), increasing antibody titer was only measured by the index value, which may be inaccurate. to reduce inaccuracy, we tested acute and followup specimens simultaneously. in conclusion, our study reaffirms challenges associated with diagnosing covid- in areas hyperendemic for tropical infections with overlapping presentations such as dengue. the known potential for repeat dengue infections and the possibility for repeat sars-cov- infections add further complication. when molecular diagnostic testing for denv is not available, we recommend the use of a validated ns and igm/igg rdt. addition of ns will improve the specificity of identifying acute dengue cases. detection of denv igm and/ or high igg titer should not be considered an exclusion of covid- . past infection with denv with acute covid- or even acute denv and sars-cov- coinfection would remain possibilities. hence, evaluation for covid- should be conducted when dengue ns or rt-pcr (when available) is negative. e-mails: unurhayati@ina-respond.net, gsalim@ ina-respond.net, dpepy@ina-respond.net, hkosasih@ina-respond.net, wwahyunawang@gmail.co, amenur@ina-respond.net, yuanita_djajady@ yahoo.com, and rasari@ina-respond.net. dona arlinda and muhammad karyana a novel coronavirus from patients with pneumonia in china coinfection between dengue and covid- : need for approach in endemic zones covert covid- and false-positive dengue serology in singapore force of infection and true infection rate of dengue in singapore: implications for dengue control and management dengue viral infection in indonesia: epidemiology, diagnostic challenges, and mutations from an observational cohort study clinical and laboratory diagnosis of dengue virus infection the epidemiology, virology and clinical findings of dengue virus infections in a cohort of indonesian adults in western java prolonged persistence of igm against dengue virus detected by commonly used commercial assays accuracy of dengue clinical diagnosis with and without ns antigen rapid test: comparison between human and bayesian network model decision covid- and dengue co-infection in a returning traveller this is an open-access article distributed under the terms of the creative commons attribution (cc-by) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord- - vim wno authors: zandi, keivan; teoh, boon-teong; sam, sing-sin; wong, pooi-fong; mustafa, mohd rais; abubakar, sazaly title: antiviral activity of four types of bioflavonoid against dengue virus type- date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: vim wno background: dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. in the present study, antiviral activity of four types of bioflavonoid against dengue virus type - (denv- ) in vero cell was evaluated. anti-dengue activity of these compounds was determined at different stages of denv- infection and replication cycle. denv replication was measured by foci forming unit reduction assay (ffura) and quantitative rt-pcr. selectivity index value (si) was determined as the ratio of cytotoxic concentration (cc( )) to inhibitory concentration (ic( )) for each compound. results: the half maximal inhibitory concentration (ic( )) of quercetin against dengue virus was . μg ml(- )when it was used after virus adsorption to the cells. the ic( )decreased to . μg ml(- )when the cells were treated continuously for h before virus infection and up to days post-infection. the si values for quercetin were . and . μg ml(- ), respectively, the highest compared to all bioflavonoids studied. naringin only exhibited anti-adsorption effects against denv- with ic( )= . μg ml(- )and its related si was . . daidzein showed a weak anti-dengue activity with ic( )= . μg ml(- )when the denv- infected cells were treated after virus adsorption. the si value for this compound was . . hesperetin did not exhibit any antiviral activity against denv- . the findings obtained from foci forming unit reduction assay (ffura) were corroborated by findings of the qrt-pcr assays. quercetin and daidzein ( μg ml(- )) reduced denv- rna levels by % and %, respectively. there was no significant inhibition of denv- rna levels with naringin and hesperetin. conclusion: results from the study suggest that only quercetin demonstrated significant anti-denv- inhibitory activities. other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of denv- virus replication. these findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. this group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication. dengue virus (denv) is a member of the genus flavivirus of the flaviviridae family. it is a significant human pathogen which causes a wide spectrum of clinical illnesses ranging from a silent or mild febrile infection, self-limited dengue fever (df) to the severe dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). there are four dengue virus genotypes, denv- , denv- , denv- and denv- which are transmitted to humans mainly by two species of mosquitoes, aedes agypti and aedes albopictus [ ] . all four denv can cause dengue. to date there are no effective vaccine or antiviral treatment for dengue. dengue patients are usually supportively-treated until they recover without any specific treatment measures. several studies have shown that the level of viremia correlates with the severity of disease with high viremia often seen in severe dengue. hence, antivirals that can reduce the level of viremia or the viremic phase could possibly reduce the severity of dengue. plants and plant's derived compounds remain an important source for the discovery and the development of new antiviral drugs because of their expected low side effects and their high accessibility in the nature [ ] [ ] [ ] . there have been numerous reports on the antiviral activity of various phytochemicals against dengue viruses and these include various flavonoids [ ] [ ] [ ] [ ] . flavonoids are basically low molecular weight phenolic compounds found widely in different kinds of plants. different types of flavonoids can be found in fruits, roots, nuts, seeds, bark, steams and flowers of plants. these include quercetin which can be found in some foods and fruits such as green and black tea, apple, onion, citrus, tomato and some other plants [ , ] . antiviral activities of various other flavonoids have also been reported against some viruses including human cytomegalovirus (hcmv), hsv- , hsv- and some types of human adenoviruses [ ] [ ] [ ] . in the present study, we are interested to examine the anti-dengue virus properties of quercetin, hesperetin, naringin and daidzein. hesperetin is a flavonone and its glycoside form, hesperidin is water soluble and it could be found in various citrus fruits. after ingestion of hesperidin, its sugar moiety is released from the backbone of this compound and the aglycone form known as hesperetin can be released. in vitro antiviral activities of hesperetin have been reported against some rna viruses [ ] [ ] [ ] . naringin on the other hand, is a flavonone glycoside found abundantly in grapefruit juice. antiviral activity of naringin were reported against hsv- and hsv- but this finding remains controversial [ , ] . daidzein is an isoflavone found in soybeans and its antiviral activity against influenza viruses has been reported [ ] . currently, there is no published data on the possible anti-dengue virus activities of quercetin, hesperetin, naringin and daidzein. therefore, in this study we evaluated these compounds activities on denv- (ngc strain) replication in cell culture system. the effects of each compound were evaluated against different stages of dengue virus replication including virus adsorption, intracellular replication and direct virucidal activities. four different types of bioflavonoid, quercetin, naringin, hesperetin (sigma-aldrich, st. louis, mo, usa) and daidzein (indofine chemical co. inc., hillsborough, nj, usa) were evaluated for their potential activities against dengue virus replication. dimethyl sulfoxide (dmso) (sigma-aldrich, st. louis, mo, usa) was used to dissolve the lyophilized form of compounds and prepared stock solutions ( mg ml - ) were stored at - c. stock solution was diluted using cell culture medium and sterilized by a syringe filter with . μm pore size (millipore, ma, usa) right before each experiment. c / mosquito cell line derived from aedes albopictus and vero (african green monkey kidney) cell line were used in this study. both cell lines were maintained and propagated in eagle's minimum essential medium (emem) (gibco, ny, usa) containing % fetal bovine serum (gibco, ny, usa). cultured c / and vero cells were incubated at c and c, respectively in % co humidified chamber. at the time of virus propagation, serum concentration was reduced to %. dengue virus type- (denv- ) new guinea c strain (ngc) was propagated using c / cell line and harvested after cpe presentation on day seven post-infection. after titration, viral stock was stored at - c. cell lines and virus were provided by virology laboratory of the tropical infectious disease research and education center, faculty of medicine, university of malaya (kuala lumpur, malaysia). mtt assay was performed following the manufacturer's instructions (promega, wi, usa). briefly, confluent vero cells in -well cell culture microplates were treated with different concentrations of each compound in triplicate. the treated cells were incubated for four days at c followed by the addition of μl of mtt solution to each well. the microplate was incubated at c for h. then, μl of the solubilisation/stopping solution was added to each well. the optical density (od) of wells was measured at using -well plate reader (tecan, mannendorf, switzerland). dose-response curves were plotted using graph pad prism (graph pad software inc., san diego, ca). in order to determine the prophylactic anti-dengue activity of compounds, different concentrations of compounds were added to the vero monolayer cells in triplicate at different times, h before virus infection. after h of pre-infection treatment, the cells were washed twice with sterile pbs and then ffu of denv- was inoculated to the cells and incubated at c for h. to determine the effects of continuous treatment, different concentrations of each compound were added to the vero cells, h pre-infection and continuously for days post-infection. in a separate experiment, antiviral activity of compounds against intracellular replication of denv- was performed by inoculation of ffu of virus to each well in triplicate. after adsorption of virus to the cells for h at c, the cells were washed with pbs to eliminate the unabsorbed viruses. then, different concentrations of each compound were added to the cells, followed by days of incubation at c. denv rna was then quantified using quantitative rt-pcr. in another experiment, vero cells at % confluency were infected with ffu of denv- in the presence or absence of different concentrations of each compound. the microplate was kept at c for h for virus adsorption. then the cells were washed two times by sterile pbs and incubated at c for four days. direct virucidal effects of the bioflavonoids were investigated by incubating denv- suspension containing ffu with an equal volume of the different concentrations of each compound for h at c. then, vero cells were infected with the treated viral suspension in triplicate. after h adsorption at c, cells were washed twice with pbs in order to remove unabsorbed viruses. then the microplate was incubated at c for days. antiviral activities of the tested compounds were evaluated by measuring the reduction in number of viral foci. briefly, confluent monolayers of vero cells were prepared in wells cell culture microplate. then the infected cells treated using different procedures described above were overlaid with . % of carboxy methyl cellulose (cmc) (sigma-aldrich, st. louis, mo, usa) containing emem. viral foci were visualized using peroxidase-based foci staining assay four days post infection [ ] . the numbers of denv- foci were counted using a stereomicroscope and the titer of virus was expressed as foci-forming-unit (ffu). the percentage of viral foci reduction (rf %) compared with controls was calculated as follows: rf (%) = (c-t) × /c. where, c is the mean of the number of foci for negative control well (without compound) and t is the mean of the number of foci in treated wells. reduction in the number of viral foci was further verified using quantitative rt-pcr (qrt-pcr). quantitative rt-pcr was performed to determine the effects of bioflavonoids on denv replication by quantifying denv- genomic rna copies based on a method described previously with some modifications [ ] . briefly, intracellular and extracellular denv- rnas were harvested from the denv-infected vero cells. viral rna was extracted using two types of rna extraction kits (qiaamp viral rna mini kit and rneasy mini kit) (qiagen, hilden, germany). quantitative rt-pcr assay was performed by adding μl of extracted denv rna to the sensimix sybr green reagent (quantace, watford, united kingdom) which contained . μl ddh o, μl x sensimix one-step, . μl x sybr green solution, units of rnase inhibitor, pmol of forward (dnf) and also reverse (d r) primers [ ] . all samples were assayed in triplicate. the amplifications were performed using the dna engine opticon system (mj research/bio-rad, hercules, ca) with the following thermal conditions: reverse transcription at °c for min, initial denaturation at °c for min, followed by cycles of °c for sec, °c for sec and °c for sec. melting curve analysis was subsequently performed at temperature from °c to °c to verify the assay specificity. for absolute quantitation of the viral rna, a standard curve was established with a serially diluted rna extracted from denv- stock with known titer. graph pad prism for windows, version (graph pad software inc., san diego, ca, ) was used to determine the cytotoxic concentration (cc ) and inhibitory concentration (ic ) values of bioflavonoids. selectivity index value (si) was determined as the ratio of cc to ic for each compound. mtt assay was used to determine cytotoxicity of each bioflavonoid on vero cells and the cc value of each compound was calculated (table and figure ). vero cells were treated by bioflavonoids for days which was the same duration used for antiviral activity assay. results showed that hesperetin with cc = . ± . μg ml - is the most cytotoxic compound for vero cells compared to the other tested compounds. quercetin and daidzein showed lower toxicity against vero cells at cc . ± . and . ± . μg ml - , respectively. meanwhile, naringin with cc = . ± . μg ml - showed the least cytotoxic effects against vero cells. cells treated with vehicle control, % dmso did not show any cytotoxicity against vero cells. results of vero cells pre-treatment with the compounds showed that μg ml - of quercetin could decrease the number of denv- foci by % ± . when compared to the non-treated cells. however, there was no prophylactic activity against denv- from other compounds (data not shown). in post-adsorption assay, quercetin exhibited the most significant antiviral activity against denv- amongst the bioflavonoids tested with ic = . μg ml - (figure a) . the si value for quercetin in post-adsorption assay was relatively high at . . it was also demonstrated that the level of denv- specific rna production in the presence of μg ml - of quercetin decreased by more than % ± when compared to the non-treated infected cells (figure b ). daidzein showed very weak anti-dengue activity with ic = . μg ml - when the infected cells were treated after denv- adsorption (figure a ). its related si value was . . the levels of denv- rna production in the presence of μg ml - of daidzein decreased by only . % ± . when compared to the non-treated infected cells (figure b ). naringin and hesperetin did not exhibit any anti-dengue activity when they were used after adsorption of denv- to the vero cells ( figure ). although there was no significant direct virucidal activity against denv- by quercetin, continuous treatment of cells from h before virus infection up to days post-infection exhibited anti-dengue activity with ic = . μg ml - (figure a) . the si value for continuous treatment with quercetin was . and higher than the si value ( . ) for post-adsorption assay. in addition, the level denv- rna production decreased by more than . % ± . when vero cells were treated with μg ml - of quercetin, h before virus infection and up to days post infection ( figure b ). there was no significant change in the antiviral activity of daidzein when cells were treated continuously from h before virus infection up to days post infection comparing to its anti-dengue activity for postadsorption treatment (figure ). no significant antiviral activity for naringin and hesperetin was observed for the continuous treatment against denv- ( figure ). however, naringin exhibited anti-adsorption activity when it was added to the cells at the same time of virus adsorption. the ic value for naringin was . μg ml - and its related si was . ( figure a ). there was a reduction of . % ± . in denv- rna production in the presence of μg ml - of naringin (figure a) . the majority of the viral foci in cells treated with μg/ml quercetin appeared smaller, less intensely stained and more diffused within the focus (figure b) , compared to the larger, well-defined and more intensely stained foci of the untreated cells (figure a ). this observation is consistent with the reduction of the percentage of foci and rna copy number. results from the direct virucidal activity evaluation of each compound showed that there was no extracellular inhibitory activity against denv- for all the tested figure anti-adsorption activity of flavonoids against denv- . flavonoids were added directly to virus inocula for h at c. the inocula were used to infect vero cell monolayers in wells cell culture microplates. the reduction in foci forming unit was calculated relative to the controls maintained in parallel (a) and the respective denv- rna copy numbers were quantified using qrt-pcr (b). data from triplicate experiments were plotted using graph pad prism version (graph pad software inc., san diego, ca). the reduction of intracellular replication of dengue virus by . % and % following treatment with μm of glabranine and -o-methyle glabranine, respectively [ ] . similarly, other synthetic flavonoid derivatives also showed antiviral activity in hepg cells [ ] . whereas, pinostrobin was reported to inhibit denv- ns b/ns protease an enzyme important in dengue virus replication in an in vitro study [ ] . these suggest that flavonoids as a group could consist of select compounds that possesses inhibitory activities against denv. to investigate which of the many flavonoids could affect denv infection, in the present study, we examined the potential effects of quercetin, naringin, hesperetin and daidzein on dengue virus infection of vero cells. unlike the previous studies which evaluated antiviral activity of flavonoids only after adsorption of virus to the cells [ , ] , the present study evaluated antiviral activity by different treatment procedures tailored to determine prophylactic, post adsorption, continuous treatment and direct virucidal activities of quercetin, naringin, daidzein and hesperetin. our findings demonstrated that quercetin was the only compound among all tested flavonoids that consistently showed significant antiviral activity against denv- in vero cells. selectivity indices for quercetin when the infected cells were treated or when uninfected cells were treated continuously h before infection until days post-infection were . and . , respectively. the noted differences between si values for quercetin could be due to the intracellular accumulation of quercetin during continuous treatment. a weak effect for prophylactic activity of quercetin however, was also observed. these findings suggest that the main anti-dengue activity of quercetin is likely due to its activity against the different stages of intracellular replication of denv- instead of early stages of its replication cycle such as virus attachment or entry. although, no direct virucidal activity or anti-attachment activity of quercetin was observed in the present study, antiviral activity of quercetin against human cytomegalovirus was reported with ic = . μm [ ] . quercetin was also reported to be effective against herpes viruses where it is more specific against hsv- with si = compared to hsv- with si = . [ ] . the mechanisms of how quercetin exerts its antiviral effects are not known. however, the effects of other flavonoids against cellular rna polymerases and formation of the complex with rna have been reported suggesting that quercetin could also affect the similar replication enzymes [ , ] . sylimarin, a flavonoid found effective against hepatitis c virus (hcv), another member of flaviviridae family [ ] , inhibits virus replication by inhibiting the activity of viral rna polymerase. in our study, results from the qrt-pcr supported the findings from the viral foci reduction assays that quercetin inhibits denv- replication and the significant reduction in the denv specific rna suggests that quercetin may also target the virus replication machinery, namely by inhibiting the rna polymerase. antiviral activity of naringin has been evaluated against few herpesviruses and rotavirus but their reported antiviral activities against hsv- and hsv- are inconclusive [ , ] . in addition it was reported that naringin did not exhibit any antiviral activity against another rna virus, sindbis virus [ ] . in the present study, the only anti-dengue activity of this flavonoid was demonstrated against adsorption and attachment of virus to the vero cells and based on its antiviral activity (ic = . μg ml - ) and its related selectivity index (si = . ), it may not be a good candidate for further development as anti-dengue drug. similarly, daidzein activity against denv- was not significant compared to quercetin (si = . ). continuous treatment of the infected vero cells from h before virus infection up to days post infection did not improve its anti-dengue activity significantly. this compound therefore, could not be a suitable candidate for further development as anti-dengue drug. hesperetin, the other flavonoid evaluated in our study, did not show no anti-dengue activity in any stages of virus infection and replication processes and this is despite the previously reported antiviral activity of hesperetin against sindbis virus [ ] . therefore, hesperetin is also not recommended for further investigations for anti-dengue drug development. in all our experiments, we showed that . % of dmso, the highest concentration of solvent used in the bioflavonoid treatment did not exhibit any antiviral activity against denv- and this eliminated any probable antiviral activity from dmso. findings from our study, suggest that there are select flavonoids including quercetin and fisetin, which are both flavonol, that exhibited significant denv replication inhibition properties [ ] . while the flavonoids in general share common basic molecular base structure, flavone ( -phenyl- , -benzopyrone), we showed here that the flavanone, hesperetin, and flavanone glycoside, naringin, showed no significant anti-denv replication activities. in addition, we had earlier shown that naringenin [ ] , another flavanone metabolized from naringin and here, daidzein, an isoflavone also had no significant denv replication inhibition properties. while quercetin was shown here to be effective in inhibiting denv replication, its glycoside form, rutin (quercetin- -o-rutinoside) showed no significant inhibition properties [ ] . these suggest that while flavonol could be the basic molecule that possesses anti denv replication properties, specific structural properties of the different flavonol derivatives would have different effects on the efficacy of the compounds against dengue. the demonstration in vitro that flavonols including quercetin and fisetin possess anti denv replication properties does not necessarily translate into immediate use of these compounds as antivirals against denv. further studies will be needed to demonstrate the antiviral activities of these compounds against different genotypes of dengue virus and in appropriate animal model. there is also a need to address the issue of the low bioavailability of quercetin especially for therapeutic use [ ] [ ] [ ] . several strategies to increase the bioavailability of quercetin that include using lipids and emulsifiers, co-crystalization of quercetin or using ester-based precursors have been investigated [ ] [ ] [ ] . the other topic of research would be combination drug study. at its current calculated ic values, the antiviral efficacy of quercetin can be further improved possibly by combining it with other potential anti-dengue compounds. this is exemplified in a study that reported the synergistic effect of αglucoside in combination with a standard antiviral drug, ribavirin is effective against dengue infection [ ] . in conclusion, the present study demonstrates that the bioflavonoid quercetin exhibited significant anti denv replication properties. we further showed that quercetin affects intracellular denv virus replication but not the denv attachment and entry processes. these results together with the earlier findings reporting the anti denv properties of fisetin, suggest that these 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quercetin in rats cocrystals of quercetin with improved solubility and oral bioavailability ester-based precursors to increase the bioavailability of quercetin combination of α-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo antiviral activity of four types of bioflavonoid against dengue virus type- we thank the ministry of science, technology authors' contributions kz designed and carried out the antiviral and cytotoxicity studies and drafted the manuscript. btt carried out the virus propagation and antiviral studies. sss participated in the quantitative rt-pcr. wpf participated in the design of the study, performed statistical analyses and edited the manuscript. mrm participated in study design and provided all bioflavonoids. sab conceived the whole study and edited the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -md u va authors: shrivastava, gaurav; valenzuela leon, paola carolina; calvo, eric title: inflammasome fuels dengue severity date: - - journal: front cell infect microbiol doi: . /fcimb. . sha: doc_id: cord_uid: md u va dengue is an acute febrile disease triggered by dengue virus. dengue is the widespread and rapidly transmitted mosquito-borne viral disease of humans. diverse symptoms and diseases due to dengue virus (denv) infection ranges from dengue fever, dengue hemorrhagic fever (life-threatening) and dengue shock syndrome characterized by shock, endothelial dysfunction and vascular leakage. several studies have linked the severity of dengue with the induction of inflammasome. denv activates the nlrp -specific inflammasome in denv infected human patients, mice; specifically, mouse bone marrow derived macrophages (bmdms), dendritic cells, endothelial cells, human peripheral blood mononuclear cells (pbmcs), keratinocytes, monocyte-differentiated macrophages (thp- ), and platelets. dengue virus mediated inflammasome initiates the maturation of il- β and il- , which are critical for dengue pathology and inflammatory response. several studies have reported the molecular mechanism through which (host and viral factors) dengue induces inflammasome, unravels the possible mechanisms of denv pathogenesis and sets up the stage for the advancement of denv therapeutics. in this perspective article, we discuss the potential implications and our understanding of inflammasome mechanisms of dengue virus and highlight research areas that have potential to inhibit the pathogenesis of viral diseases, specifically for dengue. dengue fever is an acute febrile disease triggered by the dengue virus (denv). denv is positive sense, single stranded, rna virus, belongs to the flaviviridae family, genus flavivirus. aedes mosquitoes, aedes aegypti and occasionally aedes albopictus transmit denv to humans.the disease is caused by mainly four denv serotypes, denv - . denv imposes risk for more than . billion people with likely million infections per year of infection, majorly in tropical and subtropical countries (guha-sapir and schimmer, ; brady et al., ; bhatt et al., ) . constant circulation of different serotypes of the virus further leads to cross-reactivity and imposes added risk on successive dengue infections (secondary infection). denv infection leads to several clinical symptoms including dengue fever (df) that is asymptomatic mild flu, to coagulopathy, enhanced vascular fragility, and thrombocytopenia, well-known as dengue hemorrhagic fever (dhf) , that lead to hypovolemic shock called dengue shock syndrome (dss), a more severe condition (murphy and whitehead, ; afroz et al., ) . several factors contribute to denv pathogenesis such as substantial cytokine secretion ("cytokine storm"), host genetics, heterotypic secondary infection, and antibodydependent enhancement (ade) (pang et al., ; katzelnick et al., ) . although, dengue pathogenesis remains elusive, the cytokine storm has been considered to be one of the primary and crucial causative factors (hatch et al., ; rothman, ) . il- β, a very strong cytokine that is intensely regulated and stimulated by denv infected macrophages and monocytes, considered as an important component in cytokine storm (chang and shaio, ; dinarello, ; wu et al., b) . several evidences have demonstrated the elevated level of il- β in serum cytokine and gene expression profiles in severe dengue patients suggesting the contribution of il- β in the severity of dengue pathology (bozza et al., ; jaiyen et al., ) . il- β increases the vascular permeability, especially in concurrence with tnf-α and ifn-β in many severe dengue profiles (netea et al., ; dinarello, ) . as a part of its role as an endogen pyrogen (fever-causing), il- β stimulates lymphocytes and foster leukocytes infiltration to the inflammation site, thereby regulating local and systemic inflammation (dinarello, ) . two-step activation pathways are required in the production of il- β: "priming signal" fuels the induction of transcription and synthesis of pro-il- β, and subsequent "activation signal" assembles and activates inflammasome and caspase- (schroder and tschopp, ) . "inflammasomes" are multimeric protein complexes of innate immune system that form in the cytosol after detecting pamps (pathogen associated molecular patterns) or damps (damage associated molecular patterns). inflammasome protein complex comprises of a sensor proteins; nlr [nucleotide-binding domain (nbd) and leucine-rich-repeat-(lrr)-containing] family, aim like receptor, alr, an adaptor protein asc (apoptosis-associated speck-like protein containing a card), and an inactive zymogen, procaspase- (martinon et al., ; rathinam et al., ) . activation of inflammasomes in response to pamp and damp signals leads to autocleavage process, which in turn process pro-caspase- to active caspase- . activated caspase- then converts pro-il- β and pro-il- into their active forms (il- β and il- , respectively) (martinon et al., ) . inflammasome plays a vital function in the host innate immune system by regulating the secretion of proinflammatory cytokines (il- β and il- ) and subsequent induction of "pyroptosis, " a form of programmed cell death, activated by inflammatory caspases (fink and cookson, ; lamkanfi and dixit, ) . although a variety of pathogen senses the pamp and damps and activate diverse inflammasomes, study shows a range of virus infection that activates inflammasomes such as the nlrp , aim , and rig-i and mediate the robust host immune response . inflammasomes protect the host from the attack of microbial pathogens and endogenous danger signals, thereby playing a crucial role in host defense. irrespective of the location in cytosol, inflammasome creates an efficient immune response against extracellular and intra-cellular foreign invaders. although the optimum inflammasome activation is extremely favorable to the security of the host, dysregulation of inflammasome activation can leads to the worsening of symptoms in infectious diseases and results in the autoimmune and inflammatory disorders development (lamkanfi and dixit, ) . it has been shown that increased serum levels of il- β and il- correlates with the gravity of dengue infection (mustafa et al., ; bozza et al., ) . this finding suggests that inflammasome play a critical role in the pathogenesis of dengue infection. in this perspective, we will discuss the inflammasomes role during denv infection and the mechanism through which inflammasome imparts a central role in dengue severity. numerous studies have emphasized the significance of inflammasome activation in the regulation of virus infection. a range of upstream sensing receptors comprising the aim -like receptor, the nod-like receptor, and the rig-i-like receptor determine the inflammasome activation during viral infection. these receptors regulate inflammasome activation while present in different cellular compartments i.e., cytoplasm and the nucleus (schroder and tschopp, ) . among all the known inflammasomes, the nlrp inflammasome is the most significantly studied in viral infections and suggested to play a decisive role in both inflammation and antiviral responses (chen and ichinohe, ) . viral components as well as several stimuli by virus infection are sensed by host innate immune system and activate nlrp inflammasome. there are several stimuli which induce cytosolic damp signals, such as protein aggregates, mitochondria injury, and aberrant ion mobilization. two-step process is prerequisite to trigger the nlrp inflammasome. the priming step: tlr, nlr, or rig-i-like receptor families, ifnar or by a cytokine receptor activate the priming step that leads to transcriptional upregulation of nlrp , pro-caspase- , pro-il- β, and pro-il- . the activation step: signal for inflammasome activation is triggered in response to infection, tissue damage, metabolic imbalances or diverse stress signals coupled with host sterile cell/tissue damage. stress signal include pore-forming toxins, nucleic acids, ca ++ mobilization, k + efflux, atp lysosomal damage, crystalline substances and invading pathogens (lamkanfi and dixit, ; swanson et al., ) . several studies have demonstrated that nlrp senses the known activators indirectly due to their structural variability (bauernfeind et al., ; latz, ) . structurally, nlrp inflammasome is made of nlrp , asc (adaptor protein apoptosis-associated speck-like protein containing card) and procaspase- . structurally, nlrp has three components i.e., pyd-nod-lrrs in the order of its domains from n-terminus to c-terminus. during nlrp inflammasome activation, nod domain arranges the nlrp as a homo-oligomer and further links with asc through its pyd domain. asc binds with procaspase- through its card domain to form the complete structure. thus, nlrp inflammasome activation leads to the activation of caspase- and secretion of mature il- β and il- (latz, ) . subsequently, il- β potentially recruits neutrophils to the site of inflammation to assist in the removal of infecting viruses (niu et al., ) . furthermore, il- β and il- are also involved in the consequent initiation of the adaptive immune response joosten et al., ) . although the nlrp inflammasome provides the host antiviral status, irregular nlrp inflammasome activation leads to severity of pathological injury during virus infection. as an example, in an influenza a virus (iav) infection model, nlrp inflammasome activation in juvenile mice leads to severe lung injury independent of viral titer, while sustained elevated levels of type i ifns exist (coates et al., ) . iav infection also shows the high mortality in il- ri − / − mice as compared to wild type mice (schmitz et al., ) . likewise, il- − / − shows mortality with increased viral load as compared to wild type mice (liu et al., ) . in herpes simplex virus (hsv- ) infection, increased viral load was observed in il- β − / − mice as compared to wild-type mice with decreased immune response (sergerie et al., ) . in other studies, il- administration prior to hsv- infection protect the hsv- -infected mice and increase survival (fujioka et al., ) , revealing the immune control mechanism of il- β and il- against the hsv- infection. on the other hand, il- β and il- protect the host against hsv- -induced encephalitis. in addition, nlrp inflammasome mediates the chronic intrahepatic inflammation and liver injury during hcv infection (negash et al., ) . inflammasome leads to immunopathology progress and recruits excessive inflammatory cells that leads to pyroptosis mediated cell death (mcauley et al., ; haque et al., ) . numerous studies have established the activation of nlrp inflammasome by viruses with in vivo relevance for control of virus infection (chen and ichinohe, ; zhao and zhao, ) . (lupfer et al., ; shrivastava et al., ; zhao and zhao, ) . thus, the outcomes of nlrp inflammasome activation are very much virus specific (in vivo and in clinical settings) and deliver substantial defense against some pathogenic viruses however can also play a detrimental role in immunopathology of others. dengue infected patients shows a plethora of clinical manifestations including sudden-onset fever, hepatosplenomegaly, headache, muscle and joint pain (setiawan et al., ; wu et al., ) . endogen pyrogens (ep) mediate the fever onset which suggests that denv may trigger high amounts of endogenous pyrogens (e.g., il- β and tnf-α) and lead to high fever onset in the patients. studies have demonstrated that macrophages (mφ) serve as major target cells for denv infection, replication and the major source of inflammatory cytokines (chen and wang, ; jessie et al., ; balsitis et al., ) . mφ are heterogeneous in nature and during denv infection, m-mφ (resting macrophages) and gm-mφ (inflammatory macrophages) display distinctive cytokines expression profiling and innate immunity receptors/sensors. high expression of clec a, mannose receptor, and nlrp were found in gm-mφ as compare to m-mφ, supporting the hypothesis that these two subsets display distinct functions (wu et al., a) . in the continuation, a striking study has suggested that the functions of human macrophage subsets and corresponding signaling events are instrumental in the production of il- β and il- . this study demonstrated that clec a serves as a myeloid prr for denv, and denv replicates more competently in gm-mφ (inflammatory macrophages) than in m-mφ (resting macrophages) (wu et al., b) . denv infection activates the nlrp inflammasome in gm-mφ that results in caspase- activation and subsequent il- β and il- release. interestingly, denv infection in gm-mφ induces the increased expression of nlrp without affecting other sensors i.e., nlrc and nlrp , while sirna targeted to nlrp obstructs denv stimulated il- β and il- secretion. additionally, denv infection in gm-mφ leads to increased level of tnf-α with decreased level of il- . moreover, released cytokines increase body temperature and stimulate the release of soluble factors that result in vascular permeability during dengue infection (wu et al., b) . these results suggest the important function of clec a in dengue severity as another study also shows that clec a is critical for dhf and dss (chen et al., ) . the study also indicated the crucial role that clec a plays in nlrp activation, as blockade of clec a blocks the nlrp inflammasome activation, and subsequent ablated denv induced il- β production and caspase- mediated proptosis in gm-mφ (wu et al., b) . apart from facilitating innate immune response, il- β and il- display a crucial function in fostering adaptive immune response during denv infection. il- β secretion further enhances the production and release of il- and il- and the association of il- , il- β, and il- stimulate th /γδ t cells to generate pro-inflammatory cytokines (gm-csf, il- a, il- f, il- ,) that create the stage for host adaptive immune responses during denv infection (wu et al., a) . thus, the macrophage is also considered as the primary target of denv since denv induced thrombocytopenia releases il- via the activation of nlrp inflammasome. epidemiologic study also supports this notion as the severity of df/dhf were correlated with elevated level of gm-csf in the plasma of denv patients (bozza et al., ) . this study indicates the probable switch mechanism of resting macrophages to inflammatory macrophages by gm-csf during denv infection, that affects the gravity of clinical outcomes. this warrants further investigation as the inhibition of clec a may diminish the severity of dengue induced clinical symptoms in patients. notably, a study demonstrated the importance of immunosuppressive status based on different age group that influences the differential induction of il- β by monocytes during denv infection (valero et al., ) . monocytes/macrophages from neonatal, adult, and elderly was infected with all four denv types and cytokine profile was analyzed. the study observed the highest cytokine production (tnf-α, il- , and il- β) by adult monocytes as compare to neonates and elderly subjects depicting the presence of immunosuppressive condition at both sides of life and may be due to different physio pathological mechanisms (valero et al., ) . denv also targets, human dendritic cells (dcs), a proficient antigen-presenting cell with immune sensors that mounts the immune response in the peripheral blood and in the lymphoid (ho et al., ) and trigger ifn signaling (hsu et al., ) . it has been reported that tlr in endolysosomes in dc senses "self-dna, " binds to it and triggers signaling events, including nf-κb and mitogen-activated protein kinase (mapk) p signaling pathways (sasai et al., ) . mitochondria plays a crucial role in tlr mediated pathways as damps released from mitochondria during tissue injury, stress conditions, and cytokine production due to trauma, trigger inflammatory responses through activation of the tlr signaling pathway (zhang et al., ; caielli et al., ; lood et al., ) . mtdna that serves as a damp, engages several pattern-recognition receptors present on diverse cell types and initiates potent inducement of the proinflammatory immune response and ifn production (west and shadel, ) . another study demonstrated that denv infects dc and induces nlrp inflammasome activation, triggering ros production in mitochondria that leads to the discharge of mitochondrial dna (mtdna) into the cytosol. mtdna further triggers the production of interferons (ifns) by activating tlr signaling pathways (lai et al., ) . these incidents results in the productions of several anti-viral cytokines ifns, such as ifn-λ , ifn-λ , ifn-λ . inhibition of inflammasome diminished the release of mtdna in the cytosol during denv infection subsequent tlr activation. these data support the crucial roles of denv-induced inflammasome activation and further denv induced mtdna-tlr axis mediated ifn production and their following incidents. during denv infection, increased il- β also correlates with thrombocytopenia (bozza et al., ) . a study reports that denv infects the platelets via dc-sign receptor that further leads to platelets activation, mitochondrial dysfunction and activation of the apoptosis caspase cascade, that leads to the development of thrombocytopenia in patients with dengue (bozza et al., ) . another study has tested the hypothesis, whether platelets activation by dengue induce the synthesis and processing of il- β, that can be capable of disrupting the endothelial cell barrier function. denv infection activate the nlrp inflammasome, and subsequent activation of caspase- , and caspase- dependent secretion of il- β in platelets microparticles from patients with dengue as well as when platelets get exposed to denv in vitro. denv induced inflammasome activation and subsequent platelet released il- β-rich microparticles associated with increased vascular permeability and this increase were found to be il- β dependent. in addition, rip /rip -mediated mitochondrial ros generation are required for nlrp inflammasome activation and subsequent release of mature il- β cytokine (p ) and the shedding of il- β mps, indicating the role of rip /rip mediated mitochondrial ros generation in dengue vasculopathy (hottz et al., ) . a new mechanism has been demonstrated regarding the role of platelets in dhf. the study demonstrated that denv activates mouse and human platelets via the clec- receptor, thereby triggering the release of extracellular vesicles (evs), including microvesicles (mvs) and exosomes (exos). further, exos (dv-exos) and mvs (dv-mvs) upregulate the clec a and tlr on neutrophils and macrophages, resulting in the formation of a neutrophil extracellular trap (nets) and subsequent release of proinflammatory cytokine. in vivo mouse model, denv induced nets formation and inflammasome activation was impaired in clec a −/− and tlr −/− mouse neutrophils. furthermore, denv triggered systemic vascular permeability and lethality were reduced dramatically. further, in vivo study demonstrated the role of nets in complex interface among macrophages, platelets, and neutrophils during denv infection as dnase i mediated exclusion of nets provided certain defensive effect in stat −/− mice ( % survival). furthermore, dnase i has no effect in the survival of denv-infected stat −/− clec a −/− mice, suggesting the nets formation induced pathogenic effect in denv infection by increasing systemic vascular permeability (sung et al., ) . nets induce the interruption of vascular endothelial cell layer and vascular leakage in dengue infection, due to the presence of metalloproteinases and histones in the nets (saffarzadeh et al., ; carmona-rivera et al., ) . nets formation outcome varies greatly in virus to virus infections and specific mechanism for nets formation and the factors that decide the functions of nets in viral infection are still elusive. specifically, while nets show their beneficial roles in preventing viral dissemination (murine pox virus) (jenne et al., ) and trapping (hiv) (saitoh et al., ) , formation of disproportionate nets turn in exacerbated allergic airway inflammation during rhinovirus (rv) infection (toussaint et al., ) and airway obstruction during respiratory syncytial virus (rsv) infection (cortjens et al., ) . another in vivo study also demonstrated the neutrophil activation and neutrophil extracellular trap (nets) formation during an acute denv infection. in vitro incubation of nets results in reduced denv infectivity. notably, nets components were more predominantly present in the serum patients with hemorrhagic fever (dhf), but not acute phase of the infection (opasawatchai et al., ) . other evidence that demonstrates the excessive inflammation during denv infection indicates that neutrophils activation and nets formation can contribute significantly in disease pathogenesis (costa et al., ) . this suggest that somehow nets are inhibited during acute phase, however, in the more severe phase, nets formation is induced, implying the dual roles of nets in denv infection. although, the nets formation directly by denv particle is still debatable due to different views from different studies. where, in a study, nets formation has shown to be found in healthy neutrophils during denv infection (yost et al., ) , another study suggests the inhibition of nets formation due to denv- (moreno-altamirano et al., ) . there could be a link of inflammasome and neutrophil activation and nets formation, as a study found the overexpression of cd b in neutrophils, a marker of granulocytes activation and increased ros production during denv infection. as ros production is directly linked to trigger inflammasome, the study observed the increased ros production in granulocytes, in response to ex vivo pma stimulation during acute dengue infection. this phenomenon is suggested due to tnfα and il- cytokines that present during acute denv infection. ros is instrumental in rosdependent nets formation, neutrophil antimicrobial activity and inflammasome activation (opasawatchai et al., ) . another independent study demonstrates the role of gsdmd (gasdermin d), a pore-forming protein and an initiator of pyroptosis with nets formation. it has been reported that in non-canonical inflammasomes activation, neutrophils trigger the release of nets and it was found to be gsdmd dependent. it is reported that neutrophils get several unknown stimuli that activate the protease that further cleaves gsdmd to implement netosis. furthermore, ros is required during classical netosis that activates serine proteases that too cleave gsdmd (chen et al., ) . further, gsdmd acts as a feed forward loop and includes protease activation and nuclear expansion . how these mechanisms exist during denv infection is not known and more research understanding regarding the neutrophil activation, nets formation, inflammasome activation and their interconnection functions are warranted. overall, this data unravels the central role of clec a/tlr and clec in "neutrophil-platelet interactions" that fuels the enhanced inflammatory reactions during denv infection (sung et al., ) . another study emphasizes the inflammasome role in mounting the immune response in dengue virus infection. it has demonstrated that primary human γδ t cells (freshly isolated) react promptly to denv infected dc (dendritic cells) and produces ifn-γ and upregulates cd a. this anti-denv ifnγ is controlled by denv infected dc that induces type ifn and il- (tsai et al., ) . to note, dengue infected patients plasma demonstrated the elevated levels of type i ifn and il- (mustafa et al., ; gandini et al., ) and these cytokines (type i ifn and il- ) by producing ifn-γ by γδ t display a physiologically phenomenon in fostering an effective anti-denv th adaptive-immune response and an important determinant of dengue disease severity. further, the inhibition of inflammasome activation (due to extracellular atp) weakened the ifn-γ response of γδ t cells. notably, monocytes serve as an accessory cells as it induces the il- that further triggers the γδ t cells during infected dc response to denv (tsai et al., ) . a wide range of cells shows the activation and expression of both cc and cxc chemokines due to monocytes/macrophages induces il- that augment neutrophil activity, ifn-γ production and natural killer (nk) cell cytotoxicity by nk cells . therefore, to gain the full anti denv activity of γδ t cells, two activation checkpoints are required: secretion of type i ifn through irf-dependent pathways (e.g., detection of viral rna by tlr and rig-like receptors) (loo et al., ; nasirudeen et al., ) and the recognition of released extracellular atp (due to cell damage) lead to activation of inflammasome (dinarello and fantuzzi, ) . monocytes activation and aberrant inflammasome activation have demonstrated a central role in the advancement of severe form of dengue disease that also serves as a biomarker of dengue patients. recently, a study investigated plasma biomarkers from dengue virus infected patient including chemokines, cytokines, along with additional inflammatory mediators that forecast the severity of dengue disease according to the who classification. this study identified il- , lbp (lps binding protein) and scd as a best predictive value, particularly at the febrile phase as their presence is significantly higher than any other marker tested (yong et al., ) . the phenomenon of microbial translocation (mt) has been described where lps translocate from gut into the blood stream under inflammatory conditions. microbial translocation play a vital role in hiv disease (brenchley et al., ) , inflammatory bowel syndrome (rojo et al., ) , chronic liver disease (pinzone et al., ) and even end stage kidney disease (wang et al., ) . more recently, mt has been reported among dengue patients (van de weg et al., ) and levels of lps were accompanied with progression of dengue disease as lps has been found to act along with denv in triggering il- , paf, and tnf-α (van de weg et al., ; kamaladasa et al., ) . in the context of lbp, lbp is a soluble acute phase protein that initiates immune responses by binding directly to bacterial lps and relocate it to monocyte/macrophages expressing scd and tlr on cell surface (opal et al., ) , as lps stimulation to monocyte and macrophages triggers scd secretion (landmann et al., ) . the study, therefore, advocates the central role of mt in intensifying the inflammatory response in dengue infection. as systemic immunity and non-canonical inflammasome is thought to be activated by lps, an increase of lps in plasma in dws+ (dengue with symptoms) and sd (severe disease) patients further supports the lps induced inflammatory responses. this indicates that apart from viral factors, elevation of lps in plasma also contributed its part to boost cytokine storm along with other danger-associated molecule patterns (damps). therefore, prediction of biomarkers early in dengue patients fosters proficiently patient triage and permit improved healthcare support for the population specially during dengue outbreaks. ade (antibody-dependent enhancement) has been proposed to be one of the most important reasons for cytokine storm. ade develops when preexisting antibodies from a primary (first) the inflammasome complex is formed and initiated by a secondary stimulus, sensed within the cytosol such as ade activation, clec a-dap- -syk mediated response, k + efflux, ros generation, mtdna release, cathepsin b release, ca ++ mobilization, denv viral proteins (e, m, ns a, ns b) mediated damps. activated nlrp inflammasome stimutates the auto-cleavage of pro-caspase- . activated caspase- further triggers the proteolytic process of pro-il- β, pro-il- into its active form as well as gasdermin d (gsdmd) that induces vascular leakage and cell death (pyroptosis). (c) denv induced il- β also stimulates the generation of il- and il- . il- β, il- , along with il- stimulates th /γδ t cells to release pro-inflammatory cytokines that mounts adaptive immune responses to denv infection. (d) denv activates platelets and induces the secretion of evs (dv-exos and dv-mvs), that results in the clec a and tlr mediated release of proinflammatory cytokines and nets formation in neutrophils contributing to vascular leakage during dengue infection. ns protein, through autocrine loop triggers tlr signaling on platelets and thereby amplifies the platelets mediated inflammasome response to denv infection. figure generated with biorender.com. denv infection, present in the body, bind to an infecting heterologous denv particle during re-infection. the antibodies from the primary infection do not have neutralizing capacity against the virus, rather they form ab-virus complex that binds to fcγ receptors (fcγr) on circulating monocytes. this results in the receptor mediated endocytosis of the ab-virus complex, further augmenting the dengue infection and resulting in dengue severity (whitehead et al., ; raj kumar patro et al., ) . during secondary dengue infection, antibody-dependent enhancement (ade) has been an anticipated mechanism in explaining dengue hemorrhagic fever (dhf; guzman and vazquez, ) and studies have observed higher il- β level in dhf patients as compared to df patients (cui et al., ; kamaladasa et al., ) . a study has demonstrated the ade triggered cytokines including il- β were studied in primary human monocytes utilizing the anti-denv mabs from patient. the data demonstrated that denv- infection in monocytes mediated by ade induces mature il- β secretion in h, independent of denv replication although it requires the stimulation of spleen tyrosine kinase (syk). pharmacological blockade of caspase- as well as genetic blockade of nlrp drastically ablated denv derived il- β secretion (callaway et al., ) . of note, weak nlrp inflammasome activation was observed in monocytes in this study as compared to inflammatory macrophages (wu et al., b) , and it may be due to differential expression of clec a in monocytes vs. inflammatory macrophages as later shows significantly very high expression of clec a (batliner et al., ) . denv immune complexes induced activation of syk can have wider effects as activation of syk also upregulates the tnf and il- expression. it is important to note that activating erk / by syk elevates il- β secretion, and inhibition of syk and erk / diminished the ade-triggered il- β release (callaway et al., ) . in addition to the role of syk activation during ade in denv infection, downstream to c-type lectin receptor activation of syk activation play a crucial role in activating inflammasome during fungal pathogens and helminths infection (gross et al., ; poeck and ruland, ; ritter et al., ) . in addition, malarial hemozoin upon being engulfed by phagocytes, induces syk mediated inflammasome activation (tiemi shio et al., ) . this suggest that syk could be a potential therapeutic focus to restrict the "pathogenic cytokine storm" of severe dengue. furthermore, a recent study has setup a secondary infection experiment to mimic ade to demonstrate the immune response of human monocytes to the denv infection. human individual monocytes were selected based on their past exposure to severe disease or non-severe dengue and further exposed to each four denv serotypes subsequent incubation with autologous serum. results exhibited the marked increased viral load, viral sensing gene expression (nlrp , rig- , ifn-β) and production of inflammatory cytokines, specifically il- β in the monocytes of individuals with past severe dengue when compared to monocytes of individuals with past nonsevere dengue advising the influence of initial innate immune responses and inflammasome components nlrp , il- β in disease outcome (kamaladasa et al., ) . although we have discussed above the role of inflammasome and dengue severity, a recent study demonstrated the mechanism regarding the relationship between augmented inflammatory response and damage of vascular barrier integrity. the study supports that inflammasome induced il- β plays a central role in tissue injury and vascular leakage. denv infection mediated il- β activation are found in infected patient blood samples, human peripheral blood mononuclear cells (pbmcs) and monocytedifferentiated macrophages (thp- ) as well in c bl/ mice and mouse bone marrow derived macrophages (bmdms). evidence demonstrates that il- β augments tissue injury and leakage in ifnar-/-c bl/ mice, although il- receptor antagonist (il- ra) protects from this injury (pan et al., b) . together, the collected results reveal the mechanism of denv pathogenesis and establish the contribution of il- β in denv-associated pathology and recommend the consideration of il- ra for effective therapeutics in denv patients' treatment. the summary of the discussed mechanisms is shown in figure . pyroptosis is a programmed cell death (inflammatory) that engages the inflammasomes activation and subsequent caspase- activation. caspase- facilitates the processing of pro-il- β, pro-il- into the mature il- β and il- forms that are released to outside of the cell (man and kanneganti, ) . caspase- further cleaves between c and n domains of gasdermin d, and the n-terminal domain further attaches to membrane lipids and forms pores that results in cell lysis that facilitates the influx of water molecules leading to cell swelling and subsequent rupture (pyroptosis). pyroptosis cell death involves the damage of cell membrane integrity and upregulation of the caspase- and an upsurge in il- β and il- production (zhao et al., ) . during dengue infection, apoptosis mediated cell death has been reported, however, evidence has also demonstrated the pyroptosis (inflammatory cell death) in monocytes. monocytes displayed the activation of caspase- and subsequent production of il- β accompanied with release of cellular content by h during denv- infection (tan and chu, ) . as we have discussed above, denv infection induces the inflammasome activation and subsequent cell death. cell death has been linked to the upregulation of clec a in inflammatory macrophages (wu et al., a) . of note, upregulation of clec a has been reported in murine monocytes after denv infection (cheng et al., ) , supporting the notion that clec a plays a crucial role in monocyte cell death by pyroptosis. another study demonstrated a unique mechanism where denv- infection to primary macrophages activates caspase- and caspase- , and subsequent release of il- β. further, loss of cell viability along with the release to the lactate dehydrogenase was observed in extracellular medium. this study demonstrated that caspase- activates the caspase- and that caspase- serves as upstream activator of caspase- as inhibitor of caspase- ablated both caspases activity and subsequent secretion of il- β (figure ) . this study shows the new role of caspase- in activation of inflammasome and pyroptosis during denv- infection (cheung et al., ) . this study shows that denv- infection in macrophage results in decreased viability and increased ldh, revealing cell damage and lysis; however, macrophages as beneficial or pathogenic role in cell death remains controversial. several viral proteins have displayed the strong influence on inflammasome activation and elicited the host immune response; as demonstrated in the influenza a virus m protein and encephalomyocarditis virus (emcv) b protein (ichinohe et al., ; ito et al., ) . the wide range of viral proteins with their modulating effects on inflammasome have previously been discussed in detail (lupfer et al., ; shrivastava et al., ) . in the context of denv, ediii is regarded as a highly immunogenic protein. in thp- , ediii activates nlrp inflammasome via nf-κb pathway resulting in caspase- activation and subsequent il- β and tnf-α secretion. increased ros production and potassium was suggested to be the mechanism behind ediii protein mediated il- β production and release (khan et al., ) . moreover, denv m protein that plays an important role in denv packaging and egress (junjhon et al., ; lin et al., ) , also displays a role in imparting host innate immune response. this study shows that denv m induces nlrp inflammasome and il- β release that further induces the endothelial permeability and vascular leakage in mice. more importantly, m protein stimulates tissue injuries in wild-type (wt) mouse tissues, however the effect of m protein was inhibited in nlrp knockout (nlrp − / − ) mouse tissues, indicating a vital role of m protein-stimulated vascular leakage. the effects of m protein in triggering inflammasome activation and il- β secretion and subsequent pathological effect is suggested due to the interaction of m protein with nlrp (pan et al., a) . however, the molecular mechanism through which m protein binds to nlrp is still elusive and warrants more investigation. ns is a non-structural protein of denv and plays a role in denv replication complex along with other nonstructural proteins (muller and young, ) . ns is specific in a particular way; this is the only non-structural protein secreted in extracellular milieu (thiemmeca et al., ) and increased ns in patient serum is considered as a marker of severe dengue (libraty et al., ) . furthermore, ns serves as viral pamp that triggers tlr on the surface of endothelial cells and leukocytes which leads to endothelial dysfunction as well as inflammation (beatty et al., ; puerta-guardo et al., ) . recently, ns translation and secretion has shown to complement denv induced inflammasome. the study shows that denv triggers abortive viral infection in platelets, where denv translate and replicate its genome without releasing the viral particle. during this infection process, ns augments the platelets response via autocrine loop to the denv infection by caspase- mediated secretion of il- β and translocating and releasing the stored factors. notably, ns doesn't trigger the direct release of il- β, however ns triggers the tlr on platelets and amplifies the platelets response to denv, indicating the complex interplay between host and viral factors that leads to the dengue severity (quirino-teixeira et al., ) . in addition, denv non-structural proteins, ns a and ns b have been reported to trigger nlrp inflammasome activation. denv ns b plays a role in replication, viral protease co-factor and in degradation of cgas (aguirre et al., ) . in addition, ns b as a protease complex (ns b/ns ) inhibits mitochondrial fusion by cleaving the mitochondrial fusion proteins i.e., mfn and mfn (yu et al., ) . denv ns a, is a hydrophobic transmembrane protein with a diverse role in the virus life cycle. ns a displays a regulatory role in replication, viral assembly, and viral release and most notably by hindering the jak-stat and interferon pathways (leung et al., ; xie et al., xie et al., , . moreover, additional findings have demonstrated that denv- ns b and denv- ns a as "viroporins" permeabilize host cell membranes shrivastava et al., ) . viroporins from several virus that can increase host cell membrane permeability, have shown to activate the nlrp inflammasome through various mechanism and thereby regulate antiviral innate immune responses (guo et al., ; farag et al., ) . this study initially demonstrated that denv- induces nlrp activation, caspase- activation and subsequent il- β secretion in endothelial cells (hmec- ). ns a and ns b have shown to be co-localized in the endoplasmic reticulum and mitochondria. furthermore, ns a and ns b were sufficient to trigger the activation of nlrp inflammasome, and il- β secretion through caspase- activation in endothelial cells. pharmacological inhibition of nlrp , caspase- and crispr knockout of asc in endothelial cells showed that ns a and ns b mediated inflammasome and il- β secretion was nlrp and caspase- specific. lastly, the study also suggested that ns a triggering nlrp inflammasome activation is due to ca ++ homeostasis and/or mitochondrial disruption /ros production (shrivastava et al., ) . in summary, these evidences show the dengue virus proteins induced mechanism in activating nlrp inflammasome. furthermore, this mechanistic insight with more detailed study would shed more light on denv pathogenesis understanding (figure ) . these studies may help in designing and developing new therapeutic candidates against dengue virus. denv infection comprises immune dysfunction that leads to leakage of vascular fluids and cytokine storm which are suggested as main contributors to the pathogenesis of denv, culminating in life-threatening hypovolemic shock (culshaw et al., ) . numerous studies have aimed to understand the role of inflammasome in pathogenesis of virus. inflammasome activation has been accepted as a crucial player in the outcome of denv infection. inflammasome induced by denv facilitates caspase- activation and synthesis and secretion of il- β and il- . thus, the discussed investigation founded a promising direct linkage between inflammasome activation and dengue severity, while considering the contribution of caspase- , il- and il- β, to denv-mediated patient pathology. particularly, il- β plays an indispensable role in the severe dengue complex immunopathological condition. therefore, a question arises, whether therapeutics targeting il- β may be useful in the treatment of denv associated diseases? inflammasome activation plays a crucial role in controlling numerous pathogens, and thus loss of il- β can lead to impaired immune defense. in clinic settings, drugs directed to inhibit nlrp or il- β are now being utilized for numerous autoimmune diseases, however not enough clinical research has supported the use of such treatments in viral infection. though, the precise mechanisms underlying denv induced damage sensing are still debatable, the existing evidence suggests that nlrp ligand directly senses the denv. the precise mechanism of sensing is still elusive and warrants more research. whether nlrp senses the denv directly or by some other means and if so, how? nlrp often acts in concurrence with other inflammasomes sensors such as aim and ifi , however, whether these ligands sense denv along with nlrp needs further examination. in the context of il- in dengue infection, whether il- confers protective or detrimental role is still unknown. although pyroptosis might be relevant for viral clearance of infected cells, research regarding the role of denv-induced pyroptosis is still in a primitive phase. more studies are warranted to understand denv-induced proptosis in different cell culture models as well as in clinical settings. mitochondria have been regarded as an important component in inflammasome activation either by ros generation, mtdna release, autophagy, or cell death (west et al., ; zhou et al., ) . moreover, it has also been demonstrated that mitochondria adapter protein (mavs) localizes the nlrp inflammasome on mitochondria membranes (subramanian et al., ) . in denv infection, mitochondria have also been demonstrated to play a crucial role in regulating host immunity either by ros generation or by mtdna release (discussed above in this review). does denv induced inflammasome occur on mitochondria membrane and if so, how? another question is whether denv viral factors interact directly with mitochondria components? therefore, more studies are required to unravel the link between denv infection and mitochondria as it can provide several insights to regulate the inflammatory response during infection. research should also focus on several host molecules that have been suggested to play a crucial role in regulating the dengue severity such as clec a, tlr , tlr , inhibitors of the tyrosine kinases syk and btk (discussed above in text). in addition, a deeper understanding of immune evasion molecules evolved by viruses that can inhibit the function of inflammasome will expect to uncover novel concepts and may eventually identify targets in the treatment and prevention of denv severity. notably, a better understanding of the equilibrium between detrimental vs. favorable inflammasome activation is also indispensable, as host activation of inflammasome is a "double-edged sword" to defense viral infection. as the systematic vision of the inflammasome rises, prospects to create new therapeutic interventions for patients with denv severity enhance proportionately. it is important to mention that although inflammasome activation does appear to contribute to dengue, other mechanisms such as an early antiviral response (ifn response), also plays a major contributor along with high levels of immunosuppressive cytokines during dengue virus pathogenesis. moreover, a different and very strong approach against arbovirus has also been considered by targeting the mosquito vector. denv virus enters the host along with mosquito saliva at the skin epidermis and dermis level during mosquito bite. to date, various findings have reported the capacity of ae. aegypti saliva that enable blood feeding, pathogen transmission and regulate host innate and adaptive immune responses through its several pharmacologically active proteins that saliva contains (pingen et al., (pingen et al., , wichit et al., ; manning et al., ) . for example, kda protein from ae. aegypti was found to prevent the expression of irf- , resulting in the inhibition of type i ifn production (surasombatpattana et al., ) . aegyptin protein from ae. aegypti, decreases denv titers in mice along with increased gm-csf, ifnγ, il- , and il- concentrations in serum as compared to solitary denv inoculated to mice, displaying the impacts of aegyptin in denv infectivity through activation of the immune response (mccracken et al., ) . more research with enhanced knowledge regarding the interactions between saliva proteins and primary host immune cells will help to identify the key cell populations/molecules/pathways controlling the infection efficiently. increased characterization and improved functional 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human macrophage subsets clec a is critical for dengue virus-induced inflammasome activation in human macrophages membrane topology and function of dengue virus ns a protein two distinct sets of ns a molecules are responsible for dengue virus rna synthesis and virion assembly aberrant monocyte responses predict and characterize dengue virus infection in individuals with severe disease neonatal net-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation dengue virus impairs mitochondrial fusion by cleaving mitofusins circulating mitochondrial damps cause inflammatory responses to injury nlrp inflammasome-a key player in antiviral responses inflammatory caspases: activation and cleavage of gasdermin-d in vitro and during pyroptosis a role for mitochondria in nlrp inflammasome activation gs wrote the manuscript. gs, ec, and pv wrote the final version of the manuscript and approved the submitted version. all authors contributed to the article and approved the submitted version. we thank mrs. brigit sullivan, nih library editing service, for manuscript editing assistance. 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.this work is authored by eric calvo, paola valenzuela leon and gaurav shrivastava on behalf of the u.s. government and, as regards drs. calvo, valenzuela leon and shrivastava and the u.s. government, is not subject to copyright protection in the united states. foreign and other copyrights may apply. 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(s) 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- - giv m authors: tsai, jih-jin; liu, wei-liang; lin, ping-chang; huang, bo-yi; tsai, ching-yi; lee, pei-yu alison; tsai, yun-long; chou, pin-hsing; chung, simon; liu, li-teh; chen, chun-hong title: a fully automated sample-to-answer pcr system for easy and sensitive detection of dengue virus in human serum and mosquitos date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: giv m background: the insulated isothermal pcr (iipcr) technology enables consistent pcr amplification and detection in a simple heating device. a pan-dengue virus (denv) rt-iipcr, targeting the ’ untranslated region, was validated previously on the semi-automated pockit combo system (involving separate devices for nucleic acid extraction and pcr amplification/detection) to offer performance comparable to a laboratory real-time pcr. working on the same technologies, a compact automated sample-in-answer-out system (pockit central nucleic acid analyser) has been available commercially for iipcr, minimizing human error risks and allowing easy molecular bio-detection near points of need. here, we evaluated the analytical and clinical performance of the pan-denv rt-iipcr on the fully automated system by comparison to those on the semi-automated system. methodology/principal findings: testing sera containing serial diluted denv- , - , - , or - cell culture stock, the pan-denv rt-iipcr system had similar % detection endpoints on the two systems; i.e. at , , and pfu/ml, respectively, on the fully automated system, and at , , and pfu/ml, respectively, on the semi-automated system. furthermore, both fully automated and semi-automated pcr system can detect all four denv serotypes in mosquitos. clinical performance of the reagent on the two systems was evaluated by testing human serum samples. both systems detected the same samples (ten denv- , - , - , and - positive each) and did not detect the other ; % agreement (κ = ) was found between the two systems. conclusions/significance: with performance comparable to a previously validated system, the fully-automated pcr system allows applications of the pan-denv reagent as a useful tool near points of need to facilitate easy, fast and effective detection of dengue virus and help mitigate versatile public health challenges in the control and management of dengue disease. a a a a a dengue virus (denv), a member of the genus flavivirus in the family flaviviridae, causes dengue in humans. dengue virus infection, one of the most important and mosquito-borne viral diseases, is considered a major public health problem in developing tropical and subtropical countries mainly in asia, south america, and the caribbean with major disease outbreaks and endemic every year [ ] . four serotypes of dengue virus (denv- , - , - , and - ) have been identified, with distinct genotypes within each serotype. multiple virus serotypes have been found co-circulating in the hyperendemic regions in southeast asia and pacific [ , ] . its single-stranded, positive-sense genomic rna contains '-and '-untranslated regions (utr) and a single open reading frame encoding for a single polyprotein that could be cleaved into three structural proteins (capsid [c] , premembrane/membrane [prm/m], and envelope [e] ) and seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ). denv is primarily transmitted by the mosquito species aedes aegypti present in tropical and sub-tropical regions, and less efficiently by a. albopictus [ ] , particularly during the viremic phase of the disease in which high levels of denv viremia are present in the blood stream. positive association between denv infection in mosquitoes and human has been found; human cases were reported at about one week after appearance of denv-positive a. aegypti [ , ] . a. aegypti can also pick up denv from people showing no symptoms or oligo symptom, resulting in silent transmission [ ] . therefore, for dengue disease control, it is important to extend denv surveillance in the human and mosquito populations to near points of care (poc)/point of needs (pon), generating early alert signals to prevent and/or control denv epidemics and reducing the spread of denv infection [ ] . denv causes different clinical signs in humans, from mild febrile illness (dengue fever, df) to life-threatening condition such as hemorrhagic fever/dengue shock syndrome (dhf/ dss) [ , ] . early, accurate and rapid diagnosis of dengue is critical for confirmation of clinically suspected cases to ensure timely management of severe dengue disease [ ] . a number of ns rapid diagnostic tests are commercially available to easily detect ns antigen, but they are not sensitive or specific enough [ ] [ ] [ ] . during the acute phase of the illness when viremia levels are high, the viral rna or soluble antigens can be easily detected [ ] . for rapid and accurate diagnosis of dengue virus in early phases of acute infections, detection of denv rna is recommended as the most sensitive and specific method to diagnose dengue in the acute phase of the illness by who [ ] . after the first developed nucleic acid detection test for dengue diagnosis over years ago [ ] , there have been a number of rt-pcr assays for denv detection and serotype identification with high sensitivity and specificity [ ] [ ] [ ] [ ] [ ] . combined with automated rna extraction protocols, rt-pcr can potentially facilitate fast and accurate diagnosis of dengue suspected cases with a single sample obtained during the patient's first visit to the clinician. however, the requirement of a pre-pcr nucleic acids extraction step, an expensive and not easy to use thermal cycler, and a highly trained technician make pcr technology not accessible in resource-limited regions where dengue is endemic [ , ] . molecular diagnosis tools for dengue is still much needed at or near poc/pon, particularly for developing countries with limited public health resources. for poc/pon dengue diagnosis, several isothermal amplification methods such as nucleic acid sequence-based amplification (nasba), thermophilic helicase-dependent amplification (thda), loop-mediated isothermal amplification (lamp), recombinase polymerase amplification (rpa), and insulated isothermal pcr (iipcr) have been developed and reported previously as potential field-deployable tools for the detection of different microorganisms [ , , [ ] [ ] [ ] [ ] . based on these technologies, various all-in-one molecular diagnostics have been made available to help minimizing risks of human errors and allowing easy molecular bio-detection at or near poc/pon in the last few years, but, to our knowledge, no reagents are available for denv testing on these platforms. the pan-denv reverse transcription-iipcr (rt-iipcr), previously validated to work with the filed-deployable semi-automated pcr system, pockit combo (genereach biotech, taichung, taiwan), is a potential poc/pon tool for the detection of denv. it offered great analytical and clinical performances for rapid detection of denv in human serum; its clinical performance was comparable to that of the reference multiplex rt-pcr assay in studies testing samples collected in sri lanka and taiwan [ , ] . the field-deployable system includes one automated na extraction device (taco mini automatic nucleic acid extraction system, taco mini; genereach) and one compact pcr device (pockit nucleic acid analyzer, pockit; genereach) in a durable suitcase for easy field deployment. the iipcr system is unique in its employment of a single heating source in a thermally baffled device to drive rayleigh-bernard convection in a capillary tube, allowing the three steps of pcr (denaturation, annealing, and extension) to occur sequentially and continuously [ , ] . since the first report of the assay in , many pcr/rt-iipcrs on the pockit system have been validated for sensitive and specific detection of various parasitic, bacterial, and viral pathogens in animals and humans, including plasmodium spp, salmonella spp, influenza a virus, middle east respiratory syndrome virus, zika virus and dengue virus [ , , [ ] [ ] [ ] [ ] . requiring several manual liquid transfer steps to assemble the pcr tests, the complexity of the semi-automated pcr system is still relatively high. a compact fully automated sample-toanswer system (pockit central nucleic acid analyser, pockit central) is commercially available recently, in which modules in the existing semi-automated system (automated na extraction and compact pockit pcr modules) were integrated with a liquid handling module. this device can be set up and run with minimal set-up time and steps to allow quick and easy detection of denv in human at settings such as a local hospital laboratory or airport and in mosquitos for surveillance and monitoring of denv. therefore, the new assay has potential to enable timely early diagnosis of denv infection, facilitating effective disease management and control particularly in regions of low medical resources. in addition, the system can facilitate monitoring of the mosquitos carrying denv in the field to provide timely information for local government to take immediate measures to facilitate effective local management and control of dengue. in this study, we evaluated the performance of the pan-denv rt-iipcr reagent on the pockit central for sensitive and specific detection of denv - serotypes in human serums and mosquitos. serum samples were collected from clinically suspected dengue patients for routine diagnosis using rt-pcr methods [ ] at the tropical medicine center, kaohsiung medical university hospital, kaohsiung, taiwan in . the use of retrospective clinical specimens in this study was approved by the kaohsiung medical university hospital institutional review board (kmuhirb-f(i)- ); waiver of informed consent was obtained. all collected samples were anonymized. stocks of four denv isolates, denv- (hawaii strain; . x pfu/ml), denv- (ngc strain; . x pfu/ml), denv- (dn a strain; x pfu/ml), and denv- (dn a strain; . x pfu/ml), were prepared in the mosquito c / cell line (a. albopictus). the virus was diluted in rpmi medium (gibco-life technologies, grand island, ny, usa) containing % fcs (gibco-life technologies) and added to the cell at a multiplicity of infection of . . after incubation at ˚c, % co for - days. viruses were harvested when cytopathic effect was observed. titers of denv stocks and human serum of dengue confirmed cases were determined by plaque assay. briefly, -fold serial dilutions of the denv stock were made in mem medium (gibco-life technologies) and added in duplicate to bhk- cells in -well plates (about × cells per well). mem medium was used in mock infection. adhesion was allowed at ˚c under % co for h before addition of ml overlay medium containing . % methylcellulose. cells were incubated further for - days until plaques became visually apparent by microscopy, fixed, and stained with crystal violet. plaques were counted manually and plaque forming units (pfu) per ml were determined with the plaque quantification program [ , ] . two viruses known to also cause febrile illness or skin rash illness were tested for analytical specificity. zika virus (mr , prvabc strain) were from the american type culture collection, manassas, va, usa. chikungunya virus (ck strain) was from the taiwan center of disease control, taipei, taiwan. a total of archived serum specimens ( denv positive, of denv- , - and - each; denv negative) were collected from clinically suspected dengue patients with informed consents at the tropical medicine center, kaohsiung medical university hospital, kaohsiung, taiwan, for routine diagnosis in . all samples were stored at - ˚c until nucleic acid extraction. due to the lack of denv- positive clinical samples in the region, denv- samples were prepared by spiking of the denv-negative human serum specimens with different concentrations of the denv- dn a stock ( . x pfu/ml). adult female mosquitoes, aged - days, were cold anesthetized and inoculated using a microcapillary needle that had been pulled to a point with needle puller. the serotype of dengue virus stocks were standardized to x pfu/ml, and . μl was injected into each mosquito (approximately pfu/mosquito). infected mosquitoes were maintained in cages at ± ˚c and % ± % relative humidity with a h/ h light-dark cycle and fed with % sucrose solution. a. aegypti mosquitoes (ugal strain) were injected individually with denv- (hawaii strain, pfu), denv- (ngc strain, pfu), denv- (dn a strain, pfu), or denv- (dn a strain, pfu) by micro-injection (nanoinjector) into the thoracic cavity. whole mosquitoes were homogenized for plaque forming assay after , , , , , and days post-infection [ ] . denv was found to be detectable ( - pfu/ml) in mosquitoes, and viral titers (infectivity) were maintained in the mosquitoes that had been cultured for weeks without significant attenuation. three infected mosquitoes were collected on day post infection, and frozen at − ˚c until further use. for nucleic acid extraction before pcr analysis, each mosquito was homogenized in μl pbs with a disposable grinder and centrifuged briefly. subsequently, μl of the upper aqueous sample were transferred into the first well of the preloaded extraction plate or cartridge. the pan-denv rt-iipcr reagent, targeting the ' utr sequence, was performed as described in the user manual (pockit dengue virus reagent set, genereach biotech). the semi-automated pockit combo system includes an automated taco mini for nucleic acid extraction and a pockit device for pcr detection. nucleic acid extraction on the taco mini was done using the taco preloaded dna/rna extraction kit (genereach biotech) according to the manufacturer's instructions. briefly, μl of the sample were added into the first well of the extraction plate before the automatic extraction steps. all nucleic acids were collected individually and placed at - ˚c until further use. subsequent pcr detection was done manually on a pockit device. first, μl of premix buffer was added to reconstitute each lyophilized dengue virus premix, followed by the addition of μl of test nucleic acid extract. a μl volume of the premix/sample mixture was transferred into an r-tube, which was sealed subsequently with a cap, spun briefly in a microcentrifuge (cubee, genereach biotech), and placed into a pockit device. the default program, including an rt step at ˚c for min and an iipcr step at ˚c for about min; qualitative results were shown on the display screen in less than one hour. to run the pan-denv rt-iipcr reagent (pockit dengue virus reagent set) on the fully automated sample-to-answer pockit central system, the premix tube of the reagent was placed into the designated well in the transfer cartridge of the pockit cartridge set (gen-ereach biotech). after μl of the clinical sample were added into well one of the extraction cartridge of the pockit cartridge set, and sample and reagent information were logged into the device, the cartridges were placed into the pockit central device accordingly, and the program was started. the nucleic acid extraction, iipcr amplification and detection steps were completed automatically in minutes. qualitative results were displayed on the screen at the end of the program. to compare the performance of two methods, inter-rater agreement was assessed with a x contingency table and determined by cohen's kappa statistic using commercial software spss . (spss inc., chicago, il). the values � . , . - . , . - . , . - . , . - . , and > . were interpreted as no, minimal, weak, moderate, strong, and almost perfect agreement, respectively [ ] . to test analytical sensitivity, -fold serial dilutions ( , , , , , . , and . pfu/ml) of the denv- , , , or isolates were made separately in pooled denv-negative human serum and subjected in triplicate to the pan-denv rt-iipcr test with the semi-automated pockit combo and automated pockit central systems in parallel. the % detection end points were at , , , and pfu/ml for denv- , - , - and - with the fully automated pcr system, respectively; while the end points were at , , , and pfu/ml for denv- , - , - and - with the semi-automated pcr system, respectively (table , s table) . the results indicated that performances of the pan-denv rt-iipcr on the two pcr systems were comparable. similar to the observation with the semi-automated pcr system, the pan-denv rt-iipcr with the automated pcr system detected the four denv serotypes (denv- , - , - , and - ) and did not react with the two zika virus and one chikungunya virus strains, indicating that the assay also had great specificity for denv rna on the fully automated system ( table ) . performance of the pan-denv rt-iipcr on the semi-automated pockit combo system (see materials and methods for detail) was previously validated to be equivalent to that of the cdc denv- - real-time rt-pcr [ ] . the clinical performance of the pan-denv rt-iipcr reagent on the fully automated pockit central system was evaluated by comparison with the performance on the semi-automated pcr system. for this purpose, clinical serum samples were tested from sample to results using the pan-denv rt-iipcr reagent on the fully automated and semi-automated pcr systems in parallel. a total of archived serum specimens ( denv positive, of denv- , - and - each; denv negative) collected from fully automated sample-to-answer pcr system for dengue virus detection clinically suspected dengue patients were tested. there were no severe dengue patients in the denv-positive group. the control cases were selected from age-and sex-matched dengue-suspected cases; dengue virus infections were later ruled out clinically and by laboratory tests. all clinical samples were collected within days from the day of illness onset. based on analysis from a previous study, the median age was years old (range - ) in the case group and years old (range - ) in the control group. the percentage of male subjects was . % in the case group and % in the control group. the median viral titers in different serotypes were . x pfu/ml (denv- ), . x pfu/ml (denv- ) and . x pfu/ ml (denv- ) (s table) . denv- samples were prepared by spiking of the denv-negative human serum specimens with different concentrations of the denv- dn a stock ( . x pfu/ml). the results showed that the pan-denv rt-iipcr detected positive samples and negative samples by both pcr systems (table , s table) . the overall agreement between the two systems was % (ci % , . - %; κ = . ), indicating the reagent can work with the fully automated pcr system to provide performance equivalent to that with the semi-automated pcr system for the detection of denv rna in human serum. denv titers can reach . ± . to . ± . pfu equivalents/ml in infected female a. aegypti after oral infection with denv [ ] . the titers are higher than the sensitivity of the pan-denv rt-iipcr/pockit central system (table ) . preliminary evaluation of the performance of the fully automated and semi-automated pcr systems was done using mosquitos collected on day post intra-thoracic infection with one of the four serotypes. the results shows that both pcr systems could detect denv serotype -, -, -, and rna in denv- table . analytical specificity of pan-denv rt-iipcr on the fully automated pockit central system comparison with the semi-automated pockit combo system. infected mosquitos (table ; each serotype tested in triplicate, s table) , suggesting both systems can be a useful tool for screening denv-infected mosquitos. testing with the pan-denv rt-iipcr, the analytical and clinical performance of the fully automatic pockit central system was comparable to those of the semi-automatic pocki combo system, which was validated previously to offer performances equivalent to the cdc denv - real-time rt-pcr for the detection of denv in human serum [ , , ] . validation with serum samples prepared from dengue-suspected patients demonstrated that the pan-denv rt-iipcr performed equally well on both the semi-automated and fully automated equipment ( % agreement; ci % , . ~ %; κ = . ; table ). moreover, the pan-denv rt-iipcr can work on both systems to detect the four denv serotypes in denvinfected mosquitos (table ) . timely on-site detection of denv in human and mosquito can potentially alert front-line health professionals to allow timely implementation of intervention strategies and help focusing efforts in targeted areas to help mitigate disease outbreaks [ , ] . performance of such tests near poc/pon could help shift health service more to local levels, achieve disease diagnosis at early stages, and reduce costs and turn-around time, improving health management quality in underreached communities. currently, commercially available ns immunological test products are rapid and do not require trained personnel to detect denv in both human and mosquitos [ ] . however, they are reported as less sensitive than elisa and may cross react with other flaviviruses [ ] [ ] [ ] . moreover, their sensitivities were relatively low on days and and after day postsymptomatic onset in human, compared to that seen with the rt-pcr methods [ ] . pcr testing after an ns positive results has been recommended for confirmation of denv infection, but the samples have to be shipped to a central laboratory currently. there are needs for sensitive and specific molecular diagnostic tests for dengue at poc/pon to allow early intervention for clinical management, surveillance, and outbreak investigations [ ] . the previously available pan-denv rt-iipcr/semi-automated pcr system enabled sensitive and specific molecular testing near poc/pon. in this study, we showed that the reagent also worked well on the fully automated pcr system, which can serve as an advanced simple tool at settings with limited human resources and infrastructure to set up a pcr laboratory. one major advantages of the fully automated pcr system is its simple protocol (fig ) ; users simply have to put ul serum into a preloaded extraction cartridge, place both the extraction and reagent cartridges into the device, and start the reaction through the user interface on the screen; the results are ready in min. compared to conventional pcr methods and to the semi-automated pcr system (see fig for procedures involved) , the fully automated pcr system offers much shorter set up time; its capacity to process reactions in one test run makes it suitable for applications at near-patient settings where the sample numbers are relatively small and a sophisticated testing procedure is not possible. moreover, the pan-denv rt-iipcr reagent is available in a lyophilized format to facilitate shipment at ambient temperatures and storage at - ˚c for at least years, greatly reducing shipping and storage fully automated sample-to-answer pcr system for dengue virus detection costs. hence, this system can facilitate relatively inexpensive, rapid, and simple poc/pon detection of denv in routine dengue diagnosis. moreover, the fully automated and semi-automated pcr systems allows applications of the pan-denv rt-iipcr to detect denv in the local mosquito populations even at remote regions; the combined results can help decision making to effectively allocate control and prevention efforts into areas most in need of dengue control. therefore, the fully automated pockit central device and field-deployable semi-automated pockit combo system has potential to serve as a flexible mobile pon tool for rapid denv detection in both human and mosquito. studies to verify and validate further the performance of the pan-denv rt-iipcr reagents on both pcr systems for denv detection in mosquitos are underway. in summary, the pan-denv rt-iipcr coupled with the fully automated and semi-automated pockit platforms can serve as a rapid, accurate, and effective tool for use as a poc/ pon test system for early differential diagnosis of denv infection in human especially in local clinics, laboratories and hospitals, or for surveillance of denv in mosquitos. in the semi-automatic system, users add sample (serum, homogenized mosquitos) into the preloaded extraction plate (a) and place the plate into the nucleic acid extraction device for automatic nucleic acid extraction (b). the resulted nucleic acid extracts are added manually to the lyophilized reagent reconstituted first with provided buffer; the final pcr mixture is transferred manually to the reaction vessel (c). the reaction vessels are placed into the pcd device, which performs pcr amplification, detection and data interpretation automatically to provide qualitative results on the monitor (d). in the fully automatic system, users add serum sample to the preloaded extraction cartridge, place the extraction and reagent/consumable cartridges into the device, key in reagent and sample information, and start the assay (e). the device automates nucleic acid extraction, pcr reagent preparation, and pcr amplification, detection, and data interpretation to provide qualitative results on the monitor (f). table. sample info and test results of mosquito samples with pan-denv rt-iipcr on fully automated pockit central system and semi-automated pockit combo system. 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competence of potential dengue vectors aedes aegypti and aedes albopictus in the transmission of dengue virus serotype in southern taiwan interrater reliability: the kappa statistic. biochemia medica: biochemia medica a new paradigm for aedes spp. surveillance using gravid ovipositing sticky trap and ns antigen test kit detection of dengue virus ns antigen in infected aedes aegypti using a commercially available kit. the american journal of tropical medicine and hygiene comparison of nonstructural protein- antigen detection by rapid and enzyme-linked immunosorbent assay test and its correlation with polymerase chain reaction for early diagnosis of dengue key: cord- -ded t ai authors: tomashek, kay m.; lorenzi, olga d.; andújar-pérez, doris a.; torres-velásquez, brenda c.; hunsperger, elizabeth a.; munoz-jordan, jorge luis; perez-padilla, janice; rivera, aidsa; gonzalez-zeno, gladys e.; sharp, tyler m.; galloway, renee l.; glass elrod, mindy; mathis, demetrius l.; oberste, m. steven; nix, w. allan; henderson, elizabeth; mcquiston, jennifer; singleton, joseph; kato, cecilia; garcía gubern, carlos; santiago-rivera, william; cruz-correa, jesús; muns-sosa, robert; ortiz-rivera, juan d.; jiménez, gerson; galarza, ivonne e.; horiuchi, kalanthe; margolis, harold s.; alvarado, luisa i. title: clinical and epidemiologic characteristics of dengue and other etiologic agents among patients with acute febrile illness, puerto rico, – date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: ded t ai identifying etiologies of acute febrile illnesses (afi) is challenging due to non-specific presentation and limited availability of diagnostics. prospective afi studies provide a methodology to describe the syndrome by age and etiology, findings that can be used to develop case definitions and multiplexed diagnostics to optimize management. we conducted a -year prospective afi study in puerto rico. patients with fever ≤ days were offered enrollment, and clinical data and specimens were collected at enrollment and upon discharge or follow-up. blood and oro-nasopharyngeal specimens were tested by rt-pcr and immunodiagnostic methods for infection with dengue viruses (denv) – , chikungunya virus (chikv), influenza a and b viruses (flu a/b), other respiratory viruses (orv), enterovirus, leptospira spp., and burkholderia pseudomallei. clinical presentation and laboratory findings of participants infected with denv were compared to those infected with chikv, flu a/b, and orv. clinical predictors of laboratory-positive dengue compared to all other afi etiologies were determined by age and day post-illness onset (dpo) at presentation. of , participants enrolled from may , through may , , more than half ( . %, , ) had a pathogen detected. pathogens most frequently detected were chikv ( , , . %), flu a/b ( , , . %), denv – ( , . %), and orv ( , . %). participants with denv infection presented later and a higher proportion were hospitalized than those with other diagnoses ( . % versus . % with orv, . % with flu a/b, and . % with chikv). predictors of dengue in participants presenting < dpo included leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness, while negative predictors were irritability and rhinorrhea. predictors of dengue in participants presenting – dpo were leukopenia, thrombocytopenia, facial/neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea; presence of rhinorrhea, cough, and red conjunctiva predicted non-dengue afi. by enrolling febrile patients at clinical presentation, we identified unbiased predictors of laboratory-positive dengue as compared to other common causes of afi. these findings can be used to assist in early identification of dengue patients, as well as direct anticipatory guidance and timely initiation of correct clinical management. a a a a a as malaria incidence continues to decrease throughout the tropics, a new area of research has focused on identifying etiologies of non-malaria, acute febrile illness (afi) [ , ] . knowledge is limited in this area, in large part because afis often have similar non-specific clinical presentations early in the clinical course when most patients are likely to present for care. in addition, rapid point-of-care diagnostics are often not readily available. surveillance for afis, if done, is largely passive and relies on clinical identification of cases and voluntary case reporting. therefore, burden of disease for the etiologic agents of afi are likely underestimated. an improved understanding of the major causes of afi is important to guide clinical management, develop diagnostics, inform public health policy, and direct prevention efforts [ ] . in mexico, south and central america, and the caribbean, afi are common among patients of all age groups. in the last four decades, dengue, a mosquito-borne afi caused by four genetically distinct dengue viruses (denv - ), has become an increasingly common cause of afi [ , ] . the burden of dengue is thought to be less in latin america than in southeast asia [ ] ; however, several studies have found that the incidence of dengue is likely underestimated in latin america due to reliance on passive case surveillance [ ] [ ] [ ] . understanding region-specific etiologies of afi and estimating the true incidence of dengue is necessary to plan large scale interventional trials for assessing the impact of mosquito control measures and vaccines. in addition, collecting clinical signs and symptoms from afi patients of all ages with identified etiologic agents has utility in developing unbiased case definitions and identifying early clinical predictors to guide clinical management. prospective studies enrolling patients with afi provide a methodology to systematically identify causes of afi in a population and describe variation in the clinical course by patient age and etiologic agents. since , nine such studies have evaluated afis including dengue among both pediatric and adult patients [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while these studies are comparable in many ways, the studies differ in that several excluded either infants and young children [ ] [ ] [ ] [ ] [ ] ] , older adults [ , ] , severe or hospitalized cases [ , ] , or cases with a known source of fever [ ] [ ] [ ] [ ] ] . in addition, most studies were conducted in low resource settings in southeast asia where malaria is still endemic [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in fact, two studies enrolled based on a potential participant meeting national eligibility criteria for malaria testing [ , ] , and two other studies excluded cases based on malaria blood smear positivity [ , ] . in this manuscript, we describe a -year prospective study of afi among all age groups that used a pre-defined diagnostic testing algorithm for denv - and other pathogens. we conducted this study in puerto rico, where malaria was eradicated in [ ] and dengue has been endemic since the late s [ ] . we describe the frequency of dengue and other afis, and the distribution of these diseases in terms of person, place and seasonality. last, we describe clinical predictors of dengue by timing of presentation compared to other afis. the study was conducted in southern puerto rico at saint luke's episcopal hospital (sleh)-ponce, a -inpatient bed, tertiary care teaching hospital during may , -may , ; and sleh-guayama, a -inpatient bed hospital during february , -may , . sleh-ponce is one of four hospitals serving , residents of ponce and neighboring municipalities [ ] . sleh-ponce has about , emergency department (ed) visits and , inpatient admissions annually. sleh-guayama is one of two hospitals that serve , residents of guayama and three adjacent municipalities. sleh-guayama has , ed visits and , inpatient admissions annually. patients presenting to the ed or as a direct hospital admission were eligible for enrollment if fever was present (defined by a body temperature of ! . ˚c [oral] or ! . ˚c [axillary]) or they reported a history of fever of -day duration. after informed consent was administered, vital signs, signs and symptoms of current afi, history of exposures and chronic disease, and clinical laboratory results were recorded on an enrollment case report form (crf). a physician examined the participants and recorded the clinical diagnosis on the crf. participants discharged to home after enrollment were asked to return - days post-illness onset (dpo). at the follow-up visit, a second completed crf included a report of any healthcare services received and signs and symptoms experienced since enrollment. participants admitted to the hospital upon enrollment had their hospital course summarized on a standardized data collection form that included treatment received, results of clinical laboratory and radiologic investigations, and disease manifestations. prior to enrollment, informed consent was administered in accordance with puerto rico law (article , section , regulation of the office of patient ombudsman, act # ). specifically, written informed consent was obtained from eligible adults > years old and emancipated minors - years old. written informed assent was obtained from non-emancipated minors - years old and written informed consent was obtained from the parents or guardians. verbal informed assent was obtained from children - years old and written informed consent was obtained from the parents or guardian, and the parents or guardian of children < years old. the institutional review boards at the centers for disease control and prevention (cdc) and ponce health sciences university approved the study protocol. all study participants had blood ( ml in edta, ml whole blood), urine ( ml), nasopharyngeal (np), and oropharyngeal (op) specimens collected at enrollment. convalescent blood ( ml in edta, ml whole blood) and urine ( ml) were collected at the follow-up visit or hospital discharge. np and op specimens were placed in a vial containing viral transport medium. serum, blood, and urine specimens and inoculated vials were kept at ˚c until transported to cdc dengue branch (cdc-db) in san juan, puerto rico. molecular diagnostic testing for denv - , influenza a and b viruses (flu a/b), and other respiratory viruses (orv) including adenovirus (adv), human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), parainfluenza virus - (piv- - ), human rhinovirus (hrv), and four human coronaviruses (hcov) ( e, oc , nl and hku ), was performed at cdc-db. however, testing for hrv, piv- , piv- , and the four hcov was discontinued after the first year because of low yield (i.e., only piv- , hcov and hcov co-infections identified). in brief, rna was extracted from np and op specimens and tested for orv and flu a/b viral genome by real time, reverse transcriptase-polymerase chain reaction assay (rrt-pcr) [ ] . serum specimens collected dpo were tested by denv-serotype specific rrt-pcr [ , ] , and those collected ! dpo were tested by an antibodycapture enzyme-linked immunosorbent assay (mac-elisa) (inbios international, inc., seattle, wa) [ ] [ ] [ ] . beginning in may , specimens collected dpo were tested by chikv-specific real-time rt-pcr [ ], and those collected ! dpo were tested by anti-chikv mac-elisa [ ] . remaining serum, whole blood, and urine were stored at - ˚c until shipped to cdc in atlanta, georgia. at cdc, serum specimens collected dpo were tested in the picornavirus laboratory by a pan-enterovirus real-time rt-pcr assay that targets the vp region [ ]; positive specimens were sequenced. paired serum specimens from enrollment and the follow-up visit or hospital discharge were tested for leptospira spp., and burkholderia pseudomallei at the bacterial special pathogens branch laboratory. specimens were tested by microscopic agglutination test (mat) for leptospira reference antigens representing serogroups [ ]. all convalescent serum specimens were tested for presence of burkholderia pseudomallei and leptospira antibodies by an indirect hemagglutination assay (iha) [ ] and mat respectively, and acute specimens were tested in cases for which the corresponding convalescent specimen was positive. the first patients with leptospira spp. and burkholderia pseudomallei negative specimens and for which paired specimens were available were tested by ifa for rickettsia spp., ehrlichia spp., and coxiella spp. at the rickettsial zoonoses branch laboratory. whole blood and/or acute serum from cases with a reactive ifa were assessed for c. burnetii, r. rickettsii, r. typhi, and/or e. chaffeensis dna by pcr. a laboratory-positive dengue case had denv nucleic acid or anti-denv igm detected in a single specimen. a laboratory-negative dengue case had no anti-denv igm detected in serum collected ! dpo. a laboratory-positive influenza case was defined by presence of flu a/b nucleic acid in a np or op specimen. laboratory-positive hmpv, hrsv, adeno, piv- , piv- , piv- , piv- , hrv, and hcov cases had the respective viral nucleic acid present in a np or op specimen. a laboratory-positive leptospirosis case was defined by ! -fold increase in mat titers in paired specimens, or mat titer ! in a single specimen. a laboratory-positive melioidosis case was defined by presence of burkholderia pseudomallei nucleic acid in a clinical specimen and/or a ! -fold rise in iha titer in paired specimens. a laboratory-positive enteroviral case was defined by presence of enterovirus nucleic acid in serum collected dpo. a laboratory-positive ehrlichiosis case was defined by presence of ehrlichia chaffeensis igg reciprocal titer > : by ifa, a ! -fold rise in igg titer in paired serum specimens, or a positive pcr on an acute whole blood or serum specimen. a laboratory-positive rickettsia case was defined by presence of r. rickettsii or r. typhi igg titer > : by ifa, a ! -fold rise in igg titer in paired serum specimens, or a positive pcr in a whole blood or serum specimen. a laboratory-positive coxiella case was defined by presence of c. burnetii igg titer > : by ifa, a ! -fold rise in igg titer in paired serum specimens, or positive pcr on an acute whole blood or serum specimen. leukopenia was defined as a white blood cell count , cells/μl. thrombocytopenia was defined as a platelet count , /μl. severe hemoconcentration was defined by a hematocrit ! % above the u.s. population mean hematocrit for age and sex, and moderate hemoconcentration was defined by a hematocrit > . th percentile for age and sex to less than the cut-off for severe hemoconcentration [ ] . a skin bleed was defined by presence of skin bruising and/or petechiae. mucosal bleeds included epistaxis, gingival bleed, hematemesis, melena, hematochezia, menorrhagia, or hematuria (> red blood cells per high powered field) in a male or non-menstruating female. frequencies were calculated for demographic characteristics and medical history by study year. clinical and laboratory features were compared by sex, age group, and laboratory diagnostic groups including infection with denv, flu a/b, orv, and chikv. differences in proportions were tested by applying the chi-square test, and medians were compared using the mann-whitney-wilcox test. bonferroni correction was used to account for simultaneous multiple comparisons. the woolf test was performed to evaluate the homogeneity of odds ratio across dpo group for death among adult participants by sex, and the mantel-haenzel test was used to determine significance. multiple imputation was used to predict an independent plausible value for missing values using generalized linear regression on non-missing variables to create imputed complete data sets [ ] . to identify predictors of laboratory-positive dengue as compared to all other afi cases, stepwise akaike information criterion (aic) variable selection was used for each imputed data set. variables retained at least once in the models were included in a pooled logistic regression model [ ] . odds ratios (or) and % confidence intervals (ci) were calculated for significant early (< dpo) and late ( ) ( ) ( ) predictors. data were analyzed using the "mi" and "mass" packages from r software (v . . , r foundation for statistical computing, vienna, austria). during the study, sites recorded , ed visits of which , ( . %) patients had fever or reported fever (fig ) . enrollment was offered to , afi case-patients, and , ( . %) gave their consent/assent to participate. however, , ( . %) of those enrolled withdrew from the study or were withdrawn due to noncompliance with study enrollment procedures. of the remaining , participants, , ( . %) had follow-up forms completed and . % had follow-up specimens collected. half ( . %) of the , participants were female, and the median age was . years (range - ) ( table ). one-third ( . %) of participants reported having a chronic medical condition; a higher proportion of participants enrolled in the first year reported a co-morbidity than those enrolled in subsequent years. the most common co-morbid conditions were asthma ( . %), high blood pressure ( . %), diabetes ( . %), high cholesterol ( . %), coronary heart disease ( . %), and thyroid disease ( . %). participants resided in of the municipalities in puerto rico; however, most ( . %) were residents of five municipalities: ponce ( . %), guayama ( . %), juana díaz ( . %), salinas ( . %), and villalba ( . %). most ( . %) participants were enrolled < dpo (median dpo at enrollment = , range: - days) ( table ). the timing of presentation did not differ by sex but did differ by age, with a higher proportion of child participants (i.e., < years old) presenting < dpo than adult participants ( . % child vs. . % adult females, p < . ; and . % child vs. . % adult males, p < . ). one quarter ( . %) of participants were admitted to the hospital at enrollment. adult participants were less likely to be admitted than child participants; a higher proportion of female adult participants than male adult participants were sent home after enrollment ( . % vs. . % respectively, p < . ). however, a higher proportion of male versus female adult participants died after enrollment ( . % vs. . % respectively), in fact, adult males were five times more likely to die than adult females when adjusting by dpo (or = . , prospective study of acute febrile illness in puerto rico ci: . - . ). there were no statistical significant differences between female and male participants < years old in terms of the timing of presentation and disposition. the most common signs and symptoms (aside from fever) at enrollment were tiredness/lethargy ( . %), anorexia ( . %), chills ( . %), headache ( . %), muscle, bone or back pain ( . %), cough ( . %), red conjunctiva ( . %), rhinorrhea ( . %), nausea ( . %), and joint pain ( . %). slightly more than half ( . %, , ) of the , participants had a pathogen detected ( fig ) . chikv was detected in , ( . %) participants and was the most common pathogen the distribution of pathogens causing afi varied by age (fig ) . the proportion of chikungunya cases increased with age, accounting for . % of all afi cases in participants < years old versus . % in participants ! years old. in contrast, the contribution of orv to afi cases decreased with age, making up . % of afi cases in participants < years old, . % in participants - years old, . % in participants - years old, and . % in participants ! years old. dengue was the most common cause of afi in participants - years old, accounting for . % of all cases; . % of afi cases in participants < years old were dengue, . % in participants - years old, and . % in participants ! years old. the contribution of influenza was similar among age groups making up . % of afi cases in participants < years old, . % in - years old, . % in - years old, and . % in ! year-old participants. analysis of the temporal disease trends demonstrated that a dengue epidemic occurred in and continued through , during which a total of dengue cases were detected (fig ) . in comparison, few (n = ) dengue cases were detected in to the end of the study period in . the first chikungunya case was detected in may of , and was followed by a six-month outbreak during which , cases were detected. few (n = ) chikungunya cases were detected in . a large bimodal influenza epidemic took place in with increased case detection in the dry months of january-april (n = ), and during the rainy season, july-october (n = ). fewer influenza cases (n = ) were detected in and , and those detected occurred primarily in dry months with no obvious bimodal distribution. an increase in afi cases due to orv was detected at the same time influenza cases were detected, with the exception of when the peak time of orv case detection appeared to follow that of influenza. prospective study of acute febrile illness in puerto rico subject demographics at enrollment differed by subsequent laboratory diagnosis (table ) . a lower proportion of participants with dengue and orv illness were females when compared with participants with chikungunya. participants with orv illness were significantly younger (median age = . years, p < . ) than participants with dengue ( . years), chikungunya ( . years), or influenza ( . years). in contrast, the median age of participants with chikungunya was significantly greater than participants in all other diagnostic groups, and they were more likely to report having a chronic medical condition. a higher proportion of participants with dengue reported having a household member with dengue at enrollment than participants with other diagnoses ( . % of dengue cases versus % in other diagnostic groups, p < . ). over half of all participants reported having mosquito bites in the days before enrollment; however, a higher proportion of participants with chikungunya reported mosquito bites than participants with other laboratory diagnoses ( . % versus < % in other diagnostic groups, p< . ). clinical presentation and disposition varied by laboratory diagnostic group (table ) . participants with laboratory-positive dengue presented later (median = days), and a higher proportion were admitted at enrollment than participants with other laboratory diagnoses; nearly half ( . %) of dengue cases were admitted compared with . % of participants with orv illness, . % with influenza, and . % with chikungunya. a significantly higher proportion of participants with dengue had chills, signs of poor circulation, eye pain, headache, dizziness, anorexia, nausea, abdominal pain, and diarrhea at enrollment than participants with influenza, orv illness or chikungunya. a higher proportion of participants with dengue versus these other diagnoses had thrombocytopenia and leukopenia. compared to influenza and orv illness cases, a significantly higher proportion of dengue cases had a skin rash, pruritic skin, any bleeding, a skin bleed, and muscle, bone, back, and joint pain, whereas a higher proportion of chikungunya versus dengue cases had these findings. a higher proportion of dengue versus influenza and orv illness cases had facial and/or neck erythema and mucosal bleeding. in contrast, a significantly higher proportion of participants with influenza and orv illness than dengue had cough, rhinorrhea, and sore throat. among , participants who presented early (< dpo) in the clinical course, leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness were significant positive predictors of laboratory-positive dengue as compared to all other afi cases across all age groups ( table ). presence of rhinorrhea and irritability predicted non-dengue afi. age group had a statistically significant effect on multiple predictors (table ) . rash was a positive early predictor of dengue among participants < years old, and being male was a positive predictor among adults - years old. chills and cough were positive predictors for those > years old while cough was a negative predictor among those < years old. muscle, bone or back pain was a negative predictor in those > years old. pruritic skin as a predictor varied by age group, but most significantly between the < and + year-old groups. among the , participants who presented - dpo, thrombocytopenia, leukopenia, facial and/or neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea were significant positive predictors of dengue across all age groups ( table ) . presence of rhinorrhea, red conjunctiva and cough predicted non-dengue afi. again, age group significantly affected multiple predictors (table ) . abdominal pain was a positive predictor for participants - years old. red and/or swollen joints was a positive predictor among participants < years old but a predictor of non-dengue afi among participants ! years old. leukopenia was a prospective study of acute febrile illness in puerto rico significant positive predictor across all age groups, but to varying degrees. chills; muscle, bone, back and joint pain; and any bleeding as predictors varied depending on the age group. as a clinical syndrome, afis are a diagnostic challenge for clinicians especially early in the clinical course when anticipatory guidance and supportive care may pre-empt medical complications. our study identified the afi etiology in over half ( %) of participants and most were infected with one of nine viral pathogens. this detection frequency was higher than that of other recent prospective afi studies that tested for multiple pathogens ( % versus - %) [ ] [ ] [ ] [ ] ] . this difference may in part be explained by the greater contribution of chikungunya in our study than in the other studies that tested for this pathogen [ , , ] . however, unlike other studies, we were unable to detect any evidence of disease caused by rickettsia or coxiella spp., which made up - % of all afis in other studies [ ] [ ] [ ] [ ] ; and unlike other areas, malaria [ , [ ] [ ] [ ] ] and typhoid fever [ , , , , ] were not part of our diagnostic algorithm as they are only occasionally detected among travelers returning to puerto rico. while denv was not as commonly identified as chikv or flu a/b, participants with dengue were more likely to be admitted to the hospital at enrollment. the proportion of afi cases with dengue in our study was comparable to other recent studies which found - % of afi cases had dengue [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one exception to this was a study that found % of afi patients had dengue; however, the study's eligibility criteria likely enhanced enrollment of dengue cases [ ] . in our study, dengue incidence varied by age group with a nearly a -fold difference between participants < years old and - years old ( % versus %), which may be due to differences in likelihood of seeking medical care in primary versus secondary denv infections [ ] . of note, % of participants ! years old had dengue as a cause of afi, a finding comparable to a puerto rico study in which % of , laboratory-positive dengue cases detected by surveillance were ! years old [ ] . in contrast, other recent prospective studies chikungunya, the most commonly identified afi overall, was least likely to result in hospital admission, although two male participants with chikv infection died. these cases were older individuals (> years old) who had underlying co-morbidities which may have complicated their clinical course. nonetheless, since autopsy was not performed for either case, ascertaining whether chikv infection played a role in either fatality is difficult. however, in our study chikungunya was disproportionally identified among older participants, with positivity increasing from < % of pre-school aged children to about one-third of participants ! years old. this pattern of disease has been seen in other areas with recent chikv emergence [ ] , and may be due to older individuals having an increased likelihood of complications due to preexisting co-morbidities [ , ] . co-infections confirmed by molecular assays were detected among % of our participants, most commonly involving enteroviral or orv infections; less than one-third of all co-infections included a denv or chikv infection. another recent prospective study found that % of afi participants had co-infections involving molecularly diagnosed dengue or influenza, malaria, and positive blood culture [ ] . interestingly, we did not detect any co-infections involving chikv and denv. an analysis of island-wide surveillance data from puerto rico during the same time period found only one chikv/denv co-infection among approximately , specimens tested by rt-pcr for both denv and chikv [ ] . these findings are consistent with another prospective afi study conducted in sri lanka [ ] . although a recent study has shown that aedes aegypti can be infected with as many as three arboviruses simultaneously and can likely transmit these viruses to humans [ ] , the frequency of co-or tri-infection of mosquitoes in the wild depends upon the geographic spread and degree of circulation of each virus. during our study, denv transmission decreased significantly before chikv transmission peaked, making co-infections less likely. in addition, in puerto rico, where aedes aegypti is the sole vector for chikv and denv, viral interaction and viral prospective study of acute febrile illness in puerto rico interference within the mosquito may reduce the likelihood of co-infection [ ] [ ] [ ] . however, rt-pcr positive denv/chikv co-infections have been documented at higher rates in five countries [ ] . we identified differences in clinical predictors of laboratory-positive dengue by timing of presentation and age group highlighting the importance of considering these factors when developing prediction algorithms for clinical management [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we found, as have others [ ] , that even early (< dpo) in the clinical course leukopenia and thrombocytopenia are predictive of dengue across all age groups, and thrombocytopenia strengthened as a predictor over time. in our study, headache and eye pain were the only "aches and pains" that were predictive of dengue for all age groups [ ] . eye pain was a predictor early and later in the clinical course, a finding consistent with pediatric [ ] and adult [ ] prospective cohort studies, as well as a surveillance study conducted in puerto rico [ ] . we also found that rash among children < years old presenting early and erythema on the face and/or neck in all age groups presenting - dpo, were positive predictors of dengue. while the presence of skin rash has been found to be a predictor of dengue in several prospective studies [ ] , few studies have evaluated erythema as a predictor [ , , ] . last, like other prospective studies [ , ] , we found that nausea is an early predictor for dengue. we were also able to show that adults aged - years presenting - dpo were more likely to have abdominal pain than those with other afis, and dengue cases of all ages presenting - dpo were also more likely to have diarrhea and poor circulation in addition to nausea, findings that lend support to the idea that warning signs for severe dengue develop after the early phase of the illness. our study, which enrolled all patients presenting with fever regardless of age, sex, or clinical characteristics, may be limited in generalizability. the study was conducted in southern puerto rico which may differ from neighboring islands and other parts of the island with regard to population demographics, preexisting immunity to denv and other flaviviruses, and exposure to infections. second, while we enrolled nearly older adults (! years old), we were unable to adequately evaluate predictors of dengue among this population because we had only dengue cases and most presented early in the clinical course. last, we did not systematically collect stool and test for potential enteric pathogens, and bacterial infections were likely under recognized because blood cultures were only done on patients in whom sepsis was suspected. while our study identified an etiology in over half of all afi cases, the etiology of % of afi remained unknown even after extensive testing and the majority of diagnosed cases were caused by one of nine viral pathogens that typically do not require empiric therapy. in fact, we were unable to find any cases of rickettsia spp., ehrlichia spp., and coxiella spp., and only sporadic cases of melioidosis and leptospirosis were identified. our findings demonstrate that dengue is not only one of the leading causes of afi in puerto rico, but results in greater morbidity than other afis as measured by need for hospitalization. moreover, dengue affects people of all ages including older adults who may be at higher risk of developing medical complications. clinicians should include dengue on the differential diagnosis of afi among older adults so that timely anticipatory guidance can be offered. we found that the presence of leukopenia and thrombocytopenia were the best predictors of dengue in both time periods overall and for all age groups. our findings suggest that eye pain should be reevaluated as a predictor of dengue. future studies should focus on improving clinical diagnosis of afi including dengue by timing of presentation and age of the patient. supporting information s checklist. strobe statement. (docx) we thank saint luke's episcopal hospital patients for their participation in this study. we would like to thank physicians, nurses, clinical laboratory staff and administrative personnel at the saint luke's episcopal hospitals in ponce and guayama for their assistance to recruiting potential participants and implementing study procedures. in addition, we would like to acknowledge the medical management information offices from saint luke's episcopal hospitals for facilitating the review of medical records for admitted participants. we would also like to thank dr. brad biggerstaff from the cdc's division of vector-borne diseases for his critical review of the data analysis and manuscript. in addition, we would like to thank cdc staff members at the dengue branch, polio and picornavirus laboratory branch, rickettsial zoonoses branch, and bacterial special pathogens branch (zoonoses and select agent laboratory) for processing and testing of all clinical specimens. last, we would like to acknowledge the technical support of ponce health sciences university. without their interest in and support of this project, the sedss sites would have never been established and this study would never have been possible. mapping the aetiology of non-malarial febrile illness in southeast asia through a systematic review-terra incognita impairing treatment policies transition in the cause of fever from malaria to dengue, northwestern ecuador, - . emerging infectious diseases rapid diagnostic tests for non-malarial febrile illness in the tropics. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases the global distribution and burden of dengue infectious etiologies of acute febrile illness among patients seeking health care in south-central cambodia. the american journal of tropical medicine and hygiene etiologies of acute undifferentiated febrile illness in thailand etiology of acute undifferentiated febrile illness in the amazon basin of ecuador. the american journal of tropical medicine and hygiene causes of non-malarial fever in laos: a prospective study. the lancet global health acute undifferentiated febrile illness in rural cambodia: a -year prospective observational study dengue as a cause of acute undifferentiated fever in vietnam etiology of acute, non-malaria, febrile illnesses in jayapura, northeastern papua, indonesia. the american journal of tropical medicine and hygiene unsuspected dengue and acute febrile illness in rural and semi-urban southern sri lanka. emerging infectious diseases chikungunya as a cause of acute febrile illness in southern sri lanka contribution of dengue fever to the burden of acute febrile illnesses in papua new guinea: an age-specific prospective study. the american journal of tropical medicine and hygiene revista panamericana de salud publica = pan american journal of public health the american journal of tropical medicine 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during an outbreak of dengue fever and dengue haemorrhagic fever in jakarta, indonesia, during differences in clinical and laboratory characteristics and disease severity between children and adults with dengue virus infection in taiwan risk factors and clinical features associated with severe dengue infection in adults and children during the epidemic in chonburi clinical and laboratory features that distinguish dengue from other febrile illnesses in endemic populations dengue: guidelines for diagnosis, treatment, prevention and control. geneva: who early clinical features of dengue virus infection in nicaraguan children: a longitudinal analysis sensitivity and specificity of a novel classifier for the early diagnosis of dengue distinguishing dengue fever from other infections on the basis of simple clinical and laboratory features: application of logistic regression analysis early clinical and laboratory indicators of acute dengue illness key: cord- -vaygaqe authors: cheng, ming soon; lau, suk hiang; chan, kwai peng; toh, chee-seng; chow, vincent t. title: impedimetric cell-based biosensor for real-time monitoring of cytopathic effects induced by dengue viruses date: - - journal: biosens bioelectron doi: . /j.bios. . . sha: doc_id: cord_uid: vaygaqe we describe an impedimetric cell-based biosensor constructed from poly-l-lysine (pll)-modified screen-printed carbon electrode for real-time monitoring of dengue virus (denv) infection of surface-immobilized baby hamster kidney (bhk- ) fibroblast cells. cytopathic effects (cpe) induced by denv- new guinea c strain (including degenerative morphological changes, detachment, membrane degradation and death of host cells), were reflected by drastic decrease in impedance signal response detected as early as ~ hours post-infection (hpi). in contrast, distinct cpe by conventional microscopy was evident only at ~ hpi at the corresponding multiplicity of infection (moi) of . a parameter that describes the kinetics of cytopathogenesis, cit( ), which refers to the time taken for % reduction in impedance signal response, revealed an inverse linear relationship with virus titer and moi. cit( ) values were also delayed by . h for each order of magnitude decrease in moi. therefore, based on the analysis of cit( ), the virus titer of a given sample can be determined from the measured impedance signal response. furthermore, consistent impedance results were also obtained with clinical isolates of the four denv serotypes verified by rt-pcr and cycle sequencing. this impedimetric cell-based biosensor represents a label-free and continuous approach for the dynamic measurement of cellular responses toward denv infection, and for detecting the presence of infectious viral particles. in recent decades, outbreaks of emerging and re-emerging diseases caused by infectious viruses such as dengue virus (denv), ebola virus, influenza virus, severe acute respiratory syndrome coronavirus and west nile virus pose ongoing threats to global biosecurity. in confronting these infectious diseases that spread well beyond the initial affected regions, their surveillance and control often create serious challenges for public health organizations. hence, the development of rapid, effective and safe virus detection tools has become a major priority of the global community. current clinical laboratory virus detection tests based on real-time and multiplex pcr techniques, provide a promising platform for simultaneous nucleic acid amplification and detection of multiple target sequences in a single test (caliendo, ; gullett and nolte, ) . however, these molecular techniques are incapable of identifying live infectious viral particles since they detect the nucleic acids originating from both infectious and noninfectious viruses. virus samples usually comprise high viral particle-to-plaque-forming unit or pfu ratio , which may indicate that the minority of viruses in a given sample is infectious, or the presence of non-infectious viruses with mutated or damaged genomes, or failure of most of the viruses to successfully infect due to the complexity of the infection cycle (van der schaar et al., ) . proof of infectious viral particles is highly important and can only be accomplished by conventional cell culture assays, which is time-consuming and labor-intensive. clearly, it is essential to develop an effective and simple virus detection tool for the assessment of virulence and identification of infectious viral particles to aid in the control of an epidemic. the electrochemical impedance spectroscopic (eis) technique has recently gained popularity in cell-based assays in view of advantages such as high sensitivity, non-invasive measurement, accessibility of time-dependent and quantitative data. cell-based assays using adherent mammalian cell cultures such as fibroblast and endothelial cells have been utilized in toxicological and virological studies (peters et al., ; hofmann et al., ) . since the first reported application of eis in cell culture study (giaever contents and keese, ) , this technique has been widely employed for in vitro monitoring of dynamic responses of cells toward drugs, toxic agents and pathogens (kiilerich-pedersen et al., ; asphahani et al., ; xu et al., ) . principally, the eis cell-based analytical system involves quantitative monitoring of the spreading, morphology and viability of adhered cell cultures in real-time, by applying a constant alternating current (ac) electric field. due to the low conductivity of cellular membrane, formation of a confluent cell monolayer on the electrode surface often constricts current flow. the corresponding change in impedance signal response can be continuously monitored to obtain information on cellular growth or coverage on the electrode surface (cheung et al., ) . ultrasensitive impedimetric methods have recently reported non-invasive cell-based analyses of viral infections including herpes simplex virus infection of vero cells (cho et al., ) , human cytomegalovirus infection of human fibroblasts (kiilerich-pedersen et al., ) , and bluetongue virus infection of bovine endothelial cells (drew et al., ) . the direct, real-time investigation of denv-induced cytopathogenesis in mammalian fibroblast cells using the eis cell-based analytical system is first reported in this study. mosquito-borne dengue infections are caused by single-stranded rna-containing denv which is transmitted by aedes mosquitoes. denv exists as four antigenically distinct serotypes designated denv- , - , - and - (teles, ) . among the serotypes, secondary infection with denv- has proven to be responsible for the more severe symptoms of dengue fever (vaughn et al., ) . invasion of host cells by lytic viruses such as denv will ultimately result in degenerative changes and damage to host cells, known as cytopathic or cytopathogenic effects (cpe). such effects are generally characterized by degenerative morphological changes, detachment, membrane degradation and eventual death of cells. in general, decreased tight junctions between cells, and increased separation between cells and electrode arising from cpe may lead to decreased impedance signal response. in comparison to conventional inspection methods such as microscopy and plaque assay that rely on observable changes in the morphology and surface coverage of cells, the eis technique offers a more promising platform for studying virus-host interactions based on the real-time measurement of cpe-induced impedance response (cho et al., ) . here we report an impedimetric cell-based biosensor constructed from poly-l-lysine (pll)-modified screen-printed carbon electrode (spce) for real-time monitoring of denv infection of surface-immobilized baby hamster kidney (bhk- ) fibroblast cells. this in vitro method in which the cell-based biosensor is immersed in the culture medium, detects viral-induced cpe by gradual decrease in the impedance response. the sensitivity of the biosensing system is enhanced using spce, which constricts the current to flow within a small area of working electrode (campbell et al., ; alonso-lomillo et al., ) . furthermore, adherence of a cationic polymeric film (pll) onto spce significantly improves the attachment and spreading of cells (mazia et al., ; sanders et al., ; frey and corn, ; carrier and pézolet, ; khademhosseini et al., ; sitterley, ) . the excellent performance in terms of rapid detection time, automated analysis, high sensitivity and capability of indirectly identifying infectious viral particles are some desirable features of this impedimetric cellbased approach in real-time biosensing. spce, carbon working electrode, silver pseudo-reference electrode, and platinum auxiliary electrode on ceramic substrate were purchased from dropsens. pll (mw , - , at . % w/v) and potassium hexacyanoferrate (iii) (k fe(cn) , $ %) were purchased from sigma-aldrich. fetal bovine serum (fbs), and dulbecco's phosphate-buffered saline (dpbs) without calcium and magnesium were obtained from biowest. trypsin-edta (  ) and penicillin-streptomycin (  ) were obtained from paa laboratories. rpmi- medium was purchased from thermo scientific hyclone. avicel rc- ( . % solid) was purchased from fmc biopolymer. μl of . % (w/v) sterile-filtered aqueous solution of pll was applied onto the carbon electrode surface (with an area of . mm ) and the tip of pipette was used to spread the solution evenly over the entire electrode surface. the electrode was inverted and placed in a drying oven at °c for at least min to ensure the transformation of pll film into the stable β-sheet configuration, and adherence of pll (at a thickness of . nm) to the electrode surface (jordan et al., ) . excess pll solution was aspirated, and the electrode surface was thoroughly rinsed with sterile ultrapure water. the pll-modified spce was characterized using cyclic voltammetry (cv) in the presence of . mm fe(cn) À / À in  pbs, ph . at a scan rate of mv s À and potential range from À . to . v. bhk- cells (american type culture collection) were cultured in a t tissue culture flask containing ml of growth medium (rpmi- medium supplemented with % fbs and  penicillin-streptomycin solution). cells were grown in a humidified incubator at °c with % co . when the cells were - % confluent (after - days), growth medium was removed from the flask. cells were washed once with ml of  dpbs, ml of  trypsin-edta was added, and incubated at °c with % co for min until cells detached. the detached cells were flushed, trypsin-edta was removed, the cells were subsequently resuspended in ml of growth medium, and centrifuged at rpm for min. the supernatant was discarded, and the cell pellet was resuspended in ml of growth medium. bhk- cells were propagated at a sub-cultivation ratio of : . denv- new guinea c (ngc) strain and four clinical isolates each of denv serotypes , , and were propagated in bhk- cells supplemented with maintenance medium (rpmi- supplemented with % fbs,  penicillin-streptomycin). virus at multiplicity of infection (moi) of . was inoculated, and the tissue culture infectious fluid (tcif) was harvested - days upon the observation of cpe. moi represents the ratio of the number of infectious agents (viruses) to the number of infection targets (cells) adsorbed on the electrode surface. uninfected cells served as the control. tcif was subsequently centrifuged at rpm for min. the supernatant containing viruses was collected and diluted with rpmi- to achieve the desired moi for viral infection monitoring experiments. plaque assay using avicel ( . %) and crystal violet staining was performed to determine the virus titer expressed as pfu ml À . the experimental set-up consists of a chi d potentiostat-galvanostat (ch instruments) with data acquisition software, and a sterilized polypropylene vessel integrated with a pll-modified spce ( fig. ). total cell count was ascertained using tc automated cell counter (bio-rad). cell suspension with viable cell count of % was harvested to obtain a cell concentration of  cells ml À . bhk- cells (in μl) were seeded on the pll-coated carbon electrode surface, and allowed to attach and spread on the electrode surface for approximately h. thereafter, ml of growth medium was gently added to the vessel. cells were allowed to proliferate in an incubator at °c with % co for h. subsequently, μl of denv- stock solution was added and mixed gently with cell medium. bhk- cells infected with denv- at different moi were incubated at °c and % co for another days. proper sterilization of all apparatus and preparatory work were carried out in an antiseptic environment. the impedimetric properties of cell monolayers before and after viral infection were monitored at °c using the eis technique. electrochemical impedance spectra were recorded at open circuit potential, frequency of khz, and excitation amplitude of mv. . . rna isolation, reverse transcription (rt), polymerase chain reaction (pcr), and analysis of rt-pcr products of clinical isolates of dengue viruses the qiagen rneasy kit was employed to isolate total cytoplasmic rna from tcif collected from each denv serotype (at moi of . ) propagated in bhk- cells cultured with rpmi- medium supplemented with % fbs, and incubated at °c with % co for days. synthesis of cdna (rt) was carried out using ng of rna, u of moloney murine leukemia virus (m-mlv) reverse transcriptase (promega), u of rnase inhibitor, mm of dntps, and . mm each of four downstream primers dsp , , , and , and subjected to °c for min. pcr was carried out using ng of each cdna and . mm each of primers, dsp , , , , and dv . the pcr cycles consisted of an initial denaturation at °c for min, followed by cycles each of denaturation at °c for s, annealing at °c for s, extension at °c for s, and a final extension at °c for min (seah et al., a) . pcr products ( ml each) were electrophoresed for h in % agarose gels (in  tbe) containing sybr safe dna gel stain (life technologies). images were obtained using the chemidoc mp system (bio-rad). the specific pcr products corresponding to denv serotypes , , , and were excised and subjected to cycle dna sequencing using bigdye terminator cycle sequencing kit v . and xl genetic analyzer (applied biosystems). initially, we added cells to the bare spce and observed that the impedance signals were negligible and fluctuating between experiments compared with the baseline signals from the bare spce without cells added. the former signals remained unintelligible even when cells on the bare spce were infected with denv (data not shown). these findings alluded to poor attachment of cells to the bare spce, which led us to modify the electrode by coating it with pll to facilitate cell attachment. the carbon working electrode surface of spce was modified with a cationic polymeric film (pll) using a similar method described previously (anson et al., ) . the configuration of pll in solution changes from random coil to α-helix at about ph . subsequent heating to °c promotes the transformation of the pll film into the β-sheet configuration, which is believed to comprise chains that are partially cross-linked by hydrogen bonding. the cyclic voltammograms of the pll-modified spce obtained in the presence of . mm fe(cn) À / À in  pbs, ph . ( fig. (a) ) indicated fairly constant cv peak current responses for repetitive potential cycles with rsd values of . % and . % for anodic and cathodic peak currents, respectively. this excellent stability of current response reflected strong adherence of the pll film to the carbon working electrode surface. in comparison to other electrode surfaces such as platinum and glassy carbon, pll has been shown to be more resilient and strongly adhered onto the rough surface of screen-printed carbon (fogg et al., ) . the contrasting behavior of a bare spce and a spce modified with . % (w/v) pll toward the redox couple fe(cn) À / À is shown in fig. (b) . the cyclic voltammograms of the pll-modified spce exhibited a more reversible current response. coating of electrode surface with a layer of cationic polymer pll reduces the electrode overpotential and facilitates the redox reaction of anionic redox probes such as fe(cn) À / À at the electrode surface. this enhanced anionic exchange capability of the cationic polymer-modified electrode is indicated by a . % increase in cv peak current response compared to a bare electrode (fig. (b) ). electrode surface coating methods such as gelatin coating (campbell et al., ) and equilibration with culture medium (mccoy and wang, ; muller et al., ) are commonly employed to promote the attachment and spreading of cells on the electrode surface. despite their good biocompatibility, the utility of these methods is limited by the instability, weak adherence to the electrode surface, and poor cell capture capability. in this study, the positively charged pll coating was adopted owing to its excellent cell capture capability and strong adherence to carbon electrode surface. various studies have documented that adherent cell lines such as fibroblast and endothelial cells possess an overall negative surface charge, which facilitates their adhesion to positively charged surface (somosy et al., ; singh et al., ; hamdan et al., ) . this negative surface charge may be attributed to the carboxylic groups of aspartic and glutamic acid (pka ¼ . - . ) of proteins, carboxylic groups of sialic acid (pka ¼ . ) of glycoproteins, phosphate groups (pka¼ . ), and sulfate groups (pka¼ . ) of sulphated proteoglycans or glycoproteins (singh et al., ) . another explanation is that their extracellular matrix is rich in negatively charged glucosaminoglycans (somosy et al., ) , thus suggesting that the observed cell adhesion behavior is mainly electrostatic in nature. the enhanced cationic properties of pll-modified carbon electrode are therefore responsible for the strong adhesion of bhk- cells. in this study, the virus-host interactions between denv and bhk- cells were investigated by the eis technique. bhk- cells constitute a type of adherent fibroblast cells, derived by single-cell isolation from the kidneys of five syrian golden hamsters, mesocricetus auratus (stoker and macpherson, ) . this cell line is susceptible to infection by denv, vesicular stomatitis virus, reovirus, and human adenovirus. the bode diagram shown in fig. (a) was generated from eis spectra recorded at a frequency ranging from hz to mhz. in the absence of electron mediator or redox probe, the electrode impedance of cells is limited by stray capacitance at high frequency and electrode polarization at low frequency. moderate frequency ( - khz) is well-suited for probing the cellular morphological changes induced by viral infection or cytotoxicants (mccoy and wang, ; campbell et al., ; cho et al., ) . the intermediate frequency at which the impedance signal was most affected by the change in cellular morphology was $ khz ( fig. (a) ), where the impedance signals measured between cells prior to virus inoculation and during day of viral infection (at moi¼ ) were considerably different from each other. thus, the eis data recorded at khz appear to provide meaningful results which may correlate with the morphological dynamics of cells and the kinetics of denv-induced cytopathogenesis. this finding was further extended to the infection monitoring experiments where the impedance readings of cell adhesion and subsequent denv infection were monitored at khz every min up to h. due to the variation of the coating of cells on the electrode surface, the impedance signals of four different cell-based biosensors at hours post-infection (hpi) varied between . Ω and . Ω with a rsd of . %. normalization of the impedance signal response in the presence of virus (z virus ) against the response derived from the same biosensor in the absence of virus at hpi (z virus ¼ ) yielded a relatively low rsd of . %. normalization of the impedance signal against the blank signal enhances the reproducibility, precision of results, and avoids the need to calibrate each individual biosensor. the denv infection process involves several stages: attachment of viral envelope glycoprotein to cellular receptors such as dendritic cell-specific icam- grabbing nonintegrin (dc-sign), heparin sulfate or cell surface immunoglobulin (chen et al., ; tassaneetrithep et al., ) . after fusion with the plasma membrane, viruses are enveloped in an acidified endocytic vesicle known as endosome, and subsequently release rna into cytoplasm. virus replication would eventually trigger membrane degradation and release viral progeny into the extracellular environment. in general, viral invasion of host cells induces a series of intra-and inter-cellular remodeling processes, which ultimately lead to changes in tight junctions between cells, and the distance between cells and electrode. fig. (b) shows the normalized impedance signal response as a function of viral infection time at different moi. this plot reveals information on the attachment and growth of cells on the electrode surface, morphological dynamics of cells, and kinetics of viral-induced cytopathogenesis. due to the low conductivity of cell membranes, the flow of moderate frequency current at the cellelectrode or cell-cell interface is sensitively influenced by nanometer changes in the adhesion regions or tight junctions between cells (cheung et al., ) . thus, a gradual increase in the electrode impedance observed during cell culture indicates that cells are actively propagating and densely populating onto the pll-modified carbon electrode surface. impedance signal keeps on increasing after virus inoculation as cells continue to grow until cytopathogenesis induces a series of cellular remodeling processes that influence the morphology and adhesion of cells on the electrode surface. viral-induced cytopathogenesis often results in severe morphological and physical changes, detachment of cells from surface, and eventual cell death (campbell et al., ) . in fig. (b) , fluctuation of the normalized impedance signal signifies the onset of cpe induced by denv- ngc strain, followed by a drastic reduction in impedance signal response, which could be detected as early as $ hpi at moi of . moreover, after the observed cpe, the normalized impedance signal of the cell-based biosensor declined by % at $ hpi. technically, this particular time taken for % reduction in the cell impedance response is termed cit , a kinetic parameter typically used to characterize the viral-induced cytopathic events (fang et al., ) . to further characterize the viral-induced cytopathogenesis, cit was regressed as a function of virus titer in the logarithm of moi. fig. (c) displays an inverse linear relationship between virus titers and cit values (r ¼ . ). the resultant regression indicates that cit was delayed by . h for each order of magnitude decrease in moi. therefore, the dependence of the cpe-induced impedance signal response on the viral infectious dose permits the estimation of virus titers of viral samples based on the measured impedance signals. several control experiments were carried out to assess the performance of impedimetric cell-based biosensor for the investigation of virus-host interactions. a control experiment using bhk- cells in the absence of denv (represented by the black line in fig. (b) produced a gradual increment in impedance signal with time. clearly, bhk- cells cultured in growth medium remain viable and actively propagate on the electrode surface. the normalized impedance signal reaches its maximum after $ days of incubation with growth medium where the electrode surface is fully covered with cells. another control experiment using a pllmodified spce immersed in growth medium in the absence of bhk- cells and denv gave a fairly constant impedance signal over a period of days (data not shown). fig. shows the control experiments using different host cells infected with denv- ngc strain at moi of . in the control using biosensor prepared with vero cells, a commonly used mammalian cell line, the electrode impedance increased initially as the vero cells grew and reached a plateau from days to . another control using the c / aedes albopictus mosquito cell line produced a fairly constant impedance signal from days to , thus revealing poor attachment and spreading of c / cells on the pll-modified carbon electrode surface. in summary, denv-induced cpe could not be observed with vero and c / cell lines even at a relatively high moi of . this suggests that the impedance response is specific to bhk- cells and may not relevant to other cell lines. in summary, decrease in impedance signal could only be detected with the occurrence of denv-induced cytopathogenesis of the bhk- fibroblast cell line. for comparison, denv- ngc-infected bhk- cells seeded in a pll-coated culture flask were observed for cpe using an evos fl digital fluorescence microscope (life technologies). microscopic images were taken using a sony high-sensitivity monochromatic ccd camera, with contrast and brightness adjusted to improve the quality of images. uninfected bhk- cells served as control. microscopic imaging of bhk- cells after days of infection with denv- ngc strain at moi of displayed typical cpe including considerable changes in cellular morphology. detachment of cells from the surface was indicated by the presence of many empty spaces and rounded cells instead of the original completely confluent monolayer of spindle-shaped cells as depicted in fig. (a) . on the other hand, the monolayer of densely populated uninfected bhk- cells remained virtually intact after days of incubation with growth medium (fig. (b) ). to study the kinetics of cpe of bhk- cells infected with denv- ngc at moi of and , monitoring by conventional microscopy was performed at , , , , , , , , , , , , , , and hpi. bhk- cells mock-infected with heat-inactivated denv- ngc at "moi" of , and bhk- cells treated with denv- ngc viral rna served as additional controls. as expected, no cpe was observed for both negative controls up to hpi. however, the earliest distinct microscopic evidence of cpe was observed at and hpi for moi of and , respectively (supp. fig.) . in direct comparison to the optical method, the more sensitive cell biosensor could "sense" cpe much earlier as reflected by the drastic reduction in impedance signal response at $ and $ hpi for moi of and , respectively. the potential application of cell-based biosensor in analyzing clinical isolates was examined against four different dengue serotypes, denv- , - , - , and - . these viruses were isolated from the respective denv-infected patients. the four clinical denv isolates were verified to be denv- , - , - , and - strains by rt-pcr and cycle sequencing of amplified products (seah et al., a) , and their sequences deposited in the genbank database under accession numbers km , km , kp . fig. (a) shows agarose gel electrophoresis of the rt-pcr products of denv- , - , - , and - . their nucleotide sequences exhibit high identities to isolates rr ( %), dn/ ( %), tb ( %), and a ( %), respectively. infection of bhk- cells with the four clinical isolates of denv was monitored by using the procedures described for the ngc strain. fig. (b) displays similar cpeinduced impedance signals for the four clinical isolates (at moi of ). slight variation in cpe detection time may possibly be attributed to the varying degrees of cpe induced by these clinical denv strains. in summary, cpe induced by the four clinical isolates of denv could be detected within - days post-infection. the performance of this impedimetric cell-based biosensor underscores its utility for the analysis of clinical denv isolates. future studies are warranted to evaluate this biosensor for the analysis of actual clinical specimens from dengue patients, e.g. blood samples (seah et al., b; phanthanawiboon et al., ) . in conclusion, an impedimetric cell-based biosensor constructed from pll-modified spce has proven to be useful for realtime monitoring of denv-induced cytopathogenesis on surfaceimmobilized fibroblast cells. based on this platform, cpe reflected by the drastic decrease in impedance signal response could be detected at $ hpi at moi of . this impedimetric biosensing system outperforms conventional microscopic inspection that requires - days of denv infection to conclusively observe the changes in morphology and surface coverage of cells. other attractive features of this cell-based biosensor include the possibility of automation, non-invasive measurement, reduced timeframe for the diagnosis of live denv infection, mediatorless and label-free monitoring procedure, and capability of detecting the presence of infectious dengue viral particles to complement existing diagnostic tests such as rt-pcr and serology. in addition, virus titers of given viral samples can be estimated from the measured impedance signals by analysis of cit values. the utility of this impedimetric cell-based biosensor for detecting clinical infectious viral isolates also demonstrates its potential application in the clinical setting. proc. natl. acad. sci. usa the authors thank the singapore immunology network, agency for science, technology and research for research grant funding. cms acknowledges nanyang technological university for supporting his ph.d. scholarship. we thank dr. justin chu for providing chikungunya virus as control. supplementary data associated with this article can be found in the online version at http://dx.doi.org/ . /j.bios. . . . key: cord- -r mskri authors: magnani, diogo m.; silveira, cassia g. t.; rosen, brandon c.; ricciardi, michael j.; pedreño-lopez, núria; gutman, martin j.; bailey, varian k.; maxwell, helen s.; domingues, aline; gonzalez-nieto, lucas; avelino-silva, vivian i.; trindade, mateus; nogueira, juliana; oliveira, consuelo s.; maestri, alvino; felix, alvina clara; levi, josé eduardo; nogueira, mauricio l.; martins, mauricio a.; martinez-navio, josé m.; fuchs, sebastian p.; whitehead, stephen s.; burton, dennis r.; desrosiers, ronald c.; kallas, esper g.; watkins, david i. title: a human inferred germline antibody binds to an immunodominant epitope and neutralizes zika virus date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: r mskri the isolation of neutralizing monoclonal antibodies (nmabs) against the zika virus (zikv) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. here we describe the characterization of human mabs from the plasmablasts of an acutely infected patient. one of the mabs had the unusual feature of binding to and neutralizing zikv despite not appearing to have been diversified by affinity maturation. this mab neutralized zikv (neut( ) ~ μg/ml) but did not react with any of the four dengue virus serotypes. except for the expected junctional diversity created by the joining of the v-(d)-j genes, there was no deviation from immunoglobulin germline genes. this is a rare example of a human mab with neutralizing activity in the absence of detectable somatic hypermutation. importantly, binding of this mab to zikv was specifically inhibited by human plasma from zikv-exposed individuals, suggesting that it may be of value in a diagnostic setting. zika virus (zikv) belongs to the genus flavivirus of the flaviviridae family and is related to dengue virus (denv), yellow fever virus (yfv), japanese encephalitis virus (jev), and west nile virus (wnv) [ ] . the globally distributed mosquito species of the aedes genus, vectors for many flavivirus, can also transmit zikv [ , ] . however, zikv remained a relatively minor and obscure cause of human disease for most of the second half of the th century and was featured in a limited number of scientific reports. in fact, it was not until that autochthonous human infection was described outside africa and continental asia-in the federated states of micronesia [ ] [ ] [ ] . since then, reports from brazil have chronicled a rapidly spreading epidemic that co-exists with denv and chikungunya virus (chikv). the epidemic has spread north with mosquito-borne transmission being reported in many nations of the americas as far north as mexico and southern florida [ ] [ ] [ ] . more ominously, zikv has been implicated as the causative agent in fetal developmental problems [ , ] . there are reports of zikv-associated birth defects, primarily brain abnormalities and microcephaly in infants born to mothers infected with zikv [ ] . virus has been recovered from amniotic fluid, placental, and brain tissues [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . zikv infection has been classified as an ongoing threat by the world health organization. in the united states, the centers for disease control and prevention has issued guidance for the management of the infection in the general population, pregnant women, and infants [ ] [ ] [ ] . due to recent reports of sexually transmitted zikv infection, the cdc has also developed guidelines for prevention of this mode of transmission [ ] [ ] [ ] [ ] [ ] [ ] . more recently, zikv transmission has also been described in miami, florida [ ] , suggesting that autochthonous spread could occur in any region of the u.s. inhabited by aedes spp. treatment of a variety of human ailments using mabs is revolutionizing our ability to ameliorate human suffering. for infectious disease, the ebola epidemic highlighted the potential utility of a cocktail of three neutralizing (n)mabs that block infection by the ebola virus [ ] . most convincingly, the administration of a single nmab up to five days post infectious virus exposure prevents the development of disease in ebola-infected macaques [ ] . because mabs can be engineered to prevent antibody-dependent enhancement by incorporating the l a and l a (lala) mutations which reduce fcγr binding [ ] , they are a promising intervention in flaviviral therapies. our long-term goal is to use a cocktail of lala-mutated nmabs to prevent zikv infection in at-risk individuals, primarily pregnant women. therapeutic nmabs must be potent in order to be clinically viable, and most nmab isolation strategies are based on the identification of high-titer, antigen-selected repertoires. somatic hypermutation (shm) in germinal center (gc) b cells provides the basis for selection of b cells producing abs with increased affinity-a hallmark of the adaptive humoral response. this feature is conserved among mammals, highlighting the importance of ab affinity enhancement for evolutionary fitness [ ] . thus, it is unsurprising that the vast majority of human abs in the memory immunoglobulin (ig)g pool have undergone affinity maturation and have, on average, - nucleotide substitutions from precursor genes [ ] . the contribution of shm to ab-mediated viral neutralization is particularly clear for the chronicallyinduced broadly neutralizing antibodies to hiv [ ] [ ] [ ] [ ] . reversion of these anti-hiv nmabs to precursor germline antibodies results in a drastic reduction or complete loss of viral neutralization [ ] [ ] [ ] [ ] . although mutated mabs are found after secondary denv infection, the role of these mutations in acute virus-neutralization and clearance is less clear [ ] [ ] [ ] [ ] . still, the prevalent thought is that antiviral ab response involves the engagement of poor-or non-neutralizing germline clones generated by v(d)j rearrangement, followed by shm-mediated refinement in germinal centers to enhance neutralization potency. here we describe the isolation of plasmablast-derived human mabs, sorted days post onset of symptoms from a zikv-patient in são paulo, brazil. the patient reported a previous history of dengue infection and yellow fever vaccination (table ) . a few of the isolated abs neutralized zikv, most of them at relatively high concentrations. interestingly, one of these mabs (p f ) exhibited no nucleotide mutations when compared to its corresponding germline sequences, but still recognized a zikv immunodominant epitope and neutralized the virus. these results suggest unforeseen roles for gc-independent responses against zikv and possibly other viruses. blood samples were collected from volunteer , a -year-old woman who reported a pruriginous skin rash that started six days prior to the beginning of acute neurological deficits suggestive of gbs. zikv infection was confirmed by a positive real-time reverse-transcriptase pcr assay for zikv rna in urine samples collected at days and after the onset of the first rash symptoms. blood and cerebrospinal fluid were negative for zikv rna. previous history of a single dengue infection and yellow fever immunization were also reported. peripheral blood mononuclear cells (pbmcs) were obtained from blood samples collected days post onset of symptoms. blood samples from patient were obtained after signing a written consent form approved by the university of são paulo's institutional review board (cappesq / ). anonymized plasma samples from volunteers in brazil and us were obtained from naïve and convalescent subjects with rt-pcr-confirmed zikv or denv infection (s table) . four volunteers donated samples post yellow fever vaccination. we conducted reverse transcription followed by a nested pcr to amplify the variable region of the immunoglobulin (ig) chains using described protocols with minor modifications [ ] . briefly, cdna was synthesized in a μl reaction using the original sort plates. each reaction contained μl of ng random hexamer (idt), μl of mm dntp (life technologies), μl of superscript iii reverse transcriptase (life technologies), μl molecular biology grade water, and μl of single sorted cell sample in lysis buffer (described above). the reverse transcription reaction was performed at ˚c for min, ˚c for min, ˚c for min, c for min. after the reaction was completed, cdna was stored at - ˚c. heavy and light chains were amplified in three different nested pcr reactions, using a mix of ' v-specific primers with matching ' primers to the constant regions of igg, igl, and igk. pcr reactions were conducted using hotstartaq plus dna polymerase (qiagen). the second set of pcr reactions was carried out with primers redesigned to incorporate restriction sites compatible with subcloning into rhesus igg expression vectors, instead of the original human vectors [ ] . we sequenced the amplified and cloned products using primers complementary to the ig constant regions. sequences were analyzed using igblast and imgt/v-quest to identify v (d) j gene rearrangements, as well as shm levels [ , ] . we expressed mabs in expi f (thermofisher) human cell lines. the plasmids encoding heavy and light chains were co-transfected using the expifectamine transfection kit (a , thermofisher). after - days, we harvested the secreted mab in the supernatant. ig concentration in the supernatant was determined by an anti-rhesus igg elisa, before we proceeded with the functional assays. for the experiments with purified mabs, we used protein a plus (pierce)-containing columns to remove the impurities. the concentration of purified protein was determined by measurement of absorbance at nm (nanodrop, thermo scientific). virus capture assay and recombinant e protein elisa p f binding was determined by both virus capture assay (vca) and recombinant (r)e elisas. the vca plates were coated overnight with the mouse-anti-flavivirus monoclonal antibody g (clone d - g - - , emd millipore) followed by incubation with viral stocks (zikv or denv). the re elisa plates were coated with zikv e protein (mybiosource, mbs ) diluted to μg / ml in pbs. after the coating step, the plates were washed with pbs and mab samples diluted to μg / ml were added to designated wells and incubated for h at ˚c. subsequently, the plates were washed and detection was carried out using a goat anti-human igg hrp secondary ab (southern biotech), which was added to all wells at a dilution of : , . following a h incubation at c, the plates were washed and developed with tmb substrate at room temperature for - min. the plates were developed with tmb substrate at room temperature for - min. the reaction was stopped with tmb solution and absorbance was read at nm. the neutralizing potency of the mabs was measured using a flow cytometry-based assay [ , ] . in brief, recombinant mabs (transfection supernatant or purified) were diluted and preincubated with zikv (paraiba) or the reference denv serotypes in a final volume of μl for h at ˚c. the virus and mab mixture ( μl) was added onto wells of a -well plate of % confluent vero cell monolayers in duplicate. a new seed of vero cells (ccl- tm) was obtained from the american type culture collection (atcc) repository for this study. the inoculum was incubated in a ˚c incubator at % co for one hour with agitation of the plates every min. after one hour, the virus and mab-containing supernatants were aspirated and the wells were washed with media. fresh media was then added and the plates were incubated for a total of hours. cells were trypsinized with . % trypsin (life technologies), fixed (bd cytofix), and permeabilized (bd cytoperm). viral infection was detected with the g antibody (millipore) recognizing zikv or denv, followed by staining with an anti-mouse igg a apc fluorophore-conjugated secondary reagent (biolegend). the concentration to achieve half-maximal neutralization (neut ) was calculated using a nonlinear regression analysis with prnts were conducted as previously described [ ] . briefly, purified p f was serially diluted in optimem supplemented with % human serum albumin (vwr), % fetal bovine serum, and gentamicin. zikv paraiba was diluted to a final concentration of~ - pfu / ml in the same diluent added to equal volumes of the diluted ab. the virus/mab mixture was incubated at ˚c for min. cell culture medium was removed from % confluent monolayer cultures of vero cells on -well plates and μl of the virus/ab mixture was transferred onto duplicate cell monolayers. cell monolayers were incubated for min at ˚c and overlaid with % methylcellulose in optimem supplemented with % fbs mm glutamine + μg / ml gentamicin. samples were incubated at ˚c for four days after which plaques were visualized by immunoperoxidase staining, and a % plaque-reduction neutralization titer was calculated. inhibition of p f mab binding was determined by elisa. to begin, the elisa plate was coated with mouse anti-flavivirus monoclonal antibody g (emd millipore) diluted : , in carbonate binding buffer and incubated overnight at ˚c. the next day, the plate was washed five times with pbs-tween and wells were blocked with % skim milk in pbs for h at ˚c. after the block step, the plate was washed and virus samples were added to designated wells for h incubation at room temperature. subsequently, the plate was washed with pbs only and corresponding blocking plasma samples were added for h at ˚c. following the plasma block, the plate was washed and p f was added to corresponding wells for h at c. p f was detected using a rhesus igg-specific antibody (mouse anti-monkey igg-hrp clone sb a; southern biotech). thereafter, the plate was washed and wells were developed with tmb substrate at room temperature for - min before the reaction was stopped with tmb stop solution. absorbance was determined at nm. we isolated plasmablasts from patient who presented with suspected guillain-barré syndrome (gbs) ( table ) (first day of symptoms = d ). the patient had a previous history of dengue infection and yellow fever vaccination ( table ). the previously healthy -year-old woman presented to the emergency room (d ) reporting a progressive paresthesia mainly in the extremities of her hands, along with acute, intermittent pain in her left forearm during the previous four days. at physical examination, the patient presented with a grade iv asymmetric muscular weakness and hypoesthesia in her left limbs, with abolished deep tendon reflexes in the lower limbs. a mild weakness of her left facial muscles was also noted. the patient reported no respiratory disorders and no hoarseness, and no signs of dysautonomia were detected at the clinical evaluation. fever, conjunctivitis, and myalgia or joint pain were absent during the illness. afterwards, the patient was hospitalized with a clinical diagnosis of gbs, for which an intravenous human ig (ivig) treatment was initiated at a dosage of . g / kg / day for days. cerebrospinal fluid analysis and an electroneuromyogram were performed on fourth (d ) and fifth (d ) days after neurological symptom onset, respectively; the results were within normal limits. the electroneuromyogram was repeated on the th day of neurological symptoms, but no significant abnormalities were noted despite the persisting weakness in the patient's left leg and arm. during the treatment with ivig, the patient presented with transient worsening of her hemiparesis, but progressively recovered over the course of weeks after discharge from the hospital. at days post-neurological symptom onset (d ), a physical exam revealed significant improvement of muscular strength and abolished deep tendon reflexes in the lower limbs. the remittent skin rash cleared completely days after its initial emergence. blood, cerebrospinal fluid and urine samples were collected on the th day of neurological symptoms (d ) for detection of zikv by rt-pcr. the urine sample was zikv-positive by pcr, while blood and cerebrospinal fluid were negative. a saliva sample collected on d was negative for zikv. we isolated plasmablasts from peripheral blood mononuclear cells (pbmcs) collected on day (table ) . from wells containing single-sorted cells, we amplified, cloned, and sequenced heavy and light ab chains using ' primers complementary to the v gene segments and a ' primer annealing to the constant igg region [ ] . this resulted in paired heavy and light chains (s table) . eight of the mabs bound to zikv (fig ) . seven of these mabs exhibited cross-reactivity to one or more of the denv serotypes, and a single mab-p f -bound exclusively to zikv. interestingly, two mabs bound to denv but not zikv. we tested the neutralization potency of the zikv-specific p f mab in a flow-based neutralization assay and a plaque reduction neutralization test (prnt) and found that it neutralized zikv at approximately μg / ml (prnt ) (fig ) . analysis of the isolated antibody variable (v) domain sequences revealed five mabs with average gene mutation levels ( - nucleotide modifications), two mabs with over nucleotide substitutions, and mabs with unusually low levels of shm for isotype-switched mabs (lower than changes) (s table) . the most highly mutated mabs (p b and p c ) were not zikv-specific by binding (s table) . in fact, the eight zikv-binding mabs had the lowest shm levels, including four mabs lacking clearly recognizable mutations when compared with putative heavy and light chain germline precursors (s table, fig ) . except for junctional diversity, the zikv-neutralizer p f mab heavy chain did not exhibit signs of antigenselected ig diversification. p f had an identical sequence to the ig heavy chain variable (ighv) genes segment ighv - à up to the amino acid c , prior to the cdr-h (international immunogenetics information system [imgt]) [ ] . however, position g -the site of the junction between ighv and the igh diversity (ighd) genes-differed from the germline reference. interestingly, this region is part of a segment (n ) with non-germline nucleotides corresponding to six amino acids identified between the ighv and ighd genes (fig c) . this segment is likely the result of n nucleotide additions generated during b cell ig gene rearrangement, prior to antigen selection. because of the lack of mutations elsewhere in the sequences, it is likely that the r g substitution was also generated during this developmental step. the downstream sequence corresponding to the junction between ighd - à and the igh joining (ighj) ighj à genes also revealed similar nucleotide insertions. likewise, the kappa (k) chain junction between the igkv - à and igkj à genes also contained one insertion. although we cannot rule out the possibility of shm-mediated nucleotide changes in the n insertion regions, no mutation was identified in the remainder of the regions of the heavy and light chains. thus, the p f mab is likely very close or identical to the original v-(d)-j gene rearrangement in the naïve b cell before antigen contact. to investigate whether p f recognizes an immunodominant zikv epitope, we used a serological blocking assay. in brief, this assay detects the presence of competing abs that can inhibit the p f mab binding to its epitope. because p f did not bind to recombinant e protein (fig ) we used whole virus in our binding assays. we captured zikv on the plate using the g mab (pan-flavivirus), and incubated zikv with plasma from patients with diverse histories of denv and zikv exposure (s table) . we added unlabeled p f (engineered with rhesus igg constant regions) and detected binding of the mab using a hrplabeled mouse anti-rhesus mab (fig ) . nine of ten plasma samples from individuals that had been infected with zikv blocked the binding of p f in a blinded test (fig , s table) . similar blocking activity was observed regardless of whether individuals had been previously infected with denv or had been vaccinated for yellow fever. in contrast, little or no blocking activity was observed by denv+ plasma in the absence of prior zikv exposure (fig ) . furthermore, this recognition was specific in that it was not observed in of denv-only infected individuals. thus, the p f serological blocking assay accurately predicted previous zikv exposure, as confirmed by rt-pcr, in all but one of the patient plasma samples tested. although this patient, donor , had a positive urine rt-pcr result for zikv, plasma from did not block p f binding to zikv (s table) . interestingly, the plasma did not exhibit detectable zikv-neutralizing activity, suggesting that this patient did not mount a measurable antibody response against zikv. in conclusion, only the plasma that inhibited zikv infection of vero cells contained p f -blocking antibodies. here we show that a igg mab with no detectable shm was generated against zikv early in infection. remarkably, despite being germline-encoded, this mab is zikv-specific and does not bind to any of the four denv serotypes. furthermore, this mab not only neutralizes zikv, but it also binds to an immunodominant epitope on the virus. remarkably, despite being germline-encoded, p f binds specifically to zikv and does not cross-react with any of the four denv serotypes. our results also suggest that p f recognizes a unique epitope on zikv. it is unclear how this ab developed such specificity without shm. finally, these findings suggest that affinity maturation is not necessary for the generation of isotype switched virus-neutralizing abs. low levels of shm in abs possessing neutralizing activity have been previously reported in mice and humans [ ] [ ] [ ] , supporting the idea that germline-encoded mabs can indeed neutralize. abs with low levels of shm have also been reported during the acute phase of human denv infection, but it was not clear that these abs contributed to the antiviral neutralization activity [ ] . in studies in mice, vsv-specific mabs lacking shm have been isolated previously [ ] . interestingly, secondary, but not primary, mouse abs against vsv had mutations [ ] . furthermore, the reversion of these mutated abs to non-mutated precursors reduced, but did not abrogate, vsv binding and neutralizing activity. the binding differences between the mutated and germline abs were much less pronounced than might be expected [ ] . additionally, mice that cannot conduct shm due to aid knockout still mounted neutralizing ab responses against friend virus, a strain of murine leukemia virus [ ] . it has been suggested that these abs lacking extensive shm undergo a gc-independent developmental pathway [ ] , although the mechanistic basis for this phenomenon remains to be elucidated. rapid, gc-independent responses might be particularly relevant in the control of acute cytopathic viruses [ , , ] . the gc-independent abs would arise quickly after infection and then curtail viral replication, preventing virus-mediated damage [ ] . even more provocatively, hangartner et al. have argued that cytopathic viruses specifically evolved to retain binding to these germline sequences to decrease host lethality and increase fitness. on the other hand, chronic viruses may have evolved to avoid germline-binding and development of neutralizing responses to persist [ ] . so far, these hypotheses remain unsubstantiated by the lack of evidence for strictly germline neutralizing ab responses in humans. while our experiments were not specifically designed to detect gc-independent responses, it seems likely that the isotype-switched p f originated directly from a germline precursor. we isolated p f from a zikv-infected individual that developed neurological complications compatible with gbs and was treated with ivig. underlying factors that influence the potential association of gbs and zikv infection might involve an autoimmune process, which could influence the development of immune responses [ ] . additionally, ivig may have had a role in the selection of the ab responses mounted by peripheral b cell repertoires [ ] . this is unlikely, however, since the patient initiated ivig treatment on the same day that the plasmablasts were isolated. it is possible, then, that gbs or ivig-treatment influenced the development of p f . these potential associations are difficult to determine and were outside the scope of this study. it is clear, however, that these responses were not exclusive to volunteer , as p f binding can be blocked by the serum of most zikv-infected individuals (fig ) . recently [ ] . these mabs were isolated from memory cells sorted with soluble and monomeric zikv e proteins and, in contrast to p f , bind to the recombinant protein [ ] . in contrast, we isolated zikv-specific mabs from circulating plasmablasts at d . the peak recall of memory b-cell derived plasmablasts is thought to occur within the first week post-secondary infection [ , ] . thus, it is probable that most of the isolated mabs did not have a memory-b cell origin, and it remains possible that some of the plasmablasts were sorted from the basal population that circulate in low frequencies in the blood. in conclusion, the isolation of mabs using different b cell methods suggest that anti-zikv mabs with germline characteristics are not limited to specific b cell subtypes [ , ] . notably, the anti-zikv mabs isolated to date are less mutated than the mabs isolated after related denv infections [ ] [ ] [ ] [ ] . together, these findings suggest possible differences in the development of ab responses against zikv. unfortunately, despite our efforts, we were unable to map p f 's binding site. we first employed an in vitro escape assay [ ] , which did not result in a single mutated consensus sequence. also, p f did not bind to the prm/e proteins expressed in cells, precluding our ability to map this interaction using an ala-mutated envelope panel [ , ] . characterizing this interaction will, thus, require a significant effort that is beyond the scope of the current manuscript. because the p f mab retains the ability to bind virions, our conclusion is that it binds to a conformational epitope. based on the cohort of human plasma samples tested in this study, it appears that most zikv-infected individuals mount ab responses against the epitope recognized by p f . this epitope is recognized by abs in individuals previously infected by zikv, thereby preventing the binding of p f . by contrast, abs in the plasma from individuals previously infected by any of the denv serotypes, do not prevent binding of p f . p f may, therefore have potential as a diagnostic. several diagnostic options for testing for zikv exposure exist, including rt-pcr, igm elisa, and prnt methods [ , ] . while it is relatively straightforward to detect zikv nucleic acid during the acute phase in blood, urine, saliva, and semen, it has proven more difficult to design rapid and effective diagnostics for zikv exposure in the chronic phase. for samples collected after the first week of symptoms, the initial test is an anti-zikv, anti-denv, anti-chikv virus igm elisa [ ] . however, in patients who have received a flaviviral vaccine (denv, yfv, or jev) and/or have been infected with any flaviviruses in the past, these assays may be difficult to interpret due to the cross-reactivity of the abs [ ] [ ] [ ] [ ] [ ] [ ] . thus, a positive igm test needs to be confirmed with a laborious prnt assay. igm antibodies persist for - weeks in serum, and sera from individuals previously infected for more than weeks would also have to be confirmed with a virus neutralization-based method [ ] . our plasma inhibition assay may, perhaps, provide an alternative to these other techniques. in 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neutralizing human antibodies prevent zika virus replication and fetal disease in mice interim guidance for interpretation of zika virus antibody test results fields virology. th ed. philadelphia: wolters kluwer health/lippincott williams & wilkins structural basis of potent zika-dengue virus antibody cross-neutralization cross-reactivity and function of antibodies elicited by zika virus infection high specificity of a novel zika virus elisa in european patients after exposure to different flaviviruses diagnostics for zika virus on the horizon we thank the patients for their volunteer donations. we also thank the laboratory and clinical personnel responsible for collecting the plasma samples used in this study. conceptualization: dmm diw egk. key: cord- -vfznyyz authors: dauner, allison l.; mitra, indrani; gilliland, theron; seales, sajeewane; pal, subhamoy; yang, shih-chun; guevara, carolina; chen, jiann-hwa; liu, yung-chuan; kochel, tadeusz j.; wu, shuenn-jue l. title: development of a pan-serotype reverse transcription loop-mediated isothermal amplification assay for the detection of dengue virus date: - - journal: diagn microbiol infect dis doi: . /j.diagmicrobio. . . sha: doc_id: cord_uid: vfznyyz during dengue outbreaks, acute diagnosis at the patient's point of need followed by appropriate supportive therapy reduces morbidity and mortality. to facilitate needed diagnosis, we developed and optimized a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay that detects all serotypes of dengue virus (denv). we used a quencher to reduce nonspecific amplification. the assay does not require expensive thermocyclers, utilizing a simple water bath to maintain the reaction at °c. results can be visualized using uv fluorescence, handheld readers, or lateral flow immunochromatographic tests. we report a sensitivity of . % ( % confidence interval [ci], . – . %) and specificity of . % ( % ci, . – . %) using a panel of clinical specimens characterized by denv quantitative reverse transcription–polymerase chain reaction. this pan-serotype denv rt-lamp can be adapted to field-expedient formats where it can provide actionable diagnosis near the patient's point of need. dengue is one of the fastest growing global health problems today, caused by a single-stranded, positive-sense enveloped rna virus that belongs to the flaviviridae family (trent et al., ; rico-hesse, ) . the human immune system recognizes antigenically distinct serotypes of dengue virus (denv) (i.e., denv - ) (george and lum, ; gubler, ) . symptoms can range from mild fever, headaches, myalgia, and rashes (henchal et al., ; trent et al., ) to severe disease characterized by plasma leakage and hemorrhage. severe dengue can be fatal, especially without intensive medical care. the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes and is endemic in most tropical and subtropical areas where these vectors are found. an estimated million denv infections are believed to occur each year, resulting in million symptomatic cases (bhatt et al., ) . neither vaccines nor antiviral treatments are available to reduce denv-associated morbidity or mortality. the only available treatment option is supportive bed rest, fluids, and symptomatic relief with analgesics. diagnosing dengue is important for guiding appropriate supportive care and for alerting the physician to disease-specific warning signs that may require hospitalization; however, it is not possible to make an accurate differential diagnosis of dengue based on clinical features alone, as many symptoms of dengue resemble those of other diseases, such as malaria, chikungunya, measles, influenza, and rickettsial infections (teles et al., ; shu and huang, ) . traditional laboratory techniques for dengue diagnosis include viral isolation followed by indirect immunofluorescence assay or serological assays such as plaque reduction neutralization test, hemagglutination inhibition, and igm antibody capture enzyme-linked immunosorbent assay (teles et al., ; shu and huang, ; kao et al., ) . some of these techniques can take days or weeks to complete. furthermore, serological methods require convalescent samples demonstrating an increase in antibody titer from pre-exposure samples in order to make a definitive diagnosis, making it impossible to confirm a diagnosis in the acute stage, limiting the value of serological assays for informing patient management. reverse transcription-polymerase chain reaction (rt-pcr) and quantitative rt-pcr (qrt-pcr) can be used to identify denv rna, which enables diagnosis during the acute phase of dengue infection. this can be an advantage because it facilitates earlier detection of dengue (within - days post onset of symptoms), before an immune response has been mounted and serology-based diagnostics can detect igm or igg levels (typically detectable - days post onset of symptoms). however, pcr equipment is expensive, requires trained personnel, and is best suited to diagnostic reference laboratories. other nucleic acid detection strategies based on isothermal amplification such as nucleic acid sequence-based amplification (nasba) and reverse transcription loop-mediated isothermal amplification (rt-lamp) have been developed to address these diagnostic microbiology and infectious disease j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / d i a g m i c r o b i o challenges and move molecular assays closer to point-of-care use (jittmittraphap et al., ; neeraja et al., ) . here, we have explored a denv diagnostic solution using the rt-lamp technology. the technology was originally developed by eiken chemical company (notomi et al., ) and is based on the principle of strand displacement. briefly, bst polymerase has both polymerase and strand displacement activity and can be used to amplify nucleic acids using a series of primers that initiate a specific stem loop structure following binding to a target sequence (notomi et al., ; nagamine et al., ) . this structure allows the enzyme to polymerize nucleic acids continuously at permissive temperatures beyond - °c. a variety of methods can be used to visualize the amplified dna product or the magnesium pyrophosphate by-product. the dna amplicon can be labeled with an intercalating agent such as sybr green and visualized using the naked eye or under uv light. alternatively, the magnesium pyrophosphate causes turbidity, which can be visualized by the naked eye or using a turbidimeter (mori et al., ) . another benefit of this assay is that it does not require expensive instrumentation and takes less than an hour to perform, making lamp and rt-lamp suitable for diagnostic applications in low-resource settings. the technology has been adapted to detect a variety of microorganisms ranging from bacteria such as mycobacterium (iwamoto et al., ) ; parasites such as plasmodium (sattabongkot et al., ) ; and viruses such as human immunodeficiency virus (curtis et al., (curtis et al., , , severe acute respiratory syndrome-coronavirus (hong et al., ) , and hepatitis b virus (nagamine et al., , . rt-lamp has also been evaluated for diagnosing other members of the flaviviridae family, including yellow fever virus (kwallah et al., ) , japanese encephalitis virus (parida et al., ; toriniwa and komiya, ) , west nile virus , and tick-borne encephalitis virus (hayasaka et al., ) . in this study, we developed an rt-lamp assay to detect all serotypes of denv in a single reaction. other groups have explored this concept, with several assays detecting the serotypes of denv in separate reactions (neeraja et al., ; parida et al., ) . some groups have developed pan-serotype denv rt-lamp assays (teoh et al., ) , and others have expanded this concept to a pan-flaviviral assay that detects denv - , japanese encephalitis virus, and west nile virus in a single reaction (li et al., ) . we report a pan-serotype denv rt-lamp assay that has been optimized and evaluated with an extensive panel of clinical samples obtained from febrile patients in south america. we have additionally explored a method of reducing assay variability in a denv quantitative rt-lamp (qrt-lamp) that uses a quencher to reduce false positives. finally, we have also examined methods for streamlining sample preparation and evaluated visualization of the qrt-lamp product using lateral flow immunochromatographic tests (icts) and a handheld fluorescence reader. together, this represents a complete assay, from sample to result, which can be used to identify denv with high accuracy in low-resource settings. the procedures applied in this study were done in accordance with the ethical standards of the naval medical research center (nmrc) institutional review board and with the helsinki declaration of , as revised in . study protocols were approved by the nmrc institutional review board (nmrcd. . and nmrc. . ) in compliance with all applicable federal regulations governing the protection of human subjects. clinical serum samples of denv viremic patients or those determined to have other febrile illness were collected during ongoing febrile surveillance studies (years - ) at the naval medical research unit- peru at regional sites in piura, tumbes, madre de dios, and iquitos. the samples were deidentified and shipped to the nmrc. additional serum samples from denv-negative individuals were obtained from a dengue vaccine trial at the nmrc and a blood bank at the walter reed army medical center, both in the united states. all of these specimens were obtained with institutional review board approval. tissue culture-derived laboratory virus stocks propagated in vero african green monkey kidney cells at the nmrc were used as positive controls. the following denv strains were used to cover all serotypes: denv , wp ; denv , ; denv , ch ; and denv , . rna extraction for these denv stocks as well as clinical serum samples was performed using qiaamp viral rna mini kit (qiagen, valencia, ca, usa) following the manufacturer's protocol. rna was eluted in μl of nuclease-free water. samples were characterized as positive or negative for denv using a qrt-pcr assay, modified from one previously described (mcavin et al., ) . briefly, the sequences of the qrt-pcr primers used were as follows: denv forward: ′-ggttagaggagacccctc- ′, denv reverse: ′-cagagatcctgctgtctc- ′, probe: ′fam-cagcatattga cgctggga-tamra ′. we utilized the taqman® ez rt-pcr core reagents kit (life technologies, carlsbad, ca, usa) with a final volume of μl, with the following reaction concentrations: . μmol/l forward primer, . μmol/l reverse primer, . μmol/l probe, . mmol/l dntp, mmol/l mn(oac) , x abi buffer, . u rtth polymerase, and . u amperase. the reaction was held at °c for minutes and °c for minutes, followed by cycles of denaturation at °c for seconds and polymerization at °c for seconds. to quantify rna copies, the denv ′utr was cloned into the pcr_script amp vector (stratagene, la jolla, ca, usa), and the transcript was transcribed in vitro using the riboprobe t transcription kit (promega, madison, wi, usa). rna was quantified using a spectrophotometer, and the rna copy number was calculated. this rna standard with known copy numbers was serially diluted and run alongside all qrt-pcr reactions to enable quantification of rna copies in the reaction. rt-lamp primers were designed against the conserved regions of denv using eiken's primerexplorer version (http://primerexplorer. jp/e/) utilizing denv genome (genbank accession number fm . ) as the initial template. these primers were then aligned with denv , , and , and degenerate primers were manually created to improve sequence consensus. these included outer primers (f and b ), inner primers (forward inner primer [fip] and backward inner primer [bip]), and loop primer (loopb). these primers were then aligned with up to published strains each of denv , denv , and denv . six additional primers were added to broaden detection of these denv strains: fipdegen / , fipdegen , bipdegen / , bipdegen , b degen / , and b degen . the rt-lamp reactions were carried out in a -μl volume with the following concentrations: pmol/l fip, fipdegen , bip, and bipdegen ; pmol/l fipdegen / , bipdegen / , and loopb; . pmol/l f and b (integrated dna technologies, coralville, ia, usa); x thermopol buffer (new england biolabs, ipswich, ma, usa); . mol/l betaine (sigma-aldrich, st. louis, mo, usa); . mmol/l each dntp (life technologies, grand island, ny, usa); mmol/l total mgso (sigma-aldrich); u bst dna polymerase (new england biolabs); . u amv rt (life technologies, grand island, ny); and μl of template. reactions were held at °c for minutes unless otherwise indicated. a positive control template corresponding to copies/reaction denv rna was used in rt-lamp reactions, unless otherwise stated. completed reactions were run on a % agarose gel containing x sybrsafe (life technologies). the rt-lamp product banding pattern was also compared against those from true positives. to visualize rt-lamp product in the tubes, x sybrsafe was added. the agarose gels and tubes were visualized using an e-gel imager system (life technologies). fluorescence in reaction tubes was also measured using the picoflour tm model number - (turner biosystems, sunnyvale, ca, usa). the method of visualizing rt-lamp product on lateral flow icts was adapted from previous reports (puthawibool et al., ; ding et al., ) . a home-made ict strip, which had streptavidin on the detection zone and the mouse anti-fitc antibody conjugated with colloidal gold in the sample pad of the strip, was used for detection of the lamp product. for visualizing the rt-lamp product on this system, the reaction was performed as described above except with the addition of biotin- -dutp (life technologies) and fitc- -dutp both to a final concentration of μmol/l. following a lamp reaction, the mixture was heated to °c for minutes and then cooled on ice for minutes. approximately . μl of the biotinylated rt-lamp product was applied to the binding pad of the lateral flow ict. running buffer ( μl of . % tween ) was added to facilitate capillary flow, and the lateral flow icts were visually inspected after minutes. the presence of a visible purple line was considered reactive. sequence-specific detection was performed as above, but incorporating a fam-labeled-loopb primer instead of the unlabeled primer. to reduce visualization of nonspecific amplification, pmol/l of black hole quencher (bhq) -labeled antisense loopb primer was added to the rt-lamp reaction tube following isothermal amplification. both the fam-and bhq-labeled primers were obtained from integrated dna technologies. the tubes were quenched for approximately hours at room temperature, followed by visualization using a uv source as described above. we designed four sets of lamp primers to detect all serotypes and multiple strains of denv in a single reaction. among these, set of primers was down-selected based on faster time to positivity in a pilot experiment (data not shown). table summarizes the primers used. the primers target a ′ untranslated region of denv that is highly conserved between all serotypes and multiple strains. unlike traditional lamp assays, which require - primers, this pan-dengue rt-lamp uses primers, including fip, bip, and b degenerate primers. using a positive control template, the rt-lamp reaction was tested within a range of temperatures ( - °c, fig. a ) using a simple water bath as the heat source. nucleic acid amplification took place at all the temperatures tested, and subsequent experiments were performed at °c. next, various assay characteristics were systematically optimized, including primer, enzyme, and betaine concentrations, in order to achieve the fastest time to result (data not shown). under optimized conditions, copies/reaction of denv rna was detectable in approximately minutes when using gel electrophoresis or uv as a readout (fig. b) . the assay was designed to be reactive to all serotypes of denv, and this was confirmed using tissue culture-derived positive strains (fig. c) . using a serial dilution of denv , we determined a limit of detection of copies/reaction where samples were detected reliably a majority (n %) of the time, comparable to qrt-pcr ( fig. a) . fig. b illustrates how time to positivity is affected by the number of copies of denv template present in the reaction. based on these results, we selected a reaction time of minutes to enable reliable detection of n copies/reaction for all denv serotypes while minimizing nonspecific amplification. we also saw positive rt-lamp reactions using denv-positive samples that had been heat treated for minutes at °c. boiling samples can eliminate the need for formal rna extraction steps as has been previously reported with dna-based pathogen targets (mikita et al., ; hopkins et al., ) . a panel of clinical specimens pcr confirmed as denv positive was used to determine the clinical sensitivity of our optimized rt-lamp reaction. the panel included denv , , and naturally circulating in northern peru at the time of sample collection. denv-negative samples, including human serum samples from individuals without fever symptoms as well as samples from individuals presenting fever symptoms of unknown origin that tested negative for denv by qrt-pcr, were used to determine clinical specificity (fig. ) . our optimized rt-lamp assay demonstrated . % sensitivity ( % confidence interval [ci], . - . % using binomial confidence interval (clopper and pearson, ) ) and . % specificity ( % ci, . - . %). the false-negative samples consisted of denv and denv specimens. overall, the assay demonstrated a . % agreement with denv qrt-pcr, underscoring the potential clinical utility of this assay. as has been reported by others, we occasionally observed falsepositive results when using our denv rt-lamp assay (curtis et al., ). precautionary measures were implemented to reduce risk of contamination from template, and these substantially reduced initial false-positive results. however, another kind of nonspecific amplification was also infrequently observed, which was distinguishable from contamination-derived false-positive results based on a different banding pattern on agarose gels (fig. ) . it has been hypothesized that this is due to non-template-driven self-priming. other groups were able to limit this phenomenon by attaching a fluorescent label to their loop primer, followed by the addition of a probe with a fluorescence quencher (curtis et al., ) . as the increased number of primers in our pan-serotype assay may facilitate nonspecific amplification, we examined whether sequence-specific detection was a feasible solution. at the end of the rt-lamp reaction, any unbound fam-loop probe was quenched by bhq-labeled probe (which consists of the reverse complement sequence of the fam-loop probe with a bhq modification.) we optimized this formulation and found no difference in the permissible temperature range, time to positivity, or number of serotypes detected when including a fluorescently labeled loop primer ( fig. a-c) . in fig. . characteristics of a pan-dengue rt-lamp assay. amplicons generated via rt-lamp were visualized via the incorporation of sybrsafe using agarose gel electrophoresis or uv illumination. a) copies/reaction denv at - °c, b) copies/reaction denv held at °c for - min, c) copies/reaction denv , , , or held at °c for min. ntc = no template control. fig. . comparison of limit of detection between pan-serotype rt-lamp and rt-pcr. ten-fold limiting dilutions of denv beginning at copies/reaction were used to compare the limit of detection of our optimized pan-dengue rt-lamp to an in-house rt-pcr assay. addition, this formulation was able to reduce the nonspecific amplification (fig. d ). in our hands, rt-lamp readout was not prone to operator error, and the positives and negatives were always clearly distinguishable. however, various methods to visualize rt-lamp results including real-time pcr thermocyclers, agarose gel electrophoresis, turbidity measurements, and uv illumination are not practical in resourcelimited settings. we thus explored the feasibility of determining rt-lamp positivity via an inexpensive lateral flow ict. we found % correlation between our agarose gel and ict results when using denv -, denv -, or denv -positive specimens. one specimen that was false negative when visualized via agarose gel electrophoresis was also negative via ict (fig. ) , suggesting that ict has sensitivity comparable to other methods. our data demonstrate that rt-lamp results can be reliably determined without the aid of expensive or bulky equipment. we further evaluated the ability of our rt-lamp assay to be visualized using a battery-operated handheld fluorescent reader and observed the kinetics of quenching over time. true-positive samples remain above a fluorescent threshold of units following hour of quenching. diagnosing dengue accurately during acute infection is critical for informing patient management decisions. lateral flow icts targeting the denv nonstructural protein (ns ) antigen have had some success in enabling early diagnosis at the point of need; however, nucleic acid tests (nats) may detect denv rna earlier than ns antigen, and nats can be more sensitive and specific. the primary disadvantage with pcr-based nats involves the need for thermocyclers, which are often bulky and too expensive to be used in resource-limited settings. commercially available pcr kits cost $ - /test and are generally unsuitable for high-throughput usage in developing economies (dineva et al., ; fiscus et al., ) . in addition, thermocyclers can cost $ , -$ , , which may be prohibitive in many dengueendemic countries (dineva et al., ) . training personnel to operate the instrument and interpret results also requires resources typically only found in a reference laboratory. pcr is also subject to inhibition by substances such as heme compounds, heparin, and edta often present in a clinical samples and blood collection tubes, leading to falsenegative results (klein et al., ; fredricks and relman, ) ; removing pcr inhibitors can lead to increased overhead costs and turnaround time to results. conversely, pcr is also often subject to reporting false positives from the presence of contaminating nucleic acids (fredricks and relman, ) , necessitating the use of template-free "clean rooms" for preparing a master mix, which is impractical in the field. to develop a diagnostic solution that overcomes these limitations, we developed a pan-denv rt-lamp assay. we demonstrate that the reaction can proceed continuously within a wide temperature range - °c, making it suitable for a water bath or electricity-free heater (labarre et al., ) . in addition, we have explored various methods of visualizing the assay results. electrophoresis gel confirmation of reaction products may be performed in a reference laboratory, but in the field, the reaction can be visualized in real time by measuring an increase in turbidity from magnesium pyrophosphate by-products (mori et al., ) . we propose a method involving the measurement of color change by the incorporation of sybr green adapted from a previous report, which was easier to read with the naked eye (parida et al., ) . we also explored visualization using lateral flow icts and demonstrated % concordance between rt-lamp visualized on a gel versus those visualized using icts. this format is likely to be easier to read in resource-limited settings where a uv light source is not available. finally, we explored the feasibility of using a handheld uv fluorescence reader, which when coupled with a quencher, can provide higher specificity detection of denv. the handheld uv reader that we used was approximately $ , and such readers could improve accuracy of readout as well as provide some level of quantitation to rt-lamp results. lamp has several other advantages over traditional pcr, including less sensitivity to inhibitors such as ethanol, isopropanol, edta, and sodium acetate (mori et al., ; kaneko et al., ) . the use of loop-specific primers can reduce assay times to provide faster sample to result than pcr, and other groups have reported higher sensitivity (nagamine et al., ) . lamp also does not require the template to be denatured , and contrasted with nasba (a similar isothermal nat), lamp does not require a separate nucleic acid extraction step (francois et al., ) . this robustness further reduces the cost as well as time to result. like qpcr, qlamp is amenable to multiplexing (tanner et al., ) , and we adapted it to detect all serotypes of denv in reaction. our data demonstrate that the use of degenerate primers is a feasible method by which to design assays for targets of interest even when sequence conservation is limited. the assay was able to detect a majority of denv pcr-positive clinical samples obtained from south america (sensitivity of . %), representing multiple circulating strains of denv - ; however, the clinical performance is limited with only dengue-positive clinical specimens from region and lack of inclusion of denv -positive specimens. the assay was also specific to denv and did not react to other pathogens that cause similar fever-like symptoms. this demonstrates the assay's effectiveness for enabling differential diagnosis in a clinical situation with multiple strains and serotypes. since this is a molecular assay, it will likely be able to detect denv in mosquito samples and have further applications for vector surveillance. due to the robustness of lamp, it may be used without explicit sample preparation in applications where having false negatives for low copy number samples are acceptable and with integrated sample preparation where it is not. acute denv viremic patients have - plaque-forming units/ml (vaughn et al., ) , and such high titers are detectable by lamp without explicit sample preparation. the assay reagents can also be lyophilized for field expediency and provided in a single tube to which one may add hydration buffer and test sample. the assay can be run at a range of temperatures and can be performed using a water bath or other available heat source. we also envision the assay readout adapted to specific applications and present uv visualization, handheld fluorescence readers, and lateral flow icts as viable alternatives. in conclusion, this work adds to the growing body of isothermal amplification assays available to detect denv. more than previous reports, we have explored all aspects of denv detection from sample to result, with an emphasis on visualizing the rt-lamp product, as readout is critical for ensuring accuracy. the assay can be adapted to use in dengue-endemic countries where it is most needed to reduce morbidity and mortality associated with this widespread disease. the views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the department of the navy, the department of defense, or the us government. dr allison dauner, ms indrani mitra, mr theron gilliland jr, ms sajeewane seales, and dr subhamoy pal are (or were) employed by the henry m. jackson foundation for the advancement of military medicine and were funded to do this work by the us government. dr shuenn-jue wu and dr tadeusz kochel are employees of the us government or members of the us military. this work was prepared as part of their official duties. title u.s.c. § provides that "copyright protection under this title is not available for any work of the united states government." title u.s.c. § defines a us government work as a work prepared by military service members or employees of the us government as part of those persons' official duties. this work was funded by the military infectious diseases research program, l _ _nm. the work was supported by work unit number .rad .l.a . fig. . sequence-specific detection. amplicons generated via sequence-specific rt-lamp were visualized via agarose gel electrophoresis or uv illumination minutes after the addition of a reverse complement loop quencher. a) copies/reaction denv at - °c, b) copies/reaction denv held at °c for - minutes, c) copies/reaction denv , , , or held at °c for minutes, d) replicates each of ntc (lanes - ) or denv (lanes - ) after minutes at °c. reactions were quenched for hours prior to uv illumination. e. quenching reaction measured using picflour shows detection of rt-lamp product following quenching. ntc = no template control. the global distribution and burden of dengue the use of confidence or fiducial limits illustrated in the case of the binomial rapid detection of hiv- by reverse-transcription, loopmediated isothermal amplification (rt-lamp) sequence-specific detection method for reverse transcription, loop-mediated isothermal 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simultaneous multiple target detection in real-time loop-mediated isothermal amplification trends in dengue diagnosis detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification rapid detection and quantification of japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification the molecular epidemiology of dengue viruses, genetic variation and microevolution the molecular epidemiology of dengue viruses, genetic variation and microevolution dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity this work would not have been possible without the generous support of the ministry of health peru, direccion general de epidemiologia and diresa piura, tumbes, madre de dios, and iquitos. key: cord- -slmlhqnb authors: yap, sally s. l.; nguyen-khuong, terry; rudd, pauline m.; alonso, sylvie title: dengue virus glycosylation: what do we know? date: - - journal: front microbiol doi: . /fmicb. . sha: doc_id: cord_uid: slmlhqnb in many infectious diseases caused by either viruses or bacteria, pathogen glycoproteins play important roles during the infection cycle, ranging from entry to successful intracellular replication and host immune evasion. dengue is no exception. dengue virus glycoproteins, envelope protein (e) and non-structural protein (ns ) are two popular sub-unit vaccine candidates. e protein on the virion surface is the major target of neutralizing antibodies. ns which is secreted during denv infection has been shown to induce a variety of host responses through its binding to several host factors. however, despite their critical role in disease and protection, the glycosylated variants of these two proteins and their biological importance have remained understudied. in this review, we seek to provide a comprehensive summary of the current knowledge on protein glycosylation in denv, and its role in virus biogenesis, host cell receptor interaction and disease pathogenesis. most denv infections are asymptomatic or remain as mild febrile illness. a classical den fever is diagnosed when the patient shows self-limiting high fever, headache, and muscle/joint pain - days after a mosquito bite. a small proportion of den patients may develop den hemorrhagic fever and/or den shock syndrome (dhf/dss) which are life-threatening. the clinical manifestations of dhf/dss include hemorrhagic fever, vascular permeability and plasma leakage, thrombocytopenia and circulatory failure in dss. to date, there is no specific treatment for den and no licensed anti-denv drug is available. for severe den cases, clinical complications are managed by supportive therapy to avoid mortality. the progression to severe den (dhf/dss) has been linked to a phenomenon known as ade of infection (halstead, ) . the ade hypothesis postulates that during a secondary heterologous denv infection, preexisting anti-denv antibodies bind to but fail to neutralize the virus, and promote increased uptake of sub-neutralized virions by fcgamma-receptor bearing cells such as dc, macrophages, and monocytes (kliks et al., ; boonnak et al., ) . in addition, ligation of fc receptor stimulates production of interleukin (il)- which in turn suppresses the cellular anti-viral response (suhrbier and la linn, ) . these events lead to increased viral loads which are believed to correlate with disease severity (vaughn et al., ) . to reduce den morbidity and eventually eliminate the disease, an effective vaccine is urgently needed. however, the development of den vaccine has been greatly hampered by the potential risk of ade. the only licensed den vaccine (cyd-tdv) available is a tetravalent, recombinant, live attenuated den vaccine developed by sanofi pasteur (guy et al., ) . the vaccine has shown varied efficacy against different serotypes and in different age groups, with safety issues in children below years of age (capeding et al., ; villar et al., ) . in addition, large scale efficacy studies have suggested that this vaccine works best in people with pre-existing denv immunity (capeding et al., ) . thus, who recommendations have limited the use of the cyd-tdv vaccine in geographical settings with high den burden and in age group - years old (who, ) . clearly, while this first-in-human tetravalent den vaccine will certainly provide a wealth of knowledge and improve our understanding of immune correlates of protection, a better vaccine is needed to protect the . billion people that are at risk of den infection. several promising vaccine candidates are currently under development; some have entered the clinical pipeline [reviewed in (schmitz et al., )] . it is hoped that they will address the shortcomings of the cyd-tdv vaccine. denv belongs to the family flaviviridae of which the members are well known as human pathogens, including wnv, zika virus, yellow fever virus, tick-borne encephalitis virus, jev, and hepatitis c virus (hvc). they are enveloped viruses with positive sense, single stranded rna and many of them are arthropod-borne viruses. among all flaviviruses, denv has the highest impact on global disease burden. the virus particle is about nm in size and the rna genome (∼ . kb) is encapsulated by a protein shell which consists of three structural proteins, namely capsid (c), envelope (e), and (pre)membrane protein (prm/m) (kuhn et al., ) . in order to establish infection, denv first binds to the host cell receptors via e proteins on the cell surface. the ligand-receptor interaction initiates uptake of the virion through receptor-mediated endocytosis (acosta et al., ) . inside the acidic late endosome, membrane fusion occurs as the virion envelope fuses with the endosomal membrane (allison et al., ; modis et al., ) , followed by uncoating of the nucleocapsid and then release of the viral rna into the cytoplasm. the rna genome of denv is translated into a single polyprotein by host ribosomes and is made of three structural (c, e, prm/m) and seven non-structural (ns) (ns , ns a/b, ns , ns a/b, ns ) proteins. the polyprotein is then cleaved by host and viral proteases to release individual viral proteins (acosta et al., ) . the viral genome replication process within the host cell is mainly driven by the ns proteins. ns anchors the replication complex to the er membrane and interacts physically with ns b (youn et al., ; muller and young, ) . ns a is responsible for viral rna synthesis and virion assembly (xie et al., ) . ns functions as a serine protease, rna helicase and nucleotide triphosphatase/rna triphosphatase, and its protease activity is dependent on the cofactor ns b (falgout et al., ) . ns a has been reported to induce membrane rearrangement within the host cell, thereby assisting the formation of replication vesicles (miller et al., ) . ns is a multifunctional enzyme with a methyltransferase (mtase superfamily) domain and a rnadependent rna polymerase domain (acosta et al., ) . during virion assembly, the newly synthesized viral rna interacts with c proteins to form the nucleocapsid. a spiky immature virion is formed when e and prm proteins encounter the nucleocapsid acosta et al., ) . the maturation process takes place in the trans-golgi network where prm is cleaved by host furin to generate a smooth surfaced mature virion (stadler et al., ; yu et al., ) , which is released into the extracellular environment through the secretory pathway. glycosylation is the post-translational modification of biomolecules such as proteins or lipids through the enzymatic attachment of complex oligosaccharide structures to the peptide backbone or lipid anchor (varki et al., ) . over % of the eukaryotic proteome is glycosylated (dell et al., ) . the range of complexity of these structures is reflected by their covalent attachment to the protein, the monosaccharide composition of the glycan and combinations of anomeric ring linkages between these monosaccharides. this complexity affects the branching, antennae and topology of the glycan structures, which translates to the overall tertiary and quaternary structure of the glycoprotein. there are two types of protein glycosylation distinguished by their site of attachment on the protein backbone; n-linked glycosylation where the glycan is covalently attached to the asparagine (n) (consensus motif; nxs/t except where x is a proline); and o-linked glycosylation where the glycan is linked to the oxygen from some serine (s) or threonine (t) residues of the protein backbone (chandler et al., ; varki et al., ) . n-linked glycans share a common chitobiose core structure. furthermore, n-linked structures fall under three different classes: ( ) high mannose, where the non-reducing composition of the glycans are dominated by mannose sugars that extend from the core, ( ) complex, where the branching and extension of the glycans from the core is initiated by n-acetyl glucosaminyl transferases; and ( ) hybrid structures where the core is extended by both high mannose arm and a complex structure on the other arm (chandler et al., ; varki et al., ) (figure ) . such complexity can be found with the isomerism/anomercity of a sugar. whilst two glycans may have the same composition, the differences of their isomeric linkages can affect the selectivity of their host receptors and thus biology. for example, in the context of avian influenza, the haemagglutinin specifically and exclusively recognizes , -linked sialic acids that are found in the avian host. this is in contrast to , linked sialic acid normally found in the human host receptors with very low , -linked sialic acid expressed in the lower respiratory tract. cross reaction between avian h n and such sialic acids caused the recent human epidemics (shinya et al., ; walther et al., ) . glycosylation is a highly organized process that involves a network of glycotransferases and glycosidases in the er-golgi complex that enzymatically synthesize the glycan as well as trim down the structures so as to achieve the refined structure. in mammalian cells, n-glycosylation takes place predominantly within the er. briefly, the initial stages of glycosylation involve the enzymatic synthesis of dolicholphosphate-nacglcnac man glc which usually takes place across the membrane of the er. this involves firstly the enzymatic synthesis of dolichol-p-p-glcnac man , before this is "flipped" across the membrane into the er lumen where further monosaccharides are added (shi and jarvis, ; dell et al., ) . the nacglcnac man glc is transferred to the appropriate nxs/t motif of the protein via the oligosaccharyltransferase as the incipient protein is being translated. as the n-glycans transition through the er-golgi complex, a series of glycosidases trim down the mannose residues before glycotransferases present in the golgi extend the antennae of the glycans to produce larger hybrid or complex structures (varki et al., ) . in contrast to n-glycosylation, o-glycosylation occurs entirely in the golgi apparatus. it does not involve any glyco-lipid intermediates and no glycosidases appear to be involved in their synthesis and processing. within the insect kingdom, glycosylation is far simpler yet interestingly insect are able to produce elaborate protein glycosylation in a restricted fashion compared to glycosylation of higher eukaryotes (rendić et al., ) . most our knowledge in insect glycosylation was derived from studies performed on drosophila melanogaster and baculoviral-insect systems. in fact, the bulk of mammalian glycoproteins expressed and/or purified in insect cells have used cell lines from spodoptera frugiperda (sf , sf ) or trichoplusia ni (high five) (rendić et al., ) . glycoproteins derived from these insect cell systems display glycans that contain predominantly high mannose type structures. however, the long belief that these high mannose and paucimannosidic n-linked structures are the dominant forms in insect-cell derived glycoproteins has been recently challenged by the advent of high resolution glyco-analytical tools which were able to identify glyco-epitopes such as [alpha] - fucosylation (hsu et al., ; takahashi et al., ; rudd et al., ) and double core fucosylated structures ([alpha] - and [alpha] - fucosylation on the core glcnac) (staudacher et al., ; rendic et al., ) . there were also reports of the extension of the [alpha] , -arm of the chitobiose core as opposed to the [alpha] - arm extension found in most mammalian cell lines (kubelka et al., ) . higher complex n-glycans can be found in many insectderived n-linked glycoproteins, however, if grown in serum free media, lysed cell extracts from sf and high five cells do lack the nucleotide donors for sialic acid (cmp-neuac) (rendić et al., ) . further to this, the presence of a truncated trimannosyl n-glycan with an [alpha] , -linked fucose was reported (shi and jarvis, ; rendić et al., ) . extensive work has shown that such structures are the result of the action of an endogenous hexosaminidase specific for the nacglcnac[beta] , -man structure rather than low activity of the [beta]- , nac glcnac transferase ii responsible for the extension of the mannose arms of complex structures (kubelka et al., ) . studies performed with mosquito cell lines a. albopictus and a. aegypti showed that glycoproteins produced in these cell lines display predominantly high mannose and pauci-mannosidic structures (hsieh and robbins, ) . interestingly, within these initial experiments, the presence of mannosidase-resistant structures was observed (rhomberg et al., ) . glycomics as a field has experienced a significant maturation from low to high resolution analysis. in the past decades, scientists have mostly relied on digestive enzymes (johnson et al., ; mondotte et al., ; hacker et al., ) , chromatography (johnson et al., ) , lectin-binding assay (johnson et al., ; hacker et al., ) and radioactive labeling (smith and wright, ) to study the glycan structure on glycoproteins. enzymatic digestion by endo h and peptide:n-glycosidase (pngase f) remains the most popular method due to its simplicity and allows the investigator to determine whether the glycan structure is asparagine linked (n-linked). in this approach, purified glycoprotein is subjected to specific enzymatic digestion prior to separation on sds-page. after cleavage of the attached oligosaccharide chains, the digested protein migrates ahead of the undigested form due to a lower molecular weight. enzymatic digestion of glycoproteins reveals, however, relatively little information on the glycan structure. over the recent decade, glycan analysis has dramatically improved through developments in fluorescent labeling, lc and mass spectrometry. there is an abundance of techniques such as lc, ce and mass spectrometry which are available for glycomic analysis. for lc and ce, the field has benefited from labels such as -ab, -aa (lc separation) and -aminopyrene- , , -trisulfonic acid (ce separation), whereby glycans are tagged at a glycan's reducing end and the identity of these glycans is assigned based on their retention time behavior across a hilic or ce, respectively (rudd and dwek, ; callewaert et al., ) . approaches coupling fluorescence with mass spectrometry (flr-ms) have helped increase the efficiency of glycomic approaches. in such a platform, the retention time and fluorescence are usually coupled with mass detection to add further confirmation (houel et al., ; zhang et al., ) . the use of exoglycosidase arrays adds further confidence to a glycan's structure elucidation and can help to identify co-eluting glycan species (marino et al., ) . the evolving development of glycan labeling has meant that newer, faster labeling and highly sensitive labels such as procainamide or rapidfluor mass spectrometry (rfms) label can increase the throughput and efficiency of a glycomic analysis. various modes of mass spectrometry have been applied to the analysis of released glycans. the most common are maldi and electrospray ionization. maldi is a straight-forward method that often requires methods such as permethylation and esterification to increase signal intensity and stabilize labile glycans that contain sialic acids. electrospray ionization involves a milder desolvation technique and coupled with lc methods and further fragmentation of the molecule, provides high resolution techniques to qualitatively characterize a glycome (nguyen-khuong et al., ) . detection of glycan fragments which result from fragmentation along the glycosidic bonds (detected in positive mode) and cross-ring (detected in negative mode) (harvey et al., ; everest-dass et al., ) help to understand the composition and topology of the glycan without the need to adulterate the glycan through derivatization. glycoproteomics allows investigators to understand the degree of glycosylation on various sites of the glycoprotein. the platform is adapted from proteomics and as such relies heavily upon mass spectrometry and substantial data analysis. whilst most glycoproteomic methodologies follow similar approaches to proteomics such as trypsin digestion and analysis, key to any glycoproteomics method is the enrichment of glycopeptides (mysling et al., ; kolarich et al., ) . this is important to reduce the ion suppression from peptide mass spectrometry signals without enrichment. this can be performed via hilic chromatography, in which the enrichment is centered upon exploiting a glycan's hydrophilicity. fragmentation data is vital to glycopeptide identification and data analysis must be able to exploit the information which can come from fragmentation modes such as collisionally induced dissociation (cid), high collisional dissociation or electron transfer dissociation/electron transfer high collisional dissociation (etd/ethcd) available to the investigator (scott et al., ; thaysen-andersen et al., ; stavenhagen et al., ) . depending on the strength of fragmentation, information such as glycan composition, site of attachment and peptide backbone are all able to be divulged from a single spectrum (yang et al., ) . glycans either directly or indirectly have diverse biological functions which span but are not limited to inflammation, immunology, infectious diseases, metabolism, embryogenesis, cancer biology and neurodegeneration. at the protein level, glycosylation is responsible for correct protein folding/structure, protein trafficking and stability, receptor/ligand recognition as well as increasing its half-life in the blood stream (park et al., ; bork et al., ; sumer-bayraktar et al., ; hart, ; palmisano et al., ) . at the cellular level, complex sugar structures modulate receptor functions and thus are integral to regulate normal cell-cell, cell-substrate communication and adhesion (vercoutter-edouart et al., ; varki et al., ) . glycosylation disorders can adversely affect immunity and cancer development. from an immunological perspective, all living cells are covered by a dense glycocalyx and indeed, pathogens and foreign objects must deal with this complex forest of cell surface glycoconjugates upon entering the host (pickles et al., ; rodrigues et al., ) . viruses do not possess their own glycosylation machinery and by virtue of their opportunistic nature, are heavily dependent upon the glycosylation machinery of the host cell to glycosylate their proteins. hiv, influenza virus, hendra virus, severe acute respiratory syndrome coronavirus (sars-cov), hepatitis viruses and wnv are examples of viruses for which glycosylation was shown to be critical to their stability, infectivity and antigenicity (mir-shekari et al., ; vigerust and shepherd, ; medina et al., ; doores, ) . firstly, glycosylation can be involved in receptor binding. this is exemplified by hiv and denv which rely on high mannose type glycosylation to bind to their mrs or dc-sign that are present on host immune cells (cambi and figdor, ; cambi et al., ) . furthermore, glycosylation is required to facilitate proper protein folding and trafficking of the viral membranes using the host chaperones such as calnexin and/or calreticulin proteins (meunier et al., ; land and braakman, ; slater-handshy et al., ) . importantly, glycosylation is a means to evade immune recognition within the host by changing glycan sites (medina et al., ) , which in turn can increase the diversity of the glycosylation on the virus. in addition, the glycan structure has been reported to mask particular antigenic sites from recognition by neutralizing antibodies (doores, ; walls et al., ) . the external protein shell of den virion consists of copies of e ( - kda) and prm glycoproteins whereby only e proteins are exposed on the surface (kuhn et al., ) . extensive research over the years has revealed multiple functions of e protein in host receptor attachment, cellular uptake of virion and membrane fusion. e protein forms dimers on the virion surface (kuhn et al., ) . the ectodomain of each e monomer without the transmembrane domains and membrane-associated "stem" region displays an elongated structure under cryo-em, which is further defined into three distinct domains (domains i, ii, and iii) (rey et al., ) . the central n-terminal di separates the dimerization dii from the c-terminal diii. diii has been proposed to be the receptorbinding domain (kuhn et al., ) whereby neutralizing monoclonal antibodies against diii most efficiently block virus initial attachment to mammalian cells (crill and roehrig, ) . the fusion peptide located at the tip of dii is essential for endosomal membrane fusion and is essential for virus entry (allison et al., ; kuhn et al., ; huang et al., ) . dimerization of e proteins at neutral ph positions the fusion loop into a hydrophobic pocket formed by di and diii of the adjacent e monomer. this helps to prevent premature exposure of the fusion loop before endocytosis of the virion by a new host cell. di forms part of the flexible hinge region which facilitates structural rearrangement of e protein during virion maturation and fusion process (zhang et al., ) . inside the acidic endosome, the ph-dependent hinge at the di-dii interface (allison et al., ; modis et al., ) allows e dimer to dissociate and rearrange into a trimeric form which serves as a pre-fusion intermediate promoting membrane fusion (modis et al., ) . in the er lumen of the host cell, membrane-associated e protein is generated after co-translational processing of the viral precursor polypeptide by host signalase (acosta et al., ) . the newly synthesized e protein rapidly heterodimerizes with prm (lorenz et al., ) and three prm-e heterodimers further oligomerize to form a total of sixty heterotrimeric prm-e spikes per subviral particle (konishi and mason, ; zhang et al., ) . this higher-order oligomer has been proposed to represent the preassembly complex (wang et al., ) . translocation of this complex from er to golgi is critical as the transition from immature to mature virion is completed only in the trans-golgi network, where the spiky prm-e trimers are rearranged into flat dimers in a head-to-tail orientation on the virion surface (kuhn et al., ; zhang et al., ) . in the er, denv e protein undergoes n-linked glycosylation at two asparagines, n and n located in dii and di, respectively (chambers et al., ; johnson et al., ; hacker et al., ) . the n glycosylation site is unique to denv and has been proposed to interact directly with dc-sign, one of the host cell receptors (pokidysheva et al., ) (see virus attachment to cell surface and cell entry process). in contrast, n (n in other flaviviruses) represents the conserved glycosylation site in the family flaviviridae. high resolution crystal structure of tick-borne encephalitis virus e dimer shows the n -oligosaccharide chain projected overhead of the hydrophobic groove where the fusion loop fits in, suggesting that it functions as an "epitope shield" over the fusion loop to stabilize the dimer contacts (rey et al., ) . consistently, denv and denv mutant viruses lacking n glycans due to a single point mutation within the glycosylation motif displayed elevated fusion ph threshold compared to their parental counterpart (guirakhoo et al., ; lee et al., ) . the authors proposed that the altered fusion activity of these mutants was likely due to instability of the e dimers. the glycan structure on denv e protein has been studied using digestive enzymes (johnson et al., ; mondotte et al., ; hacker et al., ) , chromatography (johnson et al., ) , lectin-binding assay (johnson et al., ; hacker et al., ) and radioactive labeling (smith and wright, ) . endo hand pngase f-enzymatic digestion revealed the presence of n-glycosylation in denv e protein. no o-linked glycan has been detected to date (johnson et al., ) . in mosquito cell-derived virions, the n-glycans attached to e protein display heterogeneity in structure and sugar composition where high mannose and paucimannose with terminal mannose residues are the dominant glycoforms (figure a ) (smith and wright, ; johnson et al., ; hacker et al., ) . recently mass spectroscopy has been applied to denv glycoprotein studies to provide a comprehensive and detailed profile of the glycan moieties (dubayle et al., ; lei et al., ) . using an integrated mass spectroscopy strategy consisting of lectin microarray and maldi-time of flight mass spectrometry (maldi tof-ms), lei et al. ( ) have successfully determined the detailed composition of n-glycans attached to the e protein from mosquito cell derived mature denv . among the distinct n-glycans detected, contain terminal galactosylation while the remaining glycans were identified as high mannose type, complex type, fucosylated and sialylated n-glycans. in a separate study, the n-glycans from denv - (vaccine cyd-tdv) produced in mammalian vero cells have been reported to consist of high mannose, complex and hybrid glycans with complex glycans as the major glycan species (figure a ) (dubayle et al., ) . by performing in-gel proteolysis of e-protein, site specific n-glycans have been determined. sialylated complex glycans and high mannose ( - residues) glycans were detected at n in all denv except for denv . besides, most of the complex or hybrid glycans at n were found fucosylated. interestingly, fucosylated glycans were detected only at n but not at n across all four denv serotypes. since high mannose binding dc-sign interacts only with n glycans on the viral surface (pokidysheva et al., ) and n -glycan is dispensable for virus production in mosquito and mammalian cells (bryant et al., ) , this suggests that n glycans may serve a distinct function from n glycans in den pathogenesis possibly via interaction with an unknown fucose binder or act as a viral glycan shield. for n specific glycans, denv was reported to have a different sugar composition from the other three denv serotypes (dubayle et al., ) whereby a higher content of complex or hybrid glycans was found in denv . high mannose glycans were detected as the main glycan species for denv , and . in addition, sialylated n-glycan was detected only in denv at this site. the differential glycosylation pattern between denv and denv , , may impact on various aspects of dengue pathogenesis including virus tropism, virus fitness, and induction of host responses (see role of glycosylation in denv life cycle). non-structural protein (ns ) was first identified as a nonhemagglutinating, soluble complement-fixing antigen in the brain and serum from denv -infected mice (brandt et al., ; smith and wright, ) . ns has a molecular weight range of - kda depending on its glycosylation status. it is a multifunctional glycoprotein which presents in different oligomeric forms and locates at various cellular compartments (westaway and goodman, ; flamand et al., ) . non-structural protein monomer consists of three structural domains namely a β-roll dimerization domain, a wing domain and a β-ladder domain (akey et al., ) . the monomer structure is stabilized by six intramolecular disulfide bonds and no intermolecular disulfide bond has been identified in dimeric ns (winkler et al., ) . however, any one of the three cysteine residues at the c-terminal has been reported to be important for dimer formation (pryor and wright, ) . the β-roll domain and part of the extended wing domain form a hydrophobic protrusion surface that acts as the er membrane and replication complex (ns b) interacting site, which is critical for viral rna replication (youn et al., ; akey et al., ) . ns dimer is formed when two β-roll domains dimerize at the center and these dimers tend to trimerize resulting in hexameric ns (flamand et al., ; gutsche et al., ; muller et al., ) . the ns hexamer crystal structure revealed a barrel-shaped oligomer with a central open channel. three dimers are arranged symmetrically in a way such that the β-roll domains are entirely facing inwards and the channel interior is lined by the hydrophobic protrusion surface contributed by each dimeric component (akey et al., ) . the hydrophobic lining allows the ns hexamer to be secreted as a lipoprotein whereby the lipid cargo is loaded into the central channel (gutsche et al., ) . in contrast to the β-roll domains, glycosylation sites and most of the linear epitopes of ns identified are facing outward, representing the most accessible parts of ns hexamer by host antibodies (akey et al., ) . intracellular ns is predominantly in dimeric form whereas secreted ns is mainly in hexameric form ( figure b ) (flamand et al., ) . during protein synthesis, ns is cleaved from the viral polypeptide and translocated into the er lumen. in er, newly synthesized e protein is glycosylated and heterodimerizes with prm protein to form a higher order oligomeric preassembly complex. the immature virus particle with prm-e spikes is formed when the nucleocapsid associates with prm-e-rich membranes which buds into the er lumen ( ). the glycans remain of high mannose type on the immature virus particle as it is translocated to golgi apparatus along the secretory pathway ( ). the conformational rearrangement of prm-e spikes and cleavage of prm by host protease furin occurs in the golgi to produce a mature, smooth virus particle. in mammalian cells, the glycans are further processed and modified into complex glycans before the virus particle is released to the extracellular milieu (route a). in mosquito cells, majority of the glycans are high mannose or galactosylated due to the different glycosylation enzymes expressed in insect cells (route b). high mannose glycan on the e protein, particularly the n -glycan facilitates dc-sign(+) cell infection and virus propagation. the function of complex glycan on e protein is currently unknown. the glycosylated pr peptides are bound to e protein after furin cleavage and only dissociate at neutral ph in the extracellular milieu. (b) monomeric ns protein is glycosylated with high mannose glycans at n and n . the monomer rapidly dimerizes in the er and membrane-associated ns dimers ( ) are involved in virus rna replication. three ns dimers form a soluble hexameric ns but the exact location of hexamer formation remains unknown ( ). in mammalian cells, the n glycans are modified into complex glycans before the soluble ns hexamer is secreted out of the cells (route a). in mosquito cells, generation of complex glycans doesn't happen and the lack of complex glycans (n ) on ns hexamer affects hexamer stability and greatly reduces its secretion (route b). a subset of dimeric ns are found on the infected cell surface but the trafficking pathway has yet to be determined (route c). high-mannose glycan at n stabilizes ns dimer. the soluble monomer undergoes dimerization to gain partial hydrophobicity (flamand et al., ) , allowing membrane association of ns dimer in the absence of a transmembrane domain (winkler et al., ) . the exact mechanism of ns hexamer formation remains unclear and two possible locations have been proposed including along the golgi secretory pathway, or immediately after dimerization at the er (muller and young, ) . the functions of ns are closely associated to its cellular location throughout the virus replication cycle. er membraneassociated dimeric ns has been found to co-localize with viral dsrna (mackenzie et al., ) . circulating hexameric ns is able to bind to the plasma membrane of mammalian cells via the interaction between its n-glycans and cell surface glycosaminoglycans, heparin sulfate and chondroitin sulfate e (avirutnan et al., ) . recently, it has been reported that hexameric ns contributes to disease pathogenesis of severe den (beatty et al., ; modhiran et al., ) . the soluble protein acts as a viral toxin that induces pro-inflammatory cytokine response and vascular leakage via toll-like receptor expressed on immune cells and endothelial cells (modhiran et al., ) . beatty et al. ( ) showed that ns vaccination protects mice from ns -induced vascular leakage which was independent of complement components. glycosylation of denv ns occurs right after its cleavage in the er (winkler et al., ) at two asparagines, n and n (putnak et al., ; winkler et al., ; flamand et al., ) . these two n-glycosylation sites are conserved in the family flaviviridae. recently, a less conserved glycosylation site at n has been reported in wnv, st. louis encephalitis virus and murray valley encephalitis virus but is absent in all four serotypes of denv (akey et al., ) . intracellular and extracellular denv ns display different types of n-glycans as the oligosaccharides undergo modification during the maturation process (winkler et al., ; pryor and wright, ; flamand et al., ) . intracellular dimeric ns n-glycans are of high mannose composition regardless of the host cell type (mammalian or mosquito cell) (mason, ) . on the other hand, in extracellular hexameric ns , the n -glycans consist of complex oligosaccharides whereas the n -glycans are made of high mannose type sugar chains (mason, ; pryor and wright, ; flamand et al., ) . as dimeric ns passes through the golgi apparatus, two n -glycans are further modified into the endo h-resistant, multi-branched complex type before the protein is released (winkler et al., ) . the differential modification at these two sites is due to the inaccessibility of n -glycan by golgi-resident enzymes after the dimerization of ns (flamand et al., ) . in the denv replication cycle, prm interacts with e protein and acts as a chaperone to ensure proper e protein folding (lorenz et al., ) and to prevent premature fusion of the virus particle along the secretory pathway by concealing the e fusion loop yu et al., ) . glycosylation of the prm/m glycoprotein in denv has not been extensively studied. the protein is glycosylated at n (table ) with circumstantial evidence for n-linked glycosylation at sites , , and (courageot et al., ) . it was found that α-glucosidase inhibitor reduced the amount of prm-e heterodimer, suggesting the n-glycans are required for productive folding pathway of these glycoproteins (courageot et al., ) . triglucosylated n-glycan at n of denv affects the folding of prm by causing a delayed formation of prm-e heterodimer (courageot et al., ) . n-glycosylation on both e and ns proteins has been shown to play important roles throughout the denv infection cycle from virion attachment, entry, maturation, assembly to secretion. carbohydrate chains on the denv e proteins play a critical role in host cell infection at the early step of host receptor binding. indeed, virus attachment and penetration into mammalian and mosquito cells were blocked by pre-incubation of virus with concanavalin a, a plant lectin that binds to alpha-linked terminal mannose of high mannose or hybrid glycans (hung, ) . lectins are a group of proteins that recognize carbohydrates through a carbohydrate recognition domain [reviewed in ±the n-glycosylation site is located at residue to depending on the serotype. (zelensky and gready, ) ]. to date, various lectin families such as c-type, p-type, l-type, galectin and calnexin have been shown to interact with viral components . c-type lectins are particularly important for denv infection as they have been shown to be involved in host cell attachment and disease pathogenesis (see disease pathogenesis). the cell membrane-anchored c-type lectin dc-sign has been identified as host cell receptor for many viruses , among which denv infects dc and monocyte via dc-sign (navarro-sanchez et al., ; tassaneetrithep et al., ) . the interaction between dc-sign and denv can be inhibited by the addition of mannose and mannan and has been further examined at the molecular level by structural analysis. cryo-em data of denv/dc-sign complexes reveals that the carbohydrate recognition domain of dc-sign interacts directly with the n -glycan of e dimers (pokidysheva et al., ) . consistently, lectin (hha)-resistant denv which lacks both n-glycosylation sites on e protein failed to infect dc-sign(+) dc, in contrast to productive infection and replication in dc-sign(−) and carbohydrate-independent cells such as vero, huh , c / and baby hamster kidney fibroblasts (bhk- ) (alen et al., ) . the presence of n -glycan on e protein also allows denv to infect endothelial cells in liver and lymph node via dc-sign-related proteins known as dc-signr and l-sign, the close homologues of dc-sign (tassaneetrithep et al., ; alen et al., ) . in addition to dc-sign, mr has been identified as another c-type lectin utilized by all four serotypes of denv to infect macrophages and dc (miller et al., ) . both mr and dc-sign bind to denv e protein with high affinity (k d in the sub-nanomolar range) (lo et al., ) , despite a different ligand specificity for these two host receptors (miller et al., ) . mr shows a preferential binding to terminal mannose, fucose and n-acetyl glucosamine while dc-sign binds to high-mannose oligosaccharides (miller et al., ) . as dc-sign and mr have been proposed to be the primary host receptors for denv during infection (lo et al., ) , the engagement to these c-type lectin receptors with diverse glycoforms of e protein may allow denv to infect a wide range of host cells. in contrast and interestingly, n deglycosylated (n − ) denv and denv were found to display enhanced infectivity in mosquito cells (c / ) compared to wild type (wt) (ishak et al., ; lee et al., ; alen et al., ) . for mosquito cells, the entry mode employed by flavivirus (denv and jev) was shown to involve membrane fusion instead of receptormediated endocytosis (hase et al., a,b) . hence, it is possible that absence of the n -glycans from the virion surface reduces steric hindrance and therefore promotes cell membrane attachment and membrane fusion. finally, n deglycosylated (n − ) denv mutant displayed reduced infectivity ( -fold lower) in both mammalian and mosquito cells compared to wt, possibly due to impaired virus entry process (lee et al., ; hacker et al., ) , whereby loss of the n -glycan affected the conformational stability of e proteins and led to premature exposure of the fusion peptide (yoshii et al., ) . early studies on denv e protein showed that n-glycosylation is not essential for virus replication in mosquito cells (bryant et al., ; mondotte et al., ) . instead, loss of the n glycosylation site through site directed mutagenesis (n q) in e protein was sufficient to render denv (strain ) growth defective in bhk- cells, a dc-sign(−) cell line (bryant et al., ) . direct transfection of n q mutant rna into bhk- cells neither produced intracellular viral antigen nor released new virus progeny. the lack of virion release may be due to impaired virion secretion along the er-golgi secretory pathway in the absence of n -glycan tag on e protein. however, the same mutant replicated and grew comparably to wt counterpart in c / mosquito cells in vitro and in a. aegypti mosquito in vivo (bryant et al., ) , thus supporting that n-glycosylation of e protein at position n is essential for productive infection in mammalian cells only, consistent with earlier studies (bryant et al., ; mondotte et al., ) . further investigation on the importance of the n-glycosylation motif was done by lee et al. ( ) through extensive point mutation within the conserved n-x-t/s motif of denv (strains puo- and ngc), whereby the conserved residue t was replaced with residues of different side chain propensity. replacement of t by a larger and more hydrophobic residue (leucine and valine) either by molecular cloning (lee et al., ) or by passaging the virus under selection pressure (alen et al., ) generated viable virus that retained efficient growth in bhk- and vero cells in the absence of n glycan. in addition, n q/d mutant virus generated in strain puo- propagated in mammalian cells at reduced growth rate, which is inconsistent with previous studies carried out with denv strain (bryant et al., ; mondotte et al., ) . the differential and virus strain-dependent outcome led to the hypothesis that in the absence of n glycosylation, the aminoacid composition of the dii region determines virus survival in mammalian cells (lee et al., ) . multiple sequence alignment showed indeed that strain differed from strains puo- and ngc at two positions, arginine (r) in dii and t in di (figure ) . uncharged polar threonine replaces charged r and hydrophobic isoleucine (i) replaces t in strains puo- and ngc. it is possible that the presence of hydrophobic residues facilitates protein folding even without the glycan tag for chaperone-assisted folding and followed by productive protein secretion. nevertheless, structural comparison of the wt strains and their respective mutants needs to be carried out to confirm this hypothesis. in contrast to the varying outcomes obtained with n mutant viruses and their ability to grow in mammalian cells, it has been consistently reported that these virus variants replicate, propagate in mosquito cells but produce lower virus titers compared to wt (bryant et al., ; mondotte et al., ; lee et al., ) . n -glycan on e protein is important but not essential for denv survival in mosquito cells. successive passages (as low as two passages) of denv in c / cells in vitro resulted in mutation at t which ablates the n -glycosylation motif; however, the non-glycosylated variant was able to propagate in mosquito cells (lee et al., ) . n − denv grows in both mammalian cells and c / cells and produce lower virus titer than its wt counterpart (bryant et al., ; lee et al., ) , which could be due to defective virus budding. consistently, in a study using transmission electron microscopy, it was shown that virus budding of wt wnv occurs at the plasma membrane while the mature progeny of n − mutant scatters at the smooth membrane vesicle within swollen er lumen without budding (li et al., ) . n a ns mutant virus of denv (tajima et al., ) and denv (ngc) (pryor et al., ) failed to generate viable virus in both mammalian and mosquito cells. mutation at n in denv ns caused reduced viral growth in mammalian cells and c / cells (pletnev et al., ) . however, n q ns mutant of denv ( ) produced infectious virus with a similar titer as the wt virus in mammalian cells but with a reduced titer in c / cells (crabtree et al., ) . removal of glycan from n in denv and denv ( ) ns protein produced similar growth and virus titers compared to wt in mammalian cells despite a delayed cytopathic effect (crabtree et al., ; tajima et al., ) , which is not consistent with the observation on denv (ngc) (pryor et al., ) . double mutation attempts (n q/n q and t n/t n) failed to generate genetically stable mutant viruses (crabtree et al., ) . taken together, the findings suggest that at least one of the two n-glycosylation sites (probably n ) in ns protein is essential to produce viable virus. similar to e protein, the impact of deglycosylation at this site varies depending on the virus strain and amino acid residue used for replacement. in denv, n -glycosylation is important but not essential for ns secretion in mammalian cells (despres et al., ; jacobs et al., ; pryor and wright, ; crabtree et al., ) . single and double ns mutant proteins are secreted from infected cells even though a reduced secretion yield has been observed (crabtree et al., ; somnuke et al., ) . the impact of deglycosylation on secretion is thought to be associated with the stability of the ns oligomer. mutation of n or n does not affect dimerization of the protein but compromises the stability of the dimer (winkler et al., ; pryor and wright, ) . the dimer appeared more heat sensitive when the n-glycan was removed from the protein especially for n a mutant. the use of tunicamycin, an enzyme targeting the host glycosylation enzymes, allowed confirm that absence of n-glycan was solely responsible for the instability of ns oligomers instead of changes in the polypeptide backbone in the genetically deglycosylated mutants (pryor and wright, ; flamand et al., ) . furthermore, the secretion of ns was reduced when complex glycans maturation was blocked by glycosylation inhibitors swainsonine and -deoxymannojirimycin (flamand et al., ) . consistently, low levels of ns with solely high mannose glycan are secreted from infected mosquito cells, which lack the enzymes to generate complex type glycans. these findings support the proposal that n-glycosylation and complex glycan are important for ns secretion (hsieh and robbins, ; mason, ; thiemmeca et al., ) . in addition, the majority of the secreted wt and n q ns proteins are hexamers (somnuke et al., ) , whereas secreted n q and n /n q ns proteins showed reduced hexamer population and increase in higher order oligomer (> kda) population. as compared to mammalian cell-secreted ns , mosquito cell-secreted ns is less stable and undergoes degradation more rapidly at body temperature (thiemmeca et al., ) . hence, the presence of complex glycan at n is critical for both ns secretion and ns hexamer stability (flamand et al., ; somnuke et al., ) . similarly, it has been proposed that high-mannose glycans at n stabilizes ns dimer (pryor et al., ; flamand et al., ; somnuke et al., ) . reduced levels of denv n q and n q/n q ns proteins were observed in culture supernatant (somnuke et al., ) which could be explained by two possible scenari: ( ) stability of the secreted mutant forms is compromised due to a different protein conformation (somnuke et al., ) . the misfolding of the protein may lead to a less effective secretion of functional hexamer. ( ) transport of the protein from the perinuclear region is affected which in turn compromises the maturation and secretion of the protein (crabtree et al., ) . deglycosylated ns mutant viruses (n − ) are less neurovirulent as evidenced by the reduced mortality observed with mice infected intracranially with the denv and denv mutants (pletnev et al., ; crabtree et al., ) . the reduced neurovirulence of these viruses which lack the complex type glycans may be linked to the reduced levels of extracellular hexameric ns (crabtree et al., ) . the virulence phenotypes observed with n − mutant viruses varied depending on the denv strain. denv n − mutant displayed decreased virulence (pryor et al., ; crabtree et al., ) , whereas the denv n − mutant showed enhanced virulence in mice (pletnev et al., ) . the low to undetectable levels of ns specific antibodies in mice infected with the denv n − mutant suggests that the enhanced neurovirulence could be attributed to the reduced immunogenicity of the virus (pletnev et al., ) . the complement cascade is the central defense mechanism of innate immunity which triggers the immune effector function to remove infectious pathogens and modified self cells upon activation. the activation and amplification of the complement pathway involves a series of sequential events and the whole process is tightly regulated. complement can be activated through three major pathways, namely the classical, lectin and alternative pathways [reviewed in (ricklin et al., ) ]. the classical pathway is triggered by antibody-antigen complexes whereas the lectin pathway is activated by carbohydrate moieties on the microbial surface. the alternative pathway is activated through direct binding of c b at the surface of pathogens, which results from the constitutive basal cleavage of c (ricklin et al., ) . mannose binding lectin (mbl) in the lectin pathway triggers antibody-independent activation of complement (thielens et al., ) . the proposed mbl-mediated virus elimination mechanisms include ( ) direct virus neutralization, ( ) c /c deposition on virus surface and ( ) interference of host cell lectin receptor binding . mbl differentiates self-from non-self-antigens based on a sugar density-dependent recognition mechanism (dam and brewer, ) , and the micro pattern of the oligosaccharides structure in addition to the spatial geometry of the macro sugar pattern (takahashi and ezekowitz, ) . it was proposed that the additional n -glycan in denv (which is absent in other flaviviruses) could promote a more efficient recognition and binding by mbl . this hypothesis is supported by improved mbl binding and in vivo virus clearance of a genetically engineered wnv with additional n -glycosylation site . mbl is reactive to high mannose oligosaccharides and thus can efficiently recognize insect cell-derived denv with high mannose glycans present on its e proteins. hence, the change of n-glycan profile of e protein after one round of replication in mammalian host cells may provide an opportunity to the virus to escape from effective mbl recognition (fuchs et al., ) . however, mammalian cell-derived denv was found to be effectively inhibited and neutralized by mouse mbl . a separate study instead reported preferential binding of recombinant human mbl to insect cell-derived denv , whereas virions produced in monocyte-derived dc were not neutralized by human mbl . it therefore remains unclear whether mbl-mediated virus clearance is optimally engaged during denv infection in humans. furthermore, studies have shown that ns interferes with the complement pathway through binding to a number of its components (muller and young, ) . n q ns was found to bind to c s proenzyme, c , c , and c b with reduced affinity compared to wt and n q ns (somnuke et al., ) , indicating that the n-glycan is required for effective interaction with complement components. the role of ns glycosylation in the ability of the protein to interfere with the complement activation, however, has been largely ignored. the n-glycans were proposed to be involved in ns binding to c (avirutnan et al., ) although direct experimental evidence has been missing. a recent study reported that secreted ns binds directly to c bp, a major inhibitor of the c b component (thiemmeca et al., ) . this binding leads to the recruitment of c bp on cell surface via ns and inactivates c b thereby interfering with the formation of the membrane attack complex (mac). furthermore, the work has demonstrated a competitive binding of ns to mbl, which prevents mbl-mediated denv destruction. the presence of secreted ns in the saliva of aedes mosquito suggests that secreted ns protein could help denv to escape the host innate immune surveillance during virus transmission (thiemmeca et al., ) . severe den (dhf/dss) is characterized by increased vascular permeability and plasma leakage, thrombocytopenia, hemorrhagic fever and circulatory failure in dss (who, ) . the current paradigm proposes that viral-induced proinflammatory cytokine storm drives the disease progression to dhf/dss (pang et al., ) . direct interaction between denv and clec a expressed on macrophages indicates that virus glycosylation plays a role in den pathogenesis wu et al., ) . similar to the engagement to dc-sign, denv binding to clec a relies on sugar moieties and can be inhibited by exogenous fucose and mannose . however, clec a binding does not mediate viral entry into the host cell, instead it serves as a cooperative signaling receptor to mr/dc-sign that activates macrophage inflammasome and triggers the production of pro-inflammatory cytokines wu et al., ; lo et al., ) . consistently, anti-clec a monoclonal antibody reduced denv-induced vascular leakage in a mouse model , which further supports that targeting the viral glycoprotein-host lectin receptor interactions represents a potential therapeutic approach to counteract the excessive inflammatory responses involved in severe den. glycosylation is a post-translational modification which significantly affects the conformation of a protein. it is a heterogeneous process that is highly host-cell specific. viruses have evolved to utilize their host's glycosylation machinery so as to optimize their fitness, infectivity, replication and virulence. the role of glycosylation and glycan structures in denv virulence has yet to be reported with evidence of attenuated phenotypes in symptomatic mouse models. given the impact of glycosylation in virus entry and virus fitness in mammalian cells, it is highly likely that deglycosylated denv mutants will display reduced virulence in vivo. the ability of celgosivir treatment, a bicyclic iminosugar that inhibits glycosylation through negatively binding to er [alpha]-glucosidase ii, to protect mice from a lethal denv challenge indirectly demonstrates the importance of glycosylation in denv virulence (perry et al., ; sayce et al., ; warfield et al., ) . to date, out of the eight denv potential glycoproteins (table ) , only e and ns proteins have been characterized from a glycosylation standpoint and not across all the denv serotypes. despite the biological importance of these structures being recognized, efforts to characterize the nature of the glycan structures in denv have remained timid. there is for example little understanding of how glycosylation impacts denv cell tropism where different glycan variants may influence binding of denv to various host cell receptors and subsequent cell infection. characterization of denv glycoforms has been mainly performed in the mammalian cell line bhk- but no study has been conducted in more relevant primary mammalian cell types including langerhans cells, monocytes, hepatocytes, and endothelial cells. furthermore, in-depth characterization of the glycan structures using the latest glycomics technologies has yet to be reported for denv. associating the contribution of these glycans to the structure and ultimately function of the virion glycoproteins indeed requires glycomics and glycoproteomics. such data need to be modeled using computational approaches as methods for crystallizing glycoproteins remains a complicated feat. furthermore, the role of glycosylation is also very important to recognize in biotherapeutic strategies. while substantial efforts have been devoted to developing neutralizing antibodies against denv, the potency of these antibodies is largely dictated by the accessibility of the epitope that they target which can be influenced by the glycosylated status of the protein (smith et al., ) . consistently, e protein glycosylation site has been reported to modulate the binding of neutralizing antibodies against a highly conserved, serotype cross-reactive epitope (dejnirattisai et al., ) . these challenges have prompted substantial investment into elucidating the three-dimensional conformation of the protein-antibody complexes and more importantly how glycosylation contributes to the tertiary and quaternary arrangements of the different glycoproteins on the virion. this approach is critical for the development of new therapeutics with broader activity and increased efficacy. in conclusion, the den field as a whole would benefit greatly from in-depth understanding and characterization of the glycosylation patterns of den virions. with the recent technical advances in the fields of glycomics and glycoproteomics, this has become possible and will depend on productive interactions between glycobiologists and den virologists. sy, tn-k, and sa wrote the manuscript. pr provided suggestions and edited the manuscript. sa holds a research grant from the national medical research council (nmrc/mohiafcat / / ) to develop dengue mouse models. revisiting dengue virushost cell interaction: new insights into molecular and cellular virology flavivirus ns structures reveal surfaces for associations with membranes and the immune system crucial role of the n-glycans on the viral 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jejuni protein n-glycosylation in the baculovirus-insect cell system avian flu: influenza virus receptors in the human airway hcv e glycoprotein: mutagenesis of n-linked glycosylation sites and its effects on e expression and processing synthesis of proteins and glycoproteins in dengue type virus-infected vero and aedes albopictus cells the potent and broadly neutralizing human dengue virus-specific monoclonal antibody c reveals a unique cross-reactive epitope on the bc loop of domain ii of the envelope protein n-linked glycosylation of dengue virus ns protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement proteolytic activation of tick-borne encephalitis virus by furin alpha - (alpha - )-difucosylation of the asparagine-bound n-acetylglucosamine in honeybee venom phospholipase a site-specific n-and o-glycopeptide analysis using an integrated c -pgc-lc-esi-qtof-ms/ms approach suppression of antiviral responses by 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interactions efficacy of a tetravalent dengue vaccine in children in latin america glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy glycomic analysis of human respiratory tract tissues and correlation with influenza virus infection prm-and cell-binding domains of the dengue virus e protein inhibition of endoplasmic reticulum glucosidases is required for in vitro and in vivo dengue antiviral activity by the iminosugar uv- variation in distribution of the three flavivirus-specified glycoproteins detected by immunofluorescence in infected vero cells newly synthesized dengue- virus nonstructural protein ns is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization evidence that the mature form of the flavivirus nonstructural protein ns is a dimer clec a is critical for dengue virus-induced inflammasome activation in human macrophages two distinct sets of ns a molecules are responsible for dengue virus rna synthesis and virion assembly hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity n-linked glycan in tick-borne encephalitis virus envelope protein affects viral secretion in mammalian cells, but not in tick cells evidence for a genetic and physical interaction between nonstructural proteins ns and ns b that modulates replication of west nile virus association of the pr peptides with dengue virus at acidic ph blocks membrane fusion structure of the immature dengue virus at low ph primes proteolytic maturation the c-type lectin-like domain superfamily challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs structures of immature flavivirus particles conformational changes of the flavivirus e glycoprotein 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 © yap, nguyen-khuong, rudd and alonso. this is an open-access 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) or licensor 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- -piv h oq authors: paemanee, atchara; hitakarun, atitaya; roytrakul, sittiruk; smith, duncan r. title: screening of melatonin, α-tocopherol, folic acid, acetyl-l-carnitine and resveratrol for anti-dengue virus activity date: - - journal: bmc res notes doi: . /s - - - sha: doc_id: cord_uid: piv h oq objective: infections with the mosquito transmitted dengue virus (denv) are a significant public health burden in many parts of the world. despite the introduction of a commercial vaccine in some parts of the world, the majority of the populations at risk of infection remain unprotected against this disease, and there is currently no treatment for denv infection. natural compounds offer the prospect of cheap and sustainable therapeutics to reduce the disease burden during infection, and thus potentially alleviate the risk of more severe disease. this study evaluated the potential anti-denv activity of five natural compounds namely melatonin, α-tocopherol, folic acid, acetyl-l-carnitine and resveratrol in two different cell lines. results: screening of the compounds showed that one compound (acetyl-l-carnitine) showed no effect on denv infection, three compounds (melatonin, α-tocopherol and folic acid) slightly increased levels of infection, while the th compound, resveratrol, showed some limited anti-denv activity, with resveratrol reducing virus output with an ec( ) of less than μm. these results suggest that some commonly taken natural compounds may have beneficial effects on denv infection, but that others may potentially add to the disease burden. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. dengue is currently considered as the most important arthropod-borne viral disease [ ] . it is caused by dengue virus (denv) which is transmitted by female aedes mosquitoes, particularly aedes aegypti [ ] . using the population data of and a geostatistical model, bhatt and colleagues estimated that approximately million dengue infections occur globally every year, with million symptomatic cases [ ] . other studies have estimated that more than , deaths are reported annually [ ] . overall, an estimated . billion people are at risk of dengue virus infection in almost countries in tropical and subtropical regions [ ] . dengue infection can occur with one of four denv serotypes (denv - ), and cause clinical manifestations ranging from self-limiting febrile illness, dengue fever to life-threatening dengue hemorrhagic fever which can progress to dengue shock syndrome (dss) [ ] . there is no specific drug to treat denv infection, and care is mainly supportive [ ] . studies have shown a relationship between viral burden and disease severity [ ] , and thus there is interest in compounds that have anti-denv effects, as these may serve to reduce severity if administered early in infection. de novo drug discovery is both prohibitively expensive [ ] and time consuming, and so studies in a wide range of fields have started to explore natural compounds as a way of cutting short the drug development process and additionally developing therapies that are more affordable. this study investigated the anti-denv effects of commonly used natural compounds that have been shown to have activity against other viruses, or to have the potential to have anti-viral activity, namely the human hepatocellular carcinoma cell line hepg (atcc ® hb- ™ ) and the human embryonic kidney cell line hek t/ (atcc ® crl- ™ ) were cultivated in dulbecco's modified eagle's medium (dmem, merck kgaa) supplemented with % heat inactivated fetal bovine serum (fbs, fbs; gibco, merck kgaa), u/ml penicillin and µg/ml streptomycin (paa laboratories, pasching, austria) in a cm tissue culture flask at °c in % co . rhesus monkey kidney cells llc-mk (atcc ® ccl- ™ ) cells were cultivated in dmem supplemented with % fbs and units/ ml of penicillin and μg/ml of streptomycin at °c in an incubator with % co . c / (aedes albopictus, atcc ® crl- ™ ) cells were cultivated in minimum essential medium (mem; merck kgaa) supplemented with % fbs, units/ml of penicillin and μg/ml of streptomycin at °c. denv (strain ) was propagated in c / cells and the viral titer was determined by standard plaque assay in llc-mk cells essentially as described elsewhere [ ] . cytotoxicity of the natural compounds in the range . - mm (melatonin, alcar and α-tocopherol) or . - mm (folic acid and resveratrol) was evaluated by the mtt assay (merck kgaa) both in infected and uninfected cells. hepg and hek t/ cells were seeded in -well culture plates at a density that allowed % confluence to be reached within h. the cells were washed in × pbs and incubated with µl of denv containing the desired multiplicity of infection (moi) for h. hepg cells were infected at moi and , while hek t/ cells were infected at moi . and at °c. mock infection (no virus) was undertaken in parallel. after h of virus infection, medium was replaced with concentrations of each compound in dmem with % fbs and then incubated at °c, % co . after -h incubation, cells were collected for determination of the degree of infectivity. for some compounds, cell supernatant was collected and levels of virus production determined by standard plaque assay. flow cytometry to determine percentage cell infection was undertaken using a pan specific anti-dengue e protein antibody hb [ ] as a primary antibody and a goat anti mouse igg conjugated with fitc as secondary antibody as described elsewhere [ ] . cells were run on a bd facscalibur (becton-dickinson, bd bioscience, san jose, ca) and data was analyzed using the cellquest pro software. all data were analyzed using the graphpad prism program (grappad software inc., san diego, ca). statistical analysis of significance was undertaken by one-way anova on raw data reads using spss (spss inc., chicago, il). ec values were calculated using the freeware ed plus (v . ) software (http://scien cegat eway.org/ proto cols/cellb io/drug/data/ed v .xls). to undertake screening of compounds for anti-denv activity, two cell lines were selected, the human embryonic kidney cell line hek t/ , and the human hepatocellular cell line hepg . both cell lines are commonly used in analysis of denv infection, although het t/ cells do not represent a normal tissue involved in denv infection. five natural products (melatonin, α-tocopherol, folic acid, acetyl-l-carnitine and resveratrol) were selected for screening for possible anti-denv activity. all compounds were initially screened for cytotoxicity in both cell lines using the mtt assay. cytotoxic effects were determined for both uninfected cells and denv infected cells in the range of . - mm ( . - , μm) for acetyl-l-carnitine, melatonin and α-tocopherol, and the range of . mm ( . - μm) for folic acid and resveratrol, with treatment being undertaken for h. results showed little difference in cytotoxicity profiles of the compounds between denv infected and uninfected cells. thus there was no cytotoxic synergism between the compounds and denv infection. melatonin (fig. a, b) , α-tocopherol (fig. a, b) and acetyl-l-carnitine (additional file : figure s a , b) all showed evidence of cytotoxicity at compound concentrations of mm. folic acid (additional file : figure s a , b) and/or the vehicle showed a slight positive effect in both hek t/ and hepg cells at concentration of mm, but this was not seen at a higher concentration. cytotoxicity was observed for resveratrol at concentrations above . mm in hek t/ cells, and above mm in hepg cells (fig. a, b) . based on the cytotoxicity profiles, two concentrations were selected for each compound for assay of antiviral activity, which were and μm for acetyl-l-carnitine and melatonin, . and . mm for α-tocopherol, . and . mm for folic acid. for resveratrol which showed significant cell type variation in cytotoxicity, values of and μm were used for hek t/ cells, and and μm were used for hepg cells. for all experiments, infections were undertaken at moi . and for hek t/ cells and and for hepg cells due to different cell susceptibility to infection [ ] . melatonin (n-acetyl- -methoxy tryptamine) is a natural hormone that is primarily produced by the pineal gland and regulates the circadian rhythm [ ] , and exogenous melatonin is often administered to adjust the sleep-wake cycle [ ] . however, studies have shown that melatonin also has significant antiviral effects. melatonin has been shown to have some effects in animal models of venezuelan equine encephalitis virus [ , ] , semliki forest virus [ ] , west nile virus [ ] , respiratory syncytial virus [ ] and its use has been proposed in humans infected with ebola virus [ ] . in this study, melatonin showed a slight proviral effect upon denv infection in hek t/ cells (fig. c) under conditions of the lowest moi used, and the highest concentration of melatonin, but no effect was seen in hepg cell (fig. d) . vitamin e refers to compounds classed as both tocopherols and tocotrienols. there are four vitamin e tocopherols, termed α-, β-, γ-and δ-, and while γ-tocopherol is the most abundant form of vitamin e, α-tocopherol is the second most abundant and the most biologically active form [ ] . vitamin e has well known antioxidant properties, through its ability to donate a hydrogen group from its free hydroxyl group to the free radical, with the generation of a stable free radical form of vitamin e. however, a number of non-antioxidant activities of vitamin e have been demonstrated [ ] . α-tocopherol has been reported to have some effects in dengue patients [ ] and has proposed benefits for influenza virus a infection [ ] , as well as possible activity against hepatitis b [ ] and c [ ] . in this study, α-tocopherol showed proviral effects, particularly in hek t/ cells (fig. c) , where the percentage of infection was increased significantly under most condition combinations tested. however, only a small effect was seen in hepg cells under conditions of the highest concentration and lowest moi tested (fig. d) . acetyl-l-carnitine (alcar) is the acetylated form of l-carnitine a molecule naturally produced by the body and the acetylated form of l-carnitine is able to cross the blood brain barrier [ ] . l-carnitine functions primarily to transport activated long chain fatty acids into mitochondria for degradation by β-oxidation, and β-oxidation has been proposed to be important in denv replication [ ] . in addition, l-carnitine has been shown to have activity against hepatitis c virus [ ] . in this study, alcar was shown to have no effect on denv infection in either cell line (additional file : figure s c , d). folic acid is the synthetic form of vitamin b , while folate is the naturally occurring form found in food. folic acid is essential for nervous system development, and is a commonly administered supplement to pregnant mothers to prevent neural tube defects [ ] . folic acid is also important in the synthesis of rna and dna, and is particularly important in rapidly dividing cells [ ] . folic acid additionally plays a role in the formation of red blood cells [ ] . folic acid has not previously been explored for anti-viral activity, although the metabolically active form of folic acid, folinic acid has been described as having pro-flaviviral effects through the thymidine synthesis pathway [ ] . in this study, folic acid had little effect upon denv infection in either cell line (additional file : figure s c, d) , although a slight proviral effect was seen at the highest concentration examined in hepg cells (additional file : figure s d ). resveratrol ( , , ′-trihydroxy-trans-stilbene) is a hydroxylated derivative of stilbene, and a phytoalexin which is produced in response to pathogen attack in some plants. it is found in nature in several foods including grapes and blueberries [ ] . considerable attention has been focused on resveratrol for potential anti-cancer [ ] , anti-obesity [ ] , anti-neurodegeneration [ ] and cardio-protective activities [ ] . studies have shown that resveratrol has activity against a number of viruses including hiv [ ] , middle east respiratory syndrome coronavirus (mers-cov) [ ] , influenza a virus [ ] , poxvirus [ ] and enterovirus [ ] . as noted earlier, resveratrol showed significant cytotoxicity in hek t cells, but that hepg cells were somewhat less sensitive to the cytotoxic effects of the compound (fig. a, b) . however, infection with denv in the presence of resveratrol (at and μm) in hek t/ cells showed a significant reduction in the level of infection (fig. c) , while a slight increase in infection levels was seen in hepg cells (fig. d) with the lowest moi and μm resveratrol. we observed some background in the flow cytometry with resveratrol ( fig. ) and this might be related to the protein binding ability of resveratrol [ ] . however, subtraction of the background did not alter the result. to further investigate the effect of resveratrol in denv infection, the effect on virus production was determined by standard plaque assay. the virus titer was determined at h (input virus) and at h post infection. the results (fig. e) showed that, consistent with the reduction in the level of cellular infection (fig. c) , there was a reduction in the level of virus produced with treatment with resveratrol in hek t/ cells at h post infection, but not in hepg cells (fig. e) . the reduction of virus output observed was the order of approximately log . to determine the ec of resveratrol on virus production, the experiment was repeated in hek t/ cells with concentrations of , . , , , , and μm resveratrol. results of the standard plaque assay are shown in fig. f . the ec value was determined as . μm. finally, the experiment was undertaken at a lower moi of . . results (fig. f ) showed a dose dependent reduction of virus production, with an ec value of . μm. while the reduction in level of infection and virus output is significant in hek t/ cells, it compares poorly to other natural compounds such as andrographolide, which reduces denv virus output by . log [ ] . these results suggest that some commonly taken natural compounds may have beneficial effects on denv infection, but that others may potentially add to the disease burden. . only one of the four denv serotypes was examined. . cytotoxic effects limited the concentration test range of compounds. • 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 ready to submit your research ? choose bmc and benefit from dengue and dengue hemorrhagic fever the global distribution and burden of dengue urbanization and globalization: the unholy trinity of the st century refining the global spatial limits of dengue virus transmission by evidence-based consensus dengue: guidelines for diagnosis, treatment, prevention and control. geneva: world health organization dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity the $ . billion pill-methodologic and policy considerations behavior of the dengue virus in solution dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence activity of andrographolide against dengue virus involvement of fatty acid synthase in dengue virus infection role of melatonin in the regulation of human circadian rhythms and sleep melatonin prolonged release: in the treatment of insomnia in patients aged >/= years melatonin protects mice infected with venezuelan equine encephalomyelitis virus melatonin decreases brain apoptosis, oxidative stress, and cd expression and increased survival rate in mice infected by venezuelan equine encephalitis virus protective effects of melatonin in mice infected with encephalitis viruses inhibitory effect of melatonin on lung oxidative stress induced by respiratory syncytial virus infection in mice ebola virus disease: potential use of melatonin as a treatment many tocopherols, one vitamin e. mol aspects med non-antioxidant activities of vitamin e micronutrients and dengue a nutritional supplement formula for influenza a (h n ) infection in humans possible role of tocopherols in the modulation of host microrna with potential antiviral activity in patients with hepatitis b virus-related persistent infection: a systematic review effects of vitamin e on chronic hepatitis c genotype : a randomized, double-blind, placebo-controlled study acetyll-carnitine permeability across the blood-brain barrier and involvement of carnitine transporter octn dengue virus-induced autophagy regulates lipid metabolism anti-adipogenic and antiviral effects of l-carnitine on hepatitis c virus infection folate action in nervous system development and disease folic acid: nutritional biochemistry, molecular biology, and role in disease processes flaviviruses are sensitive to inhibition of thymidine synthesis pathways a comprehensive review of the health perspectives of resveratrol the role of resveratrol in cancer therapy the beneficial effects of quercetin, curcumin, and resveratrol in obesity resveratrol for alzheimer's disease potent inhibition of hiv- replication in resting cd t cells by resveratrol and pterostilbene effective inhibition of mers-cov infection by resveratrol in vitro antiviral activity of resveratrol against respiratory viruses suppression of poxvirus replication by resveratrol resveratrol inhibits enterovirus replication and pro-inflammatory cytokine secretion in rhabdosarcoma cells through blocking ikks/nf-kappab signaling pathway resveratrol binding to human serum albumin not applicable. the authors declare that they have no competing interests. all data generated or analysed during this study are included in this published article. not applicable. not applicable. this work was supported by grants from mahidol university and the thailand research fund (brg and irn w ). ah is supported by a thailand graduate institute of science and technology (tgist) scholarship from the national science and technology development agency (nstda), thailand. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. additional file . effect of acetyl-l-carnitine on denv infection of hek t/ and hepg cells.additional file . effect of folic acid on denv infection of hek t/ and hepg cells. key: cord- -abd ej authors: lai, yen-chung; chao, chiao-hsuan; yeh, trai-ming title: roles of macrophage migration inhibitory factor in dengue pathogenesis: from pathogenic factor to therapeutic target date: - - journal: microorganisms doi: . /microorganisms sha: doc_id: cord_uid: abd ej dengue virus (denv) infection is the most prevalent mosquito-borne viral infection and can lead to severe dengue hemorrhagic fever (dhf) and even life-threatening dengue shock syndrome (dss). although the cytokine storm has been revealed as a critical factor in dengue disease, the limited understanding of dengue immunopathogenesis hinders the development of effective treatments. macrophage migration inhibitory factor (mif) is a pleiotropic proinflammatory cytokine that mediates diverse immune responses, and the serum level of mif positively correlates with disease severity in patients with dengue. mif is involved in denv replication and many pathological changes, such as vascular leakage, during denv infection. in this paper, the pathogenic roles of mif and the regulation of mif secretion during denv infection are reviewed. furthermore, whether mif is a potential therapeutic target against denv infection is also discussed. dengue virus (denv) infection is the most widespread mosquito-borne viral infection in the tropics and subtropics. it is estimated that denv causes million infections yearly, even though more than % of cases of denv infection are asymptomatic or cause mild flu-like illness, including fatigue, headache, myalgia and nausea with sudden onset of fever called dengue fever (df). unfortunately, one in twenty infected individuals may suffer from a more severe illness, which is termed dengue hemorrhagic fever (dhf), and even develop life-threatening dengue shock syndrome (dss) [ ] . in , the who issued a new guideline that classifies symptomatic cases as nonsevere dengue or severe dengue according to disease severity. nonsevere dengue cases can be further divided into two subgroups: patients with warning signs and those without warning signs. the warning signs include abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy, restlessness, liver enlargement and decreased platelet counts. the criteria for severe dengue include severe plasma leakage, severe bleeding, and severe organ involvement [ ] . the mortality of df is less than %; however, severe dengue may carry a mortality rate up to % [ ] . although there is one vaccine (dengvaxia ® ) that has been licensed to prevent dengue in several countries, this vaccine rendered only partial protection against denv infection. in addition, the vaccine is associated with an unexplained increased incidence of hospitalization for severe dengue disease in seronegative vaccine recipients [ , ] . furthermore, due to the limited understanding of the exact pathogenic mechanisms to cause vascular leakage, no effective therapeutic drugs are available, mif, a . kda protein was first identified as a cytokine that is mainly released from t cells upon antigen stimulation and inhibits the random migration of macrophages [ ] . later, mif was found to be widely distributed in various immune and nonimmune cells, including macrophages, platelets, endothelial cells, epithelial cells that mediate several immune responses with pluripotent activity. unlike most cytokines, mif is constitutively expressed and stored in preformed "intracellular pools" during normal homeostasis [ ] , which means it can be released from cells upon inflammatory and stress stimulation promptly. accordingly, mif secretion can be found even without de novo synthesis. since mif does not possess an n-terminal secretory sequence, mif is released from cells via nonconventional er/golgi secretory pathways [ ] . both intracellular and extracellular mif exhibit pluripotent functions. intracellular mif can modulate ap- activity and the cell cycle by interacting with cytosolic jun activation domain binding protein (jab- ) [ , ] . overexpression of endogenous mif significantly suppresses p -dependent and oxidative stress-induced apoptosis [ , ] . on the other hand, secreted mif can bind to some distinct cell receptors, such as cd and its coreceptors cd , cxcr , cxcr and cxcr , which activate several signaling pathways, such as the erk / and pi k/akt pathways, which are responsible for cell proliferation, survival, and immune regulation [ ] [ ] [ ] . in addition, mif also has endocrine activity to regulate insulin secretion [ ] and tautomerase activity to manipulate cell growth [ ] . under normal physiological conditions, mif is constitutively expressed at a low level between - ng/ml, while the mif concentration in human plasma fluctuates in response to stress stimuli such as sepsis, infection and different inflammatory disorders [ ] [ ] [ ] . high concentrations of mif can activate the synthesis of more proinflammatory cytokines, such as tnf, ifn-γ, il- β, il- , il- , and il- [ ] , which accelerates inflammatory disease development. furthermore, mif also contributes to the pathogenesis of various viral infections, including human immunodeficiency virus (hiv), respiratory syncytial virus (rsv), west nile virus (wnv) and denv [ ] [ ] [ ] [ ] [ ] . the first evidence of the pathogenic role of mif in dengue disease was indicated by the positive correlation between the mif level in the sera of dengue patients and disease severity [ ] . later, another group also demonstrated that denv -induced inflammation, thrombocytopenia, viral load and disease severity could be attenuated in mif −/− mice [ ] . in , ferreira et al. also showed that the serum level of mif is higher in patients with dhf than df [ ] . however, the pathogenic mechanisms and source of circulating mif were not further investigated. here, we propose three main pathogenic roles of mif in the immunoregulatory crosstalk during denv infection: ( ) facilitation of denv replication; ( ) enhancement of vascular leakage; ( ) regulation of the immune response ( figure ). it has been shown that denv infection triggers mif expression and secretion, which enhances denv replication in huh- cells [ ] . autophagy is pivotal for denv replication [ ] , and mif is required for the induction of autophagy in denv -infected huh- cells. blocking mif with inhibitors or knockdown of mif expression by shrna inhibited both denv-induced lc conversion and denv replication. to clarify the correlation between these two parallel effects, we found that incubation with recombinant mif (rmif) only enhanced viral replication in mif knockdown but not lc knockdown cells. these results suggest that autophagy is required for mif-induced denv replication in huh- cells. given that denv infection induces autophagy, which is required for denv replication, the mechanisms of denv-induced autophagy have been widely studied. the formation of autophagosomes provides a dock for the denv replication complex, which supports viral replication in nonphagocytic cells [ ] . denv infection also induces selective autophagy associated with lipid metabolism. denv-induced autophagosomes facilitate mitochondrial β-oxidation associated with host lipid metabolism, which enhances atp production for viral replication [ ] . in addition, ns a expression induces autophagosome formation and inhibits cell apoptosis during denv infection, which contributes to prolonged viral replication [ ] . previously, mif was found to induce autophagy under starvation conditions. starvation-triggered mif binds to its receptor cd in autocrine or paracrine fashions, and then autophagy is induced through reactive oxygen species (ros) generation [ ] . cd -mediated mif endocytosis can also activate erk phosphorylation [ ] , leading to autophagy [ ] . in another study, it was demonstrated that live denv -induced endoplasmic reticulum (er) stress is required for autophagy activation, viral replication and pathogenesis in huh- and a cells [ ] . although the direct correlation between denv-induced er stress and mif secretion has not been elucidated, both mif and er stress can induce autophagy through erk / phosphorylation [ , ] and ros generation [ ] . interestingly, mif is able to induce er stress in a liver injury model [ ] . therefore, it is possible that mif signal transduction may trigger er stress and erk activation upon denv infection, leading to autophagy induction and viral replication, or vice versa. although further investigation is required to understand the relationship between mif and denv-induced er stress in the future, our previous results showed that denv-induced mif secretion can induce autophagy-facilitated viral replication, which may explain why reduced viremia was found in mif −/− mice than in control mice [ ] . in addition to the pro-viral effect in hepatocytes and the lung epithelial cells, pathogenic roles of mif in endothelial cells were also found. mif is crucial in the vasculature, which has been addressed in several studies. mif expression in human endothelial cells is upregulated upon treatment with agonists that can trigger endothelial hyperpermeability, such as thrombin [ ] or oxldl [ ] . on the other hand, inhibiting mif with either inhibitors or anti-mif antibodies could rescue thrombin-induced vascular leakage [ ] , suggesting that mif plays a role in vascular leakage. moreover, treatment with rmif could directly increase permeability within min in the dermal microvascular endothelial cell line hmec- and by subcutaneous injection in a mouse model [ ] . not surprisingly, mif has also been shown to be involved in vascular hyperpermeability upon denv infection. denv infection can stimulate mif secretion from huh- cells. conditioned medium collected from denv-infected huh- cells can enhance the permeability of human endothelial cell lines by disrupting the distribution of the endothelial tight junction protein zonula occludens- (zo- ) through mif-activated phosphatidylinositol- -kinase/mitogen-activated protein kinase kinase-extracellular signal-regulated kinase/c-jun n-terminal kinase (pi k/mek-erk/jnk) signaling pathways [ ] . moreover, mif also plays an essential role in denv ns -triggered endothelial permeability. denv ns is a viral protein that is the main reason for vascular leakage during dengue infection [ ] . there are at least two mechanisms involved in denv ns -triggered endothelial permeability, both of which involve mif: enzyme-dependent glycocalyx degradation and autophagy-facilitated disruption of endothelial integrity. denv ns can activate mif secretion in endothelial cells, which triggers the release of heparan sulfate-specific heparanase (hpa- ) and endothelial glycocalyx shedding [ ] . on the other hand, denv ns can cause the disarray of endothelial tight junctions through mif-induced autophagy [ ] . taken together, these findings suggest that mif plays important roles in vascular leakage during denv infection. in addition to viral replication and endothelial hyperpermeability, mif may also regulate the immune response of different immune cells during denv infection. in phagocytic cells, mif can upregulate coagulation molecules, such as thrombomodulin (tm), in denv -infected thp- cells [ ] . tm can compete with fibrinogen to bind to thrombin and inhibit fibrin formation, thereby contributing to coagulopathy in dengue disease. as a pivotal mediator of inflammatory cytokines, mif inhibition can attenuate denv infection-induced tnf-α and il- production, and denv ns -triggered metalloproteinase (mmp- ) production as demonstrated in thp- cells [ ] . as tnf-α and il- are critical proinflammatory cytokines involved in vascular permeability [ , ] and mmp- is a key enzyme that can trim the glycocalyx layer in endothelial cells [ ] , upregulation of mif in leukocytes can further contribute to denv-induced vascular permeability. thus, mif can lead to coagulopathy and vascular leakage through denv-stimulated immune cells, which may explain why the increased hematocrit was attenuated in denv -infected mif −/− mice [ ] . on the other hand, neutrophil extracellular traps (nets) induced by activated neutrophils are thought to be a pathogenic feature that amplifies inflammatory responses in dengue disease [ ] . mif is required for net formation in immune disorders [ , ] . therefore, the release of mif from neutrophils may induce net formation and inflammation in denv infection, which contributes to dengue pathogenesis. in addition to these immune cells, activated platelets are also a source of mif. platelet-derived mif is a unique chemokine that leads to monocyte adhesion on endothelial layers [ ] . denv ns can contribute to platelet activation and aggregation, thus enhancing platelet adhesion to endothelial cells [ ] . in addition, extracellular vesicles (evs) have been identified as functional conveyors of mif in obesity [ ] , as well as crucial mediators in platelet-leukocyte interactions upon denv infection [ ] . platelet-derived mif may be involved in denv ns -induced thrombocytopenia and hemorrhage. although a previous study demonstrated that serum levels of mif are significantly higher in all dhf patients who died than in surviving dhf and df patients [ ] , the main source of secreted mif and its release mechanisms are still unclear. here, the possible mechanisms of denv-induced mif secretion and expression are discussed (figure ). as it lacks an n-terminal secretory sequence, mif is known to be released from the intracellular pools very quickly upon stimulation. it has been shown that productive denv and denv infection can stimulate mif release from human macrophages and hep g cells without the requirement of mif rna transcription at h post-infection [ ] . similarly, denv infection of huh- cells can drive two waves of mif secretion [ ] . denv infection induced the first wave of mif secretion at - h post-infection. since no obvious lactate dehydrogenase (ldh) release nor a significant increase in mif mrna occurred during this period of time, these results suggest that the first wave of mif secretion was caused by the release of mif from intracellular pools, and this secretion is independent of mif gene transcription and cell death [ ] . as it lacks an n-terminal secretory sequence, mif is known to be released from the intracellular pools very quickly upon stimulation. it has been shown that productive denv and denv infection can stimulate mif release from human macrophages and hep g cells without the requirement of mif rna transcription at h post-infection [ ] . similarly, denv infection of huh- cells can drive two waves of mif secretion [ ] . denv infection induced the first wave of mif secretion at - h post-infection. since no obvious lactate dehydrogenase (ldh) release nor a significant increase in mif mrna occurred during this period of time, these results suggest that the first wave of mif secretion was caused by the release of mif from intracellular pools, and this secretion is independent of mif gene transcription and cell death [ ] . however, mif mrna expression is increased in huh- cells at h to h post-denv infection, as shown by rt-pcr [ ] . although quantitative rt-pcr or real-time rt-pcr should be used to detect changes in the mif rna level more precisely, these results suggest that denv infection can trigger signals to enhance mif transcription. as viral rna and viral proteins can trigger cellular oxidative stress, which is a strong stimulus for mif secretion and expression [ ] , various cellular oxidative stresses, such as er stress, the unfolded protein response (upr), hypoxic response and mitochondrial ros production, might be the upstream stimulators for mif production during denv infection [ , ] . among them, two hypoxia-related transcription factors, hypoxia-inducible factor (hif- ) and camp-response element-binding protein (creb), were found to facilitate denv infection [ , ] . in addition, mif transcription can be driven by hif- in response to hypoxia or by creb in response to er stress. therefore, live denv induces a hypoxic response and er stress, followed by mif rna transcription through hif- or creb activation, which may lead to the second however, mif mrna expression is increased in huh- cells at h to h post-denv infection, as shown by rt-pcr [ ] . although quantitative rt-pcr or real-time rt-pcr should be used to detect changes in the mif rna level more precisely, these results suggest that denv infection can trigger signals to enhance mif transcription. as viral rna and viral proteins can trigger cellular oxidative stress, which is a strong stimulus for mif secretion and expression [ ] , various cellular oxidative stresses, such as er stress, the unfolded protein response (upr), hypoxic response and mitochondrial ros production, might be the upstream stimulators for mif production during denv infection [ , ] . among them, two hypoxia-related transcription factors, hypoxia-inducible factor (hif- ) and camp-response element-binding protein (creb), were found to facilitate denv infection [ , ] . in addition, mif transcription can be driven by hif- in response to hypoxia or by creb in response to er stress. therefore, live denv induces a hypoxic response and er stress, followed by mif rna transcription through hif- or creb activation, which may lead to the second wave of mif secretion. however, in another study, mif transcription was only marginally affected by denv infection in the human hepatoma cell line hep g [ ] . a possible explanation for this discrepancy is that different infection time points and virus strains were used in these two studies. we showed that mif transcription was gradually increased at h post-infection and peaked at h post-infection in huh- cells, while in another study, the mif rna level was only analyzed within h post-infection. in addition, viral infection-induced mif secretion and production seem to be virus-and cell-dependent. for instance, influenza a virus infection does not induce mif gene transcription but causes the release of preformed mif from lung epithelial cells due to necrotic cell death [ ] . however, infection of macrophages with sindbis virus resulted in mif release from intracellular pools without a significant increase in mif transcription or cell death [ ] . accordingly, the mechanisms of mif production and the kinetics of mif secretion induced by denv infection in different types of cells require further investigation. compared with the classical pathway of cytokine secretion, mif can be released from preformed pools shortly after stimulation. secretion of preformed mif has been shown to be mediated by the atp binding cassette (abc) transporter [ ] , as well as vesicle or exosome secretory pathways [ ] . in addition, denv infection may stimulate mif secretion mediated by the golgi-associated protein p via golgi vesicle trafficking, which is similar to the effect of bacterial infection [ ] . in our study, since uv-inactivated viral particles could not induce mif secretion or expression, it is possible that denv infection triggered rna sensing or pattern recognition receptor (prr) activation, which was followed by the release of preformed mif from the cytosol through the vesicle trafficking secretory pathway. the release of preformed mif may further initiate many cellular signal transduction pathways through binding to membrane-expressed mif receptors, including cd /cd , cxcr , cxcr and cxcr [ , ] . initiation of autophagy signal transduction induced by mif binding to its receptors leads to activation of the pi k/akt/src and mapk/erk pathways [ , ] , which may explain why the first wave of mif secretion occurs before denv -induced autophagic flux in huh- cells [ ] . to reduce the burden of dengue disease, effective drugs to treat denv infection are urgently needed until a satisfactory vaccine becomes available. based on the proposed dengue pathogenesis described above, current denv therapeutic development has focused on two major targets: ( ) viral factors and ( ) host factors. by targeting viral factors, many approaches to protect against different viral life cycles have been tested, such as inhibition of receptor-dependent viral entry [ ] , ph-dependent viral fusion [ ] , interaction with the capsid protein [ ] and viral particle assembly [ ] . neutralizing abs against conserved regions of structural proteins, including e and prm/m, are widely used due to their specificity. however, ab use is often challenging due to the enhancement of viral infection in fcγ receptor-bearing immune cells with subneutralizing doses, as described by the ade theory [ , , ] . in addition to ab therapies, some off-patent drugs and antibiotics have also been tested for repurposing by high-throughput screening, which is beneficial for development time and cost. for instance, prochlorperazine, a dopamine d receptor (d r) antagonist that is used to treat nausea, schizophrenia, migraines, and anxiety, was shown to inhibit viral entry [ ] . considering another perspective, aberrant host immunity also plays vital roles in dengue pathogenesis. excessive secretion of proinflammatory cytokines (cytokine storm) induced by denv infection may trigger robust innate and adaptive immune responses, leading to plasma leakage, hemorrhage, and coagulopathy in dhf/dss patients. appropriate regulation of host immunity provides different insights into therapeutic targets, including host restriction factors, host dependency factors, and host-mediated pathogenesis pathways [ , ] . compared with viral targets, host-targeting antiviral approaches are believed to avoid rapid drug resistance or mutations that arise during viral evolution. since mif is involved in dengue pathogenesis, the therapeutic potential of blocking mif to protect against dengue disease has also been studied. in , assuncao-miranda et al. first used the mif inhibitor iso- and a mif neutralizing antibody to test the involvement of mif in denv -induced macrophage activation in vitro. although blocking mif did not affect viral replication in denv -infected human macrophages, both the secretion and mrna synthesis of tnf-α and il- were reduced. moreover, prostaglandin e (pge ), a well-known inflammatory mediator in many inflammatory diseases [ ] , was attenuated as well. furthermore, they demonstrated that the concentration of ifn-γ in sera, the level of il- in the spleen, and leukocyte infiltration in the lungs of mif −/− mice were significantly lower than those in denv-infected wild-type mice. most importantly, the study showed delayed mortality in denv -infected mif −/− mice. these results suggest that mif controls amplification of denv-induced inflammatory responses. treatment with mif neutralizing antibodies or inhibitors may provide protection against dengue disease. previously, minocycline, a us food and drug administration (fda)-approved antibiotic, was found to reduce dengue viral output through downregulation of erk / activation and upregulation of interferon stimulated genes (isgs) in hep g cells [ ] . in our recent study, we found that minocycline can block not only denv -triggered autophagy but also mif secretion. autophagy could be activated by mif through erk / phosphorylation [ ] , and the anti-denv effect of minocycline was abolished in either mif or lc -deficient huh- cells during denv infection. it is possible that the protective effect of minocycline may be due to its ability to block mif secretion, which suppresses the erk / -autophagy signaling pathway. in addition, the results showed that minocycline can reduce both mif rna transcription and secretion during denv infection, but the mechanism is unclear. given that mif secretion can be triggered by the abc transporter, which is a nonconventional secretory pathway [ ] , and minocycline is able to inhibit the function of the abc transporter to block drug-drug interactions at the blood-brain barrier [ ] , minocycline may disrupt the efflux of mif via suppression of the abc transporter upon denv infection. moreover, minocycline is reported to reduce the production of tnf-α, il- , il- , ifn-γ and ccl via suppression of the transcription factor nf-κb in the brain, which confers complete protection against jev in mice [ ] . nf-κb binds to the mif promoter and drives mif transcription [ ] , and inhibition of nf-b also blocks denv infection-induced mif production in a cells [ ] ; therefore, attenuation of de novo rna synthesis and secretion of mif from denv-infected cells by minocycline treatment may be due to its inhibition of the nf-κb signal pathway and suppression of the abc transporter, respectively [ ] . however, further study is required to clarify these hypotheses. to further understand whether minocycline can protect against denv infection in vivo, we found that minocycline treatment reduced the levels of mif and viremia in sera, as well as attenuated autophagy in murine liver tissue, in ag mice. however, the protection of minocycline in ag mice was insufficient. to rule out defects in isg-related protection in this model, which lacks type i and type ii ifn receptors, immunocompetent icr suckling mice were further used. minocycline only alleviated denv -induced neurological symptoms and prolonged the survival rate but did not fully protect against denv -induced lethality in suckling mice. it is unclear whether the failure of minocycline to fully protect against denv -induced lethality in suckling mice is due to the mouse-adapted strain ngc-n being too virulent for the suckling mice or the intracerebral challenge of ngc-n inducing irreversible damage in the brains of the suckling mice. however, these results were similar to the outcome in denv -infected mif −/− mice [ ] , which suggests that other pathogenic factors induced by denv infection may also be important for denv-induced pathogenesis. mif plays crucial roles in dengue pathogenesis; however, targeting only mif secretion and expression seems to be insufficient to provide full protection against denv infection. as mentioned above, transcription factors, such as hif- and creb, may also be involved in the increase in mif expression during denv infection. it is possible that in addition to mif, these transcription factors may also induce other pathogenic responses that contribute to disease development during denv infection [ , ] . on the other hand, although mif can induce autophagy and facilitate denv replication in huh- cells, autophagy might play different or even opposite roles in denv replication in different cells [ ] . it has been reported that autophagy plays pro-viral roles in denv replication in epithelial cells but antiviral roles in immune cells [ ] . therefore, the effect of mif on the modulation of autophagy and denv replication should be further systemically investigated in different cells, and the effect of minocycline treatment on denv infection in different cells, such as immune cells, should be compared. taken together, these findings suggest that targeting upstream transcription factors that control mif expression or multiple medication combinations targeting different mif signaling pathways may help to develop better therapeutic strategies against dhf/dss in the future. denv-triggered mif secretion can not only facilitate denv replication through the regulation of autophagy but also worsen the severity of vascular leak by enhancing endothelial permeability. in addition, mif may modulate the interaction of different immune cells, which contributes to dengue pathogenesis. inhibition of mif secretion and transcription by small molecule drugs such as minocycline could attenuate both autophagy and viral replication. however, targeting mif by multiple approaches instead of a single approach may provide a better therapeutic alternative against denv infection for translation from the laboratory to the clinic in the future. the global distribution and burden of dengue evaluation of the traditional and revised who classifications of dengue disease severity dengue hemorrhagic fever and shock syndromes dengvaxia sensitizes seronegatives to vaccine enhanced disease regardless of age effect of dengue serostatus on dengue vaccine safety and efficacy flavivirus entry receptors: an update dengue virus life cycle: viral and host factors modulating infectivity dengue viruses and mononuclear phagocytes. i. infection enhancement by non-neutralizing antibody dengue virus structural differences that correlate with pathogenesis epidemiologic, clinical, and virologic observations on dengue in the kingdom of tonga origins of dengue type viruses associated with increased pathogenicity 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designing antiviral drugs against dengue fever via targeting host-based approaches drug repurposing of minocycline against dengue virus infection brain and plasma riluzole pharmacokinetics: effect of minocycline combination regulation of inflammation in japanese encephalitis involvement of nuclear factor-κb in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- β in ectopic endometrial cells dengue virus infection induced nf-κb-dependent macrophage migration inhibitory factor production minocycline targets the nf-b nexus through suppression of tgf- -tak -i b signaling in ovarian cancer dengue virus and autophagy induced autophagy reduces virus output in dengue infected monocytic cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the members of the center of infectious disease and signaling research of ncku for their invaluable input and insights throughout the course of this study. this study is part of yen-chung lai's ph.d. dissertation. the authors declare that they have no competing interests. key: cord- -eg wcd authors: zheng, zhihang; li, min; liu, zhihua; jin, xia; sun, jin title: establishment of murine infection models with biological clones of dengue viruses derived from a single clinical viral isolate date: - - journal: virol sin doi: . /s - - -y sha: doc_id: cord_uid: eg wcd dengue virus (denv) is a single-stranded rna virus transmitted by mosquitoes in tropical and subtropical regions. it causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome in patients. each year, million people are estimated to be infected by four serotypes of dengue virus, creating a great burden on global public health and local economy. so far, no antiviral drug is available for dengue disease, and the newly licensed vaccine is far from satisfactory. one large obstacle for dengue vaccine and drug development is the lack of suitable small animal models. although some denv infection models have been developed, only a small number of viral strains can infect immunodeficient mice. in this study, with biologically cloned viruses from a single clinical isolate, we have established two mouse models of denv infection, one is severe lethal infection in immunocompromised mice, and the other resembles self-limited disease manifestations in balb/c mice with transient blockage of type i ifn responses. this study not only offers new small animal models of dengue viral infection, but also provides new viral variants for further investigations on dengue viral pathogenesis. dengue virus (denv) is an arbovirus transmitted by aedes aegypti and aedes albopictus mosquitoes. it belongs to flavivirus genus of flaviviridae family and contains a positive single-stranded rna genome. denv can be antigenically classified into four serotypes (denv- - ), which are often co-circulating in the endemic regions with similar clinical manifestations. though the majority of denv infections are asymptomatic, a minority of which can result in self-limited disease including dengue fever (df), and less than % may develop into dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). because of the global warming, increased air travel, and the lack of an efficacious vaccine, denv has become the most prevalent mosquito-borne viral pathogen in recent decades (guzman et al. ) . each year, denv is estimated to infect million people globally, affecting nearly half of the world population. asia accounts for % of the dengue disease burden, followed by latin america and africa (bhatt et al. ) . as an imported pathogen in china, dengue virus had caused sporadic outbreaks in southern china before . however, an unexpected large dengue outbreak attacked guangzhou in , resulting in , national reported cases that year (jin et al. ) , in which the majority was caused by dengue virus serotype (denv- ), and the others by serotype (denv- ) (zhao et al. ; li et al. ) . since then, - confirmed cases were reported to china cdc (chinese centers for disease control and prevention) each year. in nature, non-human primates and human are the only mammalian hosts for denv infection. naturally acquired dengue virus does not infect any outbred or inbred mice, unless viruses are inoculated with extremely high dose (e.g. - pfu of denv- in -week old c /bl ) (chen et al. ) or through intracerebral injection. in some other models, mouse-brain adapted strains are used. but such infection of adapted strain or intracerebral infection usually results in neurological manifestations, which are not routine clinical signs of human diseases. and, after passaging in mice, characteristics of these mouse brain-adapted viral strains may differ from strains naturally acquired, attenuation in their ability to cause human disease was documented (sabin and schlesinger ; hotta ; sarathy et al. ) . thus, the inability of dengue virus to replicate in murine model had hampered the development of dengue vaccine and antiviral therapy. monkey models, while more useful, are not suitable for routine preclinical testing of vaccine candidates or drug prototypes because of their high cost, limited animal availability and ethical concerns. the species specificity of denv infection may be partially attributed to the differences of innate immunity between primates and mice. interferon (ifn) signaling plays an essential role in the protection against dengue disease in humans, nevertheless denv has also evolved many mechanisms to antagonize ifn signaling and ifn production in vivo (green et al. ) . one of them is the binding and degradation of human stat by dengue viral protein ns (ashour et al. ) . another is the cleavage of human sting molecules by dengue viral protease ns b (aguirre et al. ; yu et al. ) . interestingly, both of these two antagonistic mechanisms only exist in human. denv ns does not bind to murine stat , neither does ns b cleave murine sting proteins, because the target sequences on these two molecules are highly species specific (ashour et al. ; aguirre et al. ; stabell et al. ) . but deletion of mstat or msting could enhance the replication of denv in mouse or mouse derived primary cells (ashour et al. ; aguirre et al. ) . these observations also imply the necessity of ifn signaling and ifn production in restricting denv infection in mice. therefore, immunocompromised mice without complete ifn responses or humanized mice are presumed more susceptible to denv infection. as humanized mice are laborious to prepare, and often introduce mouse-to-mouse variation, they are less suitable for application in vaccine development (akkina et al. ) . in contrast, knockout mice are easier to manipulate and a series of ifn deficient mice have been tested in the development of dengue infection model, including those of ifna/br -/-, ifncr -/-, ifna/b/cr -/-, mavs -/-, stat -/-, stat -/-, irf -/and irf -/- (shresta et al. ; perry et al. ; prestwood et al. ). among them, type i/ii ifn receptors double knock out mice usually develop lethal diseases when challenged with suitable denv strains, thus are used more frequently. however, only a limited number of dengue virus strains are able to replicate in mice and reproduce the severe symptoms (e.g., vascular leakage) of human disease. these viruses are usually based on clinical isolates (denv- c / , denv- - , denv- tvp- ), obtained through alternate passage between mosquito c / cells and ag mice (denv- d s ), or isolated after plaquepurification (denv- d y p-pp , denv- s ) (sarathy et al. ) . such mouse models are valuable in testing vaccine efficacy and studying viral pathology or transmission. notably, there are an increasing number of investigations that suggest t cell immunity is an essential component for flavivirus vaccine (st john and rathore ). but, type i interferon are vital for cd ? and cd ? t cell generation and maturation (crouse et al. ) . and ifn-c is also one of the major cytokines mediating th /ctl function and t cell development. therefore, ifn deficient transgenic mice are not ideal for studies of t cell immunity or t cell vaccines in an active immunization model, for the lacking of cross-talk between ifn pathway and adaptive immunity. in this study, we have purified two dengue virus strains of serotype exhibiting different plaque sizes from one clinical isolate denv- gz. through subcutaneous injection of low doses of these viruses to type i/ii ifn receptor knock-out mice, we successfully established mouse models of lethal infection. the pathogenicity of the two variants in ag was demonstrated to be different. further, we compared the infectivity of these two viral variants in a self-limited infection model, in which type i ifn receptor of wild-type balb/c mice had been transiently blocked before infection, and found only the virus strain exhibiting larger plaque size caused infectious viral particles in sera. our research here provides two kinds of dengue virus infection mouse models, which reproduce both severe and self-limited manifestations of human diseases, expanding the current choice of dengue viral infection small animal models. dengue virus serotype clinical strain, denv- gz, was originally isolated from a patient in guangzhou in , and was kindly provided by dr. xiaoyan che of zhujiang hospital in guangzhou, china. the virus was propagated in mosquito c / cells for no more than three passages in our lab. virus titer was determined in vero cells by foci forming assay. vero cells were passaged in dulbecco's modified eagle's medium (dmem, gibco, ny, usa) supplemented with % fetal bovine serum (fbs, gibco, auckland, new zealand) and % penicillin/streptomycin (p/s, gibco, ny, usa), and cultured at °c in a % co incubator. mosquito c / cells were cultured in minimum essential medium (mem, gibco) supplemented with % fbs, % penicillin/streptomycin and % non-essential amino acids (gibco, ny, usa) in a °c incubator under % co atmosphere. to purify viruses of different plaque morphology from the original clinical isolate, virus stock of denv- gz was serially diluted, and used to infect c / cell at , , , , and / pfu per well in a -well plate. seven days post infection, supernatant of each well was harvested. the foci size of virus that produced in each well was determined subsequently in vero cells by foci forming assay in -well plate. only supernatant containing virus with uniform foci size was kept for further propagation. after three passages, two viral clones maintained different plaque sizes, herein named as denv- d - -sp (variant with small plaque size) and denv- h - -lp (variant with large plaque size), were stored in a - °c freezer. titer of virus stock and infectious viral particle in serum of mice were determined using foci forming assay in vero cells. specifically, vero cells were seeded at cells/ well in a -well tissue culture plate and incubated overnight at °c, % co . on the second day, sera of infected mice or viral stocks were serially diluted in dmem and added into wells containing vero cells for a h incubation period. subsequently, the viral inoculum was removed and dmem supplemented with . % fbs, % p/s, . % carboxymethylcellulose (cmc, sigma, mo, usa) was added. after incubation for h at °c, cells were fixed with % paraformaldehyde (pfa). following the removal of pfa, infected cells were stained with antidengue monoclonal antibody d - ( : , santa cruz biotechnology, ca, usa) for h, followed by addition of biotin labeled anti-mouse igg (santa cruz biotechnology, ca, usa), and then streptavidin-alkaline phosphatase (sigma, mo, usa). finally, the foci of dengue virus were visualized by adding bcip/nbt substrate (beyotime biotechnology, jiangsu, china). neutralizing activity of mouse sera was evaluated using foci reduction neutralization test (frnt) as previous described (sun et al. ). genome information of two viruses was acquired through sanger sequencing. briefly, viral genomic rna was extracted from the virus stocks. after synthesis of viral cdna with specific primers, fragments covering the whole coding region of genome were amplified using phusion high-fidelity pcr polymerase (thermofisher, lithuania). when the pcr product concentration was high enough, they were sent directly for sequencing. otherwise, fragments were inserted into the peasy-blunt plasmid (transgene, beijing, china), which was then used to transform e. coli bacteria. three colonies containing each of the fragments were sequenced. full-length sequence of the single open reading frame was achieved based on the consensus sequence of each fragment, and then stringed together by overlapping them with each other. after that, sequence information of these two viruses was submitted to genbank, and the access numbers are mn (denv- h - -lp) and mn (denv- d - -sp) respectively. ifn-c elispot assay was performed according to manufacturer's protocol (mabtech, nacka strand, sweden). splenocytes of infected or control mice were isolated by homogenizing and filtering through lm cell strainers. after depleting erythrocytes, cells were re-suspended in % fbs/prmi- and added to elispot plates (millipore, co. cork, ireland) pre-coated with ifn-c antibodies. then pbs, lg/ml peptide, ns or e protein or lg/ ml cona was added to each well to stimulate splenocytes, and the plates were incubated at °c for - h. after being washed with pbs for times, plates were stained with biotin conjugated antibody, and subsequently streptavidin-horse radish peroxidase (hrp). finally, immune spots were visualized with the addition of tmb substrate. the image of spots was captured and analyzed by immu-nospot analyzer (cellular technology ltd. usa). balb/c mice were purchased from beijing vital river laboratory animal technology. type i and type ii ifn receptor double knock-out c bl/ mice (ag ) were originally provided by dr. guangxun meng of institut pasteur of shanghai, and raised in our laboratory. all mice were bred and maintained in specific pathogen-free barrier facilities and used at - weeks of age. for virus infection experiments, all procedures were completed under bsl- and absl- laboratory conditions as approved by the biosafety committee of institut pasteur of shanghai. female balb/c mice of - weeks old were injected with mg anti-type i ifn receptor monoclonal antibody (mar - a , bioxcell, usa) intraperitoneally (i.p.) at one day prior to challenge with . pfu parental strain, h - -lp or d - -sp through subcutaneous (s.c.) injection. blood samples were collected though retro-orbital bleeding daily from the st day post infection ( dpi) to th day post infection ( dpi). whole blood was lysed and homogenized within trizol-ls reagent (invitrogen, ca, usa) for measuring viral rna copy. meanwhile, for further detection of infectious viral particles in serum, sera were isolated from the blood through centrifugation. female ag mice of - weeks old were infected with pfu, pfu or pfu denv viruses through subcutaneous (s.c.) injection on day . body weight and survival rate were monitored from dpi to dpi, during which moribund mice with weight loss over % were euthanatized and recorded as death. vascular leakage was determined following intravascular administration of evans blue dye (sigma, louisiana, usa). briefly, ll evans blue dye ( % in pbs) was intravenously injected to mice on the th days post-infection of the two viral variants. after h dissemination, the mice were anaesthetized and perfused with ml pbs. digestive tracts and livers were collected and photographed. to quantify viral genomic copy in blood, rna was extracted following the user's guide of trizol ls reagent. a two-step quantitative rt-pcr (qrt-pcr) was then performed. first, cdna was synthesized using the fas-tquant rt kit (with gdnase) (tiangen biotech, beijing, china). second, amplifications were carried out using the fastfire qpcr premix (probe) kit (tiangen biotech, beijing, china) with the primer pairs designed according to the conserved sequence in denv utr: (forward, -tgayaagcartcagacac- ; reverse, -tcac-carrctccctttgc- ; fluorescence probe -cca-gagatcctgctgtctc- ). amplification cycles consisted of an initial incubation step of °c for min, cycles at °c for s, °c for s, and °c for s. standard rna were in vitro transcribed from a dna template that contains a t promoter and the target polynucleotide fragment for the primers indicated above. to obtain the standard curve, serially diluted standard rna ( - copy) were used as templates in parallel with sample rnas. copy of viral rna was calculated according to standard curves. all data were analyzed using graphpad prism software. t test or two-way anova were used to analyze the significance as indicated in legends. all results were expressed as means plus and minor standard errors of mean (sem). p values of . or less were considered statistically significant (*p \ . ; **p \ . , ***p \ . ; ****p \ . ). during in vitro propagation of a clinical strain of denv- obtained from guangzhou, china, heterogenous foci of different plaque sizes were observed in vero cell cultures (fig. a) . through limiting dilution and culture in mosquito c / cells, two viral variants were separated. after subsequent passage for three rounds in c / cells, two virus strains with stable foci morphology were obtained. one of them forms larger foci of about . mm diameter after h culture, and it was termed as denv- h - -lp (fig. a) ; the other forms smaller foci of about . mm diameter and was termed as denv- d - -sp (fig. a) . we next compared the replication abilities of these two viruses in both vero cells and c / cells using the onestep growth curve. denv- h - -lp had a higher replicating efficiency in vero cells than denv- d - -sp during the first h after infection, followed by a decline afterwards, due to the probability of running out target cells for infection; in comparison, denv- d - -sp rose slowly to a peak at h post infection (hpi), and kept its plateau until hpi (fig. b) . distinct from what was observed in vero cells, there was no obvious growth difference between the two viruses in mosquito cells (fig. c) . taken together, these data demonstrated that two viral variants with different foci sizes are isolated from one clinical virus isolate. and these two variants have different replication abilities in vero but not mosquito c / cells. to determine whether the different replication phenotypes reflect underlying genetic divergence between the lp and sp viruses, we sequenced the whole coding sequence of denv- h - -lp and denv- d - -sp, and aligned their envelope sequence with those of representative viruses belonging to different denv- genotypes, including asian genotype, american asian genotype, cosmopolitan genotype and sylvatic genotype. additionally, denv- strains isolated in guangdong, china, from to were also included for comparison. the phylogenetic analysis showed that denv- h - -lp and denv- d - -sp belonged to the cosmopolitan genotype and were clustered together with many strains isolated in west pacific regions, with percentages of homology ranging from . % to % (fig. ) . further comparison of genomic information between these two viruses also indicated that they had sequence variations in coding regions as illustrated in table . collectively, we found sequence variations in e, ns , ns b and ns regions, including synonymous mutations and amino acid changes. among the nonsynonymous mutations, two are in domain i/ii regions of e protein, two are in peptidase domain and helicase domain of ns respectively, another is located in a transmembrane helix of ns b, and the rest two are in ns rdrp domain. all of these domains are components of viral binding, replication or processing machinery, and highly related to viral antagonisms to host immunity. the conservation of mutations was also analyzed in comparison with representative strains from different genotypes, interestingly, though most synonymous mutations were also observed in other dengue- viruses, the seven nonsynonymous mutations were unique, and these sites seem more conserved in dengue- viruses (table ) . these genomic characteristics may contribute to the different infectivity both in vitro and in vivo. as type i/ii interferon deficient mice are more susceptible to dengue virus infection, we determined the in vivo infectivity of these two viruses in type i and type ii ifn receptors double knock-out ag mice. ag mice between and weeks old were infected through s.c. injection with pfu, pfu and pfu denv- h - -lp or denv- d - -sp (fig. ) . it is observed that infection of either virus led to % mortality within - days, even at a dose as low as pfu, though denv- h - -lp caused more weight loss and faster death than denv- d - -sp at the same dosage. specifically, weight loss began on dpi, dpi and dpi in mice infected with , , and pfu denv- viruses were used to infect cells at moi . , supernatant of infected cells were collected at the indicated timepoints. titers of infectious virus in supernatant were measured using foci forming assay in vero cells. detection limits of infectious virus titer were indicated with dashed lines. significance of the differences was calculated with two-way anova test, ***p \ . , n.s., not significant. h - -lp, respectively (fig. a , b, c), and death began on dpi, dpi and dpi correspondingly (fig. d, e, f ). in contrast, weight loss was first observed - days later in mice infected with denv- d - -sp, on dpi or dpi with inoculation of - pfu or pfu, respectively. the death was also delayed, began on dpi or dpi correspondingly. as a mixture of two types of viral variants, the parental strain was also able to cause weight loss and lethal diseases in ag mice. comparing to the two purified variants, the parental quasispecies presented intermediate level of pathology, which was most obvious in the dose groups of pfu. through evans blue staining, we also detected plasma leakage in sick mice on day post infection by either denv- h - -lp or denv- d - -sp viruses (fig. ) . consistent with the progress of weight loss and survival curves, leakage of evans blue in liver or digestive tract was more severe in mice infected with h - -lp on dpi. the evidence above demonstrated that the two viral isolates show differences on the pathology and disease kinetics, though they both have capabilities for causing lethal infection in ifn deficient mice. although both viruses can cause lethal infection in type i and type ii double knock-out mice, whether the lp and sp viruses behave differently in type ii ifn competent host is unknown. we have recently developed a zikv infection model in balb/c mice with transient blockage of type i ifn fig. phylogenetic analysis of denv- d - -sp and denv- h - -lp with representative serotype- dengue viruses of different genotypes isolated from different geographical regions. the phylogenetic tree was obtained after analyzing envelope sequences of denv- with mega x software, using the maximum likelihood method and kimura -parameter model. members of six reported genotypes of denv- were included. denv- d - -sp (mn ) and denv- h - -lp (mn ) were denoted by red dots in the tree. the scale bar denotes an evolutionary distance of . nucleotides per position in the sequence. virologica sinica receptor (ifnar) (liang et al. ) , we thus used the same strategy to examine these two dengue viruses for further comparison. wt balb/c mice were injected with antibody specific for ifnar at one day prior to s.c. infection with pfu of either viral strain, and monitored for seven days. the viral rna of denv- h - -lp could be detected throughout the week after infection, and the peak rna level of about genomic copy per microgram total rna was reached on day post infection, followed by gradually dropping to copy on the th day (fig. a) . in contrast, viral rna of denv- d - -sp was not detected until day post infection, when merely copy per microgram of total rna was transiently detected for days. consistently, infectious virus was detected in sera of denv- h - -lp infected mice from to dpi, during which peak viremia of pfu/ml appeared on day post infection. meanwhile, infectious virus was not detectable in mice infected by denv- d - -sp (lower than detection limit of pfu/ml) (fig. b) . moreover, h - -lp viral rna was also detected in liver, spleen and eyes on dpi (fig. c ), further confirming replication of h - -lp in susceptible organs. the parental strain also replicated in balb/c mice ( fig. a and b) , and the kinetics of viremia were closer to that of h - -lp, except for the delay of peak viremia to the th day post infection in both assays. to examine whether the above observed different virological properties of the two denv variants affect adaptive immunity in host, we evaluated adaptive immune responses induced by their infection in balb/c mice. denv- h - -lp infection elicited antibodies crossneutralizing the reference strain denv- (fig. a ) and it also stimulated t cell responses specific to peptides of denv- ns and zikv ns (fig. b) . similarly, neutralizing antibody and t cell responses were also detected in mice which were previously infected by denv- d - -sp, though both responses were weaker than those elicited by denv- h - -lp. considering genome similarity between the two variants, such different magnitudes of adaptive immune responses are mainly related to the different viral replication of two variants and different expression level of immunogens in vivo. in summary, we isolated from the same viral quasispecies two denv variants that showed distinct in vitro replication phenotype in vero cells, and different in vivo infectivity and pathogenicity in mice. using either of these two viral variants, we have established a lethal infection model in immune deficient ag mice. with the variant denv- h - -lp, we have also developed a self-limited infection model in wt mice pretreated with type-i ifn receptor antibodies. these two closely-related viruses not only offer new tools for in vivo studies, but also provide table sequence variation between h - -lp and d - -sp. in this study, we have successfully separated two viral variants that show different foci sizes in vero cells from a common serotype- clinical isolate of dengue virus. using these two viral variants, we established mouse infection models that reproduce severe manifestations of dengue diseases in ag mice. such lethal model with h - -lp or d - -sp viruses in ag mice can be used in pathology study, for example, to explore the viral and host factors that mediate hemorrhagic manifestation of dengue viruses. on the other hand, denv- h - -lp was used in another infection model which resembles self-limited infection of dengue virus in balb/c mice. and this transient infection model is more suitable for study of t cell responses, since the immune cell lineages are intact and the ifn-c responses are normal during the development of adaptive immunity. additionally, this self-limited infection model is also suitable for investigating how murine host resolves dengue infection. in previous investigations of dengue infection, only a limited number of viral strains were found to infect immune deficient mice, in which human illness without neurological disease can be studied, and lethal doses of these viruses ranged from to pfu (sarathy et al. ) . one often used virus is dengue serotype- strain d , which was obtained from a neuropathic strain pl through alternative passage between c / mosquito cells and ag mice. the ld of this virus in ag is - pfu (orozco et al. ) . another is d y p-pp , a double-plaque purified clone from a laboratory strain d y p, which had been passaged in c / cell for times. and the lethal dose of d y p-pp in ag mice fig. weight loss and survival curve of ag mice after infection with denv- h - -lp and denv- d - -sp. ag mice between - weeks old were infected with two viral variants and the parental isolate through s.c. injection. three different dose of viruses, pfu (a, d; n = ), pfu (b, e; n = ), and pfu (c, f; lp and sp n = , parental n = ), were inoculated into mice. mice injected with pbs were included within each panel for control (ct, n = ). weight change was recorded and presented as a ratio to the initial weight on day . moribund mice with weight loss over % were euthanatized and recorded as death. two-way anova was used in statistical analysis of differences among groups, *p \ . , **p \ . , ***p \ . ,**** p \ . , n.s., not significant. was documented higher than pfu (tan et al. ) . in contrast, the virus strains in this study had % mortality within days after inoculation in ag mice of - weeks old even when the dose was reduced to pfu, i.e., approximately times more lethal than the d and d y p-pp viral strains, and thus making denv- h - -lp virus a unique tool for studying the pathogenesis of dengue virus. through evans blue staining, we also confirmed the manifestation of plasma leakage in infected mice, which is a characteristic of severe dengue diseases in human. collectively, we show the highly virulent dengue strain h - -lp is ideal for establishing lethal infection models at low inoculum. to increase the utility of the new dengue viral variants in the context of relatively normal immune system, we have also adapted a recently developed model that transiently blocks ifnar with antibodies in balb/c mice. high level of viral rna was maintained within the first week after infection by denv- h - -lp, but only low level of transient viral rna was detected at - dpi in mice administered with denv- d - -sp. though no clinical symptom was observed, in mice infected with denv- h - -lp, we successfully detected infectious virus in sera, and viral rna in multiple organs including liver, spleen and eyes. the viruses might have been eliminated from mice after days of infection, accompanied by the maturation of denv adaptive immunity. thus, such characteristic and progress of infection is consistent with what was observed in patients with mild disease (who ). in previous animal models, denv infection of adult wt mice required extremely high inoculum, intracranially injection, or mouse adapted viruses. a portion of these models led to neurological symptoms or additional clinically irrelevant manifestations, limiting their use for pathology and therapeutic study (plummer and shresta ; sarathy et al. ) . to our knowledge, this is the first study which developed a denv infection model in wt mice, using a moderate dose of a non-adapted virus strain through physiologically more relevant s.c. injection route. it is often observed that many viruses form heterogenous plaques of different sizes in vitro, indicating that viruses exist as quasispecies during transmission or passaging (kato et al. ; moser et al. ), but the virological differences among various phenotypic variants have not been carefully examined. clinically, such viral diversity may assist arbovirus to survive under different selection pressure from two fig. vascular leakage in ag mice induced by infection of denv- h - -lp and denv- d - -sp. ag mice of weeks old were infected with pfu denv- h - -lp or d - -sp through s.c. injection. seven days post infection, mice were injected intravenously with evans blue and denv-induced vascular leakage was visualized in different tissues. pbs infected mice were used as negative controls. female balb/c mice of - weeks old were injected through i.p. with mg antibody to type i ifn receptor (mar - a ) at one day prior to infection. on the next day, pfu of viruses were inoculated into mice through s.c. route. then, blood samples were collected daily after infection. a viral rna copies in blood were measured using qrt-pcr, two-way anova was used in statistical analysis of the differences among groups, **p \ . , ****p \ . , and b titers of infectious virus in serum samples were determined through foci forming assay in vero cells; n = . t test was used in statistical analysis of the differences among groups, *p \ . , **p \ . , *** p \ . , ****p \ . . c eye, liver and spleen were collected from h - -lp virus-infected mice, and viral rna loads were determined, n = . detection limits of rna copy and infectious virus titer were indicated with dotted lines. n.d. not detected. distinct hosts, invertebrate and vertebrate. denv- h - -lp exhibits better replication capability than denv- d - -sp in vero cells, whereas their growth in mosquito cells is similar, reflecting possibility that the large-plaque variants in the original isolate contribute more to viral fitness in mammalian host. consistently, in mouse model, denv- h - -lp presents higher pathogenicity in ag mice and better replication in balb/c mice. comparing the coding sequence of denv- h - -lp and denv- d - -sp, we found amino acid variations on e, ns , ns b and ns proteins. e protein is responsible for virus attachment and entry; ns , ns b and ns are all the components of replication complex and essential antagonists for type i ifn responses (green et al. ). thus, further investigation of the underlying mechanism for the different phenotypes of these two viruses is valuable for understanding the molecular basis of dengue virulence and denv-host interaction. denv inhibits type i ifn production in infected cells by cleaving human sting humanized rag -/-gammac-/-mice support multilineage hematopoiesis and are susceptible to hiv- infection via systemic and vaginal routes mouse stat restricts early dengue virus replication the global distribution and burden of dengue both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage regulation of antiviral t cell responses by type i interferons innate immunity to dengue virus infection and subversion of antiviral responses dengue: a continuing global threat experimental studies on dengue. i. isolation, identification and modification of the virus at days post infection, frnt (foci reduction neutralization test) a and ifn-c elispot assay (b) were performed with mouse sera and splenocytes, respectively. reference strain denv- was used in frnt assay, peptides and proteins derived from denv- (d -ns , d -ns , d -e ) and zika virus (z-ns , z-ns ) were included in elispot assay to measure specific and crossreactive responses dengue fever in china: an emerging problem demands attention characterization of large and small-plaque variants in the zika virus clinical isolate zikv/hu/s /chiba/ molecular epidemiology demonstrates that imported and local strains circulated during the dengue outbreak in guangzhou recombinant zika virus envelope protein elicited protective immunity against zika virus in immunocompetent mice growth and adaptation of zika virus in mammalian and mosquito cells characterization of a model of lethal dengue virus infection in c bl/ mice deficient in the alpha/beta interferon receptor stat mediates innate immunity to dengue virus in the absence of stat via the type i interferon receptor mouse models for dengue vaccines and antivirals gamma interferon (ifngamma) receptor restricts systemic dengue virus replication and prevents paralysis in ifn-alpha/beta receptor-deficient mice production of immunity to dengue with virus modified by propagation in mice mouse models of dengue virus infection for vaccine testing murine model for dengue virus-induced lethal disease with increased vascular permeability adaptive immune responses to primary and secondary dengue virus infections dengue viruses cleave sting in humans but not in nonhuman primates, their presumed natural reservoir elaboration of tetravalent antibody responses against dengue viruses using a subunit vaccine comprised of a single consensus dengue envelope sequence subcutaneous infection with non-mouse adapted dengue virus d y p strain induces systemic vascular leakage in ag mice in: dengue: guidelines for diagnosis, treatment, prevention and control, new edn. who guidelines approved by the guidelines review committee dengue virus targets the adaptor protein mita to subvert host innate immunity epidemiological and virological characterizations of the author contributions js and xj designed and supervised the study. zhz and js carried out the experiments. zhz, js and xj analyzed the data and wrote the paper. zhl and ml assisted with animal experiments on ag mice. all authors read and approved the final manuscript. conflict of interest all authors declare that they have no conflict of interest. key: cord- -eyukbot authors: diosa-toro, mayra; urcuqui-inchima, silvio; smit, jolanda m. title: arthropod-borne flaviviruses and rna interference: seeking new approaches for antiviral therapy date: - - journal: adv virus res doi: . /b - - - - . - sha: doc_id: cord_uid: eyukbot flaviviruses are the most prevalent arthropod-borne viruses worldwide, and nearly half of the flavivirus members identified are human pathogens. despite the huge clinical impact of flaviviruses, there is no specific human antiviral therapy available to treat infection with any of the flaviviruses. therefore, there is a continued search for novel therapies, and this review describes the current knowledge on the usage of rna interference (rnai) in combating flavivirus infections. rnai is a process of sequence-specific gene silencing triggered by double-stranded rna. antiviral rnai strategies against arthropod-borne flaviviruses have been reported and although several hurdles must be overcome to employ this technology in clinical applications, they potentially represent a new therapeutic tool. the genus flavivirus of the family flaviviridae comprises more than enveloped rna viruses and depending on the virus can cause disease and mortality in humans and animals. flaviviruses are arthropod-borne viruses (arboviruses) and most of them are transmitted to vertebrates by mosquitoes or ticks (gubler, kuno, & markoff, ) . important human pathogens are dengue virus (denv), west nile virus (wnv), yellow fever virus (yfv), and japanese encephalitis virus (jev; gould & solomon, ) . these viruses cause major outbreaks with a variety of disease symptoms, including encephalitis and hemorrhagic fever. in severe cases, the mortality rates range from below % to % (who, a (who, , b (who, , c (who, , d . currently, effective vaccines are in use for yfv (barrett & teuwen, ), jev (halstead & thomas, ) , and tick-borne encephalitis virus (tbev) (heinz, holzmann, essl, & kundi, ) but not for other flaviviruses. furthermore, there is no specific antiviral therapy available for any of them. for the rational design of vaccines and therapeutic drugs, it is imperative to understand the molecular interplay between the host and the pathogen. in recent years, extensive progress has been made in this area and one of the important discoveries is the role of rnai in controlling flavivirus infections. rnai is a conserved process of gene silencing, and due to its ability to control viral and host gene expression, it is considered a key determinant in the replication machinery of viruses and consequently in the pathogenesis of disease (sidahmed & wilkie, ) . the two primary effector molecules of the rnai pathway are micrornas (mirnas) and small interfering rnas (sirnas; carthew & sontheimer, ; kim, han, & siomi, ) . rnai can act to reduce viral loads by inhibition of viral protein expression and transcription of viral genomes (sidahmed & wilkie, ) . the rnai strategy has been shown to be effective against multiple mammalian host viruses such as coronavirus (shi et al., ) , vesicular stomatitis virus (otsuka et al., ) , human immunodeficiency virus (hiv; ahluwalia et al., ) , h n influenza a virus (song, liu, gao, jiang, & huang, ) , and hepatitis c virus (hcv; pfeffer & baumert, ) . in this review, we will discuss a compilation of papers in which rnai has been suggested as a potential therapy against the most commonly distributed flaviviruses. we will begin with the description of the rnai mechanism and its antiviral features, as well as some general characteristics of the genus flavivirus. then, we will summarize the studies that have been reported in the field of rnai against flaviviruses and its vectors. finally, the challenges that need to be overcome in order to improve sirna delivery and its applicability to prevent or treat infections by flaviviruses will be discussed. the effector molecules of the rnai mechanism are small rna molecules of - nucleotides in length. there are several categories of small rnas, and in this review, i will focus on the two most described molecules, mirnas and sirnas. the processing and generation of mirnas and sirnas is depicted in fig. . . within the nucleus, pri-mirna molecules are generated by rna polymerase ii transcription (lee et al., ) . these pri-mirna molecules have a hairpin-shaped structure and are about nucleotides in length. the double-stranded rna structure within the hairpin is subsequently recognized by a nuclear protein called digeorge syndrome critical region (dgcr ; landthaler, yalcin, & tuschl, ) . thereafter, dgcr interacts with the rna-cutting enzyme drosha to generate mirna precursors (pre-mirnas). the pre-mirnas are then transported to the cytoplasm by exportin- (lee, jeon, lee, kim, & kim, ; lund, guttinger, calado, dahlberg, & kutay, ) . once in the cytoplasm, the pre-mirnas are cleaved into -nt mature mirnas by an rnase iii-like enzyme termed dicer (bernstein, caudy, hammond, & hannon, ) . the mature mirna duplex then undergoes an atp-dependent unwinding process and a single strand is loaded onto the rna-induced silencing complex (risc). the mirna binds to mirna recognition elements (mre) which are mostly located in the untranslated region ( utr) of mrnas (bartel, ). binding of a mirna to its mrna target typically leads to translational repression and exonucleolytic mrna decay, although highly complementary targets can be cleaved endonucleolytically as well (filipowicz, bhattacharyya, & sonenberg, ) . several methodologies have been developed to exploit the rnai mechanism. for example, mirna functioning can be mimicked through the introduction of a plasmid or viral vector encoding short hairpin rna (shrna) molecules into the cell (amarzguioui, rossi, & kim, ) . furthermore, it has been shown that exogenous dsrna molecules can also be incorporated in the rnai pathway. the dsrna molecules can be transfected into the cell cytoplasm, taken up from the environment, or produced in virus-infected cells as rna replicative intermediates . these dsrna molecules are then processed by dicer into sirnas. alternatively, exogenous synthetic - nt sirnas can be transfected directly into the cytoplasm (elbashir et al., ) . the sirna molecules undergo an atp-depending unwinding process and a single strand is incorporated in the risc complex, similarly as with the processing of mirnas. in contrast to mirnas, sirnas only induce degradation of the perfectly base-paired target mrna (dykxhoorn, novina, & sharp, ) . besides regulation of gene expression, rnai in plants, insects, nematodes, and fungi play an essential role in the antiviral response via virusspecific sirnas (segers, zhang, deng, sun, & nuss, ; voinnet, ; wilkins et al., ) . in virus-infected mammalian cells, small rnas have been identified (parameswaran et al., ; yeung et al., ) but it remains unclear whether they contribute to an antiviral response (haasnoot & berkhout, ; sidahmed & wilkie, ) . in contrast, mammalianencoded mirnas have been reported to target and inhibit viral gene expression. for example, mirna mir- targets two different sequences (located in the utr and the open reading frame ) in the genome of primate foamy virus type , thereby inhibiting viral translation and replication (lecellier et al., ) . furthermore, similar findings were reported for mir- and mir- in vesicular stomatitis virus infection (otsuka et al., ) ; mir- a during hiv infection (ahluwalia et al., ) ; and mir- , - , and - in influenza h n infection (song et al., ) . these findings are indicative for the importance of the host rnai are encoded in the genome and transcribed into primary mirna transcripts (pri-mirnas). pri-mirnas are processed to mirna precursors (pre-mirnas) in the nucleus by the rnase iii-like enzyme drosha and the digeorge syndrome critical region (dgcr ). pre-mirnas are transported to the cytoplasm by exportin , where they are cleaved by dicer to yield - nt mirna duplexes. one strand is then selected and loaded in the rna-induced silencing complex (risc). the key components of risc are proteins of the argonaute (ago) family which mediate translational repression or cleavage of target mrnas. furthermore, dsrna molecules are targets for the rnai pathway. these dsrna molecules are artificially introduced in the cell cytoplasm or are virus rnas. like mirna precursors, long dsrna are processed by dicer into nt sirna duplexes. one strand of the sirna is selected and loaded into risc. the binding of an sirna to its target mrna typically induces degradation. in modulating the replication efficiency of the infecting viruses and point out that harnessing this pathway could represent a powerful antiviral method. flaviviruses are spherical particles with a diameter of approximately nm. the viral genome consists of a single-stranded positive-sense rna molecule which is approximately kb in length. the rna molecule contains highly structured and utrs and has one open reading frame (lindenbach, thiel, & rice, ; markoff, ) . the rna is packaged by multiple copies of the capsid (c) protein (nucleocapsid) and is surrounded by a host-derived lipid membrane in which two structural transmembrane proteins are inserted, the envelope glycoprotein e and the membrane protein m. infection with one of the arthropod-borne flaviviruses begins when the vector takes a blood meal thereby injecting the virus into the dermis of the host. the reproductive cycle of flaviviruses starts with attachment of the virus to cell-surface receptors. upon virus-receptor interaction, the particle is internalized by receptor-mediated endocytosis and delivered to endosomes (stiasny, fritz, pangerl, & heinz, ) . within the acidic lumen of the endosome, fusion of the viral and cellular membranes is triggered by the viral e glycoprotein allowing the release of the nucleocapsid into the cell cytoplasm (smit, moesker, rodenhuis-zybert, & wilschut, ) . following nucleocapsid uncoating, the rna is translated into a polyprotein of about amino acids in length. the polyprotein is subsequently processed by viral and host cellular proteases yielding the three structural proteins (c, prm, and e) and seven nonstructural proteins (ns , a, b, , a, b, and ns ) (lindenbach et al., ; urcuqui-inchima, patino, torres, haenni, & diaz, ) . the prm and e proteins are translocated across the endoplasmic reticulum (er) membrane and form heterodimers that are oriented into the lumen of the er. the ns proteins assemble and form a viral rna replication complex (mackenzie, jones, & westaway, ; urcuqui-inchima et al., ) . rna synthesis is regulated by the - cs and - uar inverted complementary sequences that allow the circularization of the genome (villordo & gamarnik, ). after genome cyclization, the rna-dependent rna polymerase, ns , synthesizes the complementary minus-strand rna molecule from the genomic rna, which subsequently acts as a template for the synthesis of new positive-sense viral rna (davidson, ; maeda, maeda, takagi, & kurane, ) . the newly synthesized positive-sense rna molecule interacts with multiple copies of the c protein to form a nucleocapsid (ivanyi-nagy & darlix, ) . the nucleocapsid then buds into the er thereby acquiring a lipid bilayer containing heterodimers of the prm and e proteins. the prm protein acts as a scaffold to prevent premature fusion of the virus during its transport out of the cell (perera & kuhn, ) . during the secretory pathway, virion maturation occurs in the trans-golgi network through furin-mediated cleavage of the prm protein to m and a pr peptide (fernandez-garcia, mazzon, jacobs, & amara, ). the pr peptide dissociates after the release of the virion into the extracellular milieu and allows new progeny virus particles to bind and infect a new permissive host cell via receptor-mediated endocytosis. in figure . the life cycle of flaviviruses is depicted. ( ). the acidic environment within the endosomes induces fusion ( ) between the viral and cell membranes resulting in the release of the rna genome ( ). the rna genome of flaviviruses contains a single open reading frame and highly structured and utrs ends. the rna is translated into a polyprotein precursor that is processed into three structural proteins and seven nonstructural proteins ( ). the complementary sequences cs (cyclization sequence) and uar (upstream aug regions) at both ends of the genome allow its circularization a required step for replication. replication is mediated by the nonstructural proteins. virus assembly occurs in the endoplasmic reticulum (er) ( ). the resultant immature particles are transported through the golgi network where furin-mediated cleavage of prm to m generates mature infectious particles ( ) that are released by exocytosis ( ). the natural hosts of wnv are birds, but humans, horses, and other mammals can also be infected (ulbert, ) . the virus is transmitted by a vast range of mosquitoes and depending on the geographical region, different species are responsible for the transmission of the virus. the most important vectors are bird-feeding mosquitoes of the culex genus (ulbert, ) . approximately % of the infected people develop a classical west nile fever (flu-like symptoms with high fever), and in about % of the cases, severe neurological complications, such as encephalitis, meningitis, or flaccid paralysis are seen. in the latest group, the mortality rate can reach % (who, c). the prophylactic potential of exogenous sirnas against wnv infection was highlighted several years ago. mccown et al. constructed dna vectors encoding sirnas targeting the c and ns genes of wnv and transfected these into t cells h prior to wnv infection. the number of e-positive cells was % and % lower in cells transfected with the plasmid encoding the sirna against c and ns , respectively. in addition, a decrease in rna levels ( %) and virus particle production ( %) was achieved in cells transfected with the sirna against c (mccown, diamond, & pekosz, ) . similar results were observed in vero cells using sirnas targeting the ns gene (ong, choo, chu, & ng, ) and the e gene (bai et al., ) and in htb- cells when the c gene was targeted (yang et al., ) . also, wnv replication and virus particle production was found attenuated when sirnas were applied targeting the utrs of the viral genome (anthony, bai, krishnan, fikrig, & koski, ) . taken together, these observations indicate that pretreatment of cells with sirnas directed against the wnv genome results in a significant inhibition of wnv replication. the therapeutic effect of sirnas to wnv infection is limited and appears to be dependent on the transfection methodology applied. geiss, pierson, and diamond ( ) observed that sirnas targeting the c gene had no effect on virus replication when transfected into cells h after wnv infection using lipid-based reagents. in addition, no significant reduction in viral protein or rna levels was seen in wnv replicon-expressing cells transfected with sirnas targeting the ns gene using lipid-based reagents. on the other hand, however, a -fold reduction in ns protein expression and a . -fold decrease in rna levels were observed when the sirna molecules were delivered to the cell cytoplasm by electroporation. these results indicate that transient opening of membrane structures is required to induce rnai during active wnv replication. however, a word of caution is in place here as sirna transfection with oligofectamine, also a lipid-based reagent, was found to reduce hcv protein expression and rna replication in hcv replicon-expressing cells (takigawa et al., ) . the different efficacy of sirna treatment before and after wnv infection is supported by in vivo data in mice. bai et al. reported that mice are protected from fatal infection with wnv when sirnas targeting the e gene are administered h before the virus challenge, but failed to protect when the sirna is given h after virus challenge (bai et al., ) . furthermore, ye et al. observed that the administration of sirnas toward ns and c genes failed to protect mice when given days postinfection with wnv (ye, abraham, wu, shankar, & manjunath, ) . dengue is the most prevalent vector-borne disease in the world with an estimated million cases each year in (sub)tropical regions (who, a). the virus is predominantly transmitted to humans through the mosquito aedes aegypti, but aedes albopictus can act as a vector as well. four antigenically distinct viruses designated from to (denv - ) have been identified so far. infection with any of the four denv serotypes can give a broad spectrum of clinical manifestations ranging from acute self-limiting febrile illness to life-threatening complications like hemorrhagic fever and dengue shock. individuals that develop severe disease often have preexisting antidengue immunity generated during previous heterologous denv infections or received anti-dengue antibodies from their mother (ubol & halstead, ) . the immunopathogenesis of severe disease is complex and see references for reviews on this topic (green & rothman, ; ubol & halstead, ) in the past few decades, dengue disease has become a major health problem due to increased endemic activity and emergence to previously nonendemic regions (gould & solomon, ) . the first evidence of an rnai-mediated antiviral mechanism during denv infection was obtained by olson and colleagues. the authors showed that transient or stable expression of long dsrnas cognate to the denv- genome in a. albopictus (c / ) cells inhibited subsequent infection of these cells with denv- (adelman, blair, carlson, beaty, & olson, ; adelman et al., ; gaines et al., ; olson et al., ) . similar results were observed in dsrna transduced female a. aegypti mosquitoes (adelman et al., gaines et al., ; olson et al., ) . furthermore, wu et al. found that a synthetic sirna molecule targeting a sequence in the prm protein-coding region reduced the cytopathic effect in c / cells and increased cell survival more than twofold (wu et al., ) . the therapeutic potential of sirna for denv infection in mammalian cells has also been described. for example, adeno-associated virus vectors carrying sirna against the cs region (a conserved sequence in the utr of all four denv serotypes) decreased the percentage of denv-infected human monocyte-derived dendritic cells (mddc) and reduced apoptosis of the infected cells. the observed attenuated but prolonged virus particle production may be beneficial as activation of humoral and cellular immune responses is considered to help with the clearance of denv particles (zhang et al., ) . in addition, subramanya et al. reported % reduction in infection of mddc when cells were treated with sirnas targeting e gene after denv infection (subramanya et al., ) . in this study, the authors used a novel peptide-based delivery system that allows targeting of sirnas to human dcs both in vitro as well as in in vivo models (subramanya et al., ) . importantly, the effectiveness of rnai against denv infection has also been verified in vivo (stein et al., ) . retro-orbital intravenous injection of three doses of sirna molecules (targeting a conserved sequence in the cs region) in mice at h pre-and and h-postchallenge with denv revealed that sirna treatment increases the average survival, and significantly reduces viral loads in several tissues and at different time-points postinfection (stein et al., ) . in addition to the use of exogenous sirnas to restrict denv replication, the antiviral function of endogenous mirnas has also been described. in these studies, engineered viruses were used that contain mres for cellular mirnas within the viral genome (heiss, maximova, thach, speicher, & pletnev, ; lee et al., ) . it was found that the insertion of the mre for the hepatic-specific mir- in the utr of denv genome suppresses the viral replication in transfected cells (lee et al., ) . moreover, using a chimeric denv/tbev virus (c, prm, e from tbev), it was shown that the inclusion of the mre for the brain-expressed mir- and mir- a, reduces the access of the virus to the central nervous system and consequently the development of lethal encephalitis in mice (heiss et al., ) . another approach that has been explored is to reduce denv replication by modulating host gene expression. for instance, downregulation of the heat shock protein by rnai in denv-infected u cells decreased viral replication and particle production significantly (padwad et al., ) . in another study, the cell-surface receptor grp and the clathrin-mediated endocytosis pathway were silenced by rnai, and this approach resulted in a % reduction of denv-infected cells (alhoot, wang, & sekaran, ) . also, a recent report showed that sirna toward the tnf-a gene reduced cytokine response in denv-infected dcs, highlighting the potential of targeted rnai-based approaches to simultaneously decrease viral replication and the detrimental host immune response (subramanya et al., ) . although effective vaccination against jev and yfv are currently available, these viruses remain a major public health problem that threat hundreds of millions of people living in endemic regions and to people traveling to these regions. the annual estimated incidence of jev and yfv infections is , and , cases with a mortality rate of % and %, respectively (misra & kalita, ; who, d) . jev is transmitted through a zoonotic cycle between mosquitoes, pigs, and water birds. humans are only accidentally infected. the primary mosquito vector of jev is culex tritaeniorhynchus, but species such as cx. gelidus, cx. fuscocephala, and cx. annulirostris are important secondary or regional vectors (van den hurk, ritchie, & mackenzie, ). for yfv, several species of the aedes and haemogogus mosquitoes are important for transmission. the vertebrate hosts of yfv are monkeys and humans, and the mosquitoes transmit the virus from one host to another (who, d). the continuous geographical expansion of the mosquito vectors of jev and yfv and the difficulties to implement a worldwide vaccination program highlights the need for a specific antiviral therapy. the effectiveness of rnai to inhibit yfv replication was described by pacca and coworkers (pacca et al., ) . vero cells stably transfected with a shrna targeting the ns or the e gene decreased yfv infection in % and %, respectively. administration of these shrnas to mice h prior to yfv challenge increased the survival rate in % (sirna toward ns ) and % (sirna toward e) (pacca et al., ) . the effect of rnai in jev replication has been examined in vitro and in vivo. jev replication was attenuated in bhk- and hek- t cells transfected with a shrna targeting the ns gene h prior to infection (liu et al., ; qi et al., ) . in addition, kumar and coworkers reported that jev infection is almost completely abolished in vero cells and neuro a cells prior transduced with a shrna directed to the e gene. importantly, all mice survived jev infection when treated with shrna . or h postchallenge (kumar, lee, shankar, & manjunath, ) . the use of several sirnas to target multiple sites along the genome of jev has also been reported. wu et al. constructed a single polycistron to simultaneously express nine sirnas-targeting eight genes of jev (prm, ns , ns a, ns b, ns , ns a, ns b, and ns ). transfection of bhk- cells with this construct inhibited replication of three wild-type jev strains belonging to two different genotypes (wu, xue, wang, du, & jin, ) . furthermore, efficient inhibition of infection was seen using recombinant jev carrying mres. yen et al. inserted two copies of the mre for mir- in the utr of jev. none of the mice challenged with this virus died from infection, whereas virus challenged with the wild-type strain or with a virus carrying a mutated mre was lethal for all mice. interestingly, immunization with the jev carrying the mre for mir- elicited protective immunity against subsequent lethal challenge with wild-type jev (yen et al., ) . the vast majority of mosquitoes become infected with an arbovirus when they take a blood meal from a viremic vertebrate host. viral amplification begins with the infection of midgut epithelium cells which is followed by dissemination of the virus to the salivary glands and other tissues. once the virus is present in saliva, a female mosquito transmits the virus to a new host while taking blood meal (kuno & chang, ) . despite efficient virus replication, minimal pathology is seen in the mosquito vector (lambrechts & scott, ). this suggests the presence of evolutionary mechanisms that allow virus replication without jeopardizing the host and transmission. indeed, it has been suggested that rnai is a key component of the innate immune response of mosquitoes to viral infections (blair, ) . in previous sections, we have described that an exogenous trigger of the rnai pathway in mosquito-cultured cells (adelman et al., gaines et al., ; olson et al., ) and a. aegypti mosquitoes (adelman et al., gaines et al., ; olson et al., ) can mediate resistance against flaviviruses. in addition, it has been shown that wnv (chotkowski et al., ) and denv (mukherjee & hanley, ) infection (mukherjee & hanley, ) of drosophila cell lines induce functional virus-specific sirnas that promote a protective rnai response. although the production of denv-specific sirnas has also been reported in aag cells and orally infected a. aegypti mosquitoes, it seems that the virus can somehow circumvent the natural rnai response, since persistent infection is observed in both cases (brackney et al., ; sanchez-vargas et al., ) . furthermore, impairment of the rnai pathway was found to increase viral replication (brackney et al., ; sanchez-vargas et al., ) . taken together, these observations suggest that the rnai response in mosquitoes controls virus replication to such an extent that it prevents pathology in the infected host but is sufficiently high to allow transmission of the virus to a new host. furthermore, the exploitation of rnai to reduce vector competence for flavivirus infection has been suggested, especially since traditional approaches for vector control have failed (beaty, ) . significant progress has been made in this area with a. aegypti, the major vector of denv. female mosquitoes genetically manipulated with germ line transgenes to express dsrna derived from the prm gene of denv- in their midgets have a high level of resistance toward denv- infection and a reduced ability to transmit the virus (franz et al., ) . the resistant phenotype of the transgenic mosquitoes was maintained for generations; however, from g it became weaker and eventually it was lost in g . the loss of resistance was caused by the inhibition of the transgene expression, but the explanation for this phenomenon remains unclear . as the authors pointed out, considerations in the transgene design-such as the potential risk for the modified mosquitoes and the ability of the transgene to be inherited and expressed-should be taken into account if a strategy of population replacement will be applied in the field. so far we have described the antiviral effect of the rnai mechanism induced by exogenous delivery of sirna or precursors, and how cellular mirna can target sequences artificially introduced within the genome of flaviviruses. in recent years, the naturally occurring interactions between viruses and the host-cellular rnai pathway have also been highlighted. viral infections induce profound changes in the mirna expression profile of the host cell. these changes may represent an additional mechanism of the innate immune response and/or are induced by the virus in order to promote reproduction . there are only a few reports available describing how arthropod-borne flaviviruses influence the mirna expression of their host cells. skalsky, vanlandingham, scholle, higgs, and cullen ( ) , observed downregulation of mir- and overexpression of mir- in cx. quinquefasciatus mosquitoes infected with wnv when compared to noninfected mosquitoes. unfortunately, there is no evidence on the functional role of these mirnas during wnv infection . it has also been reported that wnv induces upregulation of mirna hs_ in infected neuronal cells and in the central nervous system tissue of infected mice. hs_ targets the mrna of the antiapoptotic proteins, ccctc-binding factor (ctcf), and egfr-coexpressed protein (ecop). the authors hypothesize that the upregulation of hs_ is a cellular antiviral response to the infection since downregulation of ctcf and ecop contributes to cell death (smith, grey, uhrlaub, nikolich-zugich, & hirsch, ) . in addition to cellular mirnas, many virally encoded mirnas have been identified. viral mirnas have been described to regulate both, cellular and viral gene expression (grundhoff & sullivan, ) . although initially cytoplasmic rna viruses such as flaviviruses were not believed to express mirnas (pfeffer et al., ) , hussain et al. have reported the generation of a mature mirna derived from wnv in infected aag and c / cells. the discovered mirna (kun-mir- ) upregulates the mosquito gata mrna level and promotes replication of the virus (hussain et al., ) . furthermore, as a proof of concept it was shown that functional viral mirnas can be produced if the pre-mirna sequence is incorporated into the genome of the virus. for example, tbev was able to express an epstein-barr viral mirna when the precursor was integrated into the utr of tbev genome (rouha, thurner, & mandl, ) . flavivirus infections represent a severe global public health problem with major individual, social, and economic consequences; therefore, preventive and therapeutic strategies are urgently needed. as we reviewed here, rnai can be used as a prophylactic and therapeutic agent against flavivirus infections and its associated diseases. furthermore, we highlighted the possibility to use rnai in vector control and pathogen transmission. however, many hurdles must be overcome in order to translate the positive in vitro and in vivo data to the clinic and into the fields. rnai is based on sequence homology and therefore virtually any sequence can be targeted. indeed, this review described that many flavivirus genes can be successfully targeted. in addition, mrnas encoding cellular cofactors for viral replication can be targeted to suppress viral replication. however, careful design of the small rna sequence must be done since partial homology between small rnas and unintended mrna transcripts might result in nondesirable gene silencing (jackson et al., ) . in addition, if the activation of the innate immune response is not desirable, it is critical to avoid sequences that can be recognized by toll-like receptors (robbins, judge, & maclachlan, ). moreover, during rnai silencing of viral replication, escape mutants often arise and therefore targeting of conserved regions is preferable as well as the use of multiple rna molecules targeting distinct regions (sugiyama, habu, ohnari, miyano-kurosaki, & takaku, ; wu et al., ; zhang et al., ) . bioinformatic approaches followed up with experimental validation should be applied to determine optimal sirna sequences that are complementary to the target mrna, induce minimal immune response, and avoid the appearance of escape mutants. in the case of flaviviruses, highly conserved sequences within the c, ns genes and within the utr might represent excellent targets for rnai therapies. furthermore, future research should continue to focus on the complex interplay of arthropod-borne flaviviruses and the rnai pathway in different host cells to search for novel targets for rnai-based intervention. perhaps, the biggest challenge in the development of successful sirnabased drugs lies within the design of a safe and effective delivery system targeting biological relevant cell types. this is a complex task with multiple demands, including slow excretion, high stability in blood serum, limited nonspecific accumulation in tissues, efficient cellular uptake, and intracellular release. the most commonly applied delivery systems to date are lipid-based carriers, antibodies, peptides, and nanoparticles conjugates (shim & kwon, ) . recently, a clinical trial with patients having solid cancers showed inhibition of gene expression using sirna-targeted nanoparticles that were administered systemically (davis et al., ) . this technology was also found effective in a cell culture model of hcv infection (chandra et al., ) . alternatively, as was mentioned above, subramanya and coworkers successfully linked an sirna molecule to a peptide able to bind specifically to human mddc, one of the main targets of denv (subramanya et al., ) . future research is required to further characterize the potential of these and other delivery 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effectively inhibits virus replication hypersusceptibility to vesicular stomatitis virus infection in dicer -deficient mice is due to impaired mir and mir expression rna interference inhibits yellow fever virus replication in vitro and in vivo rna interference mediated silencing of hsp gene in human monocytic myeloma cell line u revealed decreased dengue virus multiplication six rna viruses and forty-one hosts: viral small rnas and modulation of small rna repertoires in vertebrate and invertebrate systems structural proteomics of dengue virus impact of micrornas for pathogenesis and treatment of hepatitis c virus infection identification of micrornas of the herpesvirus family effective inhibition of japanese encephalitis virus replication by small interfering rnas targeting the ns gene sirna and innate immunity functional microrna generated from a cytoplasmic rna virus dengue virus type infections of aedes aegypti are modulated by the mosquito's rna interference pathway evidence that 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and associated proinflammatory cytokine production rna interference targeted to the conserved dimerization initiation site (dis) of hiv- restricts virus escape mutation suppression of hepatitis c virus replicon by rna interference directed against the ns and ns b regions of the viral genome how innate immune mechanisms contribute to antibodyenhanced viral infections west nile virus: the complex biology of an emerging pathogen recent developments in understanding dengue virus replication ecology and geographical expansion of japanese encephalitis virus genome cyclization as strategy for flavivirus rna replication rna silencing as a plant immune system against viruses water-related diseases. japanese encephalitis. who ( -west nile). west nile virus. fact sheet n. . who ( -yellow fever). yellow fever rna interference is an antiviral defence mechanism in caenorhabditis elegans inhibitory effect of small interfering rna on dengue virus replication in mosquito cells broad-spectrum antiviral activity of rna interference against four genotypes of japanese encephalitis virus based on single microrna polycistrons inhibition of west nile virus replication by retrovirus-delivered small interfering rna in human neuroblastoma cells silencing early viral replication in macrophages and dendritic cells effectively suppresses flavivirus encephalitis neurovirulent flavivirus can be attenuated in mice by incorporation of neuron-specific microrna recognition elements into viral genome pyrosequencing of small non-coding rnas in hiv- infected cells: evidence for the processing of a viral-cellular double-stranded rna hybrid efficient inhibition of hiv- replication by an artificial polycistronic mirna construct attenuation of dengue virus infection by adeno-associated virus-mediated sirna delivery this work was supported by colciencias-colombia, grant - - and convocatoria nacional para estudios de doctorados en colombia año , by jan kornelis de cock foundation (february -july ), and by the dutch organization for scientific research (nwo-vidi, earth and life sciences). key: cord- - lujp oy authors: neeraja, m.; lakshmi, v.; lavanya, vanjari; priyanka, e.n.; parida, m.m.; dash, p.k.; sharma, shashi; rao, p.v. lakshmana; reddy, gopal title: rapid detection and differentiation of dengue virus serotypes by ns specific reverse transcription loop-mediated isothermal amplification (rt-lamp) assay in patients presenting to a tertiary care hospital in hyderabad, india date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: lujp oy early and rapid detection of dengue virus (denv) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. in the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (rt-lamp) for rapid detection and serotyping of the denv targeting ns gene using the genie® ii flourometer was carried out. the performance of the rt-lamp was compared to rt-pcr, cdc - real time pcr and the ns antigen elisa, igm and igg anti denv antibodies. acute denv infection was confirmed in / patients suspected clinically of denv infection. rt- lamp and cdc - real time pcr assay was positive in / patients, while / patients were positive for anti- dengue igm and igg antibodies. the rt-lamp assay and the cdc real-time rt-pcr assay showed high concordance (k = . ). the detection rate of acute denv infection improved to % ( / ) when the results of rt-lamp were combined with ns ag, igm and igg elisa. the rt-lamp had a detection limit of copies for den- and den- , copies for den- and den- compared to copies for den- and den- , copies for den- and den- by the conventional rt-pcr. the assay showed % specificity. the rt-lamp assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of denv infection in endemic countries such as india. dengue is a mosquito borne flaviviral infection, affecting the tropical and subtropical regions of the world and is one of the major emerging global public health problems. there are four antigenically distinct dengue virus serotypes den- , den- , den- and den- and each serotype contains phylogenetically distinct genotypes (teoh et al., ) . the dengue virus (denv) infection induces a lifelong protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection by the other three serotypes. therefore, multiple and sequential infections with the four denv serotypes would be expected for people living in a region where the infection is hyper endemic due to the lack of cross-protective neutralizing antibodies. seroepidmiological studies have shown that the secondary infection is a major risk factor for dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) through antibody-dependent enhancement (halstead et al., ; monath and heinz, ) . diagnosis of denv infection on the basis of clinical signs and symptoms is not reliable as more than half of the infected individuals either are asymptomatic or have a mild undifferentiated fever (burke et al., ; endy et al., ) . early diagnosis of dengue infection can reduce the number of cases of dhf and dss. therefore, there is a great demand for the rapid detection of the infection and differentiation of denv serotypes for timely clinical management and disease control, respectively. the most common methods for laboratory diagnosis of denv include serological methods detecting antibodies (igm and igg) against denv and additionally various methods are used in detecting denv rna or antigens: non-structural protein (ns ) and envelope protein (e). the serological methods are vulnerable to cross reactions caused by antibodies against related flaviviruses and are therefore not denv-specific tests like denv ns antigen and rna detection methods. the detection of dengue specific secretory ns (non-structural protein ), a highly conserved glycoprotein represents a new approach to the diagnosis of acute denv infection, in recent times. enzyme-linked immunosorbent assays (elisa) directed against ns antigen (ns ag) have demonstrated its presence at high concentrations in the sera of dv infected patients during the early clinical phase of the disease (dussart et al., ) . assays based on the detection of nonstructural protein (ns ) tend to be specific for denv infection. ns antigen levels correlate well with viremia and it circulates at high levels during the first few days of illness especially in patients with dhf. ns antigen remains circulating in patients' blood for longer periods than does viral rna and is reported to be detectable even up to the th day of illness. the first isothermal amplification methods introduced in s included the transcription mediated amplification (tma), nucleic acid sequence based amplification (nasba) and the strand displacement amplification (sda). the loop-mediated isothermal amplification (lamp) is a novel nucleic acid amplification method and has the potential to replace pcr because of its simplicity, rapidity, specificity, sensitivity and cost-effectiveness without the need of specialized equipment (notomi et al., ; parida et al., ; tomita et al., ) . the rt-lamp assay is being increasingly used by various investigators for rapid detection and typing of emerging viruses (chan and fox, ; mori et al., ; parida et al., ) . these earlier reports, however, evaluated their rt-lamp assays for the detection of denv infection with a small clinical sample size (< ) and using the c-prm gene (lu et al., ) or serotype-specific regions of the untranslated region (utr) (parida et al., ; li et al., ; sahni et al., ) . the c-prm gene, however, was relatively less conserved among all the four denv serotypes (inter-serotype) in comparison to the utr (teoh et al., ) . however, we have targeted a highly conserved region of ns , revealing > % sequence identity among various genotypes within each serotype. as such genotyping of dengue serotypes can be done employing many gene including ns and ns (klungthong et al., ) . in the present study, the rt-lamp assay was developed for the detection and serotyping of denv infection targeting the serotype specific regions of the ns gene using a real-time flourometer (genie ® ii from optigene, u.k.). the detection sensitivity and the specificity of the reported denv ns serotype specific rt-lamp in freshly obtained blood samples from patients suspected clinically of denv infection, is compared with available test system for suitable algorithm. to the best of our knowledge this is the first report of the detection and differentiation of dengue using ns rt-lamp with real time fluorometer (genie ® ii from optigene, u.k.) from south india. the study was approved by the institutional ethics committee of nizam's institute of medical sciences (ec/nims/ / ). written informed consent was obtained from each patient. reference strains of the four dengue virus serotypes den- , rr (kf ), den- , gwl (ay ), den- , nd (fj ), den- , nd (hm ) were used in this study (dash et al., (dash et al., , neeraja et al., ) . patients suspected clinically of dengue/dhf/dss, who either reported directly or were referred to a tertiary care institute for treatment from the regions in and around hyderabad, from july to december , were included in the study. the dengue/dhf/dss case proformas prepared as per the who protocol (world health organization, ) for denv infection was filled by the treating clinicians. acute phase and early convalescent serum and plasma samples based on reporting time were collected from patients with a history of sudden onset of fever, and the presence of two or more of the symptoms viz. headache, eye pain, nausea, vomiting, rash, myalgia, abdominal pain suggestive of denv infection. samples collected within days of fever were categorized as acute phase samples and those collected after days of fever were considered as convalescent phase sample. in order to check the cross-reactivity, within serotypes and with other closely related members of flavivirus family i.e., je, wnv archived samples from drde gwalior and hcv positive samples from our tertiary care hospital were included in the study. confirmed chikungunya (chikv) rna positive samples were also included as symptoms of denv and chikv mimic each other. in addition, a panel of samples collected from healthy individuals was included as negative controls. before performing the rt-lamp assay, all the samples were also screened for denvspecific rna by rt-pcr (lanciotti et al., ; neeraja et al., ) and ns antigen by panbio dengue early elisa assay (inverness medical innovations, australia), dengue igg and igm capture elisa (pan bio, queensland, australia). denv serotype specific oligonucleotide primers were designed from the ns region of denv genome. the nucleotide sequence of the ns gene of denv, representative of respective genotype and serotype strain was retrieved from gen bank (den- , accession no. eu ; den- , accession no. af ; den- , accession no. eu ; and den- , accession no kc ) and was aligned with the available ns gene sequences from global denv strains including the circulating strains in india, to identify the conserved regions using dnasis software (hitachi, japan). the primers were selected based on criteria described by notomi et al. percent gene homology among each serotype were found to be ≥ %. the potential target region corresponding to the genome positions was selected from the aligned sequences, and the rt-lamp primers were designed from conserved region of each serotype using the primer explorer version software (eiken chemical co., tokyo, japan). a set of six primers comprising two outer (f and b ), two inner (fip and bip), and two loop primers (flp and blp) that recognize eight distinct regions on the target sequence was designed. the primers were selected based on criteria described previously by notomi et al. all the primers were assessed for specificity before use in lamp assays with a blast search with sequences in the gen bank (table ) . the viral rna was extracted from l of the serum/plasma samples by using the qiaamp viral rna mini kit (qiagen, germany). the rna was eluted from the qia spin columns in a final volume of l of the elution buffer and stored at − • c until testing. the rt-lamp was carried out in a final reaction volume of l. the reaction mixture contained l of isothermal master mix iso- (optigene, u.k.) containing, geobacillus species dna polymerase, thermostable inorganic pyrophosphatase, optimized buffer including mgcl , dntps and ds-dna dye (optigene, u.k.), l primer mix consisting of primers each for denv- , denv- , denv- , and denv- (f and b primers at . m, fip and bip primers at . m, lf and lb primers at . m), . units amv reverse transcriptase (promega, madison, wi.), . l nuclease free water and l extracted nucleic acid. the rt-lamp assay was run at temperatures between and • c and time between min and min in the real-time fluorometer (genie® ii from optigene, u.k.) to determine the optimal temperature with the shortest amplification time and the highest fluorescence reading. all the rt-lamp assays were subsequently run at • c for min followed by a heating and cooling step to • c to • c ( . • c/s) to allow re-annealing of amplified dna and display of the annealing curve. the genie ii displays amplification signals in real time and at the end of the run displays the time to positivity that is expressed in terms of plots of fluorescence signals (real time curves) and t m for each specimen. the analysis of each sample was done in a set of four tubes, with serotype specific primer mixture. the t m for denv- was . • c, denv- was . • c, denv- was . • c and denv- was . • c. positive and negative controls were included in each run, and all precautions to prevent cross-contamination were observed. amplification of the dna leads to an increase in fluorescence emitted from a dna intercalating dye. this increase was monitored in real time using the genie® ii fluorometer. following incubation at • c for min, a l of aliquot of the rt-lamp assay products was electrophoresed on % nusieve : agarose gel (biowhittaker molecular applications, rockland, maine) in trisborate buffer, followed by staining with ethidium bromide and visualization on a uv transilluminator at nm. in order to facilitate the field application of the rt-lamp assay, monitoring of amplification was done visually with an unaided eye. following amplification in genie ® ii flourometer l of sybr green i intercalating dye was added to the reaction tube. the rt-lamp amplification was visually monitored for colour change. positive reaction turned the reaction mix green and fluoresces under the white light and uv irradiation, respectively. the reaction mix remained orange and non-fluorescent in the absence of amplification. this change of color is permanent and thus can be kept for record purposes. in order to compare the sensitivity and specificity of the rt-lamp assay, one-step rt-pcr was done by employing the two outer primer pairs ( pmol of f and b ) targeting the ns gene of each serotype. amplification of the rna was carried out in l reaction volume with the pcr mix containing primescript tm step enzyme mix and its buffer along with respective sense (f ) and anti sense (b ) primer in a thermal cycler (applied biosystems, usa). the thermal profile of the rt-pcr reaction was-reverse transcription at • c for min, initial denaturation at • c for min, followed by cycles of denaturation at • c for min, annealing at • c for min, extension at • c for min and final extension at • c for min (neeraja et al., ) . the real-time rt-pcr assay from cdc was used as a standard test for the denv serotype specific identification in abi quantitative pcr system (abi, usa). the assay is based on taqman chemistry including a panel of oligonucleotide primers and dual labeled hydrolysis probe sets [d , d , d , d ] employing invitrogen super script tmiii platinum® one step quantitative kit. the amplification was carried out in a l reaction volume. instruction and standard thermal profile for sample screening was as follows, reverse transcription • c for min, initial denaturation and enzyme inactivation • c for min, cycles of extension at • c for sec and • c for min of denaturation and annealing extension respectively (chien et al., ) . briefly, the reagents include × buffer (invitrogen one-step rt-pcr kit, usa) . l, enzyme mix . l, d /d both forward and reverse primers . l ( nm), d /d both forward and reverse primers . l ( nm) and d -d probe . l ( nm) each and depc treated water added up to a total volume of l. finally, l of viral rna elute extracted from different samples was added for real-time rt-pcr assay. . . performance parameters of denv rt-lamp . . . sensitivity of serotype-specific dengue virus-specific rt-lamp assay the sensitivity of the ns serotype specific rt-lamp assay was determined through serial dilutions of in vitro transcribed denv with known copy number. the specificity of the primers for detecting denv serotypes was validated by testing samples that were positive for other flavivirus including je, wnv, hcv and chikv. in addition, the authenticities of the amplified products were also established by nucleotide sequencing of amplified products with outer (f ) and inner (b ) primers (parida et al., ) . nucleotide sequencing of the ns gene of randomly selected dengue viruses from the clinical samples, that included one den- (vl ) and two den- (vl , vl ), was carried out by employing the big dye terminator cycle sequencing ready reaction kit with an abi sequencer (applied biosystems, usa) for identifying the genotype of the denv serotype by following the standard protocol (dash et al., ) the sequences were initially subjected to blast to find the closest sequence identity. further, phylogenetic analyses based on the ns gene junction of den- and den- were carried out by including a large number of geographically diverse denv gene sequences, by using the neighbour-joining (nj) method of the mega software version . . the sequences of vl , vl , and vl were submitted to genbank under the accession numbers kc , kj , kf , respectively. inter-assay variability for reproducibility was assessed by testing sample of each serotype in separate lamp runs and recording time and t m for each serotype. the intra-assay variability for repeatability was assessed by simultaneously testing samples of each serotype that included strong positive and weak positive of each serotype. the degree of agreement between rt-lamp and the cdc real time pcr test results was measured by kappa value (k). fisher's exact test (two tailed) was done to calculate p value, p value < . was used to suggest significant results. the diagnostic performance of rt-lamp assay and the cdc real time pcr assay as compared with ns ag and ns rt-pcr assay was calculated using med calc easy to use statistical software (http://www.medcalc.org/ calc/diagnostic test.php). / patients suspected clinically of denv infection, were confirmed as acute denv infection by detection of the ns ag, anti-igm, conventional rt-pcr, and the real-time rt-pcr either alone or in combinations. / patients were positive by ns ag alone. out of the remaining patients were positive only for anti-dengue igm and igg antibodies. ns serotype specific rt-pcr assay was positive in / patients, ns serotype specific rt-pcr assay detected patients that were negative for ns ag by elisa test. rt-lamp assay detected additional patients that were negative for ns ag by elisa and ns rt-pcr assay. / patients were identified as past denv infection as dengue igg antibodies alone were tested positive among them (table ) . all the four denv serotype-specific primers were highly specific for the detection and differentiation of the appropriate serotypes with no cross reaction. none of the serotype-specific primer sets amplified or cross reacted with any of the je, wnv, hcv or chikv viral rna template and samples from healthy individuals, there by indicating their specificity. as depicted in fig. , the size of the resultant product by rt-pcr using outer primers f and b was in good agreement with the predicted size for each serotype, i.e., bp for denv- , bp for denv- , bp for denv- , and bp for den- , (fig. ) . % sequence homology was also observed between the primers and the corresponding nucleotide sequences. the sensitivity of the in house developed denv ns serotype specific rt-lamp assay was same as that of the cdc - real time rt-pcr assay. these two methods showed high concordance with kappa value of . . the diagnostic accuracy improved to % ( / , % confidence interval = . - . ) when the results of the denv rt-lamp or the cdc real time assay were combined with the results of the ns antigen and anti -dengue igg and igm elisa (table ) the diagnostic performance of rt-lamp compared to ns ag by elisa and the ns rt-pcr is summarized in table . the sensitivity of the ns serotype specific rt-lamp assay as a function of the timing of the test (days after onset of fever) was studied and it was found that the sensitivity was optimal, at . % ( % ci, . % to . %), between days and for ns serotype specific rt-lamp assay (table ) . sensitivity of the ns serotype-specific dengue virus rt-lamp assay compared to ns ag, igg + igm antibody, ns rt-pcr and the cdc real time rt-pcr assay. the cdc-real time pcr assay was considered as gold standard control. a ns rt-pcr detected samples that were negative for ns ag by elisa. b rt-lamp and cdc real time rt-pcr assay detected samples which were negative for ns ag by elisa and ns rt pcr assay. the diagnostic performance of the rt-lamp assay against ns ag and the rt-pcr assay in dengue patients in tertiary care hospital in hyderabad. table the sensitivity of the ns serotype specific rt-lamp assay related to number of days after onset of fever (n = ). the rt-lamp assay was positive more in the patients with primary infections ( / ) compared to patients with secondary infections ( / ). p value for the detection of primary infections by rt-lamp was statistically significant when compared with secondary infections (p < . , by fishers exact test). rt-lamp was positive among patients with df, patients with dhf and patients with dss. the rt-lamp assay detected copies of den- and den- and copies of den- and den- rna, respectively, as shown in fig. a and b and the sensitivity of rt-pcr was copies of den- and den- and copies of den- and den- as shown in fig. c and d. the optimized amplification time of samples from patients ( of each serotype) by denv rt-lamp was min. as shown in table . the mean time to positivity for all positives was min fig. . agarose gel electrophoresis of denv serotype-specific rt-pcr assay products on a % agarose gel employing f and b primers of respective serotypes. d -denv- rt-pcr assay product, bp; d -denv- rt-pcr assay product, bp; d -denv- rt-pcr assay product, bp; d -denv- rt-pcr assay product bp. comparative sensitivity of rt-lamp versus rt-pcr for detection of the ns gene of denv. sensitivity of the rt-lamp assay as monitored by real-time measurement of fluorescence. shown from left to right are the curves of decreasing concentrations of virus from × to × copy numbers of the template in a serial -fold dilution. the detection limit for the assay was copy numbers for denv and denv (a) and copy numbers for denv and denv (b). (c and d) sensitivity of rt-pcr for the detection of the denv ns gene as observed by agarose gel analysis with a detection limit of copy numbers for denv and denv and copy number for denv and denv . lane m, -bp dna ladder (sigma); lanes to , different concentrations of virus ranging from × to × − copy numbers in a serial -fold dilution pattern. the real-time amplification of each dengue virus serotype in genie ® ii fluorometer is shown in fig. a and b, that shows different amplification times and annealing temperatures (c and d). on %agarose gel electrophoresis the amplification product was detected as a ladder-like pattern due to the formation of a mixture of stem-loop dnas with various stem lengths and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target sequence in the same strand (fig. ) . the visual detection of the rt-lamp results is shown in fig. . the most predominant serotype documented in our study was den- ( patients), followed by den- ( patients), den- ( patients) and den- ( patients). den- and den- serotypes were confirmed by sequencing, with accession numbers kj and kf for den- and kc for den- . phylogenetic analysis of den- showed that the strain belonged to genotype iv and den- belonged to genotype iii (fig. ) . precision of the rt-lamp for identification and serotyping of denv was determined by testing samples of each serotype that included strong positive and weak positive of each serotype. the mean amplification times of the replicates for strong and weak positives for den- was . min (standard deviation [sd], . ) and . min (sd, . ), den- was . min (sd, . ) and . min (sd, . ), den- was . min (sd, . ) and . min (sd, . ) and den- was . min (sd, . ) and . min (sd, . ), respectively. to further assess the reproducibility of rt-lamp, we tested sample of each serotype in separate lamp runs and recorded the time to positivity and t m for each serotype. the difference in the amplification times for each serotype across separate runs were within . min for each serotype and sds ranging from . to . indicating that the rt-lamp for denv identification and serotyping is highly reproducible (table ) . the nonstructural protein (ns ), of dengue viral genome has been shown to be a useful tool for the early diagnosis of acute dengue infections (cdc-laboratory guidance dengue) and was found to be highly conserved for all dengue serotypes. the denv ns antigen elisa, a widely used test in recent times, is highly sensitive and specific (young et al., ; alcon et al., ) but it can be compromised by pre-existing ns -igg immunocomplexes in the acute stage of secondary denv infection, a common feature in dengue endemic regions (lapphra et al., ; hang et al., ) . although, who (world health organization, ) recommends the detection of denv rna, as the most effective diagnostic method in the acute phase of the illness, its use is limited due to lack of infrastructure and technical expertise. more recently, molecular techniques to detect virus genomic rna sequence by the reverse transcription-polymerase chain reaction (rt-pcr) and the real-time quantitative rt-pcr (qrt-pcr) are gradually being accepted as new standards over virus isolation for the detection of denv in the acute sera (lanciotti et al., ; shu et al., ) . these pcr-based methods require either high-precision instruments for the amplification or elaborate methods for detection of the amplified products. in addition, these methods are often cumbersome to adapt to routine clinical use, especially in the peripheral health care settings and the private clinics (parida et al., ) . the rt-lamp assay has emerged as a powerful gene amplification tool for rapid identification of microbial infections and is being increasingly used by various investigators for rapid detection and typing of emerging viruses, such as the west nile, severe acute respiratory syndrome, dengue, and japanese encephalitis viruses (hong et al., ; parida et al., parida et al., , parida et al., , parida et al., , . a four tube denv ns serotype specific rt-lamp assay was developed in this study for the rapid detection and differentiation of dengue serotypes in this study with high sensitivity and specificity. using the primer concentrations (f and b primers at . m, fip and bip primers at . m, lf and lb primers at . m), together with the use of commercially available isothermal master mix from optigene, uk, containing an engineered large fragment dna polymerase (gspssd), amplification time of min and temperature of c was optimized for rapid detection and serotyping of the denv. geobacillus dna pol enzyme demonstrated superior lamp amplification speed compared to bst dna pol i (parida et al., ; sahni et al., ; boon-teong et al., ) . this isothermal amplification mix allows fluorescence detection of the product on the genie ® ii platform but may also be used on generic qpcr instrumentation (www.optigene.co.uk, ). very few rt-lamp assays have been described for the detection and serotyping of denv. recently teoh et al. ( ) developed single tube rt-lamp assay by targeting utr of denv using nine sets of primers for serotyping of denv. however no information on the serotypes detected from the clinical samples was reported in fig. . phylogenetic tree of studied sequences with geographical strains of serotype and generated by neighbor-joining method. the tree is based on ns regions (nt - bp for denv- and nt - bp for denv- ) of the selected strains. this study. our experience revealed that because of emergence of many genotypes across multiple denv serotypes, the designing of pan-dengue primers is an impossible task and is most likely to miss some genotypes. the reproducibility of rt-lamp assay in this study was low and sensitivity was slightly lower than q rt-pcr. the specificity of the rt-lamp assay developed by teoh et al. was tested by single site restriction enzyme digestion that is not cost effective and also laborious. these reported methods for detection and differentiation of lamp results were done by real time monitoring of turbidity (at nm) with a loopamp real-time turbidimeter, "ladder-like feature" on agarose gel electrophoresis and color change from orange to green caused by fluorescent detection reagent. however real time monitoring of amplification for the detection and differentiation of denv serotypes in a fluorometer has rarely been reported. a few studies have reported the lamp assay for rapid detection of viruses by real-time fluorometer (genie ii from optigene, u.k.) (mahony et al., a,b) . in this study we established real-time fluorescence monitoring of isothermal method using simpler and less costly genie ® ii instrument. the most common method for real-time fluorescence monitoring of lamp reactions uses intercalating dyes such as sybr green (maeda et al., ; ohtsuka et al., ) . fluorescence detection using intercalating dyes has the advantage of allowing further analysis in terms of the temperature at which amplification products melt or anneal. lamp products contain structures of differing lengths containing catenated repeats of the target sequence which melt/anneal at a specific temperature determined by the length and g/c content of the target. after amplification, the reactions can be subjected to a gradual melting or annealing step with fluorescence monitoring to discriminate the specific amplification products from the non-specific artefacts in genie® ii instrument. this eliminates the need for gel electrophoresis or turbidity detection and allows for a closed-tube system thus reducing the cost of the assay. in our study the lack of overlap of t m allowed for the easy identification of all serotypes of dengue. the color change from orange to green in the positive control and the samples was evident in first - min in this study. the denv ns serotype specific rt-lamp assay developed in this study was significantly faster with a mean amplification time of min compared with earlier studies (parida et al., ; sahni et al., ; teoh et al., ) . the speed of the assay was probably due to the use of improved polymerase. this is the first report for evaluation of rt-lamp employing ns region of viral genome with larger clinical samples size of in a genie ® ii flourometer. the performance of the rt-lamp assay was validated by testing the samples simultaneously by the cdc real time pcr that is most sensitive and specific method for detection and differentiation of the denv (cdc dengue). both the rt-lamp and the cdc real time assay for detection and differentiation of the denv showed comparable sensitivity (k = . ). the rt-lamp scored over rt-pcr in terms of sensitivity and specificity and reproducibility compared with study done by teoh et al. in developing countries such as india where dengue is endemic and resources are limited, serological assays are most common methods used to confirm denv infection. in our study using actual clinical samples, the ns antigen by elisa was positive in / of patient's samples which were collected in acute phase of illness when antibodies were absent. the rt-lamp or cdc real time assay when used in combination with ns antigen and anti -dengue igg and igm elisa increased the diagnostic coverage of febrile patients to % ( / ). this is in concordance to the study done by teoh et al. ( ) . the most predominant serotype documented in this study was den- and den- which belonged to the genotype iii and genotype iv respectively. co-circulation of more than one serotype of denv is also known to cause hyperendemicity (dash et al., ) . the ns serotype specific rt-lamp assay developed in this study may be utilized as a rapid, simple, easy, cost effective, isothermal, highly sensitive and specific field applicable technique for detection and differentiation of the dengue virus serotypes which overcomes the deficiencies present in existing techniques. its applicability in tertiary care institutes is emphasized by its ability in viral quantification and evaluation of viraemia in patients. it can suitably be introduced in zonal/peripheral hospitals as an adjunct to existing immunological tests acting as parallel controls. the rt-lamp assay in optigene genie ii instrument may be employed as the new gold standard for timely and accurate diagnosis and serotyping of dengue in the clinical care, disease surveillance, disease prevention, and control activities in endemic countries such as india. enzyme-linked immunosorbent assay specific to dengue virus type nonstructural protein ns reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections a prospective study of dengue infections in bangkok nasba and other transcription-based amplification methods for research and diagnostic microbiology development of real time reverse transcriptase pcr assays to detect and serotype dengue viruses emergence and continued circulation of dengue- (genotype iv) virus strains in northern india emergence of dengue virus type (genotype i) in india evaluation of an enzyme immunoassay for detection of dengue virus ns antigen i human serum epidemiology of inapparent and symptomatic acute dengue virus infection: a prospective study of primary school children in kamphaengphet observations related to pathogenesis of dengue hemorrhagic fever. iv. relation of disease severity to antibody response and virus recovered diagnostic accuracy of ns elisa and lateral flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses development and evaluation of a novel loop mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome corona virus molecular genotyping of dengue viruses by phylogenetic analysis of the sequences of individual genes mega : integrated software for molecular evolutionary genetics analysis and sequence alignment rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction evaluation of an ns antigen detection for diagnosis of acute dengue infection in patients with acute febrile illness simultaneous detection and differentiation of dengue virus serotypes - , japanese encephalitis virus, and west nile virus by a combined reverse-transcription loop-mediated isothermal amplification assay rapid identification of chikungunya and dengue virus by a real-time reverse transcription-loopmediated isothermal amplification method detection of periodontal pathogen porphyromonas gingivalis by loop-mediated isothermal amplification method development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in minutes multiplex loop-mediated isothermal amplification (m-lamp) assay for the detection of influenza a/h , a/h and influenza b can provide a specimen-to-result diagnosis in min with single genome copy sensitivity flaviviruses detection of loop mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation investigation of acute dengue infection in southern india employing dengue specific serological and molecular assays loop-mediated isothermal amplification of dna detection of salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of salmonella isolates real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay development and evaluation of reverse transcription loop mediated isothermal amplification assay for rapid and real-time detection of japanese encephalitis virus rapid and real-time detection of chikungunya virus by rtlamp reverse transcription loop-mediated isothermal amplification (rt-lamp) for diagnosis of dengue development of group-and serotype-specific one-step sybr green i-based real-time reverse transcription-pcr assay for dengue virus detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification loop-mediated isothermal amplification (lamp) of gene sequences and simple visual detection of products guidelines for diagnosis, treatment, prevention and control an antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein ns in the sera of infected patients authors acknowledge financial support by the drde, gwalior, india, through sanction no. tc/ /proj /task- / to carry out this work. the authors would like to thank ms. deepal pandya of ampligene india biotech pvt. ltd. for her valuable help in the rt-lamp technique. the authors declare that there is no conflict of interests regarding the publication of this article. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . key: cord- - ah ijf authors: halstead, scott b. title: chapter pathogenic exploitation of fc activity date: - - journal: antibody fc doi: . /b - - - - . - sha: doc_id: cord_uid: ah ijf while the benefits of antibody responses are widely known, pathogens are also able to exploit antibodies to facilitate cell entry and potentially alter the cellular response via interactions with fc receptors. this phenomenon, known as antibody-dependent enhancement (ade) of disease, is a factor in numerous human and veterinary diseases. it is thought to result from innate cellular responses to fcγ receptor-facilitated entry of infectious microbial immune complexes, and paradoxically results in increased production of pathogenic organisms. ade has been described in vitro in numerous settings, but the strongest data regarding the in vivo impact of this mechanism on human disease come from human disease and animal models of dengue and leishmanial infections. this chapter reviews the literature of ade in relation to the innate immune responses to fcγ receptor ligation by infectious igg immune complexes and discusses the research frontiers regarding this harmful antibody activity. the phenomenon of antibody-dependent enhancement (ade) underlies severe diseases across the entire spectrum of both microbes and vertebrates. indeed, ade could be classified as a fifth type of immunopathology (table . ): type i, ige-mediated immediate hypersensitivity; type ii, igg-mediated acute immune complex disease; type iii, igg-mediated foreign antigen-complement-dependent immune complex disease; type iv, cell-mediated immune and autoimmune diseases; and type v, infectious immune complex enhancement of microbial infection in fcr-bearing cells. over the past four decades, different lines of scientific inquiry have coalesced to sharpen our understanding of antibody-mediated mechanisms that govern the severity of infections by a wide spectrum of microorganisms. independent studies of host responses to acute and chronic human and animal infectious diseases have generated evidence that crosslinking of immune complexes with fcγ receptors can increase intracellular infection and contribute to disease severity. , one of the earliest descriptions of this activity came from neutralization studies of murray valley encephalitis virus (mvev), in which exposure to virus in the presence of dilute avian antibodies resulted in greater clearance of chick embryo fibroblast monolayers than virus-only controls. , while follow-up research in the s suggested that this phenomenon resulted from the stabilization of mvev by antibodies, a different explanation emerged when sequential infections in humans with dengue viruses (denvs) were shown to produce severe disease (dengue hemorrhagic fever, dhf). , [ ] [ ] [ ] [ ] during initial studies on ade it had been assumed that increased virus output, which in some cases approached -to -fold, resulted from extrinsic phenomena, such as the avid attachment to monocytes and macrophage and increased internalization of infectious immune complexes via interactions with fcγi and fcγiia receptors as compared to virus alone, which was observed in numerous systems. [ ] [ ] [ ] [ ] [ ] [ ] [ ] however, this effect of igg opsonization can be complemented by a complex phenomenon involving suppression of innate cellular immunity. unexpectedly, the incubation of ross river virus (rrv) with diluted rrv antiserum resulted in enhanced infection in mouse macrophage cell lines and in primary human monocytes/macrophages via innate immune suppression involving reduced production of reactive nitrogen radicals via nos and a downregulation of tnf-α and ifn-β production through abolished interferon regulatory factor (irf- ) and nuclear factor-κb gene expression. remarkably, a marked increase in il- gene transcription and protein production was observed. , surprisingly, ligation of fcγr by zymosan-antibody complexes in the presence of rrv did not exhibit the same transcription patterns, indicating that features of the opsonized pathogen were involved in driving immune suppression. similarly, many investigators showed that igg bound to the surface of leishmania amastigotes (the intracellular mammalian host-dwelling parasite stage) can induce the cytokine il- , which, in turn, can suppress the protective immune response to the parasite by downregulating inducible no synthase (inos) and by inhibiting th cell development and ifn-γ production. , hence, the igg response, which is usually protective, is usurped by the parasite for its survival. when t-cell immunity fails, in the presence of abundant igg antiamastigote antibodies, infection starts on a lethal course. thus, ade is a complex phenomenon in which antibody-mediated interactions with fc receptors may be involved not only in altering pathogen uptake into macrophage or monocytes but also in the cellular response to internalized pathogen. antibody-enhanced viral infections require an initial adaptive immunological event, termed sensitization, which occurs in three settings: ( ) primary infections with naturally occurring heterotypic viruses of the same genera; ( ) infection by viruses that create antigenic diversity by the rapid evolution of biologic or antigenic variants during the course of a chronic infection; and ( ) immunizations that result in incomplete protective immunity (table . ) (reviewed in halstead , , and tirado and yoon ). the ade phenomenon has attracted wide interest in virology because many viruses replicate in macrophages in vivo and manifest antibody-enhanced infections/ disease. , , the dengue viruses (denvs) are a group of four closely related members of the flavivirus genus. denvs through share to % genetic homology and are inoculated by the bite of an infected aedes aegypti mosquito. initial infections with any of the four denvs raise protective but type-specific antibodies in addition to cross-reactive but non-neutralizing antibodies that may enhance infection by a different denv type. , [ ] [ ] [ ] [ ] [ ] indeed, infection-enhancing antibodies are a risk factor for enhanced dengue disease. that infants regularly acquire severe dengue disease during their first dengue infection when maternal polyclonal dengue antibodies circulate below protective levels is a unique illustration of the ade phenomenon in human medicine. [ ] [ ] [ ] [ ] [ ] [ ] [ ] in humans, secondary dengue infections follow a stereotypical course with severe outcomes, including shock or gastrointestinal hemorrhage, accompanying vascular collapse that results from capillary permeability occurring around the time of defervescence. high levels of viremia early in disease and high levels of [ ] [ ] [ ] in vitro studies indicate that the major requirement for ade activity is a subneutralizing antibody concentration. , in practice, antibodies directed at surface epitopes not involved in virus entry efficiently produce ade. in studies of ade in the thp- cell model (human monocytic fcγ receptor-bearing continuous cell line) intracellular denv production was increased as a result of idiosyncratic fcγ receptor signaling. when immune complexes ligate fcγri and fcγriia, at least two types of suppression pathways are expressed (figure . ). collectively, these pathways downregulate antiviral responses in ade-infected target cells. as a result, adeinfected thp- cells secreted reduced levels of type-i ifn and at the same time suppressed the transcription and translation of il- , ifn-γ and tnf-α, facilitating expression and synthesis of the antiinflammatory cytokines. ade infection also suppressed the innate anti-denv mediator, nitric oxide radicals, by disrupting the transcription of the inos gene transcription factor, irf- , believed to be mediated by il- . it can be concluded that in vitro ade infection not only facilitates viral entry but also modifies innate and adaptive intracellular antiviral mechanisms resulting in enhanced denv replication. critically, the same responses are observed in vivo. genome-wide transcriptomes from peripheral blood mononuclear cells (pbmcs) collected during the acute phase from children with dengue fever (df) or dengue hemorrhagic fever (dhf) indicated that patients with dhf had decreased levels of no, reduced ifn transcript, and increased il- blood levels compared to patients with milder illness. in other studies, during the acute stage of severe disease increased production of il- and downregulation of multiple ifn regulatory genes were noted. [ ] [ ] [ ] the protective role of ifn in moderating dengue infection has been demonstrated in a mouse model and suggested for humans with df. [ ] [ ] [ ] the precise role of immune complex-elicited il- production on the clinical evolution of severe dengue infections is not well understood but may be responsible for the observed th to th shift observed in dhf. results obtained by studying ade in continuous human fcr-bearing cells are subject to a critical caveat: are responses observed faithful to those occurring in vivo? when dengue ade was studied in four different primary human myeloid cells derived from the same peripheral blood leukocyte (pbl) donors, viral infection and cytokine responses differed significantly. human monocytes, activated macrophages, and mature dendritic cells (dcs) support ade, while immature dcs did not. infection of macrophages by denv alone or as fully neutralized immune complexes stimulated high levels of α and β ifn, and these were downmodulated under ade conditions and replaced by secretion of il- and tnf-α. type i ifns were neither produced nor suppressed by ade infection of monocytes with denv . , however, during ade infection of primary monocytes, il- synthesis peaked at the same serum dilution that produced peak virus yield. the precise mechanisms fueling enhanced production of dengue virus in monocytes and macrophages require further study, as neither the production of il- nor suppression of type i interferon is critical to the ade phenomenon. there is some evidence that the degree of antibody opsonization may impact fcγr interactions, resulting in differential processing of surface-bound virus. in particular, in the thp- model, large immune complexes have been found to stimulate fcγiib, resulting in inhibition of cellular uptake. in considering the contribution of fcrmediated phenomena during the course of acute infectious diseases it is important to note the differing kinetics of microbial invasion and response to infection. infections in vertebrates can be divided into afferent and efferent events. afferent events are those that relate to the invasion, propagation, and survival of infecting organisms. efferent events are those contributing to reduce, retard, and eliminate infecting organisms. for those infections that produce disease, afferent events successfully predominate during the early stages, while efferent events may culminate in elimination of invading organism and cure. in the ade phenomenon, igg antibodies residual from a previous dengue infection, whether actively produced or passively acquired, act as powerful afferent events. igm and, in the case of secondary dengue infections, igg dengue antibodies play important roles in the efferent events of dengue infections, some of which include clearing extracellular virus, activating complement, and mediating adcc. in dengue-endemic countries, second heterotypic dengue infections are responsible for up to % of dhf, while primary dengue infections in infants account for the remainder. , the in vitro ade literature makes it clear that antibodies to virtually any flavivirus can enhance dengue virus infection in fcr-bearing cells. in southeast asia, the dengue viruses co-circulate with japanese encephalitis (je); in india, with je and west nile; in pakistan, with west nile; in australia, with the kunjin strain of west nile; and, in most of the american region, yellow fever or use of yellow fever d vaccine. a direct examination of enhancement of denv infections in a human monocytic cell line by antibodies raised in humans who had experienced apparent wild-type je infections was negative. however, this study screened for ade at just a single high dilution of serum. there have been no reports of enhancement of dengue infections by prior west nile or yellow fever infections (or vaccination); however, recently it was observed that children in thailand who had either had wild-type je infections or were immunized with killed je vaccine experienced a mild overt dengue disease at a higher frequency than when the same dengue virus infected susceptible individuals. not only did dengue infections in je-immunes increase the frequency of mild overt dengue disease, but the ensuing illness lasted two times longer than dengue illnesses in susceptibles. prior to this observation it was thought that je vaccination actually reduced the severity of dhf accompanying a second dengue infection. second denv infections can occur in combinations, at least of which have been documented to result in hospitalized disease. human experimentation has established that there is a denv infection refractory period of three months following an initial denv infection. as presented briefly above, shortly after a primary denv infection abundant heterotypic antibodies may form large immune aggregates that are neutralized in solution but not eliminated by standard phagocytic clearance by macrophages. after the refractory period, second dengue infections occur and some are expressed as clinical illness. varying amounts of heterotypic neutralizing antibodies are raised following a primary dengue infection. the natural histories of antibody responses following infection by denvs , , , or including heterotypic antibodies are essentially unknown. heterotypic antibodies are critically important in modulation of a second denv infection. low dilutions of preinfection sera from children who had unapparent secondary denv infections almost invariably reduced or neutralized denv in primary human monocytes, while preinfection sera from children who developed an illness requiring hospitalization had few or no detectable heterotypic neutralizing antibodies. this observation suggests that low levels of heterotypic neutralizing antibodies (most were anti-denv ) did not prevent but downregulated denv clinical responses. around one-fifth of monotypically dengue-immune schoolchildren lacked heterotypic denv antibodies and developed dhf when infected by denv . this is virtually the same ratio for the occurrence of dengue shock syndrome (dss) during sequential denv then denv in rayong, thailand. the sequence of infection may be highly determinative of disease severity. only secondary denv infections were pathogenic in the cohort study at rayong, thailand. the specific infection sequences associated with dss cases were known from virus isolations in acute-phase sera and antibodies in pre-illness sera or by applying the original antigenic sin phenomenon to paired sera. although secondary denv infections were most common that year, dss occurred only during secondary denv infections. burmese workers came to a similar conclusion in their to longitudinal seroepidemiological study in yangon, myanmar. by contrast, in an indonesian study, dss was associated with sequences ending in denv , , and , but not denv , even though secondary denv infections were common. year-old children who were immune to denv . over time the pathogenicity of established denv infection sequences may change. notably, there was a rapid increase in pathogenic expression of secondary denv infections (in denv -immunes) month to month in cuba. complete and partial sequencing of denv viruses isolated from the beginning to the end of this outbreak identified no structural genetic mutations but consistent amino acid changes at position in the ns protein. , a more complex study in managua, nicaragua, attributed year-to-year increases in disease pathogenic expression to a combination of genetic changes in denv and a waning denv immunity in the affected children. essentially none of these field observations has been subjected to study using cultures of primary human myeloid cells. from the work cited above it must be concluded that ade studies should not be performed on continuous human myeloid cells but instead on characterized primary human myeloid cells. to date, all research has been carried out using denv . studies of ade should be extended as quickly as possible to the complete range of dengue viruses using type-specific polyclonal human antibodies rather than mouse or even human monoclonal antibodies. more observations on human dengue illnesses are needed to better understand the ade phenomenon. in this context, understanding dhf in infants that accompanies primary denv infections acquired during the latter half of the first year of life is important and has attracted recent research interest. , this unique clinical phenomenon illustrates not only the role played by dengue antibodies in modulating disease expression but also their bifurcated role-protection for several months after birth at high concentrations and enhancing infections some months later at sub-neutralizing concentrations. the contribution of immature denv particles to ade is of interest. immature den virions are not infectious for human myeloid cells, but in the presence of enhancing dengue antibodies ade infection occurs readily. , it has been surmised that immature denv are released into circulation during human infections as antibodies to prm (immature denv antigen) and are frequently observed. note has been made above of the possible role of different fcγ receptors in mediating ade in macrophages in leishmania mouse models and the interesting observation suggesting that ligation of fcγriib may affect the fate of large immune complexes during the immune clearance phase of dengue infections. the real research challenge is to document the critical fcγ receptors that are operative during human or veterinary clinical microbial disease enhancement episodes. but, more important will be to study the specific outcome of ade infection by different wild-type dengue viruses-antibody combinations including appropriate genotype variants. the possibility that differing combinations of denv and dengue antibodies (or, for that matter, antibodies to other flaviviruses) have uniquely different effects on monocyte/ macrophage innate immunity requires careful study. it is widely held that the process of eliminating dengue virus-infected cells generates a cascade of chemokines and cytokines that contribute to the pathophysiology of dengue disease syndromes. this has been termed "a perfect cytokine storm." given the evidence of increased infected cell mass in severe dengue infections, the cytokines generated by virus infection and by interactions between virusinfected cells and the host immune response would appear to be quantitatively proportional to viral load and not exaggerated or "abnormal." because dengue is not a cytophilic virus, cellular infection proceeds until the infected cell is eliminated. a wide misconception is that peak viremia, which occurs early in infection, represents peak "viral load." in fact, the quantity of virus in blood during the course of infection only describes the kinetics of extracellular virus clearance. using the sensitive technique of intrathoracic inoculation of toxorrhynchites splendens mosquitoes, , it was observed that dengue virus could be detected in nearly all plasma samples collected within hours of the onset of fever. the frequency of virus isolation declined rapidly and virus was usually not present at defervescence. viremia was of longer duration in individuals experiencing primary dengue virus infections than in those experiencing secondary dengue virus infections, and the slope of the decline in virus titer was greater in individuals with dhf than in those with df. the most important mechanism of extracellular virus clearance is thought to be igm and igg dengue antibodies, although t lymphocytes and antibody-dependent cell killing may contribute by attacking and destroying virus-infected cells. as discussed above, it is likely that elimination of dengue virus-infected cells continues well after onset of antibody production resulting in peak cellular infection (viral load) at around the time of defervescence and cellular infection is not eliminated until well after end of viremia. of interest, a major effort failed to detect circulating cd + t cells during the late acute-illness phase of dhf patients. this suggested to the authors that cd + t cells might be located only in the tissues where dengue virus replication had occurred. alternatively, as in a japanese encephalitis model, the antibody component of the adaptive immune response may play a much more important role in terminating dengue infections than heretofore thought. involving ade (table . ). feline coronaviruses circulate as two forms: feline enteric coronavirus (fecv) is a pathogen of minor significance; however, a spontaneous mutation of this virus yields fipv, which is capable of replicating in peritoneal macrophages and producing peritonitis and occasionally fip, a fatal arthus-like pyogranulomatous disease in kittens and cats. ade has been incriminated as a disease-enhancing factor. [ ] [ ] [ ] that antibodies are pathogenic is evidenced by observations that kittens who have acquired passive maternal fipv antibodies develop a more rapid and fulminant disease following challenge with fipv than do seronegatives. disease enhancement has been demonstrated in cats that were infected in the presence of vaccine-derived humoral immunity directed against the spike protein of fipv (table . ). similarly, cats immunized with a recombinant vaccinia virus refs. expressing the spike protein of fipv died earlier than control animals. in adult cats, fipv develops during chronic infections with feline coronaviruses after fecv mutates to fipv, gaining macrophage tropism. as antibody responses are mounted to fipv, infection and disease severity are enhanced. in summary, antibody responses to fipv occurring during the course of infection, passive transfer of antibodies from natural infections, and immunization with killed or recombinant vaccines all have led to enhanced fipv disease. enhanced disease severity has been attributed largely to the presence of non-neutralizing antibodies. leishmaniasis is caused by protozoan parasites of the genus leishmania which are transmitted by the bite of certain species of sandfly. human infection is caused by about of leishmania species that infect mammals. the disease exists in two major forms, cutaneous and visceral leishmaniasis. visceral leishmaniasis (vl) is the most severe form of leishmaniasis and the second largest parasitic killer in the world after malaria, responsible for an estimated , cases each year worldwide. leishmania are transmitted by sandflies as promastigotes, motile forms that parasitize macrophages and spread within hosts as amastigotes, which are obligate parasites of macrophages. in human hosts the responses to infection by leishmania vary with species and the patient's immune reaction. patients whose lymphocytes produce amounts of ifn-γ from th -type t cells often recover from cutaneous infection on their own and after recovery are immune to reinfection. patients infected with visceral forms of the parasite make high levels of anti-parasite antibody that does not contribute to host defense. without treatment, these patients are likely to succumb to vl. mouse models exist for the study of cutaneous and vl. for example, normal balb/c mice are susceptible to this disease and develop progressive non-healing lesions with numerous intracellular parasites within macrophages. despite the presence of high levels of anti-parasite antibody, mimicking human vl, these parasites often disseminate to the liver, spleen, and bone marrow. in vitro models are also available-promastigote or amastigote infections in cultures of bone marrow or peritoneal macrophages from mice or in differentiated peripheral blood human monocytes. during the s and s a number of mammalian cytokines were discovered; among them was mouse cytokine synthesis inhibitory factor produced by th cells, later renamed il- . , il- is a type ii cytokine and the "founding" member of a family of cytokines that includes il- , il- , il- , il- , il- , il- , and il- . all of these cytokines have similar intron-exon genomic organization, bind to receptors with similar structures and in some cases shared components, and all activate janus kinase (jak)/signal transducer and activator of transcription (stat) signaling pathways. il- is capable of inhibiting synthesis of proinflammatory cytokines such as ifn-γ, il- , il- , il- , tnf-α, and gm-csf. il- also displays a potent ability to suppress the antigen presentation capacity of antigen-presenting cells. there are, however, some settings in which il- is not immunosuppressive. il- can enhance nk and b-cell survival and b-cell antibody production. il- is made by macrophages, and a variety of t cells, including t reg, th , th , and th . il- plays important roles in regulating immune responses and it can also increase host susceptibility to intracellular infections. in the mouse model of cutaneous leishmaniasis susceptibility of leishmania major is associated with th responses. cd + t cells from susceptible balb/c mice produced il- and il- when infected with l. major, while cd + t cells from resistant c bl/ mice expressed ifn-γ and il- . the transient depletion of cd + cells or the in vivo neutralization of il- in balb/c mice promoted the killing of intracellular parasites and healing of leishmanial lesions. il- was not the only determinant of susceptibility, however, because susceptible balb/c mice lacking il- were fully resistant to infection. furthermore, in humans where the th /th dichotomy is not as pronounced, il- appears to be a major inducer of susceptibility. in humans with visceral leishmaniasis (vl), il- levels in plasma directly correlate with disease severity. il- was identified in lymph nodes taken from patients with vl. pbmcs from acute visceral leishmaniasis responded to stimulation with leishmania lysate by producing il- mrna. il- , when added to pbmcs, suppressed production of ifn-γ and il- but after treatment with anti-il- pbmcs from patients with acute vl demonstrated a marked increase in proliferative response to leishmania lysate. in vitro studies of leishmania-infected peritoneal and bone marrow macrophages demonstrated that the intracellular killing of this organism by classically activated macrophages could be inhibited by the administration of exogenous il- , or by endogenous macrophage production of il- . greater clarity was achieved when the reciprocal actions of il- and il- were recognized. the complementary cytokine il- acts directly on cd + t cells to enhance priming for ifn-γ production and reverse il- priming. the most potent cytokine for the induction of leishmanicidal activity in macrophages is ifn-γ. the sustained production of ifn-γ in response to infection is commonly associated with the development of specific th t-cell responses. administration of il- increased ifn-γ production and reduced the severity of leishmania major infections in mice and potentiated vaccinederived immunity and depressed il- production. however, exposure of macrophages from susceptible mice to opsonized leishmania promastigotes suppressed the expression of il- . the importance of macrophage receptors in the generation of cytokines in leishmaniainfected macrophages was recognized when il- production in balb/c mouse bone marrow macrophages in response to lps was suppressed after ligation of fcr, complement or scavenger receptors. both mrna synthesis and protein secretion were diminished to nearundetectable levels following receptor ligation. suppression was specific to il- since tnf-α production was not inhibited. also, the ligation of mouse fcγr with immune complexes was shown to enhance the production of il- . stimulation of mouse bone marrow macrophages by lps resulted in some il- production, but the addition of rbc opsonized with igg antibodies dramatically enhanced il- production. immune complexes not only induce activated macrophages to produce il- but also induce both macrophages and dendritic cells to switch off their production of il- . , the modulation of il- and il- in macrophages results from different mechanisms. whereas the abrogation of il- biosynthesis was a property shared by ligation of several macrophage receptors, the induction of il- was specific to fcγrs. additional evidence for the role of il- in supporting chronicity of infection was obtained when normal balb/c mice developed progressive non-healing lesions with numerous leishmania major parasites while il- −/− balb/c mice controlled disease progression and had relatively small lesions with -fold fewer parasites at the fifth week of infection. furthermore, in established l. donovani visceral infection in normal mice, anti-il- or anti-il- receptor monoclonal antibody (mab) treatment successfully induced intracellular parasite killing within liver macrophages. these and other studies showed that amastigotes of leishmania exploit an unusual and unexpected virulence factor, host igg. when the surface of leishmania amastigotes is coated with igg, the resultant immune complexes allow them to ligate fcγ receptors on inflammatory macrophages, preferentially inducing the production of high amounts of il- . the il- induction by iggamastigotes did not occur in macrophages from mice lacking the common gamma chain that signals through fcγrs , , and , indicating that one or all three of these receptors were involved. subsequent studies using defined immune complexes demonstrated that all three of the fcγrs that signal through gamma were capable of signaling for il- production in macrophages. the implication from these studies is that in some settings igg itself biases the immune response toward a th type response. indeed, for some species of leishmania, chronicity of infection requires that amastigotes be coated with igg. this phenomenon is now very well established. , , that il- induction by ligation of fcγr was a generic process was demonstrated with a non-microbial antigen. lipopolysaccharidetreated balb/c mouse macrophages when exposed to ovalbumin (ova) alone developed t-cell responses driven to th- and characterized by the production of ifn-γ. when the same antigen was complexed with igg anti-ova, t-cell responses were driven to th and produced il- . this th -like phenotype was stable and was retained when the t cells were subsequently restimulated under nonbiasing conditions. mice vaccinated with iggopsonized ova made high levels of igg abs of the igg isotype. the t-cell biasing and its reversal via fcγr ligation was also observed in vivo. using macrophages from gene knockout mice, the production of ifn-γ and il- by t cells was shown to be controlled by the macrophage cytokines, il- and il- , respectively. these and other studies demonstrate that the ligation of fcγr on activated macrophages reverses th biasing that accompanies innate immune responses to microbial products. importantly, in patients with vl, high levels of anti-leishmanial antibodies correlate with peak parasitemia and with negative dth responses. successful treatment of leishmaniasis with amphotericin resulted in decreased antibody titers and a restoration of dth responses. earlier observations identified polyclonal b-cell activation and high levels of immune complexes as well as rheumatoid factor in patients with vl. humans infected with leishmania donovani had higher frequencies of rheumatoid arthritis. in experimental models of vl, infected hamsters develop immune complex glomerulonephritis. these observations have been widely confirmed in mouse models. in addition to leishmania donovoni and l. major, humoral immune responses against l. mexicana were not effective at killing organisms hiding in parasitophorous vacuoles because host igg-coated amastigotes generated immunosuppressive il- responses by infected macrophages. , a similar observation, although perhaps occurring by a different mechanism, may be involved in l. amazonensis-infected jhd mice in which infections were minimized in the absence of b cells or antibodies. when these immune elements were restored leishmania lesions progressed by a process thought to involve cd + t cells. how do antibody-coated amastigotes result in the production of il- by macrophages? ligation of macrophage fcγr produces a rapid and enhanced activation of two mapks, erk and p . the activation of erk leads to the phosphorylation of serine on histone h at the il- gene, making the promoter more accessible to transcription factors generated in response to p activation. activation of both mapks was required for il- synthesis. in addition to erk activation, an inflammatory stimulus, such as low-molecular-weight hyaluronic acid from the extracellular matrix, must also be present. the combination of these two signals resulted in the superinduction of il- . macrophages lacking fcγr, or macrophages treated with an inhibitor of spleen tyrosine kinase that is activated following fcγr ligation, failed to activate erk and consequently failed to produce il- following infection with leishmania amastigotes. recently, however, it has been observed that igg and igg a/c induce il- from mouse macrophages in vitro equally well but through different fcγr subtypes: igg through fcγriii, and igg a/c through fcγri primarily but also through fcγriii. in sharp contrast, mice lacking igg develop earlier and stronger igg a/c , igg , and igm responses to leishmania mexicana infection and yet are more resistant to the infection. thus, igg , but not igg a/c or igg , is pathogenic in vivo, in agreement with prior studies indicating that fcγriii is required for chronic disease. this calls into question the assumption that mouse macrophages, which should secrete il- in response to both igg and igg a/c immune complexes, are the most important source of il- generated by igg-fcγr engagement in l. mexicana infection. the possibility that fcr-mediated enhancement of disease occurs across a broad range of microorganisms has received little attention by research communities. the possibility that ade contributes to severity or chronicity of disease exists because a large number of bacteria replicate partially or solely in human macrophages. larger microorganisms that infect macrophages, "professional intracellular pathogens," often produce chronic infections. indeed, one of the criteria of successful parasitism by microorganisms that produce systemic infections may be the ability to evade macrophage microbicidal mechanisms. these defensive mechanisms have been subject to intensive scrutiny. both chronic infections and an antigenic relatedness between microorganisms may contribute to the formation of pathogenic igg immune complexes controlling microbial survival. infections that may be analogous to the leishmania model include mycobacterium tuberculosis (mtb), mycobacterium leprae, legionella pneumophila, listeria monocytogenes, brucella spp., salmonella spp., shigella spp., coxiella burnetii, anaplasma phagocytophilum, and ehrlichia chaffeensis, as well as another protozoan, toxoplasma gondii, and fungi (e.g., histoplasma capsulatum). the role of fc receptors or immune complexes in mediating immunopathology has not been studied exhaustively for most of the organisms on the above list; however, interesting findings have been made in tuberculosis. although studies on resistance to tuberculosis have centered on t-cell immunity, in a recent experiment c bl/ mice deficient in inhibitory fcγriib showed improved bacterial control and diminished pathology at but not days following aerosol challenge with mycobacterium tuberculosis. enhanced production of il- p was observed in fcγriib −/− mice. il- and il- share the common subunit il- p and both promote the polarization of naive cd + t cells into th effectors. treatment of human macrophages with exogenous il- combined with blockade of il- reduced the burden of mycobacterial infection. these infections were characterized by enhanced protective ifn-γ responses that coincided with heightened activation of macrophages. in humans, elevated levels of il- correlate with the suppression of host defenses and exacerbation of infection. in a model of reactivation tuberculosis the presence of macrophage-derived il- in the lungs of infected transgenic mice permitted th cells to efficiently express effector functions and secrete sufficient ifn-γ to induce classical macrophage activation characterized by expression of nos and lrg- . however, mycobacteria survived and successfully proliferated within mouse macrophages with il- production under control of a human cd promoter. macrophage-derived il- appears to override ifn-γ-dependent classical macrophage activation and other effector mechanisms against mtb by inducing an alternatively activated phenotype. to date, an explicit contribution of mtb-igg antibody complexes toward modulation of infections in model systems has not been reported. successful pathogens are armed with an array of weapons, including penetration and cell attachment factors and a galaxy of defenses against innate immune responses. much is known and there is still much to learn about the offensive attributes of microbial pathogens. however, because of the ade phenomena, antibodies very likely complement and contribute a generic and thus far poorly explored afferent disease mechanism for many infectious diseases in addition to the examples cited here. cognizance of the need to balance protective and potentially harmful antibody activities remains an important concern in vaccine development. intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes how innate immune mechanisms contribute to antibody-enhanced viral infections enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls an explanation for enhanced virus plaque 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associated with a lentivirus cytotoxic t lymphocyte vaccine regimen the pathogenesis of aleutian disease of mink. ii. enhancement of tissue lesions following the administration of a killed virus vaccine or passive antibody aleutian mink disease parvovirus infection of mink macrophages and human macrophage cell line * : demonstration of antibody-dependent enhancement of infection aleutian mink disease parvovirus cross-protection and cross reactive cytotoxic t cells induced by influenza virus vaccines in mice possible role of immunological factors in pathogenesis of rs virus in lower respiratory tract disease altered reactivity to measles virus. atypical measles in children previously immunized with inactivated measles virus vaccines antibodymediated enhancement of rabies virus infection in a mouse macrophage cell line (p d ) acute rabies deaths mediated by antibody key: cord- -iapgkz p authors: el-bitar, alaa m. h.; sarhan, moustafa; abdel-rahman, mohamed a.; quintero-hernandez, veronica; aoki-utsubo, chie; moustafa, mohsen a.; possani, lourival d.; hotta, hak title: smp , a scorpine-like peptide isolated from the venom of the scorpion scorpio maurus palmatus, with a potent antiviral activity against hepatitis c virus and dengue virus date: - - journal: int j pept res ther doi: . /s - - - sha: doc_id: cord_uid: iapgkz p growing global viral infections have been a serious public health problem in recent years. this current situation emphasizes the importance of developing more therapeutic antiviral compounds. hepatitis c virus (hcv) and dengue virus (denv) belong to the flaviviridae family and are an increasing global health threat. our previous study reported that the crude venom of scorpio maurus palmatus possessed anti-hcv and anti-denv activities in vitro. we report here the characterization of a natural antiviral peptide (scorpion-like peptide smp ) that prevents hcv and denv infection. smp was purified from s. m. palmatus venom and contains amino acids with six residues of cysteine. smp antiviral activity was evaluated using a cell culture technique utilizing huh it- , vero/slam, hcv (jfh , genotype a) and denv (trinidad , type ). a potential antiviral activity of smp was detected in culture cells with an approximate ic( ) of . μg/ml. moreover, smp prevents hcv infection and suppresses secondary infection, by inactivating extra-cellular infectious particles without affecting viral replication. interestingly, smp is neither toxic nor hemolytic in vitro at a concentration -fold higher than that required for antiviral activity. conclusively, this report highlights novel anti-hcv and anti-denv activities of smp , which may lay the foundation for developing a new therapeutic intervention against these flaviviruses. hepatitis c virus (hcv) is a single-stranded rna viruses that belongs to family flaviviridae (mohammed et al. ; supanee et al. ) . around million people worldwide are chronically infected with hcv and the annual mortality from hcv-related liver diseases reach up to , individual (ministry of health and ; world health organization ; jefferies et al. ). in the past decade, interferon-based therapy was the gold standard for hcv treatment with a sustained virological response (svr) rate hovering around %. the recent approval of oral direct-acting antivirals (daas), like hcv ns protease inhibitors, ns a inhibitors and ns b rna-dependent rna polymerase inhibitors, for clinical use improved the svr rates to more than % (pawlotsky ; falade-nwulia et al. ). nevertheless, cirrhosis patients remain at risk for severe complications. in addition, treatment with daas is not affordable for many patients and they are still not readily available around the globe. therefore, uncovering novel hcv inhibitors is still a clinical priority. dengue virus (denv) is another single-stranded rna flaviviridae virus that is transmitted by mosquitoes causing dengue fever (rodenhuis-zybert et al. ) . denv is currently endemic in more than countries with the highest prevalence in south-east asia, africa and the americas (mackenzie et al. ; malavige et al. ; deen et al. ; bhatt et al. ) . each year, there are around million denv infections are recorded worldwide and among them to million patients are presented with the clinical manifestations of dengue fever (bhatt et al. ) . dengue fever leads to , annual deaths, mainly in young children (rui-feng et al. ) . to date, four denv serotypes, denv- , denv- , denv- and denv- , have been identified and infections with one serotype does not offer protection from infection with the remaining three serotypes (weaver and vasilakis ; messina et al. ; mustafa et al. ) . a major impediment to the development of vaccines is that vaccines should have a tetravalent effect, i.e. sufficient protective immune responses against all four denv serotypes. owing to the absence of specific treatments against denv and the limitations of the available vaccine (dengvaxia® or cyd-tdv), the global burden of denv infection is becoming enormous (behnam et al. ) . therefore, the development of new antiviral compounds against denv infections is urgently needed. scorpion venom is a rich source for drug discovery and prototyping ghosh et al. ) . scorpion venoms are highly complex mixture of nucleotides, enzymes, mucoproteins, biogenic amines, nucleotides, salts, as well as peptides and proteins (omran ; rodriguez de la vega and possani ; ozkan et al. a, b, c; feng et al. ; kanoo and deshpande ; ortiz et al. ) . antimicrobial peptides (amps), isolated from several venomous animals, exhibit a wide range of antibacterial and antiviral activity with direct or indirect microbicide activity (hv et al. ; ortiz et al. ) . several studies demonstrated an antiviral effect for certain scorpion venom peptides (carballar-lejarazu et al. ; el-bitar et al. ; ortiz et al. ) . in this study, we report the molecular and functional characterization of a new antiviral peptide (smp ), a scorpion-like peptide derived from an egyptian scorpion's venom, s. m. palmatus. our findings will broaden the currently known antiviral peptides and open a new avenue for the development of novel hcv and denv therapies. adult s. m. palmatus scorpions were collected from the western coastal mediterranean desert (alexandria governorate, egypt) and were housed individually in clear plastic containers. scorpions were fed small insects and were given water. crude venom was extracted using electrical stimulation ( v) and the milked venom was collected and centrifuged for min at , rpm/ °c as detailed previously (abdel-rahman et al. ) . clear supernatants were pooled, freeze-dried and stored at − °c until use. venom samples were dissolved in bi-distilled water and the total protein concentration was determined by bca protein assay kit (pierce biotechnology, rockford, il, usa) according to the standard protocols. the human hepatoma-derived cell line, huh it- , was cultured in dulbecco's modified eagle's medium (dmem; wako, osaka, japan) supplemented with non-essential amino acids (invitrogen, carlsbad, ca, usa), fetal bovine serum (biowest, nuaille, france), streptomycin ( μg/ml) and penicillin ( iu/ml) (invitrogen) in a % co incubator at °c (aoki et al. ). huh it- cells were infected with cell culture-adapted hcv (jfh strain of genotype a) and supernatants were collected at day post-infection (wakita et al. ; yu et al. ). next, supernatants were, concentrated by k amicon centrifugal filters and used for antiviral screening. denv type (trinidad strain) (hotta et al. ; hotta and homma ) was infected into vero/slam cells (ono et al. ) . following an hour of virus adsorption, the virus-infected cells were cultured with dmem medium containing % fetal bovine serum at °c in % co . supernatants were collected at to days post-infection and stored at − °c. measles virus (k strain) was inoculated to vero/slam cells and the culture supernatants was collected from the virus-infected cells as described previously (otaki et al. ). the cytotoxicity of smp was estimated using wst- assay as described previously with a some modification (deng et al. ) . briefly, huh it- cells seeded in -well plate ( . × cells/well) were treated with serial dilutions of smp ( . to µg/ml) or medium (control) for h at °c in % co . then, supernatants were discarded and replaced with fresh dmem medium containing μl of wst- reagent (roche, mannheim, germany) and incubated for h. the number of viable cells was quantified by using a microplate reader at and nm. for each dilution, the percentage of viable cells were compared to the control sample and used to calculate the % cytotoxic concentrations (cc ) values according to the following formula: hemolytic activity of smp was performed as previously described (evans et al. ). briefly, a total of μl of smp peptide was mixed with μl of diluted human red blood cells (rbcs) to achieve a final dilution / of the original venom peptide per well. alternatively, the rbcs were incubated with μl of . % triton x- or pbs to serve as both positive and negative controls, respectively. after an hour incubation period at °c, the plate was centrifuged for min at ×g and μl of supernatant was transferred to a clear -well plate. the released hemoglobin was measured on a microplate reader at : nm. the percentage of hemolysis was calculated relative to the positive control ( . % triton x ). the hemolysis concentration (hc ) value was defined as the peptide concentration that can lyse % of the rbcs. huh it- cells were grown on coverslips ( -mm in diameter; . × cells/well) day before viral infection. different concentrations of the venom fractions were mixed with hcv at multiplicity of infection (moi: ) for h at °c. then, the virus/venom fraction mixture was inoculated in huh it- cells for h at °c. medium-treated virus and cells were used as controls. the percentage of inhibition for absorbance of sample/absorbance of control × . virus infectivity was compared to the control samples and the % inhibitory concentrations (ic ) were calculated. hcv infectivity was determined as described previously (deng et al. ) . in brief, huh it- cells, grown on glass coverslips, were incubated with tenfold serially diluted virus samples for h; then, the cells were washed with free medium and cultured for another h. following fixation and permeabilization, huh it- cells were incubated for h with the serum of hcv-infected patients, followed by fitc-conjugated goat anti-human igg (medical & biological laboratories co., ltd., nagoya, japan). finally, the cells were counterstained by hoechst (molecular probes, eugene, or, usa) and mounted using vectashield h- reagent (vector laboratories, inc. burlingame, ca, usa). hcv antigen positive cells were counted under a fluorescence microscope (bz- , keyence, osaka, japan). for dengue virus infectivity, serially diluted venom fractions and smp were mixed with fixed amount of denv and incubated for h at °c. the virus/venom fractions mixture was inoculated for h at °c on vero/slam cells. the cells were washed twice after the virus inoculation and incubated with a fresh medium for h. the infected cells were incubated with mouse monoclonal antibody against dengue virus followed\alexa fluor a goat anti-mouse igg (life technologies). to determine the infectivity of measles virus, serially diluted smp was mixed separately with fixed amount of measles virus and incubated for h at °c. virus/venom fraction mixture was inoculated to vero/slam cells for h at °c and the cells were washed twice then, incubated with fresh medium for h. the plaques (virus-induced syncytia) forming on the infected monolayer cells were counted. the smp venom peptide was mixed with a fixed amount of hcv jfh for h at °c. next, the virus/smp mixture was inoculated to huh it- cells and incubated for h at °c. the cells were washed and cultured without smp for h. finally, the cells were subjected to an indirect immunofluorescence assay as previously described (el-bitar et al. ) . huh it- cells were lysed in sds sample buffer and equal amounts of protein were separated on a sds-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (pvdf) (millipore, bedford, ma, usa). the pvdf membrane was blocked by % skim milk and probed with anti-hcv ns antibody and anti-gapdh antibody (millipore). followed by horseradish peroxidaseconjugated goat anti-mouse immunoglobulin (invitrogen) as a secondary antibody and visualized using the enhanced chemiluminescence detection system (ecl; ge healthcare, buckinghamshire, uk). the amounts of hcv rna in the infected cells were determines as described previously (el-bitar et al. ) . rna was extracted by rna cell miniprep system reliaprep (promega, madison, wi, usa). the cdna was transcribed from one µg total rna using a goscript reverse transcription system (promega) with oligo(dt) primers. quantitative real-time pcr was performed using sybr premix ex taq (takara, kyoto, japan) in a microamp -well reaction plate. pcr was conducted on a abi prism fast system (applied biosystems, foster city, ca, usa) with specific primers used to amplify the ns a region of the hcv genome ′-aga cgt att gag gtc cat gc- ′ (sense) and ′-ccg cag cga cgg tgc tga tag- ′ (antisense). the expression of gapdh mrna was also measured as a housekeeping gene using the ′-gcc atc aat gac ccc ttc att- ′ (sense) and ′ tct cgc tcc tgg aag atg g- ′primers. chromatographic separation of s. m. palmatus venom was conducted using reverse phase high performance liquid chromatography (rp-hplc; waters, milford, massachusetts, united states) (abdel-rahman et al. ) .a total of mg scorpion venom was reconstituted in ml . % trifluoroacetic acid (tfa) and fractionated by a c rp-hplc column ( × mm, µm; vydac, california, united states). a gradient of buffer a ( . % tfa in milliq water) and sixty percent buffer b ( . % tfa in acetonitrile) were used to separate scorpion venom in h ( ml/min flow rate). individual venom fractions were collected manually according to the peak's absorbance (at nm). all collected fractions were dried using a rotary evaporator (savant speed vac sc a, minnesota, united states). the active fraction eluted at retention time . min was further characterized using mass spectrometry and amino acids sequencing. in addition, recombinant and synthetic smp derivatives (n-terminal aa and c-terminal aa) were prepared as described below. the average molecular mass of native smp peptide ( da), the recombinant fusion protein thioredoxine-smp ( da) and a recombinant c-terminal of smp ( . da) were determined using esi-ms, esi lcq fleet spectrometer (thermo scientific, ca, usa). the sequence of native smp (approximately pmol) was determined using edman degradation (protein sequencer ppsq- a, shimadzu scientific biotech, maryland, united states). synthetic n, c-terminal and fulllength smp peptides were manufactured by genscript japan inc. six oligonucleotides were designed to cover the c-terminal region of smp ( aa) (supplementary table ). the oligonucleotides bhek-dir and scsmp-lw included the bamhi and xhoi restriction sites, respectively. subsequently, this enabled the cloning into the pet b-thio-ek expression vector as detailed previously (jiménez-vargas et al. ; vargas-jaimes et al. ) . pcr assembly of the c-terminal peptides was carried out using vent dna polymerase (new england biolabs, ma, united states). the final concentration of external primers bhek-dir and scsmp-lw was . pmol/μl while the concentration of internal oligonucleotides was . pmol/μl. in order to express the fusion protein thioredoxine-c-terminal, pet b-thio-c-terminal plasmid was transformed into e. coli bl (de ) using electroporation. the pellet was harvested and the fusion protein was purified using the ni-nta agarose resin columns (qiagen) as previously described (vargas-jaimes et al. ) . hplc purification was further performed using a c rp-hplc column ( × mm, µm; vydac, california, united states). the purified fusion protein thioredoxine-c-terminal was digested with enterokinase (new england biolabs) in mm tris hcl (ph . ), mm nacl, mm cacl for h at °c. then, the pure recombinant c-terminal of smp was finally isolated and purified by hplc as described above. data are presented as mean ± standard error of mean (sem). the difference between data sets was determined by student's two-tailed t test. a p value of < . was considered to be statistically significant. since a whole s. m. palmatus soluble venom strongly inhibited hcv infectivity in vitro and displayed anti-hcv activity in cell culture (el-bitar et al. ) , the crude venom was fractionated to identify active molecule(s) with anti-hcv activity. accordingly, fractions from four milligrams of the venom were separated by hplc analytical method ( fig. a; table ). subsequently, the anti-hcv activity of fractions obtained from the venom of s. m. palmatus were tested against jfh strain of genotype a. based on protein concentrations, fractions were tested for anti-hcv activity. the other fractions showed very low protein concentrations and, therefore, it was not possible to check their antiviral activity (table ) . each fraction was incubated separately with fixed amount of hcv for h at °c. then, huh it- cells were infected with the virus/venom-fraction mixture (fig. a) and virus infectivity was measured by infectious center assay. the anti-hcv activity started to appear from the retention time (rt) . until . min. the fraction at rt . min showed the most potent anti-hcv activity with ic being . μg/ml ( fig. b ; table ). in order to identify the bioactive compound(s) present in rt . , lc-ms-esi analysis was performed. interestingly, the data of mass spectrometry showed that the active fraction contains a unique peptide with molecular mass of da (fig. b) . to go further into the characterization of the active peptide, the amino acid sequence was determined (fig. c) . the sequence of this peptide contains amino acids (gwinekkmqqkidekigkniiggmakavihk-maknefqcvanvdtlgnckkhcakttgekgych-gtkckcgielsy). the obtained sequence belongs to the scorpion venom antimicrobial peptides and matched with the scorpine-like peptide smp , which was identified in the scorpion venom gland of s. m. palmatus using transcriptomic analysis (abdel-rahman et al. ) . the amino acid sequence of smp was confirmed until the amino acid number by edman degradation method and the molecular mass was confirmed by mass spectrometry resulting in da (see "materials and methods"). the cytotoxic activity of smp against huh it- cells was tested using the wst- assay, and hemolytic activity was examined on human red blood cells. the cc and the hc were calculated. as shown in table , cc of smp against huh it- cells and hc were > μg/ml. these results indicate that this peptide has no cytotoxic or hemolytic effects up to μg/ml with selectivity index (si) > . since smp displayed a significant inhibitory effect at the early stage of hcv infection, we examined whether the smp peptide can also inhibit hcv ns protein production and hcv rna replication in the cells. virus at multiplicity of infection of pfu/cell was inoculated to the huh it- cells for ~ h at °c. after virus adsorption, the cells were cultured with media supplemented with . μg/ml of smp for h at °c (fig. a) . the cells were harvested and subjected to immunoblot and rt-qpcr analyses. the results showed that the post-treatment of hcv rna replication was not significantly inhibited (fig. b) or hcv ns protein synthesis in the cells (fig. c) . the above results suggest that the smp directly affects hcv particles and/or host cells in the culture medium to inhibit the viral infection and does not have an antiviral effect in the cells. we previously showed that the crude venom of s. m. palmatus inhibits denv (el-bitar et al. ) . therefore, anti-denv activity of the selected -fractions obtained from the crude venom of s. m. palmatus was tested. each fraction was incubated separately with fixed amount of denv for h at °c. after that, the virus/venom fractions mixtures were used to infect vero/slam cells and virus infectivity was measured by infectious center assay. interestingly, the results were consistent with the data of anti-hcv activity obtained in this study (table ) . also, the fraction identified at . min which contains smp showed the potent anti-denv activity with ic being . μg/ml (table and fig. b ). to determine whether the antiviral activity of smp peptide (previously described) was specific to hcv and denv, a schematic of infection assay. b amounts of hcv infectious particles. the data represents mean ± sem of two independent experiments. § below the detection limit; ‡ ≤ . %; # < . % we tested its possible effects on another enveloped virus such as measles virus (otaki et al. ) . in this investigation, the virus was incubated with smp ( - . μg/ml) for h. then, vero/slam cells were infected with the virus/smp mixture (fig. a) and virus infectivity was measured using an infectious center or plaque assay. the results revealed that while smp peptide showed strong activity against denv with ic ng/ml, it induced weak inhibition on measles virus at μg/ml (fig. b ). in an attempt to identify the active domain of smp , the antiviral activity (anti-hcv and anti-denv) of synthetic n-terminal ( aa) and c-terminal ( aa without disulfide bonds) were tested. although, the purified native smp showed strong antiviral activity with ic of ng/ml, there was no antiviral activity for both synthetic terminals (table ) . moreover, antiviral activity of recombinant c-terminal ( aa) was examined. also, no antiviral activity for recombinant c-terminal was detected (table ) . these results indicate that the full-length of smp may be required for its activity against hcv and denv. the above mentioned results imply that the full-length of smp may be required for its activity against hcv and denv. the full-length smp peptide ( aa) was synthesized but without disulfide bonds. the antiviral activity of the synthesized smp peptide was examined against hcv and denv. these results showed no antiviral activity for the synthetic full-length peptide without disulfide bonds against hcv and denv (table ) . scorpine is firstly isolated from the venom of pandinus imperator. the structure of scorpine is a hybrid between a cecropin and a defensin. the sequence of scorpine carboxyl terminal region is similar to that of β-ktx family, with cysteine-stabilized α/β fold, and three disulfide bridges. on the other hand, its amino-terminal region is identical to the cecropin family peptides (conde et al. ) . scorpine has also amino acid sequences similar to amps and k + channel blocking peptides (luna-ramirez et al. ) . scorpine homologs were thereafter identified from the venom of various scorpions such as opistophthalmus carinatus (zhu and tytgat ) , heterometrus laoticus (uawonggul et al. ) , h. gertschi ), s. m. palmatus (abdel-rahman et al. ), genus vaejovis (quintero-hernandez et al. and urodacus yaschenkoi (luna-ramirez et al. ) . importantly, all these peptides possess anti-malaria as well as antimicrobial activities (conde et al. ; carballar-lejarazu et al. ) and act also as potassium channel blockers ). the present data clearly showed that smp inhibits the ability of hcv virus to infect the host cells. indeed, our previous study demonstrated that the crude venom of s. m. palmatus venom prevents hcv infection with direct virocidal activity (el-bitar et al. ) . on the other hand, we cannot rule out the possibility that, smp peptide might has an independent effect on the receptor complexes of host cell components or interacts with components that inactivate viral entry. this possibility will be further investigated by the incubation of smp with cells in a free-virus condition prior to hcv infection. however, it worth to mention that the incubation of s. m. palmatus crude venom with cells prior to the hcv infection of cells did not impair the viral infectivity (el-bitar et al. ) . thus, the possible effect of smp on the host cell to abrogate hcv infection is unlikely. in the present study, smp prevents the early stages of life cycle of hcv and denv most probably through interacting with viral particles. the viral particle can be neutralized by targeting the envelope of the hcv or host factors related to the mature viral particle (zeisel et al. ) . notably, it has been reported in various studies that the structure of biological membranes could be altered by amps (zasloff ; harrison et al. ) . currently, the new approach for hcv infection treatment probably based on the combination of several drugs (pereira and jacobson ; sarrazin and zeuzem ; zeisel et al. ; qian et al. ). therefore, the use of smp with anti-hcv drugs for treatment of hcv infection may have synergistic effect. however, further experiments are needed to check this possibility. the present study reported distinctive data on the ability of smp peptide to protect cellular systems from attack of denv and neutralize viral infection. currently dengue virus considered as one of the most important arthropod born viral disease worldwide (botta et al. ) . despite the global efforts, there is no antiviral therapy against denv infections clinically approved and only symptomatic treatment and hospital supportive care setting are available for infected people (behnam et al. ) . it was shown that recombinantly expressed scorpine (rscrp) inhibited denv- replication in c / mosquito cells. also, it was suggested that the development of transgenic mosquitoes that overexpress and correctly secrete rscrp and could eventually break the dengue fever transmission (carballar-lejarazu et al. ). on the other hand, smp , as an infection inhibitor, has some advantages compared to antiviral drugs that target the viral replication stages inside the target cell. smp can inhibit denv infection before viral entry and is, therefore, helpful for treatment of denv viraemia. the prospective pharmaceutical potential of smp cannot be neglected, especially considering its potent antiviral activity. however, smp isolation from natural sources is ineffectual and time-consuming. synthetic smp without disulfide bonds showed no antiviral activities against hcv and denv. one possibility is that the synthetic smp peptide without disulfide bonds did not have the properly folded structure necessary for activity. recently, recombinant scorpine with antimalarial and antibacterial activities was produced by different fusion technology using small ubiquitin-related modifier (sumo) (zhang et al. ) and maltose binding protein (mbp) (zhang et al. ). these methods have improved efficiency and reduced the cost of producing scorpine and can contribute to the future production of active recombinant smp . interestingly, the recombinant smp was shown to inhibit denv and zikv infections in cultured cell lines and primary mouse macrophages. however, rsmp did not inactivate the viral particles directly but suppressed the established viral infection by upregulating the expression of ifn-β (ji et al. ). this mechanism is significantly different from the virucidal effect of native smp peptides. the exact mechanism by which smp exerts its antiviral activity against hcv and denv to inhibit infecting their target cells need further studies. in vivo studies should also assess the future role of smp in managing hcv and denv infections. venom proteomic and venomous glands transcriptomic analysis of the egyptian scorpion scorpio maurus palmatus (arachnida: scorpionidae) isolation and identification of substances with anti-hepatitis c virus activities from kalanchoe pinnata the medicinal chemistry of dengue virus the global distribution and burden of dengue drug repurposing approaches to fight dengue virus infection and related diseases recombinant scorpine: a multifunctional antimicrobial peptide 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polyadenylation and fold recognition the authors are grateful to dr. lin deng and dr. ming chen, division of microbiology, kobe university graduate school of medicine, japan, for their assistance in virological analyses. the authors also acknowledge dr. fernando zamudio, m. sc. leonel vargas jaimes and m. sc. maría teresa romero gutiérrez for their assistance in the proteomics work done in this work. we also grateful key: cord- - lrqubdl authors: badawi, alaa; velummailum, russanthy; ryoo, seung gwan; senthinathan, arrani; yaghoubi, sahar; vasileva, denitsa; ostermeier, emma; plishka, mikayla; soosaipillai, marcel; arora, paul title: prevalence of chronic comorbidities in dengue fever and west nile virus: a systematic review and meta-analysis date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: lrqubdl background: flavivirus diseases such as dengue fever (denv), west nile virus (wnv), zika and yellow fever represent a substantial global public health concern. preexisting chronic conditions such as cardiovascular diseases, diabetes, obesity, and asthma were thought to predict risk of progression to severe infections. objective: we aimed to quantify the frequency of chronic comorbidities in flavivirus diseases to provide an estimate for their prevalence in severe and non-severe infections and examine whether chronic diseases contribute to the increased risk of severe viral expression. methods: we conducted a comprehensive search in pubmed, ovid medline(r), embase and embase classic and grey literature databases to identify studies reporting prevalence estimates of comorbidities in flavivirus diseases. study quality was assessed with the risk of bias tool. age-adjusted odds ratios (ors) were estimated for severe infection in the presence of chronic comorbidities. results: we identified studies as eligible for inclusion for denv ( studies) and wnv ( studies). obesity and overweight (i.e., bmi> kg/m( ), prevalence: . %, % ci: . – . %), hypertension ( . %, . – . %) and diabetes ( . %, . – . %) were the most prevalent comorbidities in denv. however, hypertension ( . %, . – . %), diabetes ( . %, . – . %) and heart diseases ( . %, . – . %) were the most prevalent in wnv. ors of severe flavivirus diseases were about to in infected patients with comorbidities such as diabetes, hypertension and heart diseases. the small number of studies in jev, yfv and zika did not permit estimating the prevalence of comorbidities in these infections. conclusion: higher prevalence of chronic comorbidities was found in severe cases of flavivirus diseases compared to non-severe cases. findings of the present study may guide public health practitioners and clinicians to evaluate infection severity based on the presence of comorbidity, a critical public health measure that may avert severe disease outcome given the current dearth of clear prevention practices for some flavivirus diseases. a a a a a the flaviviridae is a large family of positive-strand rna viruses, that comprises four genera: flavivirus, pegivirus, pestivirus, and hepacivirus [ ] . the flavivirus genus consists of more than viruses, many of which are arthropod-borne human pathogens that cause a variety of clinical diseases, ranging from asymptomatic to mild fever to more severe diseases including encephalitis and hemorrhagic fever [ , ] most flaviviruses are transmitted through the bite of an infected arthropod vector, mainly aedes genus (aedes aegypti and to a lesser extent, aedes albopictus) and cluex mosquitos, and most were once maintained by animal reservoirs in sylvatic transmission cycles [ ] . many flaviviruses, however, such as dengue virus, yellow fever and zika virus, are now principally maintained by mosquito-borne transmission with a possible human-to-human transmission through transfusion of infected blood or transplantation of infected tissue [ ] . some flaviviruses can cause globally significant vector-borne diseases with a substantial public health impact such as dengue virus (denv), japanese encephalitis virus (jev), west nile virus (wnv), zika virus (zikv) and yellow fever virus (yfv) [ ] . other members of the flaviviridae family that have a more regional impact include murray valley encephalitis virus (mvev) in oceania, st. louis encephalitis virus (slev) in north america, and tick-borne encephalitis virus (tbev) in europe [ ] . over the past few decades, many of these flaviviruses have re-emerged for a range of reasons including decreases in mosquito control efforts, rapid changes in climate and vector's demography, dense urbanization, population growth and globalization with increased transportation and trade activities [ ] . examples include the geographic spread of denv throughout the tropical world; jev throughout south asia, australasia and the pacific; zikv into south and central america; yfv into the americas and the invasion of wnv into much of north america [ , ] . it is estimated that there are over million denv infections per year, of which million manifests clinically with varying degrees of severity [ ] and . billion people in countries are at risk of infection [ ] . similarly, high incidence rates for symptomatic cases of jev were reported over the past three decades to reach . per year per , population [ ] . epidemic waves of yfv are projected to result in , to , clinical cases per year with case-fatality rates ranging from to % [ ] [ ] [ ] [ ] . wnv, first appeared in the northeastern usa in , are spread presently across much of the usa and southern canada. for example, in , the cdc reported , cases of wnv, of which , ( %) were hospitalized and ( %) died [ ] . in the developing world, wnv incidence is likely to be underestimated due to political, psychological, and economic barriers to reporting [ , ] . although most human flavivirus infections are asymptomatic or have an undifferentiated febrile illness, a small percentage of affected individuals develop acute fever that can progress to severe clinical manifestations such as hemorrhage, vascular leakage and encephalitis [ ] . currently, our knowledge of the host-related factors that influence the pathogenesis of severe disease is inadequate to allow prediction of who will develop severe clinical illness. however, some mechanisms and etiological factors underlying inter-individual variations in response to flavivirus infections have been identified. interactions of virus-encoded proteins with human innate immune pathways [ ] ; the effect of host-cell surface molecules in virus binding and entry [ ] ; the role of viral protein nuclear localization in the host cell response [ ] ; and the flavivirus replication dynamics within multiple immune systems [ ] have all been considered as host-pathogen interaction events that may regulate viral virulence or attenuation and the subsequent disease severity. over the past few years, however, host-related factors such as preexisting chronic conditions, e.g., cardiovascular diseases, diabetes, obesity, and asthma have received attention as predictors for increased risk of progression to severe flavivirus infection [ ] [ ] [ ] . recent studies have raised the proposition that cardiovascular disease, stroke, diabetes, respiratory diseases and renal disorders may contribute, together with old age, to severe clinical manifestations of dengue [ , ] . a few studies of wnv [ ] and jev [ ] infections, and responses to yfv vaccination [ ] , have also explored the role of chronic comorbidities in the prognosis of infections. given the lack of specific medical treatment for flavivirus diseases, effective public health surveillance for vector-borne infections together with continuing vector control efforts will be critical to preventing infection. however, elucidating the impact of comorbidities to the severity of disease when infection occurs will be critical to identifying vulnerable populations, to whom effective interventions protocols and individually-tailored clinical monitoring practices should be particularly targeted. the objective of this study is to systematically review the existing literature on the prevalence of the most common non-communicable comorbidities related to the cluster of metabolic syndromes-associated diseases, such as diabetes mellitus, heart diseases, hypertension, asthma, stroke and obesity in flavivirus infections and to evaluate the difference of their prevalence in severe vs. non-severe clinical outcomes to infection. identifying and characterizing associations between comorbidities and severity of flavivirus infections will be significant factor in designing public health measures that aim to prevent the severe outcomes of infection. we carried out a systematic literature search that conforms to the preferred reporting items for systematic reviews and meta-analysis (prisma) guidelines [ ] (see s table) , in pubmed, ovid medline(r), embase and embase classic databases from inception to the last week of november (november , ). the date for searching database is the same date for the upper limit of the period considered. a grey literature search was also conducted in the american society of tropical medicine and hygiene and open forum infectious diseases-infectious diseases society of america for the two most recent years. using the pico format (acronym for "population or problem", intervention or exposure of interest", "comparison" and "outcome") [ ], the research questions were: "what is the frequency of chronic comorbidities in flavivirus infections?" and "is the severity of flavivirus infection associated with higher prevalence of comorbidities?" (s table) . the search related terms (mesh), inclusion and exclusion criteria and synonyms are shown in table . non-english language reports were excluded from this study. to identify relevant studies, we used the comprehensive fourstep search strategy of prisma (fig ) , i.e., (i) identification, (ii) screening, (iii) eligibility and (iv) inclusion of studies. we evaluated studies in denv , in wnv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and two in jev [ , ] for inclusion (see fig ) . given the small number of studies in jev, the two articles were excluded from further quantitative analysis. no studies met the inclusion criteria for yfv and zikv infections. the titles and abstracts of the identified studies were reviewed independently by two reviewers using covidence systematic review software, (veritas health innovation, melbourne, australia. available at www.covidence.org). differences and conflicts were resolved by a third reviewer and through discussions for a consensus to be reached. percentage agreement and cohen's kappa (κ) statistic [ ] were calculated and interpreted in accordance with landis and koch's benchmarks for assessing the agreement between reviewers [ ] as poor (< ), slight ( . - . ), fair ( . - . ), moderate ( . - . ), substantial ( . - . ), and excellent (> . ). the methodological quality of each study-from the standpoint of evaluating the prevalence of chronic comorbidities in flavivirus infections-was assessed as part of the data extraction. with some modification, we used the standards tool for evaluating and reporting epidemiologic studies on chronic disease incidence or prevalence, designed to assess population-based prevalence studies [ ] [ ] [ ] . to assess the risk of bias, each study was rated against each of the ten following criteria: ) clearly stating the research objective, ) distinctly defining the study population, ) homogeneity of the study subjects (e.g., age, ethnicity, gender ratio and origin of the studies population), ) sample size and power justification, ) assessing the infection prior to identifying the comorbidity, ) evaluating levels of infectious disease severity and the related frequency of comorbidity, ) standardizing the infectious disease assessment across all study subjects, ) standardizing the assessment of table . keywords for the search related terms and synonyms. population individuals with a flavivirus infections (both severe and non-severe cases), all age groups. dengue fever, yellow fever, west nile virus, zika, japanese encephalitis. diabetes, hypertension, heart disease (including: cardiovascular disease, coronary artery disease, coronary vascular disease, atrial fibrillation, chronic ischemic heart disease, acute coronary syndrome for duration less than months, cardiac disorder, cardiac heart failure, congestive cardiac failure), stroke, obesity, asthma (does not include chronic obstructive pulmonary disease; copd). proportion/rate frequency of at least one of the comorbidities, percent comorbidity, death/fatality, incidence, hospitalization, mortality, mortality rate. comorbidities across all study subjects, ) assessment of comorbidities blindly of the infectious disease status, and ) considering the potential confounders with clear and adjusted statistical analyses. each criterion was rated dichotomously (yes: low risk = point; no: high risk = point). an overall score was calculated by adding all the items rated as low risk. thus, higher scores indicated lower risk of bias and stronger method quality. grey literature (mainly conference abstracts) was not assessed in this way as the required information was unavailable. data extracted from the selected studies in duplicate by two reviewers and included the first author's name, publication date, country, dates of recruitment, total sample size (divided to males and females), age estimates (from reported mean, median or the mid-point for age range of the highest subject frequency), procedures for case identification, type of flavivirus infection, severity of infection, prevalence of clinical manifestations (mild symptoms such as fever, headache, muscle pain, rash, and malaise together with severe symptoms as described below) and percentage of comorbidities including diabetes (both type i and type ii, if mentioned), hypertension, heart diseases (due to the small sample size of individual conditions, we (table ) . all denv and wnv were confirmed cases in the selected studies and infection was identified either by rt-pcr, elisa, report reviews or from national surveillances. chronic comorbidities were either self-reported or record-based and did not allow for differentiating between those diagnosed before, after or during the infectious episodes. weighted average was used to calculate the overall age. severe cases of denv were defined as those with any form of the disease that develops major complications such as dengue hemorrhagic fever (dhf, grades i and ii), dengue shock syndrome (dss) (dhf grades iii and iv), infections developing organ failures (e.g., acute renal failure and acute respiratory failure), clinically significant bleeding, cases requiring icu hospitalization and/or fatal cases . severe cases of wnv were defined as those developed wnv neuroinvasive diseases, such as, cases of wn encephalitis (wne), wn meningitis (wnm), poliomyelitis or acute flaccid paralysis, cases requiring icu hospitalization, need for rehabilitation, wnv-associated retinopathy (wnvr), chorioretinitis or fatal cases [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . to measure the prevalence rates of comorbidities, we extracted the proportions or percentages reported in the selected studies from the total number of flavivirus cases and from non-severe and severe cases. publication bias was assessed by the visual inspection of funnel plot (s fig). egger's test [ ] was also used to assess publication bias and the tendency for the effects estimated in small sample size studies to differ from those estimated in larger studies. the results of egger's test were presented as t-value and p for publication bias for studies reporting the comorbidity of interest (s table) . significant egger's test for publication bias was based on p< . . the primary outcome measure was to evaluate the overall prevalence of comorbidities in the flavivirus infection and the estimates stratified by severity. meta-analyses were carried out to assess the pooled prevalence (and % ci) of clinical symptoms and the proportions of each comorbidity in the total, severe and non-severe cases of each infection. meta-analysis tests were conducted using comprehensive meta-analysis software, cma version . (englewood, nj, usa) [ ] . variances of raw proportions or percentages were pooled based on a binary random-effects model [ ] , given the population heterogeneity and assuming the relationships between comorbidities and flavivirus infections vary across populations. forest plots were used to illustrate the prevalence of comorbidities in flavivirus infections from the selected studies prior to stratifying the proportions by severity. comparisons of proportions for the prevalence of clinical symptoms between diseases and the pooled prevalence estimates of comorbidities in relation to severity were carried out using the chi-squared test as previously recommended [ , ] . ors adjusted for age (and % ci) for severe infection outcome in patients with comorbidities were calculated using the cochran-mantel-haenszel test as previously described [ ] [ ] [ ] . all statistical tests were two-sided and conducted using spss statistics, version . (spss inc., chicago). assessing the heterogeneity among the selected studies was carried out using the q test [ ] that informs about the presence versus the absence of heterogeneity. the q test, however, does not report the extent of heterogeneity and has inadequate power to detect heterogeneity among the small number of studies identified for some comorbidities. therefore, we calculated the i index to complement the q test to describe the degree of between-study heterogeneity [ ] . i index values were categorized as low ( - %), moderate ( - %), substantial ( - %), and considerable (> %) as recommended [ ] . we also quantified the true heterogeneity by estimating the within-study variance in the random-effects model (τ ), as previously described [ ] . all data generated or analyzed during this study are included in this published article (and its supporting information files). the original search resulted in , articles selected for title review as they satisfied our selection criteria (s table) . additional articles were identified through bibliography search from previously identified systematic reviews (fig ) . after de-duplication, a total of , original titles were selected for abstract review. abstract review resulted in the exclusion of , reports. full text assessment was conducted on the remaining articles by at least two reviewers and resulted in the selection of studies in denv [ - ], in wnv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and two in jev [ , ] (those were further excluded) for inclusion (see fig for exclusion criteria and breakdown). the agreement on the inclusion between two reviewers was . % with weighted κ = . ( % ci: . - . ). this substantial agreement ( . - . ) may relate to the extent of clarity in the assessed abstracts when reporting the rates or the presence of comorbidities at the initial stage of the selection process. no studies were identified, however, for yfv and zikv infections. after excluding the studies on jev, the quality of each of the remaining studies, involving sets of patients, was assessed. with a maximum quality score of , a good score (! points) was achieved in % of the studies ( studies on denv and on wnv); % of the studies were scored as being of fair quality ( - points including studies on denv and on wnv) and % of the studies were scored as low quality ( points including studies on denv and on wnv) ( table ) . no score was assessed for the remaining reports from the grey literature (abstracts; on denv and on wnv). more than % of the reports ( studies) were retrospective in nature with the rest being age-and sex-matched or nested case-control studies, surveillance reports, or prospective studies. in these studies, there was a wide variation in the sample size ranging from five [ ] to , [ ] patients. same cohort was likely to be reported for denv in two occasions, i.e., by chen et al., [ ] and lee et al., [ ] and also by lee et al., [ ] and lee et al., [ ] . to avoid repeated counting of the cases from the same study cohort, we only included the study with the larger sample size both in evaluating the total number of subjects and in the analysis of average age of the studied cases. furthermore, one study [ ] reported that out of the , studied denv cases, about % were classified as highly suggestive whereas the remaining % were confirmed by the diagnostic algorithm. this report did not separate or identify this small number of cases (~ cases) that represents only . % of the total number evaluated here and were included into our analysis. moreover, one study [ ] included fatal cases of denv and chikungunya virus coinfection. although these cases may represent an unusual clinical population, they were included here for our report to be inclusive as the number of subjects was extremely small (n = ) and they only had cases of hypertension (representing < . % of the entire hypertensive cases). to examine if the study estimates are related to the size of the study, publication bias was assessed by visual inspection of funnel plots (s fig) and by egger's test (s table) . funnel plot inspection demonstrated a seemingly non-symmetrical distribution of the effect size on either side of the pooled estimate, suggesting some evidence of publication bias. results of egger's test, however, for most of the associations between denv or wnv and the comorbidities showed p> . (except for the prevalence of hypertension in denv and heart diseases in wnv, p = . ), given the assumption for evidence of smallstudy effects is based on p< . as previously reported [ ] . the studies selected for denv were geographically diverse and included countries. the reports were from southeast asia, south america, eastern mediterranean, and western pacific regions. most of the wnv reports were, however, from the united states with few studies (table ). in wnv, however, meningitis and encephalitis were present in > % of the severe cases, representing the highest prevalent severe condition. obesity/overweight was the most prevalent comorbidity in denv patients ( . %, % ci: . - . %), followed by hypertension ( . %, % ci: . - . %) and diabetes ( . %, % ci: . - . %) (fig ) . heart disease, asthma and stroke were present in about . % of the denv cases. on the other hand, in wnv cases, hypertension was the most frequent comorbidity ( . %, % ci: . - . %), followed by heart diseases (~ %), diabetes (~ %) and stroke ( . %, % ci: . - . %) (fig ) . no study in wnv has reported the incidence of obesity or asthma. overall, there was low (i = - %) to moderate (i = - %) heterogeneity among the identified studies. when cases of denv and wnv were stratified by severity (table ) , there was -to -fold higher prevalence (p< . ) of diabetes and heart diseases in severe denv and wnv cases, respectively compared to their rates in the non-severe disease. hypertension, however, was about -fold more prevalent in severe cases of both flavivirus infections (p< . ) than nonsevere cases. asthma and obesity were only examined in denv as none of the selected studies reported their prevalence in wnv. asthma and obesity were~ . -fold more frequent (p< . ) in patients with severe denv than in non-severe cases. the frequency of stroke was assessed only in one study in non-severe denv [ ] and wnv [ ] , which did not permit comparison between severe and non-severe cases. based on the differences in prevalence of comorbidities between severe and non-severe cases of denv and wnv, we estimated the age-adjusted or of severe infection outcome in patients with chronic comorbidities to be . ( %ci: . - . ) in denv patients with obesity/overweight, . ( %ci: . - ) in patients with diabetes, . in those with heart diseases ( % ci: . - . ) and . ( % ci: . - . ) in patients with hypertension. the or of severe wnv was . in patients with diabetes ( % ci: . - . ) and . in those with hypertension ( % ci: . - . ) (table ). the present study evaluates the frequency of comorbidities in denv and wnv as examples to flavivirus infections that represent a major global health problem. datasets were few or unavailable for jev, zikv and yfv. only two studies were found to satisfy our selection criteria for jev [ , ] and most of the reports on zikv were related to co-existing infectious diseases such as chikungunya or hiv [ ] [ ] [ ] or to gestational diabetes in a single case [ ] . studies in yfv, however, primarily evaluated the safety and effectiveness of vaccination ( d-yf) in people with pre-existing chronic illnesses [ ] [ ] [ ] . a few systematic reviews on the frequency of comorbidities in flavivirus infection exist and none have used meta-analysis to synthesize global prevalence estimates. we observed a large difference in the volume of literature and total number of patients evaluated for each of the examined condition where studies were identified for denv [ - ] with , subjects and for wnv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] with , patients. almost % of the selected studies were assessed to have good quality score for evaluating and reporting chronic disease incidence or prevalence [ ] [ ] [ ] . however, chronic diseases were not clearly defined in the vast majority of the studies as they were either selfreported or record-based and did not allow for differentiating between those diagnosed before, after or during the infectious episodes. very few of the selected reports provided sample size justification and all had infectious disease status assessed prior to the chronic disease scoring. the development of chronic diseases following infection was not reported in any of the selected studies. furthermore, none of the selected studies reported either denv or wnv as a coinfection for one another. moreover, a few number of studies provided information on the status of more than one chronic disease comorbidity in the same patient. additionally, assessment of the chronic diseases was not blindly evaluated with respect to the infectious diseases status. these factors meant that % of the selected studies had good-to-fair quality scores. this made us choose to include studies that were assessed to be of "fair" or "low" quality in our meta-analysis to attain more comprehensive estimates with larger sample size and to avoid the bias when excluding studies with small or no effect. the geographical diversity of denv reports compared to wnv studies likely reflects the fact that dengue now is the most common flavivirus infection globally with transmission occurring in at least countries and almost billion people around the world [ ] . wnv, on the other hand, despite being an important cause of human disease worldwide with continual increase in transmission over the past years [ , ] , is spreading in a notably lesser rate than denv [ ]. only one study of the selected reports on wnv were from regions other than europe or the usa [ ] . given the repeated wnv outbreaks between and in israel and europe and its movement through north america over the last decade that resulted in sustained presence in the community [ , , ] , it was predictable to identify a limited set of wnv studies from regions other than those primarily affected. most clinically apparent acute flavivirus infections progress from a classic presentation of mild fever, headache, muscle pain, rash, and malaise to a more disease-specific syndrome of acute febrile illness to further severe states of hemorrhagic fever and encephalitis. pooled prevalence analysis of these clinical symptoms indicated their comparatively lower frequencies in , heart diseases (c), asthma (d), stroke (e) and obesity/overweight (f) in dengue fever patients and % ci. heterogeneity analysis was carried out using q test, the among studies variation (i index) and within-study variance in the random-effects model (τ ). wnv than in denv. it is known that wnv infection is commonly asymptomatic, with~ % of infected persons having clinically apparent disease [ ] [ ] [ ] . symptomatic patients mostly present with fever, headache and malaise and less commonly with myalgia, rash, neck pain, and arthralgia [ ] . on the other hand, patients infected with denv are asymptomatic in the majority of cases but a small proportion may develop an array of clinical symptoms ranging from mild flu-like syndrome, such as fever, skin rash, headache, myalgia, and arthralgia, to severe forms of the disease. only rash was -fold more frequent in denv than wnv cases. according to the who classification system [ ] , denv patients can be clinically classified as with 'probable dengue', 'dengue with warning signs' or 'severe dengue'. this deviates from the who classification system [ ] , categorizing patients as with df, dhf or dss. over % of the denv patients in the selected set of studies presented with df. the progression of df to severe clinical manifestations is mostly unpredictable. case fatality rate may exceed % if timely and appropriate differential therapy is not introduced to reduce the impact of disease complications [ ] . recent estimates for the global burden of dengue suggest the epidemics mainly affect children and young adults between the ages of and years old in terms of death, years lived with disability (ylds), years of life lost (ylls) and disability-adjusted life-years (dalys) [ ] . however, an age shift to more adult cases with severe denv (and more comorbidities) is presently demonstrated in several reports from around the world (see below) [ , , ] . in addition to this age-shift in severe denv cases, the incursion of denv into new world regions may have influenced the age-related increased incidence of the disease. advanced age, on the other hand, has long been known as a frequent risk factors for severe disease outcome of wnv [ ] . average annual incidence of severe wnv cases reported to cdc between - for cases over years old was~ . per , population per year compared to only~ . for patients < - years old [ , ] . introduction of the competent vectors into natural environments and urban areas, together with the changing societal factors (migration, industrialization, trade, urbanization and population growth), may all have facilitated the geographical expansion of flavivirus diseases into different regions of the world. examples include the expansion of denv from the caribbean islands to brazil and from the pacific islands to other regions in south asia [ , ] and wnv from africa and the middle east to europe and north america [ ] . many of the regions where these vector-borne diseases spread are also experiencing an epidemiological shift from communicable diseases to non-communicable disorders as the primary causes of the morbidity and mortality [ ] . with the ageing population, non-communicable diseases now account for nearly half of the disease burden in low-and middle-income countries [ ] . therefore, flavivirus diseases, particularly denv, may have been shown now to affect older adults, an age group with inherently more comorbidities [ , ] . the present study demonstrates that hypertension and diabetes are the most prevalent comorbidity in both denv and wnv with obesity/overweight and heart diseases being present, respectively, in about % of the denv and wnv cases. although the prevalence of diabetes, hypertension and heart diseases varied widely among the denv selected studies (ranging from -to -fold), the vast majority of the reports showed values clustering around the pooled estimated averages for each comorbidity, as evidenced by the low i index values. obesity, stroke and asthma proportions varied only by -to -fold among the studies with moderate i index values. however, in wnv studies, the among-studies prevalence of comorbidities varied by -to -fold for diabetes, hypertension, heart diseases and stroke with low to moderate i indexes. these variations in the prevalence of comorbidities in wnv cases may relate to the higher average patients' age (compared to denv) since older age is a risk factor for a number of non-communicable diseases, e.g., diabetes and heart conditions [ ] [ ] [ ] [ ] [ ] . on the other hand, the wider variation in prevalence of comorbidities in denv than wnv can be linked to the different patterns of geographical distribution between the two diseases. diabetes, hypertension and heart diseases were, respectively, -to -fold significantly more prevalent in severe denv and wnv than in non-severe cases. this observation is supported by a number of case-control studies implicating comorbidities in severe outcome of flavivirus infections. over -fold higher frequency of diabetes were found in severe denv cases than in cases with df [ , , , ] . furthermore, severe clinical presentation of denv was likely to develop in patients with diabetes than in non-diabetic subjects with the odds ratio (or) ranging from . ( % ci . - . ) [ ] to ( % ci . - . ) [ ] . this is line with the or of about to for severe denv and wnv observed here among patients with diabetes or heart diseases. diabetes risk factors such as glucose intolerance [ ] and hyperlipidemia [ ] were prevalent in % and % of severe denv cases, respectively and more frequent in elder patients [ , ] . similarly, diabetes ( - %), hyperlipidemia ( - %) and coronary artery disease ( - %) were prevalent in wnv cases than controls [ , ] with or of neuroinvasive outcome varying between . ( % ci . - . ) and . ( % ci . - . ) [ ] . diabetes was % more frequent in cases with wne and wnm than in wnv fever [ ] . developing encephalitis was more likely to occur in wnv cases who also had diabetes (or = . ; % ci: . - . ) or cardiovascular diseases (or = . ; % ci: . - . ) [ ] . furthermore, hypertension was shown to be present in % of the severe or fatal denv cases [ ] , the manifestation of acute respiratory failure [ ], and in higher proportion of elder patients [ , ] with or varying from . ( % ci . - . ) [ ] to . ( % ci . - . ) [ ]. in contrast, some studies did not show such a relationship in severe denv [ , , ] . in wnv, patients with hypertension had higher odds of developing neuroinvasive outcome (or . , % ci . - . ) [ ] and encephalitis (or = . , % ci . - . ) [ ] than non-hypertensive cases. high prevalence of hypertension ( %) was also noted in patients with wne, wnm and fatal outcome compared to those in wnv fever [ ] . in line with these findings, the results of the present study strongly suggest that diabetes, heart diseases and hypertension are more prevalent in severe denv and wnv cases than in the non-severe disease and may present significant risk factors-together with advanced age-in complication of flavivirus infection. diabetes, hypertension, cardiac diseases and obesity are interrelated as they share similar cardiometabolic risk factors that result in the development of metabolic syndrome and the subsequent manifestation of this range of chronic diseases. these metabolic syndrome related diseases may impair the immune system to increase the level and duration of viremia [ , ] and facilitate the passage of neurotropic flavivirus across the blood-brain barrier to predispose patients to neurologic complications [ , , ] . impairment of the innate immune system-that mediates the host defense to infection-render individuals more susceptible to a range of infectious diseases and severe illnesses [ ] [ ] [ ] [ ] . in fact, the metabolic syndrome related chronic conditions are linked to endothelial dysfunction, attenuation of anti-inflammatory responses and a generation of a pro-inflammatory state; features that are also common in many infectious disorders [ , , ] . for overproduction of pro-inflammatory cytokines such as ils, tnf-α, ifn-γ and tgf-β is known to occur in severe denv [ ] , wnv [ ] and yfv [ ] leading to cytokine storm and vasculopathy, hemorrhage, tissue damage and septic shock characteristic of severe flavivirus infections. cytokine synthesis shift to the th (microbicidal action of pro-inflammatory ifn-γ) from th (anti-inflammatory il- , - and - ) in severe infection, when accompanied by the increased pro-inflammatory cytokine levels arising from chronic diseases, both can lead to endothelial dysfunction and a subsequent range of complications, including allergy, vascular leakage, ascites and pericardial effusion as observed in denv [ , , [ ] [ ] [ ] . in support, mononuclear cells from diabetic patients, when infected with denv, produced significantly higher levels of il- , il- and granulocyte-macrophage colony-stimulating factor compared to their healthy counterparts [ ] . conditions such hyperglycemia and cellular insulinopenia may also impair macrophage and lymphocyte functions leading to a status of reduced acquired immune response [ ] that was linked to about % increased risk of pneumonia-related complications and hospitalization [ ] . further evidence for a possible role of altered innate immunity in mediating the association between metabolic syndrome-related comorbidities and severe clinical presentation of flavivirus infections can be also substantiated from the relatively high prevalence of asthma in severe denv cases. patients with asthma normally exhibit altered th and th responses [ , ] . in general, although the etiological relationship between chronic comorbidities and severity of flavivirus diseases is yet to be fully elucidated, it may be reasonable to suggest that in infected patients, chronic conditions may synergistically attenuate both the innate and adaptive immune systems [ ] . this may further impair critical components of immunity such as chemotaxis, phagocytosis, and the bactericidal activity of neutrophils and macrophages as well as downregulate the functions of t cells and neutrophils [ , ] to exacerbate the complications of the infectious diseases. although the present study is the first to systematically report and quantify the prevalence of comorbidities in flavivirus infections, it has several limitations. it does not address the effect of flavivirus infection on the development of chronic comorbidities or attempting to substantiate a causality between the two conditions. also, the study is not addressing the effect of integrated vector management practices on the infectious or chronic disease incidence or causal associations. we are simply reporting the prevalence of comorbidities in flavivirus severe and non-severe cases to warrant further studies investigating the effect of chronic comorbidities on the infectious disease outcome. the selected studies were mostly retrospective in nature with variable clinical and laboratory diagnostic criteria and control groups and were heterogeneous for exposures and outcomes. in the denv studies, although the majority were conducted after the who adopted the new case classification of [ ] to improve clinical management, many of the reports still used the who classification [ ] . this absence of similar endpoint measures hindered the results comparability and may limit the generalizability of conclusions to other geographical regions or older age groups since the who criteria were based on disease patterns of children in thailand [ , ] . in addition to the retrospective nature of many of the identified studies, incomplete clinical datasets, relatively small sample size, inappropriate definition or selection of control population were noted as shortcomings in various reports leading to further limitations in interpreting the findings. in fact, most of the studies were hospital-based with a high potential for selection bias of appropriate control groups. additionally, the studies retrieved in this review measured different outcomes for denv (dhf and dss-with different grades and definitions of severity) and wnv (wne, wnm, poliomyelitis or acute flaccid paralysis) making results difficult to compare, broadening the scope of the outcome and rendering the findings challenging to extrapolate. furthermore, the identified reports have shown several-fold of among-studies variance in the proportion of comorbidities which may have contributed to the significant heterogeneity observed in our report. additional sources of heterogeneity may relate to the large amongstudies variation in sample size and surveillance approaches. the heterogeneity of the selected studies was evident from the publication bias that may have been driven from the small-study effect, i.e., the possibility of including small studies with spuriously overstated estimates while discounting those without statistically significant effects that may have a lower possibility of being published. this was addressed, however, by stratifying the analysis by the flavivirus infection and including all studies related to the research question rather than excluding reports based on lack of quality. this may levy some limitations on the estimated contribution of comorbidities to severe flavivirus infections and render our results as a guide to generate more accurate estimates for national or international intervention strategies in subjects with comorbidities. when assessing comorbidities, the studies showed that the various co-existing conditions have been either self-reported or record-based and did not allow for differentiating between those diagnosed before, after or during the infectious episodes. furthermore, most of the retrieved reports did not provide clearly characterized, valid and reliably-defined comorbidities that were implemented consistently across studies. for example, the majority of the reports did not distinguish between the prevalence of type and type diabetes where both were combined despite their different characteristics, etiological factors and clinical features. given this paucity of information, it was challenging to distinguish between the two types of diabetes for their role in viral diseases severity and complication. furthermore, the lack of consistent reporting for the statuses of heart diseases made it difficult to evaluate the frequency of each heart condition. therefore, we combined the different heart conditions under a single comorbidity. the utility of pooling the clinically different and heterogeneous pathologies of heart diseases may lead to losing significance of this observation and result in misinterpretation. this observation warrants developing more rigorous studies exploring the role of various heart condition in the severity of flaviviral infections. given the possible extent of under-diagnosis for many of the assessed comorbidities, particularly in the developing world, misclassification of many co-existing medical conditions is likely substantial. together with the possible underestimation for the frequency of infectious disease where not all asymptomatic cases will be detected, these factors may have led to lower estimates of prevalence for many of the assessed comorbidities. lastly, since we identified only two studies for jev and no studies met the inclusion criteria for yfv and zikv infections, the present report may be viewed as focusing primarily on denv and wnv. this lack of available information on the prevalence of comorbidities in flavivirus diseases other than denv and wnv, calls for developing large-scale studies to cover this knowledge gap. in conclusion, the study of comorbidities in flavivirus infection is important for reducing the burden of the disease via guiding approaches for improved patient outcome or differential case management. we provided evidence for a higher prevalence of diabetes, hypertension, and heart diseases in severe cases of flavivirus infections such as denv and wnv than in nonsevere cases. these findings do not implicate causality between the chronic comorbidities and severe denv or wnv. it simply demonstrates different profiles of comorbidities at different stages of the infectious diseases. our results warrant further assessments to identify the nature and extent of the co-existence between comorbidities and infection. for example, standardized prospective case-control studies in regions of high infection prevalence would contribute to a better understanding for the etiological role of comorbidities in severe disease outcome when conducted with an agreed protocols of comorbidity assessments, infection classification, disease biomarker analysis and appropriate control groups. however, even in the absence of causal inference between the non-communicable and infectious diseases, it may be justified that once non-severe episodes of flavivirus infection are confirmed in subjects with comorbidities that they remain under close surveillance to avert complications. this may guide public health practitioners and clinicians to predict complications-at least partially-based on the presence of comorbidity. this can subsequently substantiate close observation, adequate treatment, or hospitalization following infection to avert severe disease outcome. 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cascade in dengue hemorrhagic fever: implications for pathogenesis increased production of interleukin- , interleukin- , and granulocyte-macrophage colony-stimulating factor by type diabetes' mononuclear cells infected with dengue virus, but not increased intracellular viral multiplication suggested use of vaccines in diabetes infections in patients with diabetes mellitus: a review of pathogenesis the authors thank dr. nicholas ogden for constructive discussions and comments. key: cord- -qlqavi t authors: chiow, k.h.; phoon, m.c.; putti, thomas; tan, benny k.h.; chow, vincent t. title: evaluation of antiviral activities of houttuynia cordata thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection date: - - journal: asian pac j trop med doi: . /j.apjtm. . . sha: doc_id: cord_uid: qlqavi t objective: to evaluate the in vitro activities of the ethyl acetate (ea) fraction of houttuynia cordata (h. cordata) thunb. (saururaceae) and three of its constituent flavonoids (quercetin, quercitrin and rutin) against murine coronavirus and dengue virus (denv). methods: the antiviral activities of various concentrations of the ea fraction of h. cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus (mhv) and denv type (denv- ). cinanserin hydrochloride was also tested against mhv. the ea fraction of h. cordata was tested for acute oral toxicity in c bl/ mice. results: the ea fraction of h. cordata inhibited viral infectivity up to d. cinanserin hydrochloride was able to inhibit mhv for only d. the % inhibitory concentrations (ic( )) of the ea fraction of h. cordata added before the viral adsorption stage were . μg/ml for mhv and . μg/ml for denv- with absence of cytotoxicity. the mice fed with the ea fraction up to mg/kg did not induce any signs of acute toxicity, with normal histological features of major organs. certain flavonoids exhibited comparatively weaker antiviral activity, notably quercetin which could inhibit both mhv and denv- . this was followed by quercitrin which could inhibit denv- but not mhv, whereas rutin did not exert any inhibitory effect on either virus. when quercetin was combined with quercitrin, enhancement of anti-denv- activity and reduced cytotoxicity were observed. however, the synergistic efficacy of the flavonoid combination was still less than that of the ea fraction. conclusions: the compounds in h. cordata contribute to the superior antiviral efficacy of the ea fraction which lacked cytotoxicity in vitro and acute toxicity in vivo. h. cordata has much potential for the development of antiviral agents against coronavirus and dengue infections. severe acute distress syndrome (sars) is a highly contagious respiratory illness caused by sars coronavirus, which emerged in and rapidly spread throughout the world, with a mortality rate of %- %. although the disease disappeared in mid- , its re-emergence cannot be excluded since sars-like coronaviruses are zoonotic and exist in animal reservoirs (e.g., bats, raccoon dogs and palm civets), thus posing a potential risk for future epidemics [ ] [ ] [ ] . sars coronavirus is a large, enveloped, single-strand, positive-sense rna virus. the viral genome is about kbp, containing open reading frames that encode the polymerases required for viral rna synthesis, while the remaining open reading frames encode structural proteins (i.e., spike, envelope, membrane and nucleocapsid) and accessory proteins. first identified in , the emerging middle east respiratory syndrome (mers) coronavirus causes severe pneumonia with multiorgan involvement (including acute renal failure), and a mortality rate of % ( deaths out of cases documented by who as of june ). it is considered to be a zoonotic virus being transmitted from camels in the middle east. currently, no specific treatment exists against mers coronavirus. the murine coronavirus, mouse hepatitis virus (mhv), is a coronavirus that causes an epidemic murine illness with high mortality. generally, mhv is extremely infectious to colonies of mice and causes hepatitis upon infection. sars, mers and mhv belong to the group coronaviruses and are classified under the genus betacoronavirus. in view of the relatedness of these coronaviruses, mhv was selected in this study to act as a surrogate model for sars and mers coronaviruses which necessitate bsl- facilities, whereas mhv is considered a bsl- pathogen. belonging to the genus flavivirus, dengue virus (denv) represents the most important mosquito-borne viral disease with considerable resurgence in many parts of the world. there are four antigenically-related but distinct denv serotypes, such that infection with one serotype does not confer life-long immunity against the other serotypes. denv infection causes dengue fever, and occasionally the more serious conditions of dengue hemorrhagic fever and dengue shock syndrome [ ] . antibodydependent enhancement is thought to play a central role in dengue pathogenesis, with the risk of developing dengue hemorrhagic fever and/or dengue shock syndrome being greater in secondary infections with denv- compared to other serotypes [ ] . the different manifestations of dengue may also be attributed to denv variants with varying degrees of virulence [ ] , while viral load is also a contributing factor in the development of potentially fatal complications [ ] . in addition, being a highly prevalent serotype in tropical and subtropical regions worldwide, denv- was selected for this study. although several dengue candidate vaccines are undergoing clinical trials, there are currently no effective antiviral therapies which are urgently needed to control dengue. belonging to the saururaceae family, houttuynia cordata (h. cordata) thunb. is a perennial plant native to mountainous regions of eastern asia, with an indefinite spread as a creeping rhizome in moist locations. this herb possesses very promising antiviral properties especially against clinically important enveloped viruses such as herpes simplex virus- (hsv- ), influenza virus, and human immunodeficiency virus- in vitro. interestingly, the steam distillate of h. cordata can strikingly inactivate an enveloped virus but is incapable of inactivating a non-enveloped virus [ ] [ ] [ ] [ ] [ ] . to support these observations, we and others have demonstrated that the ethyl acetate (ea) fraction of h. cordata is effective in inhibiting the infectivity of enveloped viruses such as denv [ ] . meng et al. discovered common peaks in the hplc-dad ms fingerprint of fresh h. cordata [ ] . in our previous project, we verified some of these peaks in the ea extract as polyphenols or flavonoids (chlorogenic acid, hyperoside, quercetin and quercetrin) and have investigated their antiviral efficacy against denv [ ] . flavonoids are a class of natural products with high pharmacological potency, are ubiquitous in photosynthesizing cells and hence likely to be consumed daily [ ] . flavonoids are known to display antiviral activities, e.g., glabranine and -o-methyl-glabranine against dengue virus, procyanidin and pelargonidin against hsv, and catechins against influenza virus [ , ] . using virus-specific neutralization tests, this study tested the ea fraction of h. cordata for its antiviral efficacy against mhv (for the first time) and denv- (as further verification using a different batch of h. cordata specimen). three flavonoid components of h. cordata, i.e., quercetin, quercitrin and rutin, were also investigated for their antiviral activities against both viruses. they were also selected since they are common and naturally occurring flavonoids, whose molecular structures share high degrees of similarity. in the mhv experiments, we also compared the potency of cinanserin hydrochloride which has been proven to neutralize sars coronavirus in vitro. all the compounds were also tested for cytotoxicity in vitro, while the ea fraction was also tested for acute oral toxicity in mice. . . plant material, ethanolic extraction, aqueous-ea fractionation, and flavonoids of h. cordata the aerial parts (fresh leaves) of h. cordata were collected from a farm in johor, malaysia and authenticated in the department of pharmacology, national university of singapore. the voucher specimen of h. cordata was deposited in the singapore botanic gardens and assigned the identification number sing - . prior to extraction, the herb (dry weight of g) was washed with de-ionized water, homogenized to a fine powder, and soaked overnight in % (v/v) ethanol. the next day, the ethanolic extract was removed and stored. more solvent was added to the blended herb, which was left to soak till exhaustion. the extract was filtered, concentrated with a rotary evaporator (buchi rotavapor r- ) and freeze-dried to yield . g of crude extract in powder form. the crude extract was then dissolved in ea and de-ionized water. the water and ea phases were separated with a separating funnel. the ea phase was concentrated with a rotary evaporator, and the concentrated ea fraction was subsequently stored at − c overnight and freeze-dried. the flavonoids tested were quercetin dihydrate ( % hplc), quercitrin, and rutin hydrate ( %), all purchased from sigma-aldrich. ccl . , a normal mus musculus (mouse) liver epithelial cell line, was used for propagation of mhv and for the mhv neutralization test. compounds that were tested against mhv were the ea fraction of h. cordata, its flavonoid components, and cinanserin hydrochloride (tocris bioscience). each compound was -fold serially diluted with the corresponding diluent, and the dilutions were then added to wells of a -well microtiter plate. next, tcid of mhv was added to each diluted compound and incubated at c for h with % co . also tested was treatment with diluent only at various concentrations. virus-infected controls and uninfected cell controls were included in each batch of assays. confluent ccl . cells were cultured in dmem supplemented with % horse serum, and × cells were seeded into each well of another -well microtiter plate, and incubated at c with % co until the cells were % confluent. cell culture fluid from each well was discarded. each virus-compound mixture and controls were transferred to these wells in duplicate, followed by addition of dmem with % (v/v) horse serum. the plate was sealed and incubated at c with % co , and observed daily for d. the highest dilution of compound that inhibited cytopathic effects (cpe) was considered as the minimum inhibitory concentration (mic). the new guinea c strain of denv- was propagated in the c / aedes albopictus mosquito cell line and maintained in leibovitz l medium supplemented with % (v/v) fetal bovine serum at c under % co atmosphere. the anti-denv- activity of h. cordata and its flavonoids were evaluated by plaque reduction neutralization test or prnt [ , ] . bhk- (baby hamster kidney) fibroblasts were cultured to form cell monolayers in -well plates with rpmi- supplemented with % (v/v) fetal bovine serum at c under % co . the test compounds were dissolved in the relevant diluents, and -fold serial dilutions were prepared to obtain different concentrations. denv- new guinea c neutralizing monoclonal antibody h from chemicon served as the positive control [ ] . diluent only (at various concentrations), virus, and cell controls were also included by adding the corresponding diluent, virus suspension, and medium without the treatment compounds. each experiment was performed in duplicate. denv- ( plaque-forming units or pfu) were incubated for h with various concentrations of each compound together with controls before adding to the cells. the virus-sample mixtures were incubated with the cells at c under % co for another hour with rocking at -min intervals before the cells were overlaid with % carboxymethylcellulose at c under % co for d. the cells were then fixed with % formaldehyde and stained with % crystal violet, and the number of plaques was counted. the percentage plaque reduction of the compounds at every dilution was determined as follows: (mean number of plaques in virus control) − (average number of plaques in sample) × % divided by (mean number of plaques in virus control). the percentage plaque reduction was plotted against various concentrations of the test agents to determine the concentration that causes % plaque reduction (ic ). the mtt cell proliferation assay was performed to determine the cell viability following exposure to the test compounds. various concentrations of each test compound were added to wells containing cell monolayers and incubated at c under % co for h. after incubation, mtt reagent was added to each well, and further incubated for h or until purple precipitates were visible under an inverted microscope. then, ml of g/l sds in . mol/l hcl were added to each well and incubated overnight in the dark at room temperature. the optical density (od) at nm was then read, and the cell inhibition rate calculated from the formula: [ the inhibition rates were plotted against various sample concentrations to ascertain the concentration that causes % cytotoxicity (cc ). the ea fraction and flavonoids (either individually or in combination) were tested at the same concentrations as those for neutralization tests. the assay included wells containing medium only as well as untreated control cells. each experiment was repeated, and the mean and standard deviation were calculated. this approach was adapted from the oecd guideline for testing of chemicals, : acute oral toxicity -fixed dose procedure, and relied on the observation of clear signs of toxicity or even mortality. c /bl nulliparous and nonpregnant female mice about -weeks old were obtained from the laboratory animals center, national university of singapore. upon arrival, they were kept in cages for d prior to commencement of the study. on the actual dose-feeding day, the mice were fasted for about h. doses of , , and mg/kg were prepared for administration using water as vehicle to suspend the ethanolic extract of h. cordata at a constant volume of ml/ g of body weight. in total, the five groups of mice (n = each) were fed with a single dose of the extract (including the vehicle control) by oral gavage. a sighting study was then conducted frequently from the first min, with special attention to the first h. thereafter, the condition of each mouse was observed daily to detect abnormalities such as changes in physical appearance, behavioral signs and body weight. all mice were finally euthanized after d of observation, and major organs (brain, heart, lungs, liver and kidneys) were harvested for histopathological examination after staining with hematoxylin and eosin (h&e). the ea fraction of h. cordata was evaluated at concentrations of . mg/ml down to . mg/ml. table and figure show that the ea fraction of h. cordata exhibited anti-mhv activity at a mic of . mg/ml without any apparent cytotoxic effects on ccl . cells. tested at concentrations from . to . mg/ml, the anti-mhv effect of quercetin was significantly less, with mic of . mg/ml (figure ), cytotoxicity cc value of . mg/ml, and selectivity index of . . however, quercitrin and rutin did not exhibit antiviral activity on days postinfection (dpi) at concentrations of . mg/ml and below (table ). cinanserin hydrochloride was tested at concentrations ranging from . to . mg/ml. table indicates that cinanserin exerted anti-mhv activity at mic of . mg/ml on dpi and . mg/ml on dpi (figure ) , with minimal or no cytotoxicity. however, on dpi, no viral inhibition was observed. the ea fraction of h. cordata was tested starting with the highest concentration of . mg/ml followed by -fold dilutions down to . mg/ml. by means of prnt, a distinct trend of denv- inhibition was observed at various concentrations of the ea fraction without cytotoxicity to bhk cells, with the ic being . mg/ml. the efficacy of the ea fraction in inhibiting denv- infection was clearly evident at concentrations of . mg/ml and above, at which complete viral inhibition was achieved ( figure ). individual flavonoids were tested at concentrations of . mg/ml down to . mg/ml. table table . the ea fraction, quercetrin and rutin were dissolved in % dmso. quercetin was dissolved in aqueous alkali ( . mol/l naoh) as it was not very soluble in dmso. various concentrations of ea fraction and compounds were subjected to the corresponding viral neutralization tests as well as the mtt assay. as negative controls, dmso and aqueous alkali were also tested separately by denv- prnt, mhv neutralization test and the mtt assay which revealed absence of non-specific denv- and mhv inhibition as well as lack of cytotoxicity of these solvents (data not shown). throughout the -day period of observation, the control group as well as the four groups of mice fed with varying doses of h. cordata extract showed no mortality and no significant weight loss (data not shown). moreover, all mice appeared to be active and well-groomed before euthanasia. from the histopathological sections of all major organs stained with h&e, there was no significant difference between the control group and all the test groups, as exemplified in figure which depicts the control group compared with the group fed with the highest dose of mg/kg. the experimental strategy in this study was to investigate the prophylactic antiviral effects since the compounds were allowed to interact with the viruses for h before introduction into the cells. this approach was employed as we previously found no therapeutic effect when the compounds were added after prior infection of cells with denv [ ] . overall, this study demonstrated that ea fraction of h. cordata and its flavonoid component, quercetin, could inhibit both mhv and denv- in vitro. this was followed by quercitrin which could inhibit denv- but not mhv, whereas rutin did not exert any inhibitory effect on both viruses. we and others have provided evidence to show that different flavonoid components in the ea fraction of h. cordata exert varying degrees of antiviral activity against different viruses. our findings corroborate previous evidence that among the flavonoid components, quercetin is the most effective against denv- [ ] . wleklik et al. emphasized that there is a structural basis for the distinct differences in antiviral activities of flavonoids [ ] . hence, it is noteworthy that being the most bioactive, quercetin possesses the hydroxyl group at the r position compared to the other two flavonoids tested, i.e., rhamnose in quercitrin and rubinose in rutin [ ] . quercetin is an aglycone present at high concentration in onions. this compound has virucidal activity against enveloped viruses such as mengovirus, herpes simplex, parainfluenza type , pseudorabies, respiratory syncytial, and sindbis viruses [ ] [ ] [ ] [ ] . quercetin is able to inhibit h + -atpase of lysosomal membrane and thus prevent virus coat removal [ ] . moreover, quercetin exhibits significant inhibitory effects on the atpase of multidrug resistance-associated proteins, thus increasing the bioavailability of anticancer and antiviral drugs in vivo [ ] . hence, quercetin can be considered for its potential efficacy in antiviral drug therapies. quercitrin (quercetin- -l-rhamnoside) and rutin (quercetin- rutinoside) occur as glycosides. quercitrin appears to show the highest content in fingerprint analysis of h. cordata by hplc [ ] . among the flavonoids, quercetin also exerts the highest activity on hsv- , whilst rutin has no effect at all [ ] . this characteristic implies that the substitution or addition of free hydroxyl group at certain positions may culminate in a decreased or even completely abolished antiviral effect. such the ea fraction of h. cordata was tested at concentrations of . mg/ ml down to . mg/ml. the upper plot reveals a distinct trend of denv- inhibition, with the ic being . mg/ml, and complete viral inhibition being achieved at concentrations of . mg/ml and above. the lower plot shows that no cytotoxicity to bhk- cells was evident at various concentrations of the ea fraction tested. structural-activity relationships can lead to the design and development of more active and less toxic flavonoids with appropriate pharmacokinetic properties. hence, they may pave the way for novel compounds that can match or even exceed the efficacies of existing antiviral drugs [ , , ] . another interesting aspect was the enhancement of anti-denv- activity of quercetin when combined with quercitrin. besides augmenting the antiviral effect, this combination also reduced the cytotoxicity relative to that induced by quercetin alone. this characteristic reiterates that combinations of components with greater anti-dengue efficacy but lower toxicity are potentially promising, as discovered previously using combinations of quercetin, chlorogenic acid and/or hyperoside [ ] . indeed, several reports have already documented the synergistic antiviral effects of combinations of individual flavonoids against viral pathogens, such as herpesviruses and fowlpox virus [ , ] . although cinanserin was able to inhibit mhv at . mg/ ml on dpi and . mg/ml on dpi, its effect was relatively short-lived and it could not inhibit mhv from dpi onwards. in comparison, however, we observed that the ea fraction and quercetin yielded a longer-lasting antiviral effect of up to d. this finding may be explained by superimposition of the two c-like proteinases of mhv and sars cov which illustrates that certain amino acid residues residing within Å of the active site (c ) are different (not shown). thus, these amino acid disparities may influence recognition and binding of cinanserin hydrochloride to the active site and may also lead to its lower affinity for c-like proteinase of mhv than sars-coronavirus. competitive binding of cinanserin hydrochloride to the active site of c-like proteinase hinders the processing of the precursor polyprotein to generate functional replicase necessary for viral replication [ ] [ ] [ ] . importantly, the ea fraction of h. cordata was capable of inhibiting both denv- and mhv in vitro at relatively low concentrations with negligible cytotoxicity and very good selectivity indices. moreover, the ea fraction was also more potent than all the individual compounds tested (including their combination) and did not cause any cytotoxicity to mammalian kidney (bhk) and liver (ccl . ) cell lines in vitro, nor any acute toxicity or pathology of major murine organs in vivo. this feature reiterates the synergistic effects of the combination of multiple flavonoids and other constituents within h. cordata that confer optimal antiviral activities and minimal toxicity [ ] , in keeping with the majority of successful traditional chinese medicine. our study lends support to the accumulating literature that the ea fraction of h. cordata indeed represents a highly promising prophylactic agent against coronaviruses and dengue viruses [ ] [ ] [ ] . to complement this study, future in vivo studies are warranted to assess the efficacy of the ea fraction of h. cordata against coronaviruses and dengue viruses using the relevant mouse models [ ] . however, further in vivo experiments are necessary to further assess the long-term safety of h. cordata as an antiviral agent in biological systems, including acquiring crucial information on its adsorption, distribution, metabolism and elimination. to address the important issue of batch-to-batch variation, greater efforts should also focus on the quality control of extracts derived from this medicinal plant. finally, the mechanisms of action of the ea fraction and its constituent flavonoids against viruses at the molecular level need to be further elucidated [ ] . isolation and characterization of viruses related to the 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replicase structure chemical composition and hepatoprotective effects of polyphenol-rich extract from houttuynia cordata tea immunomodulatory and anti-sars activities of houttuynia cordata in vitro and in vivo effects of houttuynia cordata on infectious bronchitis virus the inhibitory actions of houttuynia cordata aqueous extract on dengue virus and dengueinfected cell development of animal models against emerging coronaviruses: from sars to mers coronavirus houttuynia cordata thunb: a review of phytochemistry and pharmacology and quality control we thank annie hsu and s.h. lau for laboratory assistance, t. narasaraju for helpful advice, and mary ng for providing the dengue virus strain. this study was supported by a research grant from the national university of singapore. the authors declare no conflict of interest with respect to the publication of this paper. key: cord- -xu mvc authors: avirutnan, panisadee; mehlhop, erin; diamond, michael s. title: complement and its role in protection and pathogenesis of flavivirus infections date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: xu mvc the complement system is a family of serum and cell surface proteins that recognize pathogen-associated molecular patterns, altered-self ligands, and immune complexes. activation of the complement cascade triggers several antiviral functions including pathogen opsonization and/or lysis, and priming of adaptive immune responses. in this review, we will examine the role of complement activation in protection and/or pathogenesis against infection by flaviviruses, with an emphasis on experiments with west nile and dengue viruses. the complement system is comprised of soluble and cell surface associated proteins that recognize exogenous, altered, or potentially harmful endogenous ligands [ ] . complement is activated through three distinct pathways referred to as the classical, lectin, and alternative pathways depending on specific recognition molecules [ , ] . classical pathway activity is triggered by c q binding to antigen-antibody complexes on the surface of pathogens or by spontaneous tickover [ ] . the lectin pathway is initiated by mannan binding lectin (mbl) or ficolin recognition of carbohydrate structures on the surface of microbes or apoptotic cells. the alternative pathway is constitutively active at low levels through the spontaneous hydrolysis of c and also serves to amplify activation of the classical and lectin pathways. despite the distinct triggering mechanisms, the classical, lectin, and alternative pathways generate convertase enzymes (c bc a for classical and lectin, and c bbb for the alternative) which cleave c , the central component of the complement system, and expose a reactive internal thioester bond on c b necessary for covalent attachment to target surfaces. the binding of c b back to c b a and c bbb c convertases forms the classical and alternative pathway c convertases, respectively. these enzymes cleave c and promote assembly of c b- membrane attack complex (mac), which lyses pathogens or infected cells. sub-lytic amounts of c b- on a cell surface can activate granulocytes and endothelial cells, whereas soluble c b- independently induces inflammation through cytokine induction [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the release of anaphylatoxins (c a and c a) by the c and c convertases also contributes to the host inflammatory response by promoting chemotaxis of immune cells via the interaction with specific g-protein coupled transmembrane receptors (c ar and c ar) [ ] . deposition of opsonic c and c fragments (c b and c b) on a pathogen facilitates binding and phagocytosis by complement receptors (cr , cr , cr , and crig), a process called opsonization, which helps to clear microbial infections [ , ] . to limit inappropriate activation and potential tissue damage, the complement system is controlled by a group of cell surface and soluble regulators [ ] . negative regulation of complement activation is achieved by several independent mechanisms: (a) proteolytic cleavage of c b and c b by the plasma serine protease factor i in conjunction with one of the membrane or plasma cofactors (membrane cofactor protein (mcp or cd ), complement receptor (cr or cd ), factor h, and c binding protein (c bp) [ ] [ ] [ ] [ ] ; (b) dissociation of the c and c convertases, a process known as decay accelerating activity, which involves decay accelerating factor (daf or cd ), cr , c bp and factor h [ ] [ ] [ ] [ ] [ ] ; (c) mac formation is inhibited by the membrane regulator cd (protectin) [ , ] , the soluble regulator apolipoprotein clusterin (apo-j) [ ] [ ] [ ] [ ] [ ] , and vitronectin [ , ] ; (d) specific protease inhibitors (e.g., serpins and c inhibitor) limit cleavage of c and c by dissociating the classical (c r-c s) and lectin (mbl-associated serine protease (masp- )) pathway serine proteases [ ] . beyond its roles in direct recognition and clearance of microbes, complement activation is critical for generating an efficient adaptive immune response. ligation of complement receptors enhances humoral immune responses [ , ] . binding of the complement split products c d, c dg, or ic b [ ] by cr (cd ) lowers the threshold for b cell activation by cross-linking the b cell receptor with the cd /cd /cr co-receptor complex [ ] . indeed, conjugation of c d to viral glycoproteins increases their immunogenicity up to , fold [ ] [ ] [ ] [ ] , and c −/− or cr −/− mice have impaired humoral responses to t cell-dependent (td) antigens [ ] [ ] [ ] [ ] . additionally, expression of cr on follicular dendritic cells (dc) is required for b cell survival within the germinal center, affinity maturation, and the establishment of b cell memory [ ] [ ] [ ] . in addition, cr (cd ), a type i integral membrane protein that binds c b, c b, and c q, and mbl, also plays a role in establishment of b cell responses [ ] [ ] [ ] . this glycoprotein is expressed on all peripheral blood cells in humans with the exception of platelets, natural killer cells and most t cells [ , ] . in primates, cr expression on erythrocytes contributes to immune complex clearance and transfer of c b-opsonized antigens to splenic and hepatic macrophages [ , ] . in mice, cr is expressed as an alternative splice product of the cr gene and is restricted to b cells and follicular dendritic cells [ ] [ ] [ ] . profound defects in humoral immunity have been observed in cr /cr −/− mice [ , , , ] , with little effect on t cell activity [ , ] . cr /cr -mediated antigen trapping on follicular dendritic cells enhances antigen presentation to b cells, and is required for both primary and secondary humoral responses [ , ] . complement and its receptors can also augment t cell activation. cr and cr can mediate phagocytosis of ic b-opsonized antigens on antigen presenting cells, and thus, may augment antigen presentation. in the absence of complement c , t cell responsiveness to influenza virus, lymphocytic choriomeningitis virus (lcmv), leishmania, and alloantigens are reduced [ , , , ] . correspondingly, c b opsonization augments protein antigen uptake [ , ] and t cell stimulation [ , , ] . covalent c b modification can target antigen to specific mhc class ii containing vesicles [ ] and may increase lysosomal peptide-mhc stability [ ] , and the diversity of t cell epitopes presented [ ] . additionally, a deficiency of c q can lead to suboptimal antigen uptake, impaired dc differentiation and maturation, and reduced t cell responses [ , [ ] [ ] [ ] [ ] [ ] [ ] . dc present exogenous antigen in a mhc class i-restricted manner, leading to the activation of naïve cd + t cells through crosspresentation [ ] . dc uptake of complement containing immune complexes (ic) enhances the efficiency of protein antigen crosspresentation compared to free antigens [ , , ] . however, c q may not be necessary to stimulate t cell priming against pathogenderived antigens [ , ] . to minimize recognition and/or destruction by complement several different families of viruses have evolved strategies to evade or exploit complement to establish infection (reviewed in [ ] [ ] [ ] [ ] [ ] ). complement evasion mechanisms include: (a) use of complement receptors to enhance viral entry or suppress adaptive immune response (e.g., hiv, west nile virus (wnv), measles virus, adenoviruses, herpesviruses, enteroviruses, hepatitis b and c viruses ); (b) expression of viral proteins that directly inhibit complement (e.g., herpesviruses, coronaviruses, and astroviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ); (c) modulation of expression of complement regulators on host cells to prevent complement-dependent lysis (e.g., herpesviruses [ ] [ ] [ ] ); (d) incorporation of human regulators on the surface of virions to protect from complement-mediated virolysis (e.g. hiv, htlv, cytomegalovirus, and vaccinia virus [ ] [ ] [ ] [ ] [ ] [ ] [ ] ); (e) recruitment of soluble complement regulatory proteins to the virion or infected cell surface (e.g., wnv and hiv [ ] [ ] [ ] [ ] [ ] ); (f) expression of viral decoy proteins that structurally or functionally mimic complement regulatory proteins (e.g., poxviruses and herpesviruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a single virus may utilize several independent strategies to escape from recognition and targeting by complement and modulate the immune response to establish persistent infection. although complement activation inhibits infection of many viruses [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it appears to have both protective and pathogenic roles in flavivirus infection depending on the specific virus, phase of the infection, and immune status of the host. the genus flavivirus is composed of enveloped viruses containing ∼ kilobase single-stranded, positive-polarity rna genomes [ ] . within this family, several are associated with severe human diseases including dengue (denv), yellow fever (yfv), wnv, japanese encephalitis (jev), and tick-borne encephalitis (tbe) viruses [ ] . a single open reading frame is translated in the cytoplasm as a polyprotein and cleaved by virus-and host encoded-proteases into three structural (capsid (c), membrane (prm/m), and envelope (e)) and seven nonstructural (ns) proteins including ns , ns a, ns b, ns , ns a, ns b and ns [ ] . the e protein functions in receptor binding, entry, and membrane fusion and elicits the majority of neutralizing antibodies whereas prm assists in folding, assembly, and function of the e protein [ ] . viral particles assemble at the endoplasmic reticulum and are released by exocytosis following transport through the trans-golgi network [ ] . flavivirus non-structural proteins regulate viral transcription, replication, and attenuation of host antiviral immune response, including antagonizing the interferon response (reviewed in [ ] ). one non-structural proteins, ns , has been recently shown to regulate complement function (see below). ns is synthesized as a monomer and dimerizes after post-translational modification [ , ] . within the cytoplasm, ns acts as a co-factor for the ns rna-dependent rna polymerase during viral replication [ , ] . however, it is also expressed on the cell surface and secreted as a hexamer [ , ] . ns has been implicated in the pathogenesis of severe denv infection [ ] [ ] [ ] and immune evasion by wnv [ , ] . complement can limit flavivirus infection by stimulating adaptive immune responses. c −/− mice are more susceptible to lethal wnv infection and show greater viral burden and reduced antiviral antibody titers [ ] . infection studies with mice lacking c q, c , or factor b suggest that all complement activation pathways orchestrate protection against wnv infection [ ] . however, each activation pathway appears to exert somewhat distinct protective effects in response to wnv infection. humoral igm responses to wnv likely depend upon activation of c by the lectin recognition pathway. in contrast, both the lectin and alternative pathways appear necessary for efficient t cell priming as c −/− , factor b −/− , and factor d −/− mice exhibited reduced wnv-specific cd + t cell responses [ ] . the t cell defects in c −/− mice may be indirect as depressed igm responses could affect viral opsonization and antigen presentation. flaviviruses also directly trigger complement activation in vitro and in vivo. increasing concentrations of complement or serum neutralize as much as % of a given infectious dose of wnv in cell culture in the absence of antibody [ ] . complement activation by flaviviruses also has been described in vivo. c and c consumption were observed in a mouse model of wnv infection prior to the induction of a specific antibody response [ ] . c catabolism and production of complement split products during secondary denv infection correlate with increased disease severity and development of dengue hemorrhagic fever and shock syndrome, the most severe form of denv infection [ , [ ] [ ] [ ] . complement activation augments antibody-mediated neutralization of several viruses, including influenza [ , ] , hiv [ ] [ ] [ ] [ ] , respiratory syncytial [ , ] , varicella zoster [ ] [ ] [ ] , epstein-barr [ , ] , and herpes simplex viruses [ ] [ ] [ ] . complement also improves antibody-mediated neutralization of flaviviruses. complement augments immune serummediated neutralization of yfv, denv, and kunjin virus [ ] [ ] [ ] and monoclonal antibody-dependent neutralization of wnv [ ] . similarly, the protective efficacy of flavivirus neutralizing antibodies in vivo correlates with igg subclasses that efficiently fix complement [ ] . fc-␥r engagement by antibodies in vitro can paradoxically enhance replication of flaviviruses [ ] [ ] [ ] [ ] [ ] [ ] . this phenomenon, known as antibody-dependent enhancement of infection (ade), is hypothesized to contribute to the pathogenesis of secondary denv infection [ , ] . recent studies indicate that complement can restrict ade. complement minimized ade of wnv and denv infection in fc-␥r-expressing cell lines and primary macrophages [ , ] . experiments with mouse sera deficient in individual complement components indicate that c q is sufficient to restrict ade of wnv infection in vitro. this effect was igg subclassdependent, as c q restricted ade by a human igg isotype-switch variant, but had little effect on igg and igg subclass variants [ ] ; these results correlate with the known affinity of human igg subclasses for c q [ , ] . interestingly, complement-dependent inhibition of denv ade may also require c [ ] . while these studies establish that complement restricts ade by flaviviruses, the precise inhibitory mechanisms at the cellular level remain unclear. recent studies suggest that c q also limits flavivirus ade in vivo. whereas enhancement of wnv infection was not observed after passive transfer of antiviral igg a mabs that bind c q avidly in wild type mice, it was observed in c q −/− mice [ ] . the ability of c q to suppress ade may explain some of the difficulties in consistently observing flavivirus ade in animal models. further investigation is necessary to define the links between complement restriction of ade, fc-␥r specificity, and disease pathogenesis of flaviviruses. in cells that express cr , antibody-dependent complement activation may paradoxically enhance viral infection. complement activation by antiviral igm enhanced wnv infection of macrophages and monocyte cell lines [ , ] . blockade of cr abrogated the complement-dependent enhancement of wnv infection in this model system. thus, under certain circumstances, antibody and complement-dependent opsonization of flaviviruses may increase infection in cr -expressing cells. during severe secondary denv infection, a vascular leakage syndrome occurs with fluid transudation into serosal spaces [ ] . although the pathogenesis of denv infection remains controver-sial and implicates cross-reactive antibodies and effector t cells (reviewed in [ ] [ ] [ ] ), a pathological role for complement activation has been suggested. in early clinical studies, reduced levels of c , c and factor b and increased catabolic rates of c and c q were observed, particularly in patients with severe disease [ , ] . additionally, c breakdown products and anaphylatoxins accumulated in the circulation of severely ill patients and peaked at the day of maximum vascular leakage [ , ] . circulating immune complexes formed by virions and denv-specific antibodies were hypothesized to cause the pathological complement activation [ ] , although only small amounts were detected in circulation [ , ] . one alternative hypothesis is that infected cells express sufficient amounts of denv antigens (e or ns proteins) on their surface facilitating immune complex formation and complement deposition [ ] . indeed, denv-infected endothelial cells activate human complement in the presence of antibodies resulting in c b- deposition [ ] . a subsequent study implicated ns as the key surface viral protein responsible for complement activation [ ] . as soluble denv ns differentially binds to cultured endothelial and mesothelial cells [ ] , high levels of intravascular soluble ns , as observed in denv-infected patients, could promote binding and surface expression of ns on selective cells without a requirement for direct viral infection; this could contribute to tissue-specific vascular leakage that occurs during severe secondary denv infection after recognition by anti-ns antibodies, immune complex formation, and inflammatory damage [ , ] . recent evidence suggests wnv ns has immune evasion function and protects against complement activation by binding the negative regulator factor h [ ] . factor h sustains factor imediated cleavage of c b and inactivates the alternative pathway c convertase (reviewed in [ ] ). co-immunoprecipitation experiments demonstrate that soluble wnv ns binds to factor h, leading to degradation of c b in solution [ ] . additionally, cell surface ns limits c b deposition and c b- mac formation [ ] . thus, secreted or cell surface ns may minimize immune system targeting of wnv by decreasing complement activation in solution and on the surface of infected cells. this data appears to contradict early studies that suggested denv ns might be the key viral protein that triggers complement activation [ , ] . in those studies, ns was termed "non-hemagglutinating soluble complement fixing antigens (scf)" because it has activity in the traditional standard complement fixing test that requires specific antibodies to trigger guinea pig complement [ , ] . subsequent experiments indicate that denv ns does not activate complement efficiently, but instead requires specific anti-ns antibodies for complement consumption and c b- generation ( [ ] and avirutnan et al., unpublished results). additionally, denv ns has been reported to bind to clusterin, a complement regulator that inhibits mac formation [ ] . clearly, more studies are necessary to establish the significance of these findings in the pathogenesis of infection of denv, wnv, and other flaviviruses in vivo. activation of the complement system has a critical role in protection and possibly pathogenesis of infection by different flaviviruses. complement activation primes adaptive immune responses and modulates the effector functions of flavivirus-specific antibodies. recent studies suggest that flaviviruses have evolved novel strategies to limit complement activation. the balance between complement activation and evasion likely helps determine the out-come of a productive infection. a greater understanding of how complement restricts and contributes to pathogenesis of individual flaviviruses may expand strategies for developing therapeutics or vaccines to control infection. complement. first of two parts second of two parts continual low-level activation of the classical complement pathway platelet-activating factor and kinin-dependent vascular leakage as a novel functional activity of the soluble terminal complement complex intracerebroventricular injection of the terminal complement complex causes inflammatory reaction in the rat brain cytolytically inactive terminal complement complex causes transendothelial migration of polymorphonuclear leukocytes in vitro and in vivo soluble complex of complement increases hydraulic conductivity in single microvessels of rat lung transient perturbation of endothelial integrity induced by natural antibodies and complement the cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity regulation of the complement membrane attack pathway the anaphylatoxins bridge innate and adaptive immune responses in allergic asthma the complement system in regulation of adaptive immunity crig: a macrophage complement receptor required for phagocytosis of circulating pathogens the regulators of complement activation (rca) gene cluster mapping of the sites responsible for factor i-cofactor activity for cleavage of c b and c b on human c b-binding protein (c bp) by deletion mutagenesis structure-function relationships of complement receptor type dissecting sites important for complement regulatory activity in membrane cofactor protein (mcp; cd ) functional properties of membrane cofactor protein of complement human alternative complement pathway: membrane-associated sialic acid regulates the competition between b and beta h for cell-bound c b structure-function analysis of the active sites of complement receptor type decay accelerating activity of complement receptor type (cd ). two active sites are required for dissociating c convertases human c -binding protein. i. isolation and characterization control of the amplification convertase of complement by the plasma protein beta h membrane defence against complement lysis: the structure and biological properties of cd human protectin (cd ), an , - , mw complement lysis restricting factor, inhibits c b- catalysed insertion of c into lipid bilayers incorporation of sp- , into the soluble membrane attack complex (smac, sc b- ) of complement molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein , a constituent of rat testis fluid molecular cloning and characterization of the novel, human complement-associated protein, sp- , : a link between the complement and reproductive systems potent inhibition of terminal complement assembly by clusterin: characterization of its impact on c polymerization clusterin, the human apolipoprotein and complement inhibitor, binds to complement c , c beta, and the b domain of c complement s-protein (vitronectin) is associated with cytolytic membrane-bound c b- complexes vitronectinmediated inhibition of complement: evidence for different binding sites for c b- and c structural and functional aspects of c -inhibitor the role of complement and complement receptors in induction and regulation of immunity regulation of b lymphocyte responses to foreign and self-antigens by the cd /cd complex identification of a , mr membrane protein as the c d receptor (cr ) of human b lymphocytes intersection of the complement and immune systems: a signal transduction complex of the b lymphocyte-containing complement receptor type and cd c d of complement as a molecular adjuvant: bridging innate and acquired immunity enhancement of antibodies to the human immunodeficiency virus type envelope by using the molecular adjuvant c d c d enhancement of antibodies to hemagglutinin accelerates protection against influenza virus challenge fusion to c d enhances the immunogenicity of the e glycoprotein of type bovine viral diarrhea virus disruption of the cr locus results in a reduction in b- a cells and in an impaired b cell response to t-dependent antigen antibody response to a t-dependent antigen requires b cell expression of complement receptors regulation of the b cell response to t-dependent antigens by classical pathway complement markedly impaired humoral immune response in mice deficient in complement receptors and b lymphocyte memory: role of stromal cell complement and fcgammariib receptors dependence of germinal center b cells on expression of cd /cd for survival impaired affinity maturation in cr −/− mice is rescued by adjuvants without improvement in germinal center development identification of the membrane glycoprotein that is the c b receptor of the human erythrocyte, polymorphonuclear leukocyte, b lymphocyte, and monocyte complement receptor /cd is a receptor for mannan-binding lectin complement receptor type (cr cd ) is a receptor for c q expression of c b receptors on human be cells and myelomonocytic cells but not natural killer cells both kupffer cells and liver endothelial cells play an important role in the clearance of iga and igg immune complexes clearance of antidouble-stranded dna antibodies: the natural immune complex clearance mechanism their use in a distribution study showing that mouse erythrocytes and platelets are cr -negative the murine complement receptor gene family. iv. alternative splicing of cr gene transcripts predicts two distinct gene products that share homologous domains with both human cr and cr a molecular and immunochemical characterization of mouse cr . evidence for a single gene model of mouse complement receptors and humoral immune responses in cr −/− mice: enhanced affinity maturation but impaired antibody persistence complement component c promotes t-cell priming and lung migration to control acute influenza virus infection complement component is required for optimal expansion of cd t cells during a systemic viral infection expression of complement receptors and on follicular dendritic cells is necessary for the generation of a strong antigen-specific igg response evidence for an important interaction between a complement-derived cd ligand on follicular dendritic cells and cd on b cells in the initiation of igg responses local synthesis of complement component c regulates acute renal transplant rejection natural antibodies and complement are endogenous adjuvants for vaccine-induced cd + t-cell responses c b covalently associated to tetanus toxin modulates tt processing and presentation by u cells covalent binding of c b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific b cells antigen-bound c b and c b enhance antigenpresenting cell function in activation of human t-cell clones modulation of antigen processing and presentation by covalently linked complement c b fragment b cell receptors and complement receptors target the antigen to distinct intracellular compartments complement c b fragment covalently linked to tetanus toxin increases lysosomal sodium dodecyl sulfate-stable hla-dr dimer production c b complexation diversifies naturally processed t cell epitopes maturation of dendritic cells abrogates c q production in vivo and in vitro complement protein c q induces maturation of human dendritic cells immune modulation of human dendritic cells by complement t cell-dependent immune response in c q-deficient mice: defective interferon gamma production by antigen-specific t cells immune complex processing in c q-deficient mice a novel role of complement factor c q in augmenting the presentation of antigen captured in immune complexes to cd + t lymphocytes cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens antigen-antibody immune complexes empower dendritic cells to efficiently prime specific cd + ctl responses in vivo immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine protective immune responses against west nile virus are primed by distinct complement activation pathways complement contributes to protective immunity against reinfection by plasmodium chabaudi chabaudi parasites hiv and human complement: inefficient virolysis and effective adherence viral mimicry of the complement system role of complement in immune regulation and its exploitation by virus virus complement evasion strategies complement evasion by human pathogens cutting edge: productive hiv- infection of dendritic cells via complement receptor type (cr , cd b/cd ) complement dependent trapping of infectious hiv in human lymphoid tissues decay-accelerating factor (cd ), a glycosylphosphatidylinositol-anchored complement regulatory protein, is a receptor for several echoviruses opsonization of hiv- by semen complement enhances infection of human epithelial cells interaction of west nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement complement receptor mediates enhanced flavivirus replication in macrophages immune tolerance split between hepatitis b virus precore and core proteins a function of the hepatitis b virus precore protein is to regulate the immune response to the core antigen virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions cr (cd ) and cr (cd ) complement c receptors are expressed on normal human thymocytes and mediate infection of thymocytes with opsonized human immunodeficiency virus b cellmediated infection of stimulated and unstimulated autologous t lymphocytes with hiv- : role of complement mechanism(s) promoting hiv- infection of primary unstimulated t lymphocytes in autologous b cell/t cell co-cultures the human cd molecule is a receptor for measles virus (edmonston strain) hepatitis c virus core selectively suppresses interleukin- synthesis in human macrophages by interfering with ap- activation epstein-barr virus receptor of human b lymphocytes is the c d receptor cr cd is a cellular receptor for group b adenoviruses b lymphocytes in lymph nodes and peripheral blood are important for binding immune complexes containing hiv- detachment of human immunodeficiency virus type from germinal centers by blocking complement receptor type c a and c a(desarg) enhance the susceptibility of monocyte-derived macrophages to hiv infection mechanism of suppression of cell-mediated immunity by measles virus interaction between complement receptor gc qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation functional modulation of human macrophages through cd (measles virus receptor): production of il- p and nitric oxide in association with recruitment of protein-tyrosine phosphatase shp- to cd in vitro and in vivo interactions between the hepatitis b virus protein p and the cellular protein gc qr hepatitis c virus core protein inhibits interleukin and nitric oxide production from activated macrophages inhibition of interferon-mediated antiviral activity by murine gammaherpesvirus latency-associated m protein cd is a cellular receptor for all species b adenoviruses except types and binding of human immunodeficiency virus type to the c b/c b receptor cr (cd ) and red blood cells in the presence of envelope-specific antibodies and complement. national institutes of health aids vaccine clinical trials networks ligation of cr (c b receptor, cd ) on cd + t lymphocytes enhances viral replication in hiv-infected cells identification of gp as the viral glycoprotein mediating attachment of epstein-barr virus (ebv) to the ebv/c d receptor of b cells: sequence homology of gp and c complement fragment c d complement-mediated enhancement of hiv- infection in peripheral blood mononuclear cells identification of a cellular protein specifically interacting with the precursor of the hepatitis b e antigen cd is a cellular receptor for human herpesvirus adenovirus type uses cd as a cellular receptor selective suppression of il- production by human herpesvirus in vitro analysis of complement-dependent hiv- cell infection using a model system epstein-barr virus gp / binding to the b lymphocyte c d receptor mediates adsorption, capping, and endocytosis cr (cd ) and cr (cd b/cd ) mediate infection of human monocytes and monocytic cell lines with complementopsonized hiv independently of cd triggering of complement receptors cr (cd ) and cr (cd b/cd ) induces nuclear translocation of nf-kappa b (p /p ) in human monocytes and enhances viral replication in hiv-infected monocytic cells hcv core protein interaction with gc q receptor inhibits th differentiation of cd + t cells via suppression of dendritic cell il- production human astrovirus coat protein inhibits serum complement activation via c , the first component of the classical pathway antibody-induced and cytoskeleton-mediated redistribution and shedding of viral glycoproteins, expressed on pseudorabies virus-infected cells identification of a novel herpes simplex virus type -induced glycoprotein which complexes with ge and binds immunoglobulin herpes simplex virus immunoglobulin g fc receptor activity depends on a complex of two viral glycoproteins, ge and gi mechanism of complement inactivation by glycoprotein c of herpes simplex virus herpesviral fc receptors and their relationship to the human fc receptors herpes simplex virus type evades the effects of antibody and complement in vivo in vivo immune evasion mediated by the herpes simplex virus type immunoglobulin g fc receptor molecular mimicry between s peplomer proteins of coronaviruses (mhv, bcv, tgev and ibv) and fc receptor identification and expression of a murine cytomegalovirus early gene coding for an fc receptor mechanism of host cell protection from complement in murine cytomegalovirus (cmv) infection: identification of a cmv-responsive element in the cd promoter region altered expression of hostencoded complement regulators on human cytomegalovirus-infected cells human herpesvirus infection increases the expression levels of cd and cd in target cells acquisition of host cell-surface-derived molecules by hiv- decay-accelerating factor (cd ) protects human immunodeficiency virus type from inactivation by human complement antibodydependent complement-mediated cytotoxicity in sera from patients with hiv- infection is controlled by cd and cd mechanisms of resistance of hiv- primary isolates to complement-mediated lysis extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope human immunodeficiency virus type incorporates both glycosyl phosphatidylinositol-anchored cd and cd and integral membrane cd at levels that protect from complement-mediated destruction host cell-derived complement control proteins cd and cd are incorporated into the virions of two unrelated enveloped viruses. human t cell leukemia/lymphoma virus type i (htlv-i) and human cytomegalovirus (hcmv) west nile virus nonstructural protein ns inhibits complement activation by binding the regulatory protein factor h direct interaction of complement factor h with the c domain of hiv type glycoprotein hiv glycoprotein and complement factor h interact with each other and share functional as well as antigenic homology human complement proteins c b, c b, factor h and properdin react with specific sites in gp and gp , the envelope proteins of hiv- efficient destruction of human immunodeficiency virus in human serum by inhibiting the protective action of complement factor h and decay accelerating factor (daf, cd ) herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein cd the complement control protein homolog of herpesvirus saimiri regulates serum complement by inhibiting c convertase activity structure and regulatory profile of the monkeypox inhibitor of complement: comparison to homologs in vaccinia and variola and evidence for dimer formation regulation of complement activity by vaccinia virus complement-control protein the cowpox virus-encoded homolog of the vaccinia virus complement control protein is an inflammation modulatory protein variola virus immune evasion design: expression of a highly efficient inhibitor of human complement functional characterization of the complement control protein homolog of herpesvirus saimiri: arg- is critical for factor i cofactor activities complement regulation by kaposi's sarcoma-associated herpesvirus orf protein the relevance of complement to virus biology the effect of complement depletion on the course of sindbis virus infection in mice role of complement in viral infections: participation of terminal complement components (c to c ) in recovery of mice from sindbis virus infection the role of complement in viral infections. ii. the clearance of sindbis virus from the bloodstream and central nervous system of mice depleted of complement natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity enhancement of neutralizing activity of influenza virus-specific antibodies by serum components the role of the complement system in virus infections fields' virology flaviviridae: the viruses and their replication newly synthesized dengue- virus nonstructural protein ns is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization evidence that the mature form of the flavivirus nonstructural protein ns is a dimer genetic interaction of flavivirus nonstructural proteins ns and ns a as a determinant of replicase function immunolocalization of the dengue virus nonstructural glycoprotein ns suggests a role in viral rna replication dengue virus type nonstructural glycoprotein ns is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein ns and complement secreted ns of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate e high circulating levels of the dengue virus nonstructural protein ns early in dengue illness correlate with the development of dengue hemorrhagic fever west nile virus nonstructural protein inhibits tlr signal transduction complement activation is required for induction of a protective antibody response against west nile virus infection pathogenetic mechanisms in dengue haemorrhagic fever: report of an international collaborative study the potential pathogenic role of complement in dengue hemorrhagic shock syndrome complement and dengue haemorrhagic fever/shock syndrome neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement detection of antibody-dependent complement-mediated inactivation of both autologous and heterologous virus in primary human immunodeficiency virus type infection functional activity of an hiv- neutralizing igg human monoclonal antibody: adcc and complement-mediated lysis complement activation by human monoclonal antibodies to human immunodeficiency virus broad neutralization and complement-mediated lysis of hiv- by pehrg , a novel caprine anti-hiv- polyclonal antibody effect of complement and viral filtration on the neutralization of respiratory syncytial virus role of complement in neutralization of respiratory syncytial virus neutralization of vesicular stomatitis virus (vsv) by human complement requires a natural igm antibody present in human serum complement-enhanced neutralizing antibody response to varicella-zoster virus neutralizing antibody responses to varicella-zoster virus neutralization of epstein-barr virus by nonimmune human serum. role of cross-reacting antibody to herpes simplex virus and complement complement-dependent, epstein-barr virus-neutralizing antibody appearing early in the sera of patients with infectious mononucleosis serologic responses to herpes simplex virus in rabbits: complement-requiring neutralizing, conventional neutralizing, and passive hemagglutinating antibodies complement requirement for virus neutralization by antibody and reduced serum complement levels associated with experimental equine herpesvirus infection herpesvirus neutralization: the role of complement immune response in rabbits to virion and nonvirion antigens of the flavivirus kunjin the dengue group of viruses and its family relationships yellow fever virus. ii. factors affecting the plaque neutralization test neutralizing f(ab ) fragments of protective monoclonal antibodies to yellow fever virus (yf) envelope protein fail to protect mice against lethal yf encephalitis flavivirus infection enhancement in macrophages: radioactive and biological studies on the effect of antibody on viral fate flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry dengue viruses and mononuclear phagocytes. i. infection enhancement by non-neutralizing antibody antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever monoclonal anti-fc receptor igg blocks antibody enhancement of viral replication in macrophages antibody-mediated enhancement of flavivirus replication in macrophage-like cell lines pathogenesis of dengue: challenges to molecular biology complement protein c q inhibits antibody-dependent enhancement of flavivirus infection in an igg subclass-specific manner infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type and viruses are controlled by complement levels human monoclonal igg isotypes differ in complement activating function at the level of c as well as c q the classical complement pathway: activation and regulation of the first complement component clinical spectrum and management of dengue haemorrhagic fever immunopathological mechanisms in dengue and dengue hemorrhagic fever dengue hemorrhagic fever with special emphasis on immunopathogenesis of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (dhf/dss) crossed immunoelectrophoresis for the detection of split products of the third complement in dengue hemorrhagic fever. i. observations in patients' plasma the raji cell radioimmune assay for detecting immune complexes in human sera pathogenesis of dengue: an alternative hypothesis dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis factor h family proteins: on complement, microbes and human diseases partial purification and characterization of a dengue virus soluble complement-fixing antigen molecular size and charge relationships of the soluble complement-fixing antigens of dengue viruses secreted complement regulatory protein clusterin interacts with dengue virus nonstructural protein key: cord- -tkvfneje authors: mendez, jairo a; usme-ciro, jose a; domingo, cristina; rey, gloria j; sanchez, juan a; tenorio, antonio; gallego-gomez, juan c title: phylogenetic history demonstrates two different lineages of dengue type virus in colombia date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: tkvfneje background: dengue fever is one of the most important viral re-emergent diseases affecting about million people around the world especially in tropical and sub-tropical countries. in colombia, the virus was first detected in the earliest 's when the disease became a major public health concern. since then, all four serotypes of the virus have been reported. although most of the huge outbreaks reported in this country have involved dengue virus serotype (denv- ), there are not studies about its origin, genetic diversity and distribution. results: we used bp corresponding to the carboxyl terminus of envelope (e) gene from colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. analyzed denv- colombian isolates belonged to the formerly defined genotype v. only one virus isolate was clasified in the genotype i, likely representing a sole introduction that did not spread. the oldest strains were closely related to those detected for the first time in america in from the caribbean and were detected for two years until their disappearance about six years later. around , a split up generated lineages that have been evolving separately, although not major aminoacid changes in the analyzed region were found. conclusion: denv- has been circulating since in colombia. yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype v, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. we report the rapid spread patterns and high evolution rate of the different denv- lineages. dengue virus infection has been an important impact on humans over the last several years, with an estimated million dengue infections and an average of million cases reported annually in more than countries in tropical and subtropical regions [ ] [ ] [ ] [ ] [ ] . this mosquitoborne flavivirus causes a wide spectrum of clinical manifestations in humans, which include an acute self-limited flu-like illness known as dengue fever (df). df is characterized by headache, myalgia, arthralgia, retro-orbital pain and sometimes maculopapular rash. dengue haemorrhagic fever (dhf) is a severe illness documented by haemoconcentration (haematocrit increase by %) and evidence of plasma leakage such as pleural effusion and ascites as the major pathophysiological features. in some patients, dhf may progress to hypovolemic shock (dengue shock syndrome, dss) with circulatory failure [ , [ ] [ ] [ ] . dengue virus (denv) is an enveloped virus with a positive sense ssrna of about kb coding a single open reading frame for three structural proteins, core (c), premembrane/membrane (prm/m) and envelope (e), and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b, ns ). based on serological analysis, denv can be differentiated as four distinct serotypes (denv- , denv- , denv- and denv- ), each one with the capacity to infect and cause even the more severe manifestation, although some serotypes have been isolated more frequently in dhf epidemics. on the other hand, evolution studies and molecular epidemiology using nucleotide sequences from the denv genome have demonstrated the occurrence of genotype clades within each serotype [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for this reason, genetic characterization of denv has become a critical issue for understanding epidemic patterns of viral spread. at the same time, the important role of denv itself in disease severity has also been proposed rather than the immune enhancement developed after subsequent infection with heterologous serotypes [ , , ] . the increase in virus transmission over the last years has possibly increased its adaptive potential. in addition, host factors such as the age, race, presence of non-neutralazing cross-reactive antibodies and possibly chronic diseases could act as selective pressures, resulting in more virulent genotypes that may be associated with dhf/dss [ , , [ ] [ ] [ ] [ ] [ ] [ ] . four denv serotypes have been involved in colombian epidemics, although denv- and denv- have the higher circulation rate since [ , , ] . moreover, since the first case of dhf in colombia at the end of , these two serotypes have been associated with severe disease. to date, denv- falls into five clades designated as genotype i (southeast asia, china and east africa), genotype ii (thailand), genotype iii (malaysia), genotype iv (south pacific) and genotype v (america, africa). additionally, the existence of lineages with distinctive geographical and temporal relationships had been suggested [ , , , , [ ] [ ] [ ] [ ] . due to the importance of denv in public health, the particular goals of this research were to reconstruct the phylogenetic history of denv- and to date the phylogenetic tree using isolation time as calibration points to establish date of introduction of virus and rate evolution patterns of virus in colombia. seventy four viruses obtained from symptomatic patients were isolated in mosquito cell culture and subsequently identified as denv- serotype by monoclonal antibodies and confirmed by rt-pcr methods. from the samples, it was not possible to obtain the exact geographic origin of samples. the remaining isolates are listed in table indicating locality, isolation year, accession number and genotype. sequences from the carboxyl terminus of the envelope (e) gene from the colombian denv- isolates were aligned in clustal w [ , ] and compared with previously reported sequences elsewhere, resulting in a trivial alignment as long as there were no indels in the sequences alignment. the maximum likelihood analysis comparing sequences is presented in figure . previously reported genotypes were represented in the tree and placed most of the colombian isolates nesting in the genotype v clade (america, africa) and were closely related to argentina, brazil and paraguay virus strains. nevertheless, the oldest sequences denv- /co/ _atlantico/ , denv- /co/ _choco/ and denv- /co/ _choco/ were slightly distant from the remaining strains and appeared in close proximity to some caribbean island and other american isolates (trinidad, french guinea). interestingly, the isolate denv- /co/ _valle/ appeared in a different clade, as the sister branch of japan (denv- /jp/mochizuki/ ), china (denv- / cn/gz- / ), ethiopia (denv- /dj/ethiopia/ ) and cambodia (denv- /kh/ ) strains, which have been defined as lineages of the genotype i [ ] . to allow a better resolution of the tree, we performed a phylogenetic reconstruction using only the colombian isolates. again, the oldest isolates were more divergent representing the first entrance of virus in colombia. although the genotype v was the only represented in the tree, two different lineages may be defined based on cladal distribution (figure ). we used a bayesian inference based on mcmc to reconstruct colombian denv coalescent history. beast allowed the use of isolation year as calibrating point to estimate divergence time and then generated a posterior probability (pp) distribution of trees instead of a bootstrap value [ ] [ ] [ ] [ ] . the resulting tree clearly placed the genotypes of denv- already circulating globally before the first appearance of this serotype in the americas between and (figure ). according to the % highest posterior density (hpd) beneath the strict clock model, the estimated root for this phylogeny was and the substitution rate was . × - substitutions per site, per year. to increase resolution, we use the strict molecular clock model to reconstruct colombian isolates ( figure ). under the assumption of a constant substitution rate, the estimated root indicates as the date of the more recent common ancestor. in addition, there was a split up around between denv- /co/ _guaviare/ isolates and the remaining strains (pp = , ). as the time goes by, we can see a sustained increase in number of isolates and a rapid spread of viruses, which included few changes among them as seen with the branch lengths. by the year (approximately), another remarkable partition event occurred to generate well defined clades (pp = , and ), evolving independently since the early ′s until recent time. emerging and re-emerging diseases have become a public health major concern in developing countries, where dengue is perhaps the most important vector-borne viral disease in terms of morbidity. in colombia, df and dhf had been associated to the four denv serotypes with denv- and denv- predominating since after the re-appearance and spread of aedes (stegomyia) aegipty [ ] . denv- circulated for a short time in and then it was not detected until when re-introduction occurred probably from venezuela [ ] . denv- has been detected sporadically every year since , when it was involved in several df cases. the huge genetic diversity of denv has been vastly documented, starting perhaps with the rico-hesse proposal of different "genotypes" comprising serotypes and [ ] , following by several studies and genotype definition of denv- and denv- . in this way, five different genotypes has been previously defined for denv- (genotypes i to v) suggesting a significant genetic variation. in fact, various lineages had been proposed based on time-spatial clustering and clade distribution [ , , [ ] [ ] [ ] [ ] . in the present study, colombian denv- sequences were analyzed to try to reconstruct the phylogenetic history of the virus in this country. different genome regions have been used to infer denv phylogeny including those with short fragments [ , , ] . here we employed a sequence from the carboxi terminal of the envelope (e) protein which has demonstrated to provide a useful phylogenetic signal to define genotype clustering [ ] . it is important to note that the better resolution of evolutionary patterns should be obtained from complete genomes. however, it was not possible to obtain largest fragments from the oldest isolates, probably because of rna degradation across the time. as expected, all strains were clustered with those from brazil, paraguay, argentina, and different caribbean islands, corresponding to the formerly named genotype v (america/africa), showing a well supported clade clearly separated from the others genotypes. colombian strains denv- /co/ _atlantico/ , denv- /co/ _choco/ and denv- /co/ _choco/ , were separated from the remaining isolates and appeared closer to those from the caribbean islands, which represent the entrance of serotype into the americas. it was reported for the first time in in jamaica and rapidly spreading to the antilles including cuba, antigua & barbuda, aruba, bahamas, barbados, curaçao, dominica, grenada, guadaloupe, guyana, haiti, martinique, montserrat, puerto rico, st. kitts, st. martin, st. vincent and the grenadines, trinidad, turks and caicos, and the virgin islands [ ] . in , denv- was implicated in large mainland outbreaks perhaps occurring at the same time in colombia, venezuela, surinam, french guyana, and eventually centro america and mexico. in colombia, denv- was isolated between and , so the strain denv- / co/ _atlantico/ represents perhaps the first virus entrance to the country. it rapidly spread until the next isolation in choco (denv- /co/ _choco/ and denv- /co/ _choco/ ) and then it fades away (or at less it was not reported) probably displaced by denv- (maintaining denv- in a silent low circulation) until when it established in different localities. it is important to note that even with the mobility between countries and increasing opportunity of viral introduction, only one denv- genotype is circulating in america, different to denv- and denv- of which at least genotypes has been detected (america/asia genotypes and i/iii genotypes respectively) suggesting phylogenetic analyses were conducted in paup* perhaps, dissimilar patterns of viral spread and transmission between denv genotypes and even different adaptation capacity. many researchers have categorized denv in non official taxonomic levels beneath genotype, based specially in cladal distribution or geographical clustering. circulation of these "lineages" has been particularly defined for denv- in india, where at least different lineages had been proposed (india- close to american strains, india- related to singapore isolate, india- in south india and india- from delhi and gwalior) [ , ] . in our study, a remarkable cladogenesis event occurs around according to the molecular clock, generating two well supported clades corresponding to putative colombian denv- lineages. despite the eco-epidemiology similarities between colombia and neighbor countries were dengue is a major concern, lineages have not been previously demonstrated for denv- . in fact, according to ml phylogeny, most of the american strains (argentina and brazil) correspond to the lineage- , leaving the lineage restricted to colombia. although geographic distribution of these lineages is not clearly delimitated, it is evident that they are evolving independently and most likely in parallel at the same localities. despite the emergence and rapid diversification of denv has been a matter of special concern, precise mechanisms of evolution remain unclear [ ] [ ] [ ] [ ] [ ] [ ] . it is a fact that human rna viruses including influenza, hiv, coronavirus, etc., have particularly increased mutation and evolution rates mostly because of the lack of proofreading activity of rna-dependent rna-polymerase [ , ] . nevertheless, arthropod-borne viruses (arboviruses) have demonstrated slower mutation rates comparing with those infecting directly human host, probably because of the trade-off effect occurring when the virus is obligated to adapt alternatively into the invertebrate vector and vertebrate host [ ] . this resulting constrain has been experimentally assessed in vivo to venezuelan equine encephalitis [ ] and in vitro to denv [ ] demonstrating that fitness improves when virus specialize in a single cell line but decreases in virus undergoing alternative passages in different cells. in view of that, over all denv mutation rates have been previously inferred, ranging from . × - (denv- ) to . × - (denv- ) [ ] . in the present study, we found a mutation rate of . × - substitutions per site, per year, suggesting faster evolution rates for colombian strains, perhaps because of the high transmition occurrence especially in hyperendemic areas, where virus replicates in several human hosts, reducing the constraining effect occurred in the vector. however, this high mutation rate does not necessarily reflect a fitness advantage or a successful adaptation process. actually, positive selection for denv seems to be serotype/genotype dependent and even more, protein specific. in fact, envelope (e) protein apparently exhibits some adaptation evidence in denv- , denv- and various denv- genotypes, but not for denv- , strongly suggesting a purifying selection pressure, at least over this gene. nevertheless, further studies have to be done to try to understand the adaptation process in denv. on the other hand, although mostly of colombian strains belong to the genotype v, there is an isolate, denv- /co/ _valle/ placed into genotype i near to asia, china and east africa strains. the ml tree show this strain close to denv- /jp/mochizuki/ , a strain considered extinct. since we do not have this virus as reference in our laboratory, we can discard cross contamination during the assay. moreover, the presence of this virus could be explained based on the migration process occurred from asia to america, officially starting to colombia by , and sustained until the mid xx century [ ] . thus, establishment of asian colonies increased visitors and perhaps favored the entrance of viral strains. we can speculate that those viruses did not fit to the new environment and the adaptation events were constrained because of the selective pressures including different vectors and human immune response. according to natural history of denv, evolution events could bring new genetic variants and eventually increase the severity of disease. although pathogenic markers remain unclear, hemorrhagic features on some asian denv- genotypes have been demonstrated and asian derived denv- genotypes associated to dengue fever and dengue hemorrhagic fever have been reported in brazil [ ] . moreover, changes in clinical manifestation of disease (atypical dengue) such as viscerotropism or encephalitis may respond to the circulation of new denv lineages with increased pathogenic potential. consequently, epidemiological programs should include not just virological diagnosis but genotype surveillance too. this study shows in a defined time-scale, not just the first entrance of denv in colombia, but also the viral evolution process in a highly endemic area. as a major conclusion, only one genotype of denv has been circulating since the first epidemic reports in the continental area. nevertheless, two different lineages have been evolving fast since the earliest ′s according to molecular clock. as these evolution events may derive in a marked pathogenic potential, surveillance programs should include molecular methodologies. in fact, unusual presentation of disease currently reported by local health care institutions may be correlated to this evolution process. further analyses by using at least complete e gene should be done to corroborate our results. denv- strains used in this study were obtained from the virus collection of the national health institute (ins, virology lab, bogotá, colombia), and comprise isolates from outbreaks, epidemics and routine epidemiological surveillance. clinical samples were collected between and from different localities all around the country, so they represent most viruses circulating in colombia during the last years (table ) . all viral stocks were inoculated on c / aedes albopictus cells growing in eagle's minimal essential medium (e-mem) supplemented with % fetal calf serum (fsc). after days of incubation at °c, monolayer was disrupted and supernatant was then recovered by centrifugation and stored at - °c until use. the remained cells were washed with phosfate buffer saline (pbs) and dripped on slides; after fixed in cold acetone, slides were incubated with monoclonal antibodies (anti-denv- to anti-denv- , kindly donated by cdc, puerto rico) for one hour, washed with pbs and incubated again with a fluorescent conjugated antibody. additionally, denv- serotype confirmation was done by reverse transcription polymerase chain reaction (rt-pcr) using specific primers [ ] . cell culture supernatants were used to extract viral rna using qiaamp viral rna minikit (qiagen, germany) following manufacturer's instructions. briefly, μl of each supernatant was placed into μl of avl buffer with . μl of carrier rna and mixed with ethanol ( - %) before passed through a column by centrifugation. after washing with buffers aw and aw rna was finally eluted with μl of ave buffer and stored at - °c until use. five microliters from each rna extraction were used as template in a one step rt-pcr reaction (qiagen, one-step rt-pcr kit) as previously described [ ] . primers used [den s ( ′-tggctgagacc-carcatggnac- ′) and den as ( ′-caatt-catttgatatttgyttccac- ′)] were designated to amplify bp from de joining region e/ns . reactions were evaluated in % agarose gel stained with ethidum bromide and reactions observed as negative were then subjected to nested pcr as follow: μl of initial rt-pcr product, × buffer b ( mm tris-hcl ph . , mm mgcl , mm (nh ) so ), pmol of each primer [den s ( ′-ggaaaatgttygaagcaacyg ccc- ′), den as ( ′-tcctcccatgccttcc-cratgg- ′)] and . u of taq dna polimerase (invotrogene) in a final volume of μl. pcr reactions were first denaturated at °c ( minutes) and then subjected to cycles of denaturation ( °c, seconds), primer annealing ( °c, minutes), primer extension ( °c, seconds) and a final extension step at °c for minutes. nested pcr was evaluated in % agarose gel stained with ethidium bromide. amplified products (from rt-pcr or nested pcr) were purified using qiaquick pcr purification kit (qiagen, germany) and then used as template for sequencing reactions using the abi prism dye terminator cycle sequencing ready reaction kit (applied biosystems, foster city, ca). sequencing was carried out on both strands with pmol of primers used for nested pcr, and the products were analyzed using an abi model automated sequencer (applied biosystems, usa). overlapping sequences for each sample obtained from sense and antisense primers were combined to obtain a consensus sequence using the seq-man module of lasergene (dnastar inc. software, madison, wis.). a total of bp [corresponding to carboxyl terminus of envelope (e) gene] from new sequences were compared with previously sequenced strains from all over the world, available in genbank. consensus sequences were aligned using the program clustal w included in mega package version . [ , ] . phylogenetic trees were constructed with the maximum parsimony and maximum likelihood (ml) methods incorporated in the paup* . program [ ] . phylogenetic analyses were performed by using the best model of nucleotide substitution based on modeltest [ ] (analyses are available upon request). statistical significance of tree topology was assessed with a bootstrap with replicates. obtained trees were visualized using the tree view program [ ] . in addition, estimated rate of evolutionary change (nucleotide substitutions per site per year) and tree root age was obtained with the program beast (bayesian evolutionary analysis by sampling trees) [ ] , which uses bayesian markov chain montecarlo (mcmc) algorithms combined with the chosen model and prior knowledge of sequence data to infer the posterior probability distribution of phylogenies [ ] [ ] [ ] [ ] . we analyze the data using the year of isolation as calibration points to estimate divergence time in years. in order to avoid duplicates, sequences identical to other on the dataset were removed. rate variation among branches was inferred under the strict molecular clock model, whereas substitution rate among sites was calculated with the general time-reversible model (gtr) combined with the gamma parameter and proportion of invariant sites (gtr+Γ+i ) model. mcmc was run for , , steps and sampled every steps and the , first steps of each run were discarded. beast format files were obtained in the provided beauti graphical interface and the generated trees were visualized with the figtree . . . program. finally, statistical analysis was carried out in the tracer package [ ] . ct - , and from the colombian government and the instituto nacional de salud resources bogotá d.c.,colombia. viral vector core and gene therapy current address: robert koch institute at: origins of dengue type viruses associated with increased pathogenicity in the americas dengue and dengue hemorrhagic fever: its history and resurgence as a global public health problem dengue viral infections; pathogenesis and epidemiology world health organization, special program for research and training in tropical diseases: dengue guidelines for diagnosis, treatment, prevention and control. france, world health organization a timeline for dengue in the americas to december , and noted first occurrences. division of disease prevention and control pan american health organization pathogenesis of dengue: challenges to molecular biology dengue hemorrhagic fever in cuba. ii. clinical investigations genetic variation and microevolution of dengue virus in southeast asia molecular evolution and distribution of dengue viruses type and in nature phylogenetic relationships of dengue- viruses molecular epidemiology of dengue- and dengue- viruses molecular evolution and phylogeny of dengue- viruses molecular evolution of dengue type virus in thailand molecular epidemiology of dengue viruses in brazil molecular epidemiology of dengue type virus in venezuela: evidence for in situ virus evolution and recombination phylogenetic relationships and differential selection pressures among genotypes of dengue- virus molecular evolution and phylogeny of dengue type virus in the caribbean the origin, emergence and evolutionary genetics of dengue virus molecular epidemiological study of dengue virus type in taiwan genotipificación y análisis filogenético de cepas colombianas del virus dengue tipo molecular epidemiology of dengue virus type in venezuela phylogeography and molecular evolution of dengue in the caribbean basin introduction of the american/asian genotype of dengue virus into the yucatan state of mexico dengue virus genotype assosiated with dengue fever and dengue hemorrhagic fever use of a short fragment of the c-terminal e gene for detection and characterization of two new lineages of dengue virus in india simultaneous circulation of genotypes i and iii of dengue virus in colombia phylogenetic studies reveal existence of multiple lineages of a single genotype of denv- (genotype iii) in india during - dengue virus structural differences that correlate with pathogenesis effect of age on outcome of secondary dengue infections burden of symptomatic dengue infection in children at primary school in thailand: a prospective study race: a risk factor for dengue hemorrhagic fever dengue virus evolution and virulence models microevolution and virulence of dengue viruses lineage extinction and replacement in dengue type virus populations are due to stochastic events rather than to natural selection invasion and maintenance of dengue virus type and type in the americas molecular epidemiology of dengue viruses in the philippines: genotype shift and local evolution molecular evolution of dengue virus in puerto rico: positive selection in the viral envelope accompanies clade reintroduction clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions specific gap penalties and weight matrix choice mega : molecular evolutionary genetics analysis (mega) software version . beast: bayesian evolutionary analysis by sampling trees estimating the rate of molecular evolution: incorporating non-contemporaneous sequences into maximum likelihood phylogenies bayesian inference of phylogeny: a non-technical primer relaxed phylogenetics and dating with confidence rna virus mutations and fitness for survival the causes and consequences of genetic variation in dengue virus phylogenetic evidence for adaptive evolution of dengue viruses in nature selection-driven evolution of emergent dengue virus patterns of intra-and interhost nonsynonymous variation reveal strong purifying selection in dengue virus the phylogeography of human viruses mosquitoes put the brake on arbovirus evolution:experimental evolution reveals slower mutationaccumulation in mosquito than vertebrate cells arbovirus evolution in vivo is constrained by host alternation japoneses en colombia. historia de inmigración, sus descendientes en japón rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction tenorio a: a new tool for the diagnosis and molecular surveillance of dengue infections in clinical samples phylogenetic analysis using parsimony (and other methods). massachusetts, sinauer associates modeltest: testing the model of dna substitution treeview: an application to display phylogenetic trees on personal computers phylogenetic history demonstrates two different lineages of dengue type virus in colombia we thank the red nacional de laboratorios -instituto nacional de salud, colombia. we are grateful to pablo martínez and noelia reyes for technical assistance in amplifying and sequencing in the isciii; miguel andrés páez for reviewing the manuscript and lissethe pardo for technical assistance at instituo nacional de salud; rive/cyted (red iberoamericana de virosis emergentes) allowed the authors to meet with several other researchers in the field. this research was supported by instituto colombiano para el desarrollo de la ciencia y la tecnología francisco josé de caldas -colciencias grants authors' contributions jamr contributed to the experimental design, carried out the experiments and phylogenetic and molecular clock analysis, and wrote the manuscript. jauc contributed to the experimental design, carried out the experiments and provided a critical review of the manuscript. cdc participated in the experimental design, contributed to the interpretation of data and the critical review of the manuscript. gjrb contributed to the experimental design and provided a critical review of the manuscript. jas contributed with phylogenetic and molecular clock analysis and beast running and provided a critical review of the manuscript. atm conceived the study, its experimental design and provided a critical review of the manuscript. jcgg conceived the study, participated in its design and coordination and provide a final review of the manuscript. all authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. key: cord- - smnl i authors: chan, jasper f.w.; choi, garnet k.y.; yip, cyril c.y.; cheng, vincent c.c.; yuen, kwok-yung title: zika fever and congenital zika syndrome: an unexpected emerging arboviral disease date: - - journal: j infect doi: . /j.jinf. . . sha: doc_id: cord_uid: smnl i unlike its mosquito-borne relatives, such as dengue, west nile, and japanese encephalitis viruses, which can cause severe human diseases, zika virus (zikv) has emerged from obscurity by its association with a suspected “congenital zika syndrome”, while causing asymptomatic or mild exanthematous febrile infections which are dengue- or rubella-like in infected individuals. despite having been discovered in uganda for almost years, < human cases were reported before . the massive epidemics in the pacific islands associated with the zikv asian lineage in and were followed by explosive outbreaks in latin america in . although increased mosquito breeding associated with the el niño effect superimposed on global warming is suspected, genetic changes in its rna virus genome may have led to better adaptation to mosquitoes, other animal reservoirs, and human. we reviewed the epidemiology, clinical manifestation, virology, pathogenesis, laboratory diagnosis, management, and prevention of this emerging infection. laboratory diagnosis can be confounded by cross-reactivity with other circulating flaviviruses. besides mosquito bite and transplacental transmission, the risk of other potential routes of transmission by transfusion, transplantation, sexual activity, breastfeeding, respiratory droplet, and animal bite is discussed. epidemic control requires adequate clearance of mosquito breeding grounds, personal protection against mosquito bite, and hopefully a safe and effective vaccine. globalisation and urbanisation with increasingly frequent and large-scale movements of humans, animals, and commodities by aviation and water transport has led to the spread of previously geographically-restricted microbes and vectors to distant and isolated places. recent examples of emerging viruses that have spilled over to other continents from their original localities via exportation of travelrelated cases include coronaviruses (severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus), influenza viruses, and ebola virus. e moreover, global warming and climate changes have redefined the geographical distributions of important vectors of arthropod-borne viruses (arboviruses), such as the aedes mosquitoes, and facilitated the global spread of these viruses. dengue virus (denv), west nile virus (wnv), and chikungunya virus (chikv), have been introduced (wnv in and chikv in ) and/or spread rapidly in the western hemisphere in the past two decades. zika virus (zikv) is an arbovirus that was little known before it caused a large outbreak on yap island of the federated states of micronesia in . even then, zikv was not considered as an important emerging pathogen because clinical disease was generally mild. the recent report of a possible association between zikv infection and an epidemic of microcephaly among neonates in brazil has attracted global attention. the rapid spread of zikv beyond africa and asia to the americas and europe, and the potentially novel "congenital zika syndrome" outbreak have led the world health organisation (who) to declare the zikv epidemic as a global public health emergency on february . it would therefore be important to review the current knowledge on the epidemiology, virology, clinical manifestations, and laboratory diagnosis of zikv infection, and most importantly, to formulate clinical management options with special reference to perinatal care and control measures based on comparisons made with other mosquito-borne arboviruses. important historical and epidemiological events zikv (strain mr ) was first isolated from the blood of a febrile sentinel rhesus monkey (macaca mulatta), rhesus , during a study on yellow fever virus (yfv) in zika forest of uganda in april (table ) . in , zikv was isolated from aedes africanus mosquitoes caught in zika forest, suggesting that the virus might be mosquito-borne. in , zikv was isolated from the serum of a -year-old nigerian girl who had fever and headache, implying its role as a possible human pathogen. further virological and/or serological evidence of human zikv infection was reported in african (uganda, tanzania, egypt, central african republic, sierra leone, and gabon) and asian (india, malaysia, the philippines, thailand, vietnam, and indonesia) countries. e zikv infection remained relatively restricted geographically with less than sporadic cases reported in these areas in the first years after its discovery. , in , zikv emerged outside africa and asia for the first time and caused a major outbreak on yap island of the federated state of micronesia. over % of the yap residents who were ! years were infected within months. the attack rate of zikv infection in this outbreak was . per residents (range, . e . per residents). subsequently, another major outbreak was reported in french polynesia in october . an estimated , humans (> % of the french polynesian population) were infected by zikv. zikv infection then spread from french polynesia to other pacific islands including new caledonia, cook islands, vanuatu, and solomon islands. e the first cases of human zikv infection in the western hemisphere occurred on easter island, chile, in february , possibly originating from french polynesia during the annual tapati festival. phylogenetic analysis revealed that the ns gene sequence of the chilean strains had ! . % nucleotide and % amino acid identity to the french polynesian strains. the epidemic continued to expand rapidly and autochthonous human cases were reported in many latin american countries in the ensuing years. brazil stands out as the hardest hit latin american country with an estimated , e , , cases of zikv infection since march . based on the close phylogenetic relationship between the south american strains and asian and oceanic strains of zikv, the virus might have been introduced into brazil by asian travellers during the world cup or participants from the oceanic countries of the va'a world sprint championship canoe race in the summer of . , e the climate changes associated with el niño in north and eastern south america in on the background trend of global warming might have facilitated the rapid spread of aedes mosquitoes and zikv. currently, > countries in africa, asia, south america, oceania, and micronesia have reported autochthonous cases of human zikv infection. travel-related cases from endemic and epidemic regions were also reported in europe, north america, australia, and japan. e more worryingly, the brazil health ministry reported the detection of an unusual increase in the number of cases of neonates with microcephaly in northeastern brazil in october , coinciding with the expanding zikv infection epidemic. over suspected cases including some fatal cases were reported during the second half of alone. this represented a > -fold increase in the rate of microcephaly as compared to previous years. on november , the french polynesia health authorities also reported an unusual increase in the number of foetal and neonatal central nervous system malformations in and . like other flaviviruses, zikv is mainly transmitted by mosquitoes. in addition to the sylvatic (enzootic) transmission cycle between the haematophagous mosquito vectors and susceptible primary vertebrate hosts, the recent large-scale epidemics suggest that zikv is also adapting to an urban transmission cycle. , among the various mosquito species, aedes (stegomyia) mosquitoes appear to be the most important vector for zikv transmission, although some anopheles, culex, eretmapodites, and mansonia species have also been proposed as possible vectors (table ) . , , e the animal reservoirs of zikv are unclear. non-human primates including m. mulatta, cercopithecus aethiops, c. ascanius schmidti, c. mona denti, c. albigena johnstoni, chlorocebus sabaeus, colobus abyssinicus, erythrocebus patas, and pongo pygmaeus, and other mammals including zebras, elephants, and rodents, have been suggested as possible vertebrate hosts of zikv in africa and asia, based on virological and/or serological evidence of infection. , , , , e ae. africanus is the first mosquito species from which zikv was isolated, and is likely an important vector in the sylvatic transmission cycle of zikv. , inoculation of unfiltered supernatant of zikv-infected ae. africanus into mice and rhesus macaques led to clinical disease and/or neutralising antibody response. , ae. hensilli is the most commonly found mosquito species on yap island, but no virus isolate was made from field-collected mosquitoes to ascertain its role as a vector for zikv transmission during the outbreak. ae. aegypti and ae. albopictus, which have much wider geographical distributions than other aedes mosquitoes, are considered to be more important vectors in the urban transmission cycle of zikv. these aedes mosquitoes are highly susceptible to zikv infection in vitro with potential for further transmission after an extrinsic incubation period of e days. , they bite both indoors and outdoors, and mostly during daytime. non-vector-borne transmission routes of zikv have been proposed (table ) . like other arboviruses, blood transfusion-related transmission of zikv is possible, especially in endemic regions or where blood products obtained from infected travellers immediately returning from endemic regions are used. zikv rna was detected in the blood of . % of the donors in french polynesia during the epidemic. sexual transmission of zikv appears highly probable, especially in patients presenting with haematospermia with infectious viral particles and rna in semen. , notably, no other arboviruses have been associated with haematospermia or isolated from human semen. this might further complicate the control of the zikv epidemic, since most infected patients are asymptomatic. inadvertent sexual transmission of zikv to the female partner may then lead to virus transmission to the foetus, which may be potentially associated with severe congenital anomalies. besides transplacental transmission, perinatal transmission of zikv may also occur during delivery, via breastfeeding, and/or close contact after birth via exchange of saliva and other bodily fluids. zikv rna could be detected in breast milk and saliva of infected women, although replicative virus particles have not been demonstrated , perinatal transmission of other arboviruses, including denv, chikv, wnv, and yfv, has also been reported. e other suspected routes of transmission of zivk infection are those reported for other flaviviruses. these include mucocutaneous exposure to the virus in infected blood or via monkey bite, haemodialysis, or organ transplantation. e particularly, as zikv may be shed in the urine of infected patients for more than days, the risk to recipients of donated kidneys from donors at or returning from endemic areas has to be considered. , , , , it is unknown whether zikv could be transmitted via respiratory droplets as viral rna could occasionally be detected in nasopharyngeal swab and saliva samples. , , , virology and pathogenesis zikv is an enveloped, positive-sense, single-stranded rna virus belonging to the genus flavivirus in the family flaviviridae. it is closely related to spondweni virus and the viruses represent the only members of their clade within the mosquito-borne cluster of flaviviruses ( fig. ) . , phylogenetic analysis suggests that zikv has likely emerged between and in uganda. the two major lineages of zikv are the african (subdivided into west and east african) and asian lineages, which are responsible for causing the majority of infections in africa and asia (as well as the pacific and americas), respectively. , , the single-stranded rna genome of zikv has a size of , nucleotides encoding amino acids, with flanking untranslated regions ( and utrs) and a single long open reading frame encoding a polyprotein, which is cleaved into capsid (c), precursor of membrane (prm), envelope (e), and non-structural (ns) proteins , reverse transcription-polymerase chain reaction (rt-pcr) using primers targeting the e or ns gene is a key laboratory diagnostic tool for zikv infection in the recent outbreaks. , , the e protein is a major virion surface protein that is involved in receptor binding and membrane fusion. the domain iii of e protein contains a panel of antigenic epitopes that are important targets of serological assays, neutralising antibodies, and vaccines. , loss of the n glycosylation site in the e protein may be associated with adaptation to mosquito vectors and thus facilitate transmission. a single amino acid mutation in the e protein (e -a v) of chikv has been reported to be associated with increased fitness of the virus in ae. albopictus and allows chikv to disseminate in regions lacking the typical ae. aegypti vector. the recent spread of the asian lineage of zikv to oceania and the americas may be associated with significant ns codon usage adaptation to human housekeeping genes, which could facilitate viral replication and increase viral titres. mutations in the e and ns genes should be detected in zikv strains causing the current epidemic. when an infected aedes mosquito bites an infected patient, it ingests a blood meal containing zikv. as in other flaviviruses, zikv likely replicates in the midgut epithelial cells and subsequently the salivary gland cells. after an extrinsic incubation period of e days, zikv can be found in the mosquito's saliva which can then infect human. , moreover, the virus can likely be vertically transmitted transovarially as other flaviviruses. when the mosquito's saliva containing zikv is inoculated into human skin, the virus can infect epidermal keratinocytes, skin fibroblasts in the subcutaneous layer, and the langerhans cells. the keratinocytes and fibroblasts contain axl, tyro , and tim- , which can serve as attachment factors or receptors for zikv. the langerhans cells contain dc-sign, which can also serve as a receptor for virus entry. zikv infection of primary skin fibroblasts is associated with the upregulation with tlr mrna expression, and enhanced transcription of rig-i and mda , which are known innate immune responses to rna virus infection. this is followed by enhanced expression of interferon-alpha and -beta, and their downstream pathways of immune activation. both types i and ii interferons can suppress the viral load of infected cells. moreover, zikv is capable of increasing its replication by the induction of autophagy in host cells. thus, autophagy inhibitors can decrease the viral load of infected cells. infected cells of human skin explant exhibits cytoplasmic vacuolation, pyknotic nuclei, and oedema in the stratum granulosum. after replication in endemic/epidemic areas: + universal nucleic acid testing of blood donors. + temporary discontinuation of blood donation (importation of blood products from blood blank centres in non-endemic regions). non-endemic/epidemic areas: + pre-donation questionnaire to identify donors with recent travel history to endemic/epidemic areas. + deferral of blood donors who have travelled to endemic areas within the preceding ! days. + self-reporting of symptoms after blood donation ( these local tissue cells and the regional lymph nodes, zikv may then disseminate from the lymphatics and bloodstream to reach other organs/tissues, including the central nervous system, the skeletal muscles, myocardium, and perhaps transplacentally to the foetus. zikv was highly neurotropic in infected suckling mice. the brains of infected suckling mice show neuronal degeneration, cellular infiltration, and softening in the brain with virus replication in astroglial cells and neurons on histopathological examination. , , moreover, evidence of inflammation in skeletal muscles and myocardium has also been demonstrated in infected suckling mice. axl and tyro are members of the tam family of receptor tyrosine kinases (rtks). they are also present in neurons and under the influence of gonadotropin releasing hormone (grh), which in turn may affect neuronal survival and migration. furthermore, flaviviruses such as yfv may persist for up to days after intracerebral inoculation in rhesus macaques. the neurotropism and persistence of zikv may therefore partially explain microcephaly and predominantly neurological complications and foetal anomalies in this suspected entity of congenital zikv infection. most patients with zikv infection are asymptomatic. in the outbreak of zikv infection on yap island, only % of cases were estimated to be symptomatic. the incubation period of zikv infection is unclear, but is estimated to be similar to other mosquito-borne flaviviruses ( e days). , the clinical syndromes of symptomatic zikv infection can be broadly divided into zika fever and congenital infection ("congenital zika syndrome") ( table ). zika fever is an acute "dengue fever-like" illness characterized by low-grade fever ( . e . c), rash, retroorbital headache, bilateral non-purulent conjunctivitis, myalgia, and arthritis/arthralgia with periarticular oedema of the small joints of hands and feet. the rash in zika fever is typically described as a generalized, erythematous, maculopapular rash that spreads downward from the face to the limbs. less commonly, some patients may have more prominent systemic symptoms including high-grade fever, chills, rigours, sore throat, hypotension, and cervical, submandibular, axillary, and/or inguinal lymphadenopathies. digestive tract symptoms including nausea, vomiting, diarrhoea, constipation, abdominal pain, and aphthous ulcers may also be present. , , patients with genitourinary symptoms including haematuria, dysuria, perineal pain, and haematospermia often have detectable viral rna or infectious virus particles in urine and/or semen. , haematological and biochemical laboratory parameters are usually normal. however, some patients may have transient and mild leucopenia, neutropenia, lymphopenia or activated lymphocytes, monocytosis, thrombocytopaenia, and elevated serum levels of lactate dehydrogenase, aspartate aminotransferase, g-glutamyl transferase, fibrinogen, ferritin, c-reactive protein, and erythrocyte sedimentation rate during the viraemic phase. associated with restoration of normal number of peripheral immune cells and normal function of antigen-presenting cells. notably, the clinical manifestations of zika fever are non-specific and may mimic those seen in infectious diseases caused by other arthropod-borne pathogens, especially denv and chikv. some suggest that zika fever may be distinguished from dengue fever and chikungunya fever by more prominent oedema of the extremities, less severe headache and malaise, and milder degree of thrombocytopaenia seen in the former. , moreover, haemorrhagic complications seen in dengue fever have not been reported in zika fever, and arthralgia in zika fever is less severe than that in chikungunya fever. however, none of these features are pathognomonic and laboratory confirmation is required to exclude co-infections with these arboviruses and other causes of acute febrile illness in returned travellers from endemic regions, such as malaria. zika fever is usually self-limiting with most clinical manifestations resolving completely within e days. , , no death, hospitalisation, or haemorrhagic complication was reported during the outbreak on yap island. however, some patients may experience more protracted symptoms and other non-haemorrhagic complications. zika fever-related rash usually resolve within the first week, but may last for up to days and may be pruritic. other exanthematous diseases, such as denv, chikv, rubella virus, measles virus, parvovirus b , adenovirus, enterovirus, and rickettsial infection, should be excluded. the median duration of arthralgia is . days, but some patients may develop persistent or recurrent arthralgia for more than a month after symptom onset, mimicking the post-infectious chronic arthritis seen in chikungunya fever and lyme disease. , lymphadenopathies may be present for weeks after symptom onset, and alternative diagnoses such as infectious mononucleosis-like syndrome, streptococcus pyogenes infection, and toxoplasmosis should be considered in refractory cases. a post-infection asthenia appears to be frequent and further investigations may be necessary to determine possible association between zikv infection and chronic fatigue syndrome. , , immune-thrombocytopenic purpura and cardiac complication have also been reported in a few cases. jaundice was observed in patients with virological and/or serological evidence of zikv infection in eastern nigeria in the s who had co-infections (malaria and microfilaraemia) and a patient with sickle cell anaemia. , a possible association between zikv infection and severe neurological complications has been proposed during the recent epidemics in oceania and south america, during which the incidence of guillainebarré syndrome has increased by e times in french polynesia. , / ( . %) patients with suspected zikv infection in the french polynesia outbreak developed neurological syndromes after presenting with a zika fever-like illness. forty-two of these ( . %) patients were diagnosed with guillainebarré syndrome. , , similarly, guil-lainebarré syndrome has been reported among patients with zika fever-like illness in south america. , other neurological complications potentially linked to zikv infection include encephalitis, meningoencephalitis, myelitis, paraesthesia, vertigo, facial paralysis, and , , , , e suspected fatalities due to zikv-related guillainebarré syndrome have been reported. while the neurotropism of zikv may partially explain these neurological manifestations, more details and serial studies on their cerebrospinal fluid and magnetic resonance images by case-control studies are required to ascertain their association. zika fever-related death appears to be extremely rare but a number of probable cases have been reported, especially among immunocompromised patients and neonates with suspected congenital zikv infection. , , a small number of patients with coinfection with denv or hiv did not appear to have more severe disease. , further studies should be conducted to identify patients who are at risk of severe disease or death. microcephaly (head circumference ! standard deviations below the mean for sex and gestational age at birth) is the most prominent and commonly reported clinical feature of suspected congenital zika syndrome. , besides microcephaly, neonates and foetuses with suspected congenital zikv infection also had other malformations (table ). general features included low birth-weight, redundant scalp skin, anasarca, polyhydramnios, and arthrogryposis. neurological abnormalities included cerebral lesions, polymalformative syndromes, brainstem dysfunction, and absence of swallowing. ophthalmological defects included cataract, asymmetrical eye sizes, intraocular calcifications, macular atrophy (well-defined macular neuroretinal atrophy and/or macular pigment mottling and foveal reflex loss), optic nerve hypoplasia, iris coloboma, and lens subluxation. , , notably, other features characteristic of intrauterine infections, such as hepatosplenomegaly, rash, and chorioretinitis have not been reported. ultrasonographic examination revealed cerebral atrophy, intracranial calcifications especially over the white matter of frontal lobes, caudate, lentostriatal vessels, cerebellum, or around the lateral and fourth ventricles, dysgenesis of corpus callosum, vermia, and thalami, enlarged cisterna magna, asymmetrical cerebral hemispheres, severe unilateral ventriculomegaly, displacement of the midline, and thinning of the parenchyma on the dilated side, pons and brainstem. , , , zikv particles and rna may be detected by electron microscopy and rt-pcr, respectively, in autopsied samples. two important questions concerning congenital zikv infection remain unanswered. the first question is whether zikv is indeed the cause of microcephaly and other congenital anomalies in these patients. severe consequences have been reported for materno-foetal transmission of other arboviruses, such as dengue virus (preterm delivery, foetal death, low birth-weight, prematurity, acute foetal distress during labour), wnv (chorioretinitis and focal cerebral destruction), and chikv (encephalopathy and haemorrhagic fever). , , preliminary analysis in the current epidemic of microcephaly has not yet completely excluded other infectious or environmental aetiologies. moreover, there is some virological evidence to support the association between congenital zikv infection and these anomalies. zikv rna has been detected by rt-pcr in the amniotic fluid of pregnant women whose foetuses had ultrasonographic evidence of microcephaly, in the blood and foetal tissues of a neonate with microcephaly and other congenital anomalies who died within the first min of birth, and in the neonatal brain tissues of a few cases of full-term miscarriages and neonates with microcephaly. , , , however, there is still no large-scale prospective cohort or caseecontrol study to demonstrate a causal link between the presence of zikv in the foetus and the congenital anomalies after exclusion of other infectious and toxic causes. some have suggested that the apparent microcephaly surge might be attributable to the intense search for cases due to the heightened awareness of a possible association with the zikv outbreak or the use of larvicide. furthermore, detailed investigations for exclusion of other pathogens associated with congenital malformations have only been reported in a small number of cases. , microcephaly is well reported in congenital cytomegalovirus, rubella virus, and varicella zoster virus infection. chorioretinitis and intracranial calcifications are common in congenital cytomegalovirus infection and toxoplasmosis, but the latter is more commonly associated with hydrocephalus. cataract and cardiac anomalies are characteristic of congenital rubella syndrome, although cataract can also be found in congenital herpes simplex virus infection. thus, the diagnosis of congenital zika syndrome would depend on the exclusion of these "torch" infections in future studies using clinical criteria, histopathological findings, and serological, molecular and conventional cell culture techniques. if zikv is eventually confirmed to be the cause of these congenital anomalies, the second key question would be whether congenital zika syndrome actually comprises a wider spectrum of varying clinical severities than that seen in the reported cases. as with other congenital infections, it is possible that the reported cases of microcephaly represent only the tip the iceberg, focussing on the more severely affected patients, and that the timing of infection is likely to be important in determining the severity and outcome of the affected foetus. early infection during the first or even second trimester may be associated with congenital anomalies or even intrauterine death. , , indeed, preliminary data suggested that the greatest risk of microcephaly or congenital anomalies in the affected neonates appears to be associated with zikv infection in the first trimester of pregnancy. of mothers with infants born with microcephaly, % and % had a rash during the first and second trimester of pregnancy, respectively. besides neurological defects, cardiac and muscular abnormalities should also be excluded, as suckling mice infected with zikv developed evidence of central nervous system infection, myositis and myocarditis. some suspected cases of congenital zika syndrome developed severe arthrogryposis. , , , it is possible that intrauterine zikv infections that occur at a later stage of the pregnancy may present differently, either with less severe manifestations, such as mental retardation, sensorineural deafness, and/or ophthalmological lesions, or as full-term miscarriages. neonates with probable perinatal transmission of zikv infection appear to have mild disease and favourable outcome. further investigations should be conducted to better define the spectrum of manifestations in different gestational stages of congenital zikv infection. definitive diagnosis of zikv infection requires laboratory confirmation as there are no pathognomonic clinical, biochemical, or radiological features that reliably distinguish zika fever from other arboviruses, and congenital zikv infection from other infective, toxic, or genetic causes of congenital anomalies. successful isolation of zikv in viral culture, the gold-standard of laboratory diagnosis of viral infections, mainly depends on the timing of specimen collection and viral loads in the specimens. zikv has been isolated in vero and vero e cells inoculated with infected patients' serum, urine, and/or semen samples (table ) . , , however, infectious virus particles were not recovered by culture in most specimens with low viral loads. a positive serum immunoglobulin (ig) m or -fold rise in the titre of neutralising antibodies in paired serum samples collected approximately weeks apart also establishes the diagnosis of zikv infection. igm may be detected by enzyme-linked immunoassay on as early as day of symptom onset and may last for over months. , igm antibodies to denv and wnv usually persist for months and months, respectively. e the major limitation of these serological tests is possible cross-reactivity with other flaviviruses. neutralising antibodies detected by plaque-reduction neutralisation test may be more specific than igm detection by elisa for primary zikv infection, but may also have indeterminate results for secondary infection, including patients with previous vaccination against or exposed to other flaviviruses. , , , this is especially problematic in areas where there is cocirculation of multiple flaviviruses with the same aedes mosquito vectors. , , , patients with primary zikv infection and past denv infection are more likely to have higher titre (usually ! -fold) of igm and/or neutralising antibodies against zikv than against denv or other flaviviruses. , a positive serum denv ns antigen test without serial increase in igm or the combination of a positive igm response to denv and lack of an igg seroconversion in the convalescent-phase serum sample should prompt the clinician to investigate for another flavivirus such as zikv. moreover, co-infections with other mosquito-borne arboviruses, such as denv, chikv, wnv, and japanese encephalitis virus, are always possible and should be excluded by more extensive laboratory testing if clinically indicated. rapid and accurate diagnosis of zikv infection during the recent epidemics has mainly been achieved by the application of rt-pcr using primers that target the e or ns gene of zikv. , , , , alternatively, rt-pcr sequencing using universal primers that target the conserved regions in the genomes, such as the ns gene, of multiple flaviviruses, may allow simultaneous detection of > different flaviviruses. serum samples should be collected in the early phase of the disease, because viraemia is usually shortlived (usually days, rarely up to days) and may be low-level ( copies/ml). , alternatively, urine and semen samples may have higher viral rna loads table advantages, limitations, and uses of different diagnostic tests and types of specimens for laboratory diagnosis of zikv infection. , , , , , e , , , , , , , e , may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. other tissues brain, liver, spleen, and pooled visceral (kidney, lung, and heart) tissues were positive in a fatal case (an adult male with co-morbidities and immunosuppressive treatment). may be useful to exclude concomitant infections in patients with unusually severe or fatal infection. abbreviations: rt-pcr, reverse transcription-polymerase chain reaction; zikv, zika virus. (> copies/ml) than serum samples, and may be persistently positive for > days and ! days after symptom onset, respectively. , in a few cases, zikv rna has also been detected in saliva and nasopharyngeal swab samples of patients whose serum samples tested negative for zikv. these samples should therefore also be collected in suspected cases of zikv infection. , , , collection of amniotic fluid should be considered in pregnant women with positive zikv test result or if the foetuses show ultrasonographic evidence suggestive of congenital zikv infection. , , cerebrospinal fluid, placental, and/or umbilical cord tissues from neonates with suspected congenital zikv infection should be sent for virological and/or histopathological examinations to establish the diagnosis. , , zikv rna may also be detected in organ tissues in the rare cases of suspected zikv-related deaths. future studies should aim to better stratify the clinical use of these tests and to develop point-of-care tests (eg: antigen tests) that can be widely used in less developed regions without the facilities and expertise for molecular or serological tests. treatment is usually not required for patients with asymptomatic or uncomplicated zika fever. the mainstay of treatment is supportive as there are no specific anti-zikv antiviral agents. acetaminophen may be used to relieve fever and arthralgia. anti-histamines may help to control pruritus. adequate rehydration for fluid loss through sweating, vomiting, and insensible losses should be encouraged. aspirin should be avoided due to the risks of bleeding in those with thrombocytopaenia and developing reye's syndrome in children less than years of age. nonsteroidal antiinflammatory drugs are also contraindicated in cases where denv and chikv infections cannot be confidently excluded in order to avoid haemorrhagic complications. potential neurological complications, especially guillainebarré syndrome, should be diagnosed promptly to allow early use of intravenous immunoglobulins and/or plasmapheresis. the risk of immune enhancement should also be considered if convalescent-phase plasma therapy with neutralising antibodies against zikv is used for treatment of severe cases. virological testing and foetal ultrasound to exclude zikv infection and foetal microcephaly or intracranial calcifications should be offered to pregnant women who develop zika fever-like symptoms during or within weeks of travel to areas with zikv transmission. besides collecting the appropriate specimens for virological tests, serial foetal ultrasound examinations should be performed every e weeks to monitor foetal anatomy and growth in suspected cases of congenital zikv infection. foetal ultrasound and/ or aminocentesis should also be offered to asymptomatic and seropositive pregnant women with history of travel to affected areas. after delivery, serum should be collected either from the umbilical cord or directly from the neonate within days of birth for rt-pcr, igm and/ or neutralising antibodies against zikv. comprehensive physical examination including measurement of the occipitofrontal circumference, length, and weight, evaluation for neurological abnormalities, dysmorphic features, hepatosplenomegaly, rash, ophthalmological lesions, and auditory defects, and laboratory testing for torch screening should be performed. the affected child and the family should be managed and counselled by a multidisciplinary team consisting of paediatric neurologist, clinical geneticist or dysmorphologist, infectious disease specialist, medical social worker, and other relevant specialists. long-term follow-up to monitor physical, intellectual, and functional progress of the child should be offered. both vector control and personal preventive measures are important for interrupting the transmission of zikv. systematic mosquito surveillance and control programs should be established and coordinated by health authorities. mass sanitation campaigns to eliminate mosquito breeding sites in household and high-risk areas such as garbage collection points, construction sites, illegal dumping grounds, and invalid car fields should be organised. mosquitoes should be removed with a radius of at least m around areas with high population densities, such as schools, transport terminals, churches, and healthcare facilities. in areas where autochthonous or imported cases of zikv are detected, the use of adulticide through spraying to remove infected adult mosquitoes should be considered. residents in or travellers to affected areas should stay indoor with air conditioning, window and door screens if possible, wear long sleeves and pants, use permethrintreated clothing and gear, and use insect repellents when outdoor. most environmental protection agency (epa)registered insect repellents, including n,n-diethyl-mtoluamide (deet), should be safe for pregnant and lactating women ( % deet), and children ( % deet) aged > months. individuals returning from affected areas to non-affected regions should continue to use insect repellents for at least an additional days to prevent local non-infected mosquitoes from the acquisition of virus from the asymptomatically infected returned travellers. this will serve to interrupt the mosquito-human-mosquito transmission chain. hospitalised laboratory-confirmed cases should be managed in designated wards to avoid mosquito bites. the effects of other novel mosquito-control measures, such as the wolbachia biological control approach, should be evaluated. other animals such as rodents should also be investigated as potential animal reservoirs and controlled as findings indicate. non-vector-borne transmission of zikv may be prevented by specific measures (table ) . concerning blood transfusion, universal nucleic acid testing of blood donors is recommended. the use of universal primers that can simultaneously detect multiple arboviruses such as denv and zikv should be considered. temporary discontinuation of blood donation should be considered during an outbreak situation. in non-endemic areas, pre-donation questionnaire to identify donors with recent travel history to regions with reported cases of zikv infection and deferral of blood donation from these donors until at least days after returning from affected regions should be implemented. most transfusion-related transmissions of arboviruses are associated with asymptomatic infections, and symptomatic donors who were rt-pcr-positive for zikv usually developed symptoms between and days after blood donation. newer pathogen reduction technologies for blood products should be considered. similarly, donated organs, especially kidneys, from individuals with travel history to affected areas should be tested for zikv as the virus may persist in the genitourinary tract for an undetermined period. , , , , barrier methods should be used to prevent sexual transmission through infected semen. male returned travellers should continue the use of condom with pregnant sex partner throughout the whole duration of pregnancy. future studies should evaluate the duration of virus shedding in semen and the infectiousness of rnapositive semen samples, in order to determine how long barrier methods should be used by men returning to nonendemic regions. some regional authorities have advised women to avoid pregnancy until the epidemic is over. pregnant women or those planning for pregnancy should defer travelling to regions with reported cases of zikv infection. if such travel was unavoidable, they should strictly comply with personal protective measures to avoid mosquito bites. further studies are needed to determine the risk of zikv transmission by breast milk and saliva. other less common transmission routes, including mucocutaneous exposure to infected bodily fluid during laboratory and patient-care procedures, and bites by infected primates should be avoided with strict compliance to infection control measures. 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authors: romero-brey, inés; bartenschlager, ralf title: membranous replication factories induced by plus-strand rna viruses date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: aesiff f in this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) rna viruses. we discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. a general denominator is that two “morphotypes” of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (er) and double membrane vesicles, representing extrusions also originating from the er, respectively. we hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments. members of the family flaviviridae are enveloped viruses with a single stranded rna genome of positive polarity. this family contains four different genera: hepacivirus (from the greek hepar, hepatos, -liver‖), flavivirus (from the latin flavus, -yellow‖), pestivirus (from the latin pestis, -plague‖) and the recently included genus pegivirus [ , ] (figure ). hepatitis c virus (hcv) is the prototype species of the genus hepacivirus. it was first discovered by choo et al. [ ] in the serum and tissues of a chimpanzee experimentally inoculated with serum from an individual with chronic, non-a, non-b hepatitis. this virus was associated with a mild form of chronic hepatitis frequently observed in recipients of blood transfusions [ ] and was called hcv. a second species within the genus hepacivirus is gbv-b which was first identified in tamarins that developed hepatitis following inoculation with the serum from a surgeon (initials g.b.) with acute hepatitis. additional gb-like viruses were discovered later on and have been assigned to the new genus pegivirus (an acronym derived from pe, persistent; g, gb or g) within the family flaviviridae [ ] . and cms may represent the site of denv replication and rna translation/polyprotein processing, respectively [ ] . the most complete characterization of denv-induced intracellular membrane rearrangements elucidated their d architecture as well as their spatial connection with viral assembly sites [ ] . tem of resin-embedded infected cells revealed a complex collection of convoluted and vesicular structures, including cms that were usually surrounded by multiple vesicles, often appearing as longitudinal vesicle arrays. by using electron tomography (et), the latter were found to correspond to er tubules containing - nm single-membrane vesicles (ve) that result from the invagination of the er membrane into the er lumen. by conventional em, these vesicles appeared as double membrane vesicles, likely corresponding to the vps described earlier [ ] . immuno-em confirmed that the vesicles visible in resin-embedded cells were induced by denv infection and contained all ns proteins. however, only ns was detectable within the cms, which could be due to lower affinity of the antibodies or poor accessibility of the other ns proteins in the cms. double-stranded rna (dsrna) detected by immunostaining appeared as discrete electron-dense structures inside or on the cytosolic surface of a subset of vesicles, suggesting that dsrna might be present only in some of the vesicles at a given time point. furthermore, the vesicles contain rather uniform pores of ~ nm diameter towards the cytosol ( figure a ). thus, both the topology of the vesicles and the immunolabeling results support the idea that the vesicles might be the site of rna replication. moreover, these results showed that replication factories are a continuous membrane network that provides a platform for the transport of viral proteins and genomes between sites of rna replication, ribosome-containing compartments (rna translation) and virus assembly sites. in fact, virus budding sites were found in close proximity to the pores of the replication vesicles. this topological link may ensure efficient production and delivery of viral rna for the assembly of infectious virus progeny. consistent with these findings, a very recent publication using et showed that these virally modified structures were also observed in denv-infected mosquito cells, with one exception: cms were absent from denv-infected c / mosquito cells [ ] . in addition, after multiple rounds of virus replication, tubular structures were also observed in the vicinity of vps. these structures might represent a hallmark of chronically infected insect cells, since these structures are also induced by tbev in tick cells (see below). the first reports on wnv-infected cells described the visualization of virions [ ] . an extensive characterization of kunjin virus-the australian variant of wnv (wnv kun )-infected cells has been carried out more recently [ ] [ ] [ ] . three well-defined structures were found, corresponding to large cms, paracrystalline arrays (pcs) and vps that appeared as membrane sacs containing small vesicles (ve) [ , ] . based on immunolocalization studies, a distinct redistribution of the trans-golgi network (tgn) and colocalization of tgn markers with dsrna has been observed, suggesting that the replication factories of wnv kun were derived from the tgn [ ] . three-dimensional reconstructions of the wnv kun replication sites revealed an intimate association of the rough er (rer) with the bounding membrane of the vps [ ] (figure b ), resembling the vesicles observed in denv-infected cells. these results argue for an additional role of the rer in the formation of the wnv kun replication factories. similar to denv, individual necks were observed in the vesicles as well as the majority of the viral rna, as detected by immunolabeling with a dsrna-specific antibody, resided within these vesicles [ , , [ ] [ ] [ ] . in most cases, viral rna spanned the breadth of the vesicles and was juxtaposed to the necks open to the cytoplasm [ ] . . slices through tomograms of infected cells (on the left) and d top and lateral ( ° rotation) views of the same tomograms (on the right) are depicted, showing the characteristic virus-induced structures. the replication vesicles (ve) of denv, wnv and tbev (genus flavivirus) correspond to invaginations of er membranes that remain connected to the cytosol via nm-pores (highlighted with white arrows in the d lateral views), forming vesicle packets (vps). the replication factory of hcv (genus hepacivirus) is primarily composed of double membrane vesicles (dmvs) that seem to be formed aser protusions connected to er membranes via neck-like structures (highlighted with white arrows in the d lateral view). the er is shown in yellow (denv, tbev and hcv) or in red (wnv) and the replication organelles in brown (denv, tbev and hcv) or in white (wnv). the outer and inner membranes of dmvs are depicted in different shades of brown (outer membrane in dark brown and inner membrane in light brown). figure b is reproduced with permission from [ ] . in cells infected with tbev, one of the most important tick-transmitted viruses in europe and asia, virus particles and membrane-connected vesicles were also observed inside the er [ ] , similar to what was described for denv and wnv kun . the viral dsrna was only detected inside the vesicular structures within rer, suggesting that tbev rearranges internal cell membranes to generate a compartment that protects viral rna from detection by cytoplasmic pathogen recognition receptors (prrs) [ ] [ ] [ ] . this localization of dsrna might suffice to delay the onset of the ifn response [ ] . for tbev [ ] and wnv kun [ ] it was shown that treatment with brefeldin a (bfa), a drug which disrupts the golgi apparatus, did not interfere with viral replication. however, this treatment rendered wnv kun sensitive to the antiviral action of the ifn-induced protein mxa, indicating that bfa might have disrupted the membranous wnv kun replication compartments, thus leading to exposure of dsrna and its detection by prrs. in contrast, treatment of tbev-infected cells with bfa neither affected viral replication, nor the level of ifn production. these findings indicate that tbev dsrna might be stored inside bfa-resistant membrane vesicles that robustly protect the viral rna from recognition by cellular sensors. vector-borne flaviviruses like denv and tbev must replicate in both mammalian and arthropod cells. a few comparative studies have been published describing virus-induced structures such as cytoplasmic membrane proliferations and vesicle formation, also in insect cells [ , , , , ,] . a detailed comparative ultrastructural analysis of tbev-induced modifications revealed that the extent of membrane expansion and the abundance of vesicles were lower in insect cells [ ] . single-membrane vesicles, ranging in diameter from - nm were frequently found within proliferated er areas, often occurring in large groups contained within er cisternae. pore-like openings connected these vesicles to the cytoplasm and to other vesicles. apart from these vesicles, in tick-infected cells elongated vesicles or tubules were found that were much more prevalent in persistently than in acutely infected cells. these tubules were only occasionally noted in infected mammalian cells, similar to what was found with denv-infected cells [ ] . the tubular structures had a cross-sectional diameter of - nm, similar to the one of vesicles, reached up to nm in length, were closed at the ends and often arranged in fascicle-like bundles, shrouded with the er membrane. however, no pores between the tubules or towards the cytoplasm were observed [ ] . the function of these tubules is unclear and it is not known whether they represent bona fide features of replication factories, aberrant structures as a result of incorrect membrane remodeling, or the result of a cellular process to restrict infection [ ] . in any case, the tubules might be a feature of persistent infection, eventually linked to the high number of defective virus particles, because the lack of pores could prevent proper replication or packaging of the viral genome [ ] [ ] [ ] . further studies are required to shed light on the biogenesis and biological significance of these membranous tubular structures. a recent study identified nm-diameter vesicles within the er lumen of tbev-infected bhk- cells and in cells transfected with a tbev replicon [ ] . et revealed that these vesicles are invaginations of the er within a highly organized network of interconnected membranes with half of vesicles containing pore-like connections to the cytoplasm ( figure c ). however, no pore-like openings were observed between adjacent/neighboring vesicles, in contrast to what has been described for cells infected with langat virus (lgtv), a naturally attenuated tick-borne flavivirus [ ] or in wnv kun -infected cells [ ] . interestingly, in tbev replicon cells, the number of pore-containing vesicles was slightly larger (~ %) and they were found in much more fragmented er tubules as compared to tbev-infected cells. however, despite more extensive er rearrangements in replicon cells, they contained fewer vesicles, consistent with the lower level of viral replication [ ] [ ] [ ] . conventional em analysis of neurons infected with murray valley encephalitis virus (mvev) revealed several ultrastructural features, including proliferation of er and golgi complex membranes as well as the appearance of membrane-bound spherical vesicles ( - nm diameter) [ ] , similar to those observed for the related flaviviruses japanese encephalitis virus (jev) [ , ] and st. louis encephalitis virus (slev) [ ] . in the latter case, cylindrical membranous structures (or tubules) were also observed [ ] . the presence of vesicles was also detected in monkey liver cells infected with yellow fever virus (yfv) [ ] . these findings indicate that all members of the genus flavivirus utilize the er as a source of membranes for the formation of their replication factories, whereas assembly of new virions seems to occur at er sacs in close proximity to the replication sites [ , ] , thus creating an optimized membranous environment to support efficient viral replication and assembly. maturation of the newly synthesized virions takes place in the golgi apparatus, where flavirirus virions are often observed [ ] . in stark contrast to flaviviruses, hcv, the prototype of the genus hepacivirus, provokes an alternative rearrangement of intracellular membranes, originally designated -membranous web‖ (mw). this term referred to compact vesicle accumulations embedded into a membranous matrix [ ] as detected in cells inducibly expressing the hcv polyprotein. by using different em methods, we and others have recently found that the mw is primarily composed of double membrane vesicles (dmvs) [ ] [ ] [ ] . the fact that the kinetics of their appearance correlates with hcv replication suggests that these structures play an important role for viral rna amplification [ ] . indeed, immunolabeling of purified dmvs revealed an enrichment for viral proteins as well as dsrna [ , ] . importantly, dmvs contain enzymatically active viral replicase [ ] and they originate from er membranes, similar to what has been found for other members of the family flaviviridae. et analysis showed that most of the dmvs remain connected to the er via their outer membrane [ ] ( figure d ). although dmvs are primarily closed structures, ~ % of them have an opening towards the cytosol. late in infection, multi-membrane vesicles (mmvs) with an average diameter of nm are generated, likely originating from dmvs by secondary enwrapping events [ ] . by using huh . cells infected with the highly replicative hcv strain jfh- , ferraris and coworkers observed three different types of membrane alterations: vesicles in clusters (vics), contiguous vesicles (cvs) and dmvs [ ] . the vics were small single-membrane vesicles of variable size ( - nm) , grouped together in well-delimited areas. most of them had an internal invagination. the cvs were small single-membrane vesicles, present in large numbers and widely distributed throughout the cytoplasm, with a more homogeneous size (around nm). they were tightly associated to each other and tended to form a collar around lipid droplets (lds). dmvs were heterogeneous in size ( - nm) and had a thick, electron-dense membrane consisting of two closely apposed membranes. the increase of cvs' number correlated with an increase of intracellular hcv rna levels, arguing for a possible role of cvs in the early stages of viral replication. the presence of ns a in cvs, as demonstrated by immunogold staining, is consistent with this hypothesis. alternatively, cvs might constitute the membranous platform for viral assembly. in fact, the core protein is present in these structures ( %) as well as on the ld surface ( %). however, so far visualization of virus particles in infected cells has not been possible, making this hypothesis difficult to prove. while most of the dsrna signal was located within dmvs or at dmv membranes, vics were free of viral components and rna and these structures as well as cvs were very rarely observed in cells with a subgenomic jfh- replicon [ ] or absent in cells infected with a jfh- variant designated jc [ ] . the first d reconstruction of a complete hcv-infected cell revealed that all these three membrane structures were tightly connected and closely associated with ld clusters [ ] . taken together, these findings indicate a fundamental role of dmvs in hcv replication. an in-depth comparison of the study by ferraris and coworkers [ ] and our publication [ ] suggests that cvs might be also dmvs for several reasons: first, cvs have electron dense tightly apposed membranes; second, by using correlative light and electron microscopy, we also detected dmv accumulations around lds, reminiscent of the cvs described by ferraris and coworkers [ ] ; third, taking into consideration the density of content and morphology, some of the structures described as dmvs by ferraris and colleagues might correspond to mmvs according to our nomenclature. this might account for the differences in size between the dmvs reported in both studies (up to nm versus nm, respectively). alternatively, the difference might be due to the use of distinct virus strains (jfh- and jc ) that differ in their capacity to produce infectious virus particles by ~ orders of magnitude [ ] , which might also explain the presence of vics only in jfh- infected cells. much less about membranous replication factories is known for pestiviruses. tem-based studies from the times in which the genus pestivirus was still belonging to the family togaviridae reported that pestivirus-infected cells exhibited ultrastructural modifications of rer and contained small numbers of virus-like particles (vlps) [ , ] . gray and nettleton ( ) reported that border disease virus (bdv)-infected cells contained several profiles of er and many dense lamellar bodies, which when transversely sectioned appeared as multiple rows of tubules, nm in diameter [ ] . these lamellae were often found in association with rer and in one occasion vlps appeared to be budding within them. bovine viral diarrhea virus (bvdv)-infected cells contained rer modified into tubules, in which electron-dense vlps were present. more recent studies on bvdv-infected cells revealed cytoplasmic vacuolization and vlps in dilated er cisternae [ , ] . in addition, membrane structures consisting of vesicles of various sizes enclosed in much larger vesicles have been reported [ ] . these structures that morphologically resemble multivesicular bodies (mvbs) are distinct from the hcv-induced membranous web and more reminiscent of the flavivirus-induced vps. studies on the morphogenesis of pestiviral particles were hampered by a low rate of virion production. in a recent study, schmeiser and colleagues have overcome this problem by using high multiplicity of infection in mdbk cells with a distinct virus strain, the giraffe- strain [ ] . obtained results define the er as the site of pestivirus particle assembly, where budding of virions was observed. virus particles were also found inside the lumen of the golgi and in vesicles associated with the golgi compartment, suggesting that virus egress occurs via the conventional secretory pathway. interestingly, replication kinetics of pestiviral rna did not correlate with distinct membrane rearrangements and only slight dilatation of the er lumen was noticed. the absence of significant membrane rearrangements argues for a major difference between pestiviruses and other members of the flaviviridae family. interestingly, the authors detected the capsid protein and dsrna, the marker for viral replication intermediates, mainly in mvbs, indicating that pestiviruses are either using this compartment for replication or that viral rna and proteins are transferred to this compartment for degradation. similar assumptions have been made for hiv [ ] and marburg virus, a member of the filoviridae family [ , ] . alternatively, pestiviral rna and protein in mvbs might be intermediates of the entry process, prior to fusion of the envelope with the endosomal membrane. indeed, particles inside mvbs matching the morphological criteria of pestivirus virions were detected [ ] . however, mvbs of non-infected cells also contain vesicles for lysosomal degradation termed intraluminal vesicles (ilvs) that display a very similar morphology to pestiviral virions. thus, unambiguous discrimination between ilvs and pestivirus particles will require detailed immunolabeling approaches. the first visualization of the d architecture of a (+) strand rna virus replication factory was reported for flock house virus (fhv), a member of the family nodaviridae [ ] . this insect nodavirus induces the formation of invaginations at the outer mitochondrial membrane (omm) with an average diameter of ~ nm [ , ] ( figure a ). the interior of these vesicles (called spherules) is connected to the cytoplasm by a necked channel of ~ nm diameter, which is wide enough to allow import of ribonucleotides and export of synthesized rna (diameter < nm) [ ] . furthermore, metabolically labeled fhv rna localized between inner and outer mitochondrial membranes inside these spherules, thus validating the spherules as bona fide fhv-induced compartments for viral rna synthesis [ ] . conventional tem analyses of coronavirus-infected cells identified large numbers of isolated dmvs [ ] . at least in case of the severe acute respiratory syndrome (sars)-coronavirus, these dmvs are part of an elaborate reticulovesicular network (rvn) of modified er that consists of convoluted membranes, numerous dmvs (diameter - nm) ( figure b ) [ ] , and -vesicle packets‖ apparently arising from merging of dmvs. the cms were most intensively immunolabeled for viral replicase subunits whereas dmvs labeled abundantly for dsrna. while this result argues that dmvs might be the site of viral rna synthesis, et analyses failed to detect dmv connections to the cytoplasm to allow transport of nascent rna. instead, dmvs are connected to each other, to cms and to the er via their outer membranes. also, in case of another coronavirus, the mouse hepatitis virus (mhv), er membranes are thought to be the lipid donor of the membranous replication compartment [ , ] . qualitative and quantitative analyses by (immuno)-electron microscopy of mhv-induced membrane rearrangements revealed the appearance, in strict order, of dmvs (diameter - nm), cms, large virion-containing vacuoles, tubular bodies and cubic membrane structures [ ] . the recently identified coronavirus middle east respiratory syndrome coronavirus (mers-cov) induces extensive membrane rearrangements in the perinuclear region, including the formation of dmvs and cms [ ] . the diameter of mers-cov induced dmvs ranged from - nm, comparable to the sars-cov induced dmvs. in addition, cms were always surrounded by dmv clusters and were only observed in cells that appeared to be more advanced in infection. this observation strengthens the notion that dmv formation precedes the development of cms, as postulated previously for sars-cov [ ] . in addition to the betacoronaviruses (sars-cov, mhv and mers-cov), er-derived dmvs with a diameter of ~ nm have been observed in primary avian and mammalian cells infected with infectious bronchitis virus (ibv), an important poultry pathogen belonging to the genus gammacoronavirus [ ] . however, the most striking structures induced by ibv are zippered er membranes ( figure c ). the zippered er was associated to - nm diameter spherules, structures that are not present in cells infected with betacoronaviruses. et showed that these ibv-induced spherules are tethered to the zippered er and contain a . nm long channel connecting their interior to the cytoplasm of the cell, making them the ideal candidates for the site of ibv rna synthesis. er membranes are also targeted by nidovirales that belong to the family arteriviridae. cells infected with the prototypic arterivirus, equine arterivirus (eav), also contain dmvs associated to er tubules. these dmvs are ~ - times smaller (~ nm) as compared to coronaviruses [ ] . a recent in-depth ultrastructural analysis revealed that the outer membranes of eav-induced dmvs are interconnected with each other and with the er (figure d ), thus forming a reticulovesicular network (rvn) resembling the one previously described for the distantly related sars-cov [ ] . despite significant morphological differences, a striking parallel between the two virus groups, and possibly all members of the order nidovirales, is the accumulation of dsrna, the presumed intermediate of viral rna synthesis, in the dmv interior. along these lines, dmvs visualized by means of electron spectroscopy imaging contained phosphorus in amounts corresponding on average to a few dozen copies of the eav rna genome. like in sars-coronavirus infected cells, connections between dmv interior and cytosol could not be unambiguously identified, suggesting that dsrna is compartmentalized by the dmv membranes. in addition, et revealed a network of nucleocapsid protein-containing protein tubules, intertwined with the rvn. this potential intermediate in nucleocapsid formation, which was not observed in coronavirus-infected cells, suggests that arterivirus rna synthesis and assembly are spatially coordinated. membrane remodeling in picornavirus-infected cells has been studied for more than years. massive virus-induced membrane modifications have been reported already in [ ] , but the origin of these membranes is still a matter of controversy. several lines of evidence, including biochemical and structural data, suggest that the er must play a major role in the formation of those structures [ ] [ ] [ ] . early in infection membranous replication factories contain markers of the golgi [ , ] , whereas markers of the er, golgi and lysosomes were all found to be associated with poliovirus replication sites late in infection [ ] . initial reports identified membrane rearrangements as u-bodies because of their horseshoe-like shape [ ] . later, bienz et al. described rearranged membranes as clusters of single-membrane vesicles [ , ] , while other reports [ , ] noticed the double-membrane morphology of picornavirus-induced vesicles. the single-versus double-membrane morphology of the vesicles was first interpreted as two different models of their formation. however, several recent publications suggest that picornavirus-induced membrane rearrangements might occur in a consecutive manner. thus, early in poliovirus infection small clusters of single-membrane vesicles predominate that are transformed into bigger irregularly shaped single-membrane structures ( figure g ) and, late in infection, replaced by either round or irregularly shaped dmvs [ ] . interestingly, the small clusters of single-membrane vesicles of ~ - nm diameter contain gm , a cis-golgi marker, but did not stain positive for calnexin, an er marker. however, this does not exclude a role of the er for biogenesis of these vesicles, because er-resident proteins might be sorted out as these membranes are transformed. although dsrna and metabolically labeled viral rna were detected in single-membrane vesicles and dmvs, the exponential phase of viral rna synthesis correlates with the appearance of single-membrane and intermediate structures [ ] arguing that these structures are most relevant for high level poliovirus rna synthesis. similar results have been obtained with another member of the family picornaviridae, coxsackie b virus (cvb ) that also induces the formation of single-and double-membrane compartments, whose relative abundance correlates with the stage of the replication cycle [ ] (figure f ). based on the observation that the golgi apparatus disappears in cvb -infected cells, the membrane rearrangements might originate from this organelle (montserrat bá rcena, personal communication). similar to poliovirus, single-membrane tubular clusters occur predominantly early in infection, whereas the number of dmvs increases as infection progresses. a budding event could account for the formation of the tubules, depicting an average length of ± nm and an average diameter of ± nm. a subsequent enwrapping of these single-membrane tubules via an -autophagy-like‖ mechanism could then lead to the formation of dmvs that have an average diameter of nm ± nm. this transformation may require several steps: (i) membrane pairing; (ii) induction of curvature; and (iii) membrane fusion [ ] . this scenario would be consistent with the membrane surface of dmvs as an average-sized dmv with a diameter of nm would be equivalent to a tubule with a length of nm and a diameter of nm. er membranes were found near dmv clusters. however, in contrast to previous observations in nidovirus-infected cells, these dmvs were not connected to neighboring structures. in addition to these compartments, a third type of modification was detected in cvb -infected cells: multilamellar structures, which are typical for the late phase of infection and that correspond to enwrapped dmvs: despite their various shapes and degrees of complexity, in all instances they contained one dmv, surrounded by one or several layers of curved cisternae. in conclusion, these results, and similar observations made for foot and mouth disease virus (fmdv) (genus aphthovirus) [ ] suggest that members of the picornaviridae family induce singleand double-membrane vesicles. they appear in a time-dependent manner and seem to evolve from each other, possibly in coordination with the progression of the viral replication cycle [ ] . these membrane rearrangements occur independently from the used virus strain and cell line. rubivirus (family togaviridae) [ ] . rubv anchors its rna synthesis machinery to membranes of a cell organelle known as -cytopathic vacuoles‖ (cpvs) that is derived from modified endosomes or lysosomes and has an average diameter of - nm [ ] [ ] [ ] . freeze-fracture and et analysis of rubv-infected cells revealed a high complexity of cpvs that are composed of stacked membranes, rigid sheets, small vesicles and large vacuoles ( figure i ) [ ] . the cpvs are interconnected and linked to the endocytic pathway, as deduced from labeling experiments with endocytosed bsa-gold. furthermore, rer cisternae, mitochondria and golgi stacks are recruited around cpvs to build up rubv factories. cpvs have several contacts with cellular organelles: they are coupled to the rer through protein bridges of ~ - nm and closely apposed membranes and they are attached to golgi vesicles, whereas contacts with mitochondria were not detected [ ] . it has been proposed that rna synthesis occurs on vesicular membranes within the cpvs, which are linked to the cytosol and that the viral replicase molecules are associated with vesicles that transform with time into large vacuoles and straight elements [ ] . this is supported by immunogold labeling revealing replicase components and dsrna within the cpvs [ , , ] . the modification of late endosomes and lysosomes is a feature that rubv shares with alphaviruses, the other genus of the family togaviridae [ , ] . cells infected with alphaviruses like semliki forest virus (sfv), sindbis virus and western equine encephalitis virus (weev) contain large cpvs with a diameter ranging between and nm. the inner surface of these cpvs is covered with small invaginations or spherules that originate at the plasma membrane [ ] [ ] [ ] ( figure j ). these spherules are comprised of a single membrane forming a vesicle with a diameter of ~ nm. in addition, the spherules were shown to be the site of viral rna synthesis as deduced from metabolic labeling and detection by em [ , , ] . importantly, the inside of the spherule is connected to the cytoplasm by a pore with a diameter of - nm. the spherules are formed at the plasma membrane by the concerted action of the viral nonstructural proteins (nsp -nsp ) and genomic viral rna [ ] . furthermore, froshauer et al. [ ] showed that the cpvs that contain the spherules possess endosomal and lysosomal markers. time course studies revealed that the spherules of sfv undergo an unprecedented large-scale movement between cellular compartments [ ] . the spherules first form as blebs (exvaginations) at the plasma membrane. then, they are internalized by an endocytic process requiring a functional actin-myosin network. the spherules therefore represent an unusual type of endocytic cargo. after endocytosis, spherule-containing vesicles, namely cpvs-i fuse with acidic endosomes and move along microtubules. this leads to the formation of a very stable compartment, where the spherules accumulate as invaginations on the outer surface of unusually large, acidic vacuoles localized in the pericentriolar region [ ] . members of the genus norovirus (novs, family caliciviridae) are major agents of acute gastroenteritis [ ] . ultrastructural examination of murine norovirus (mnv- )-infected cells revealed a striking change in their overall morphology and intracellular organization [ ] . structures resembling virus particles were observed within or next to single-or double-membrane vesicles in the cytoplasm. the vesiculated areas increase in size with time and by hpi, large numbers of these vesicles and viral particles occupy most of the cytoplasm and displace the nucleus ( figure h ). in addition, a complete rearrangement of the er and loss of intact golgi apparatus was observed. both dsrna and mnv- nonstructural protein , the rna dependent rna polymerase, localize to the limiting membrane of individual vesicle clusters by immuno-em [ ] . immunofluorescence-based double-labeling showed that mnv- appears to recruit membranes derived from multiple cellular organelles and/or compartments: the er, trans-golgi apparatus and endosomes. however, despite extensive efforts, human norovirus cannot be grown in cultured cells [ ] . thus, detailed studies have not been possible, but it is assumed that replication structures are similar to those of mnv- . feline calicivirus (fcv), a member of the genus vesivirus within this family, is a major agent of respiratory disease in cats, which replication originates also membranous rearrangements and vesicles [ ] . brome mosaic virus (bmv, family bromoviridae) generates its replication factory by hijacking er membranes, similar to what has been described for other plant viruses like tobacco mosaic virus (tmv, family virgaviridae) [ ] , tobacco echt virus (tev, family potyviridae) [ ] and red clover necrosis mosaic virus (family tombusviridae) [ ] . however, other plant viruses such as alfalfa mosaic virus (amv) [ ] and cucumber mosaic virus (cmv), both belonging to the family bromoviridae, and turnip yellow mosaic virus (tymv, family tymoviridae) [ ] anchor their replication sites on chloroplasts. cucumber necrosis virus (cnv), family tombusviridae, utilizes peroxisomal membranes as replication platforms [ ] , while other plant viruses replicate on the surface of mitochondria [ ] . although the d architecture of these membranous replication sites remains largely unknown, their characteristics are strikingly similar to those for fhv (family nodaviridae). best studied is bmv that induces spherules, of similar size as the insect nodavirus fhv, in the er close to the nucleus, where viral rna synthesis and viral replication proteins are localized [ ] [ ] [ ] . in the case of beet yellows closterovirus (byv, family closteroviridae), tem of infected plant cells revealed the formation of ~ nm-diameter dmvs and multivesicular complexes (single-membrane vesicles surrounded by a common membrane) ( figure e ) [ ] . these multivesicular complexes often reside next to stacks of aligned filamentous byv particles [ , ] and resemble the dmvs and vps produced by nidoviruses and flaviviruses. several byv replication-associated proteins (l-pcp, mtr and hel) colocalize with dmv and vp membranes, supporting the role of these structures as replications platforms [ , ] . the membranes in closterovirus dmvs and vps are likely to be derived from er for members of the genus crinivirus [ ] or mitochondria in case of ampelovirus [ , ] . whether these structures are -closed‖ or -necked‖ remain unknown. based on amino acid sequence homologies of their rna-dependent rna polymerases, (+) rna viruses have been classified into three large supergroups [ , , ] : supergroup i (picornavirata or picorna-like group), including picorna-, corona-, arteri-and nodaviruses; supergroup ii (flavivirata or the flavi-like group), including tombus-, diantho-, pesti-, hepaci-and flaviviruses as well as single-strand rna bacteriophages; supergroup iii (rubivirata or the alpha-like group), including tobamo-, hordei-, alpha-and rubiviruses as well as hepatitis e virus (hev). these higher-order taxonomic units encompass diverse viruses infecting different hosts from almost all kingdoms of life. as discussed earlier [ ] , amongst these viruses two main architectures of remodeled membranes (morphotypes) can be found that may reflect two alternative strategies to induce the membranous microenvironments required to allow virus replication (summarized in table ). the first morphotype involves the formation of negatively curved membranes, initiated by invaginations of the pre-existing membrane bilayer and giving rise to spherules, vesicles or vacuoles towards the lumen of the targeted cell organelle. these structures have been identified in a broad range of mammalian, plant and insect cells infected with viruses belonging to supergroups ii and iii. the second strategy involves the formation of membranes with positive curvature, giving rise to double-membrane structures that are the predominant characteristic of the replication factories of the picorna-like virus supergroup. the conservation of these two sorts of morphotypes in distantly related viruses supports the assumption of an evolutionary conserved mechanism. a striking finding in this regard was the observation that hcv, despite belonging to the flavi-like supergroup, induces dmvs whereas flaviviruses induce the formation of negatively curved membranes. to our current knowledge, hcv is the only member of the family flaviviridae inducing the formation of membrane structures with positive curvature, suggesting that hcv might share common host cell pathways to induce membranous replication compartments with distantly related viruses such as corona-, arteri-, picorna-, calivi-or closteroviruses (table ) . however, it still remains to be elucidated whether other members of the family flaviviridae, belonging to the genera pestivirus and pegivirus, also utilize a picorna-like membrane remodeling strategy. a common feature associated with the spherule/vesicle/vacuole/-type of rearranged membranes is their size ( - nm diameter) and the presence of a pore connecting the interior of the vesicle with the cytoplasm [ , , , ] . since rna replication occurs in the vesicle interior, the pore allows exchange of nucleotides and rna products with the cytoplasm. the size of the pore is variable, ranging from . nm in case of ibv-induced spherules to ~ nm in case of membrane invaginations induced by flaviviruses. in contrast, in the majority of dmvs no such channel or pore has been detected. nevertheless, as exemplified with nidoviruses, the inner compartments enclosed by interconnected dmvs contain the bulk of dsrna [ ] , and in some cases they depict an electron-dense core assumed to correspond to viral rna (ibv and eav) [ , ] . although this represents a functional enigma in terms of rna synthesis and transport, the presence of dsrna in the dmv interior does not necessarily indicate active rna replication. assuming a temporal regulation, it is possible that dmvs might be sites of rna synthesis as long as they are linked to the cytoplasm, but replication would stop upon closure of the vesicles. yet, another strategy appears to be used by enteroviruses, where active rna replication has been detected on the cytosolic side of the membranous structures [ , ] , consistent with the membrane topology of the nonstructural proteins catalyzing rna replication [ ] [ ] [ ] . as described above, studies conducted with picornaviruses revealed that the exponential phase of viral rna synthesis coincides with the accumulation of single-membrane tubules [ , ] . importantly, pulse-radiolabeling experiments localized sites of active rna replication to the outer surface of single-membrane tubules [ ] and isolation of the membranous replication factories and their subsequent visualization by em revealed that they form rosette-like structures composed of virus-induced cytoplasmic vesicles [ ] . rna replication is thought to occur at sites where the vesicles cluster, whereas rna translation probably takes place on the exposed periphery of the vesicles. this raises the question, what the role of dmvs in the replication cycle of picornaviruses might be. it is possible that dmvs either support rna synthesis or serve as rna storage sites (especially in case of closed dmvs). in this manner, dmvs might be involved in regulating viral rna replication: by complete sealing of the viral replicase inside the vesicle, it would be inactive, thus regulating overall rna copy number in the infected cell. alternatively, dmvs might be an epiphenomenon, resulting from the over-expression of membrane-active proteins that accumulate especially during the late stages of infection. the mechanism responsible for dmv formation is not clear. in case of picornaviruses, it is thought that single-membrane structures are the precursors of dmvs [ , ] . nevertheless, dmv formation might also involve the autophagy machinery, or at least several components thereof, by a process analogous to the formation of autophagic vacuoles [ ] . this hypothesis is supported by the morphological resemblance of dmvs and autophagosomes. it has been shown that the inhibition or stimulation of autophagy results in a modest inhibition or stimulation of poliovirus and coxsackie b virus yield, respectively, and there are also data supporting the involvement of autophagy in the replication of rhinovirus and [ , ] . however, brabec-zaruba et al. [ ] reported that replication of rhinovirus was insensitive to pharmacological manipulation of autophagy and did not induce detectable modification of lc . this discrepancy might be due to the use of different cell types and experimental conditions. the mechanism of dmv formation in case of hcv and coronaviruses is also unclear. biochemical analysis of isolated host cell membranes associated with hcv rna and proteins identified markers of the autophagy machinery, including lc -ii, the lipidated form of lc (lc -ii) that is generated upon activation of the autophagy machinery [ ] . however, the role of autophagy in the hcv replication cycle is also a matter of controversy. for instance, immunolabeling did not identify lc -ii at those sites where nonstructural proteins accumulate [ ] . moreover, different roles of autophagy for the hcv replication cycle have been proposed. these include a role of autophagy in hcv rna translation [ ] , initiation of rna replication [ , ] , production of infectious virus particles [ ] or suppression of the innate antiviral defense [ , ] . to clarify these discrepancies, future studies should combine biochemical and cell biological approaches with ultrastructural analyses. the autophagy machinery might also be involved in the formation of virus-induced membrane invaginations/spherules. for instance, lee and coworkers provided evidence that denv infection enhances autophagolysosome formation and that inhibition of the autophagy machinery by -methyladenine ( -ma) reduces denv particle production [ ] . however, the effects were moderate, arguing that this pathway may contribute to denv replication to only a minor extent. moreover, autophagy includes membrane wrapping, leading to double-membrane compartments involved in lysosomal degradation whereas denv-induced vesicles are invaginations. finally, immunolabeling experiments failed to detect lamp- at these vesicles [ ] , arguing against the involvement of lysosomes in the formation of the denv replication vesicles. it remains to be determined whether autophagy is actively induced by these viruses to provide a compartment favoring replication or induced as a bystander defense against infection leading to degradation of the replicase proteins [ ] . another membrane compartment frequently induced by (+) rna viruses are convoluted membranes (cms) that were observed e.g., in sars-cov-, mhv-, wnv-or denv-infected cells [ , , , , , , ] . morphologically, cms resemble smooth er membranes, lack ribosomes and in case of denv are induced by the sole expression of ns a [ , ] . cms are often associated with late stages of infection, suggesting that dmv formation might precede the development of cms. in sars-cov-infected cells, dmvs appear to be connected with cms [ ] , while in mhv-infected cells no such connections have been observed [ ] . the role of cms for the viral replication cycle is not well understood. in case of wnv kun , cms are supposed to be the site of polyprotein processing [ , ] . this conclusion is based primarily on the strong immunolabeling for ns b and ns and the absence of ns and ns b. since polyprotein cleavage occurs co-translationally and thus, should happen at the rer, this model would require the formation of rather stable processing intermediates that are transferred from the rer to the cms where further cleavage would occur. alternatively, at least in case of denv, cms might represent a storage site for proteins and lipids involved in viral replication that can be recruited to vesicles upon demand. the fact that cms are physically linked with er-containing invaginations and contain ns would be consistent with this assumption [ ] . along these lines, the fact that insects are cholesterol auxotrophs and lack several enzymes in the cholesterol biosynthesis pathway [ ] , suggests that cholesterol might be a key component of cm structures, which would explain their absence in infected insect cells [ ] . viral replication complexes are targeted to the respective membranous organelle primarily by nonstructural (ns) proteins rather than viral rna [ ] . these ns proteins seem to have some specificity in recognizing organelle subpopulations and often contain multiple hydrophobic domains implicated in membrane targeting and rearrangement. the molecular mechanisms orchestrating the formation of these complex structures are still poorly understood, but it is clear that ns proteins, often working in a concerted action, are key players in replication factory biogenesis. one well-studied example among the single-membrane vesicle inducers is bmv where it was shown that the sole expression of the ns protein a is sufficient to induce the formation of single-membrane spherules resembling the ones observed in infected cells. these spherules had a diameter of - nm, resided in the er lumen and were shown to be the site of viral rna synthesis [ ] . in case of the insect nodavirus fhv, protein a and replication competent rna were required for induction of the spherules. expression of protein a alone induced only -zippering‖ of the surfaces of adjacent mitochondria, but did not induce spherules. thus, protein a is necessary, but not sufficient for spherule formation. moreover, spherules were not formed when replication-competent fhv rna templates were expressed with a protein a mutant lacking polymerase activity or when wild-type protein a was expressed with a replication-incompetent fhv rna template. thus, the membranous fhv replication compartment requires both a viral protein and active rna synthesis [ ] . feline calicivirus (fcv) infection results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. expression of individual fcv nonstructural proteins revealed that p induces significant reorganization of the er into large, fenestrated membrane networks, resembling the structures found in infected cells [ ] . moreover, expression of p and p , two additional fcv ns proteins, induced extensive reorganization of the er and the nuclear envelope suggesting that the er is the primary source of the membranous replication factory [ ] . flavivirus membrane rearrangements are mainly induced by ns a, as suggested by recent studies with wnv kun and denv [ , ] , but it is unknown whether the same applies to ns a of tbev. in case of denv, ns a is thought to contain a central peripheral membrane domain that intercalates into the luminal leaflet of the er membrane [ ] . it is tempting to speculate that ns a oligomers [ ] might dilate the luminal leaflet, resulting in membrane invaginations towards the er lumen. however, the sole expression of ns a is not sufficient to induce er membrane invaginations that have been detected in infected cells. instead, expression of ns a lacking the c-terminal k fragment (corresponding to fully processed ns a) induced er membrane rearrangements reminiscent of cms, whereas unprocessed ns a/ k did not induce membrane alterations [ ] . these results provide strong evidence that processing at the ns a- k site is required for the induction of membrane alterations. the critical role of polyprotein cleavage for induction of membrane rearrangements is supported by studies conducted with wnv kun . there it was shown that a regulated cleavage of a ns a/ k/ b precursor by the viral ns b/ protease is needed for induction of membrane rearrangements [ ] . however, the same study reported that expression of full-length (uncleaved) wnv kun ns a/ k led to membrane alterations similar to those induced in infected cells whereas the k fragment impaired the ability of ns a to induce membrane rearrangements. whether this reflects a biological difference between wnv kun and denv ns a or is due to the use of alternative experimental approaches remains to be determined. one of the most fascinating mechanisms employed by (+) rna viruses to induce their replication factories is used by sfv. it was shown that the spherules of sfv arise by blebbing at the surface of the plasma membrane [ ] . these blebs are internalized and after fusion with lysosomes give rise to large cytoplasmic vacuoles. formation of these membrane alterations requires the viral protein nsp , which has several functions. it has guanine- -methyltransferase and guanylyltransferase activities and thus is critically involved in capping of the viral rnas [ ] [ ] [ ] , but at the same time has affinity to lipids [ ] . in fact, of the four ns proteins of sfv, only nsp has affinity for membranes, and when expressed alone, it is specifically targeted to the inner surface of the plasma membrane [ ] . nsp is a monotopic membrane protein and its affinity for membranes is dictated by an amphipathic α-helix, located in the central region of the protein [ , ] . nsp has a specific affinity for negatively charged phospholipids, which might account for its prevalent localization to the plasma membrane, where such lipids are enriched. membrane binding of nsp via its amphipathic α-helix is essential for alphavirus replication [ ] . however, nsp is not sufficient for cytoplasmic vacuole formation. for instance, it was found that nsp contributes to the transport of the replicase polyprotein from the plasma membrane to the surface of endosomes [ ] . these results indicate that nsp has to cooperate with other viral and cellular factors to allow formation of the cytoplasmic vacuoles. furthermore, in a recent study kallio et al. [ ] have shown that the size of the spherule is dependent on the length of the rna template, in contrast to what has been observed for fhv, another spherule-inducer [ ] . these results indicate that in addition to the ns proteins, the viral rna template itself critically determines the morphology of the membranous vesicles. in order to induce the variety of membrane alterations observed in cvb -infected cells ( figure f ), several membrane-remodeling mechanisms are required: induction of membrane curvature, membrane fusion and membrane-membrane interactions [ ] . these rearrangements require the enteroviral proteins bc and a; their coexpression generates er membrane-derived structures mimicking those observed during viral infection [ ] . importantly, b and c both contain an amphipathic α-helix [ ] [ ] [ ] , a well-known curvature-inducing motif [ ] . along the same lines, fmdv b and bc locate to the er when expressed on their own and cause a swelling of er cisternae [ ] . in case of hcv, we recently found that a concerted action of ns / a, ns b, ns a and ns b is required to generate the membranous web. furthermore, all these replicase components were capable of inducing membrane vesiculation with ns a having the highest potential to trigger membrane curvature. importantly, some of these ns a-induced structures corresponded to dmvs [ ] . in addition, ns b also plays an important role in triggering rearrangements of intracellular membranes [ ] . ns b is an integral membrane protein containing two n-terminal amphipathic α-helices, a highly hydrophobic central core domain composed of four putative transmembrane segments, and a highly conserved c-terminal domain that is thought to harbor two α-helices (reviewed in [ ] ). a recent study has demonstrated that ns b oligomerizes through multiple conserved determinants and that oligomerization appears to be required for membranous web induction [ ] . indeed, mutations affecting the highly conserved c-terminal domain impairing ns b self-interaction resulted in the formation of aberrant dmvs arguing for a central role of ns b in formation of functional replication compartments [ ] . studies on arteriviruses revealed that the sole expression of eav nsp and nsp is sufficient to induce membrane structures similar to those generated during eav infection [ ] . mutations within nsp , which is a tetra-spanning integral membrane protein, alter membrane rearrangements, highlighting the importance of this protein for the biogenesis of eav-induced dmvs [ ] . in case of sars-cov, nsp , nsp and nsp were found to be sufficient to induce the formation of dmvs that are similar to those observed in sars-cov-infected cells [ ] . these dmvs were, however, smaller in diameter, suggesting a role for other viral proteins or the presence of viral rna in determining the dmv morphology. importantly, em analysis of nsp mutants that are impaired in rna replication and virus growth, revealed an aberrant morphology of dmvs as well as an increased prevalence of cms [ ] . another important viral protein involved in inducing the sars-cov membranous replication factory is nsp , which is predicted to contain seven transmembrane segments and a hydrophilic cytoplasmic domain [ ] . nsp was shown to induce vesicles containing atg and lc -ii as well as phosphatidylinositol- -phosphate, thus sharing many features with omegasomes, which are omega-shaped membrane compartments that are formed during activation of autophagy [ ] . this result suggests that autophagy might contribute to the formation of the membranous replication site of sars-cov. in the last couple of years, our knowledge of the architecture of the replication factories induced by (+) rna viruses has increased substantially. this is primarily due to the more widespread use of et and other high-resolution imaging methods. nevertheless, our knowledge is still rather restricted to descriptions of the morphologies of these complex structures, whereas our understanding of their biogenesis in most cases is very rudimentary. more efforts are required to elucidate the role of the viral proteins in the formation of the replication vesicles, to identify the involved cellular components and the mechanisms used by these proteins to subvert and exploit cellular pathways to establish membranous replication factories. this includes determination of the d structure of involved (viral) proteins as well as evaluation of host cell factors and lipids contributing to biogenesis and activity of the replication compartment. in addition, further studies are needed to understand how viruses utilize these compartments to coordinate the different steps of their life cycle (replication, assembly and release) in space and time to achieve efficient replication. work in the authors' laboratory was supported by the deutsche forschungsgemeinschaft (sonder-forschungsbereich the gb viruses: a review and proposed classification of gbv-a, gbv-c (hgv), and gbv-d in 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nsp proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate the authors are very grateful to erik j. snijder (leiden university medical center, leiden, the netherlands) and kè vin knoops (european molecular biology laboratory, grenoble, france) for providing the unpublished pictures depicted in figure b and d and to montserrat bá rcena (leiden university medical center, leiden, the netherlands) and tero ahola (university of helsinki, helsinki, finland) for providing the unpublished pictures depicted in figure f and j, respectively. figures a, c , e, g, h and i are reproduced with permission from [ ] , [ ] , [ ] , [ ] , [ ] and [ ] , respectively.we also would like to thank jason m. mackenzie the authors declare no conflict of interest. key: cord- -spxgox authors: yu, jianhai; li, xujuan; he, xiaoen; liu, xuling; zhong, zhicheng; xie, qian; zhu, li; jia, fengyun; mao, yingxue; chen, zongqiu; wen, ying; ma, danjuan; yu, linzhong; zhang, bao; zhao, wei; xiao, weiwei title: epidemiological and evolutionary analysis of dengue- virus detected in guangdong during : recycling of old and formation of new lineages date: - - journal: am j trop med hyg doi: . /ajtmh. - sha: doc_id: cord_uid: spxgox the incidence of dengue is increasing in guangdong, china, with the largest outbreak to date in . widespread awareness of epidemiological and molecular characteristics of the dengue virus (denv) is required. in , we isolated the virus from patients and sequenced its genome. the sequences of denv isolated from guangdong and other countries screened since were studied to establish molecular evolutionary databases along with epidemiological data to explore its epidemiological, phylogenetic, and molecular characteristics. causes underlying the occurrence of the dengue epidemic included importation and localization of the virus. the number of indigenous cases significantly exceeded that of imported cases. dengue virus is the most important serotype and caused the long-term epidemic locally. based on the data available since , denv was divided into three genotypes (i, iv, and v). only genotypes i and v were detected in . in , an epidemic involving old lineages of denv genotype v occurred after years of silence. the genotype was previously detected from to . genotype i, which caused recent epidemics, demonstrated a continuation of new lineages, and a predictive pattern of molecular evolution since among the four lineages was present. the denv isolated from guangdong was closely related to those causing large-scale epidemics in neighboring countries, suggesting the possibility of its import from these countries. the lack of sufficient epidemiological data and evidence on the local mosquito-borne denv emphasizes the importance of studying the molecular evolutionary features and establishing a well-established phylogenetic tree for dengue prevention and control in guangdong. dengue virus (denv), a mosquito-borne flavivirus, is transmitted primarily by aedes aegypti and aedes albopictus, causing an acute infectious disease named dengue fever (df), which gives rise to public health problems in tropical and subtropical regions worldwide, such as china, singapore, and brazil. [ ] [ ] [ ] with the recent revision of the who dengue classification scheme, dengue patients are classified as having either dengue or severe dengue. the former refers to patients who recover without major complications, whereas the latter points to those who have any of the following conditions: plasma leakage resulting in shock, accumulation of serosal fluid sufficient to cause respiratory distress, or both; severe bleeding; and severe organ impairment. before , only nine countries had experienced severe dengue epidemics, but the disease is now endemic in more than countries. in recent decades, the spreading disease causes rapid upsurge in morbidity. one recent estimate indicates million dengue infections per year, of which million manifest clinically. , hence, wasting a lot of health resources and causing the growing global burden of disease, df is regarded as the most widely distributed vector-borne disease with the highest morbidity and great harm. the genome of denv is a single-stranded, positive-sense rna, and its single open reading frame encodes a polyprotein consisting of three structural proteins, which are as follows: the capsid, membrane-associated, and envelope (e) proteins. in addition, seven nonstructural proteins, ns , ns a, ns b, ns , ns a, ns b, and ns , are also present in its structure. four distinct denv serotypes (denv , denv , denv , and denv ) have been identified, and the extensive diversity within denv enables it to recognize different genotypes, such as genotype i (southeast asia and east africa), genotype ii (thailand), genotype iii (malaysia), genotype iv (south pacific), and genotype v (america/africa). dengue genotypes are phylogenetically distinct clusters of viruses, often associated with specific geographical regions, that are linked to epidemics of varying intensities and disease severity. , phylogeny is a science that makes use of a set of relationships among groups of genes or organisms and reflects their evolutionary history. the maximum likelihood method is used to describe and analyze biological sequences. alignment of the nucleic acid sequences of the denv followed by its phylogenetic analysis and subsequent generation of phylogenetic trees revealed information regarding its genetic evolution and epidemiology of the disease worldwide. [ ] [ ] [ ] the e protein of the denv is responsible for its tropism and virulence. the gene encoding the e protein has demonstrated its usefulness for decades for the phylogenetic reconstruction of the denv. thus, complete analysis of the coding region can help assign the correct denv genotype and infer the relationships within genotypes and lineages accurately. therefore, the e protein provides adequate resolution to characterize genetic relationship and evolution of the denv. guangdong province is located in the southern mainland of china and experiences tropical and subtropical monsoon climates with a hot and rainy environment that supports breeding of mosquitos, leading to epidemics and very high incidences of df. , the first outbreak of dengue in the foshan city of guangdong province occurred in , after which periodic infections and transmission of all four serotypes of dengue have occurred in the past years. the denv found in these areas may have different origins. there were two denv outbreaks in foshan in and , respectively. phylogenetic analysis revealed that isolates from the epidemic and denv from the epidemic and the epidemic were closely related to those from the epidemic in thailand, epidemic in indonesia, and the epidemic in the philippines, respectively. since , however, denv has been mainly isolated from the infected cases, and its continued existence in guangdong province indicated that endemic infectious agents of dengue may be circulating locally. sequence analysis of the viruses causing the epidemics at different time points revealed that the isolates were closely related to each other, implying that denv had probably circulated locally and caused the epidemics. since , all four serotypes were derived from autonomous patients from different outbreak localities in guangdong province. in , a total of , cases of dengue were reported, which exceeded the total number of cases reported over the previous years. although three serotypes of the denv (denv , denv , and denv ) were identified, denv was found to be the major causative agent responsible for . % of all , laboratory-confirmed cases diagnosed during this outbreak. a recent study revealed that the detected sequences belonged to viruses of multiple origins, but the strain isolated in possibly originated from the isolates of . it can be reasonably speculated that the infectious agents of denv from the endemic, which were circulating locally, played a crucial role in causing the dengue epidemic in guangdong province. however, the data mentioned previously are from the studies based on the outbreaks of the particular year and have the limitation of space and time. as a result, comprehensive evaluation of the epidemiological situation and molecular evolution of the viral agents is of significance to warn against their risks and establish preventive and control measures for df. based on denv isolated from the outbreak in , we systematically collected the e protein gene from to from genbank. with the epidemiological data since supplied by the guangdong provincial cdc, we studied phylogenetics, molecular characteristics, and epidemiology to strengthen the foundational research of denv for the prevention of large-scale dengue epidemics, providing preventive and control measures of df with important evidence. ethics statement. as this research involved human blood, the aims of our study were explained to all the dengue patients involved (all were adults) and all provided written informed consent. the collection methods of clinical samples and epidemiological data were reviewed and approved by the institutional ethics review board of southern medical university and were carried out in accordance with the approved guidelines. samples were selected randomly based on the laboratory diagnosis and clinical signs. sample collection and epidemiological data. dengue virus rna samples (n = ) were obtained from the guangdong provincial maternal and child care service. all samples were extracted from patients suspected of dengue and were confirmed by reverse transcription-polymerase chain reaction (rt-pcr) using specific primers. the steps included an initial denaturation ( °c, minutes); cycles of denaturation added to make a comprehensive evaluation of the epidemiological situation and molecular evolution in the large dengue outbreak in guangdong. at the same time, e gene sequences of denv since , comprising sequences from guangdong and , from other countries, were downloaded from genbank. after excluding several sequences with uncertain epidemiological data, representative epidemic strains in every lineage were screened using phylogenetic methods. since then, a molecular evolution database for the denv e gene in guangdong and other countries has been established. based on representative strains of the e gene in lineages of the outbreak, as well as the molecular evolution database, we analyzed molecular characterization and possibility of local circulation for denv since in guangdong. three-dimensional ( d) structure prediction of denv protein e and molecular docking with -kda glucoseregulated protein (grp ). amino acid (aa) sequences of denv protein e were translated by using dnastar. protein d structures were simulated by discovery studio . (ds . ) (http://accelrys.com/products/collaborativescience/biovia-discovery-studio/) through the homology modeling method based on the modeler program. the optimal protein d model was selected by combining the probability density function and discrete optimized protein energy, and the reliability of the model was evaluated using the ramachandran plot and profile- d. the optimal protein structure model was used to perform protein docking calculations with the grp protein (pdb number: ldp) using the zdock algorithm in ds . , and rdock was used to further optimize the docking configuration to minimize energy. finally, we analyzed aa sites in the binding interface of the optimal docking model. epidemiological findings. since , epidemics of dengue in guangdong have been characterized by periodic outbreaks, the coexistence of importation and localization, and the significantly increased number of indigenous cases compared with that of imported cases ( figure a and c). the incidence peaked in and . especially, during the large outbreak in , a total of , cases of dengue were reported. in , , cases were reported, with a continuous rising trend in comparison to cases in ( figure a ). more seriously, as of november , , a total of , cases were reported, an increase of . % over the same period in . among them, cases were imported, which was . % higher than those in the same period in ; , cases were local cases, which was . % higher than those in the same period in . based on the map of guangdong province, we diagrammed the distribution of the cumulative number of cases since . this showed that guangzhou, foshan, zhongshan, and chaozhou were the main epidemic areas. the trends of the epidemics were anastomotic, with periods of fluctuation in guangdong province ( figure ). after , the epidemic trend featured the gradual coexistence of various serotypes, with up to four serotypes emerging in recent years. each denv serotype was identified. dengue virus was the major cause of the outbreak of , and it continued to be detected in guangdong province as the primary serotype, except in . it is worth noting that the proportion of dengue virus in the total cases also gradually increased ( figure d ). data of samples were used to analyze the overall epidemiology. all patients who provided the samples had mild dengue and had no travel history (table ) . generally, the collection date distribution was concentrated in september and october ( figure b ). the endemic areas were primarily located in guangzhou, followed by zhongshan and chaozhou, accounting for . % of all samples. heyuan, huizhou, and shanwei were included in the collection areas ( figure e ). dengue virus was the major causative agent of this outbreak. the serotype detected through type-specific primers (table ) was denv , which is similar to the results from guangdong cdc that of df cases involved, cases were due to denv infection ( figure d , table ). phylogenetic analysis of denv outbreak in . all e genes were sequenced to construct phylogenetic trees. the dengue outbreak in involved asian genotype i and american/african genotype v. phylogenetic analysis showed that most of the sequences were clustered into a unified clade whose distribution differed in each city. among them, e gene sequences were identified as genotype v with . - % similarity. they clustered into the same clade, which was closely related to the denv sequences in malaysia, singapore, and india and evolved into a lineage of genotype v. during - , this lineage was involved in a co-epidemic in guangdong province, malaysia, and pakistan, with a continuous high-level local prevalence especially in singapore. significantly, this lineage was detected in six cities in our collection; sequences from shanwei, chaozhou, and huizhou all belonged to genotype v. in addition, another nine sequences of the e gene were identified as genotype i, with two different lineages. lineage i involved eight sequences in our collection and other sequences in singapore and thailand in recent years, with a . - % similarity. only one sequence in guangzhou contributed to lineage ii, which had been involved in local epidemics in malaysia, singapore, and indonesia for many years ( figure ). phylogenetic analysis of guangdong since . we downloaded e gene sequences since , comprising from guangdong and from other countries. databases of e gene molecular evolution were created via a screening process based on epidemiologic and phylogenetic methods. among them, strains in guangdong constituted the local database, whereas strains in other countries made up the imported database. based on the evolutionary lineage of denv detected in in guangdong, seven sequences (p , p , p , p , p , p , and p ) were added to the local we endeavored to clarify the relationship between guangdong and other countries concerning denv outbreaks. for this, we illustrated the origin of molecular evolution lineages and the potential of in situ evolution. representative strains from each lineage were added to the imported database for phylogenetic evolution analysis. in our collection, the molecular evolutionary lineage of the imported database proved to be highly consistent with the local database. the only distinctions were that the evolution time of lineages i and ii changed in genotype i, and denv in guangzhou, , formed a clade alone, named clade a ( figure a ). genotype i, which has extensively circulated in guangdong and has been detected in each year of the epidemics, displayed some differences with epidemic countries surrounding china. after , lineage i was not found in guangdong. lineage i was very homologous with lineages in singapore, thailand, and sri lanka in the same epidemic years. however, lineage i spread in neighboring countries, such as malaysia, myanmar, and new guinea, which contributed to cyclic epidemics ( figure b ). lineage ii in guangdong presented a more complicated epidemic situation in countries around china: for example, lineage ii extensively circulated in vietnam, singapore, thailand, malaysia, and cambodia, with vietnam and cambodia being hot spots. clade a only formed a cluster with denv in vietnam ( figure b ). recently, lineage iii in guangdong showed a complicated epidemic situation in surrounding countries as well. its sequence was related to sequences in thailand, laos, singapore, malaysia, and elsewhere. in addition, it was related to the co-epidemic in australia that occurred in and ( figure b ). there were few countries with lineage iv whose epidemic originated from the sequences in . only malaysia, singapore, and especially indonesia shared clustering in denv with long-term cyclic epidemics ( figure b ). in addition, the occurrence of genotypes iv and v was inconsistent in different years. genotype iv sequences in shared extensive homology with those in indonesia, whereasthe strains in were related to those in the philippines with long-term cyclic epidemics ( figure c ). genotype v was related to the strains in india and maldives and was divided into clade and and clade and ; the sequences shared long-term coepidemics with india and singapore, respectively ( figure d ). protein conformational changes caused by mutation of e protein-specific aas between denv genotypes i and v. a total of eight uniform aa substitutions in the ectodomain of the e protein were mainly concentrated in domains i (five substitutions) (di) and iii (two substitutions) (diii) between genotypes i and v, whereas domain ii (dii) has only one. among them, two substitutions caused the protein's secondary structure to change from β-sheet to coil, e (i → l) in dii and e (t → s) in di, whereas only e (d → n), e (s → t), e (t → i), and e (t → s) of di were observed on the d conformation surface of the protein ( figure a and b, table ). meanwhile, e (t → i) and e (t → s) of di were also found in the binding interface between the e protein of genotypes i and grp , and other substitutions were not observed in either serotypes ( figure e ). in the docking model with the grp protein, all three domains of genotype i were involved, whereas genotype v had only di and diii. we found three identical docking sites of diii in the binding interface of the two genotypes: a- p, t- d, and e- k ( figure c and d) . the distribution characteristic of denv in guangdong was determined from its long-term epidemic history. dengue virus was first detected in zhongshan, guangdong province. since then, denv epidemics have occurred sporadically in specific regions over - years. until , denv was the leading serotype, which triggered massive outbreaks in guangzhou. thereafter, denv was circulated continuously across multiple geographies in guangdong, and the isolated strains branched into several stable molecular evolutionary clades. , since , it has spread to different provinces of guangzhou, and imported cases were no longer primary. at indicated that although three denv serotypes (denv , denv , and denv ) were identified, denv was the major causative agent of this outbreak which had circulated continuously in multiple geographies. , so far, the results mentioned were consistent with those of our study. the term "lineage" has been used to denote the viruses clustered in clades at a taxonomic level beneath the genotype. the appearance, change, and reappearance of specific lineages are closely linked to the transmission of those viruses. [ ] [ ] [ ] here, strains of viruses differing in their e genes were obtained, of which nine and strains belonged to genotypes i and v, respectively. unlike the strains of genotype i which were linked to two lineages, each strain in genotype v branched into the same clade, forming a stable lineage. however, several lineages from different origins were responsible for the denv outbreak in . moreover, strains belonging to genotype v were detected in six areas evaluated in this study. all strains from shanwei, chaozhou, and huizhou gathered together also belonged to this genotype, illustrating that the same lineage was prevalent in multiple regions. coincidentally, some epidemiological data indicate that denv was restricted only to the local epidemics since , a phenomenon which was reported by guo et al. overall, the aforementioned evidence strongly opposes the conventional belief that df in guangdong was completely imported. lee et al. used the term "in situ evolution" to characterize denv in singapore through phylogenetic analysis. results of the analysis explained the correlation between its genetic and evolutionary aspects, suggesting that denv had lurked locally and reappeared after some time. rajarethinam et al. described that the dengue in singapore from to demonstrated cyclic epidemic patterns dominated by serotypes and . however, this in situ evolution, demonstrated by the step-ladder pattern of branching within each clade over time, has not been observed in guangdong since . another possible transmission and evolution pattern of denv broke out in parts (switch of the lineage), followed by silence (change of the lineage), and was then prevalent on a large scale (continuation of the lineage), achieving continuous evolution of a new lineage and the silent circulation of old lineages. in , when denv first became the leading serotype, it was due to a "switch" from the lineages in genotype i, which branched into lineages i-iii at the beginning. , from to , it was observed that the prevalence of denv was low and it frequently interchanged between the three lineages, leading to the occurrence of epidemics caused by denv belonging to each lineage. , however, since , the severity of dengue is increasing because of a continued epidemic caused by lineage iii. in , a switch in lineage iv, which was similar to the evolutionary process of lineage iii, was observed. the lineage of genotype iv was first formed in , which reappeared in , and was never detected in recent years. , it is reported that genotype v had circulated continuously from to , followed by an intermittent silence and further reappearance in . this type of molecular evolution was similar to the silent circulation prevalent figure . phylogenetic analysis of denv detected from the outbreak in guangdong. fifty-six envelope gene sequences were isolated in our study. these and reference sequences from genbank were used to construct the phylogenetic tree. asian genotype i and american/african genotype v were involved in this outbreak. forty-seven sequences identified as genotype v clustered into the same clade, whereas genotype i was divided into lineages i and ii. thirteen sequences from shanwei (blue circles), chaozhou (green circles), and huizhou (violet circles) all belonged to genotype v. this figure appears in color at www.ajtmh.org. in indonesia, brazil, etc., , , indicating that the same might happen in guangdong. castonguay-vanier et al. also described the active circulation of denv in laos and the concurrent multiple introductions of new strains from neighboring countries, whereas moore et al. claimed that the cocirculation of denv - in png provides molecular evidence of its endemic transmission. meanwhile, compared with denv , denv , and denv , denv , which is the leading serotype in guangdong, rarely caused severe df. [ ] [ ] [ ] in addition, because guangdong is the most densely populated province in southern china, which is surrounded by a large number of dengue-endemic countries and has a subtropical climate providing optimal environmental, social, and biological conditions for mosquito breeding and reproduction, a detailed evaluation of the current dengue epidemic is significant. nevertheless, there is still a lack of evidence on the local mosquito vector regarding its harboring of denv during epidemic and nonepidemic periods which can support the evidences of its molecular evolution revealed by the phylogenetic analysis. three genotypes (i, iv, and v) in denv from a variety of origins were identified. data obtained from the characterization of local or imported viruses with similarity in lineages found in guangdong demonstrated that these viruses, which were identified based on their molecular evolutionary analysis, were also present in many other countries, particularly those neighboring china. continuous occurrences of the epidemic in indonesia, malaysia, and singapore were consistent with the outbreaks in our country. the epidemiological data demonstrated that the early imported cases in guangdong had high correlation with those found in the neighboring countries, which indicated that these countries might be the source for the migration of denv to guangdong. , however, specific switches in different genotypes were observed, which are as follows: myanmar, thailand, vietnam, and laos for genotype i; the philippines for genotype iv; and india and maldives for genotype v. the outbreaks of severe epidemics over the years in the neighboring countries and frequent mobility of their populations to china may be able to explain the pandemic pattern of the spread of denv, which began with its spread to guangdong province and then broke out in other parts of the country. a similar conclusion was presented in the study conducted by sun et al. regarding the epidemiological characteristics and genetic diversity of denv in guangdong in . nevertheless, since , with an extremely high incidence of cases, denv spread more rapidly and affected a wider range of populations. the co-epidemic between other countries and guangdong and the dissemination of different genotypes and serotypes in multiple regions might not align totally with the theory that only the imported cases caused the epidemic. lee et al. reported a theory which stated that multiple factors are involved in addition to the ones described in in situ evolution that can explain this phenomenon. however, we still need more evidence to prove the applicability of this hypothesis to the epidemic outbreaks in guangdong province. a prediction of the algorithm of the secondary structure of proteins suggests that it is closely related to the distribution of protein epitopes. the high chemical bond energy of the α-helix and β-sheet enables folding of the protein, making it difficult to bind to the antibody, whereas the β-turn and coil, because of their loose structure, are easily displayed on the surface as antigenic epitopes, facilitating binding of the antibody. [ ] [ ] [ ] only two substitutions causing changes in the secondary structure from the β-sheet to coil were observed in our study, suggesting that its antigenicity was enhanced. however, e (t → s) located on the surface is more likely to be associated with protein function than e (i → l), which was located inside the protein. although di, the structure found in the central region of the e protein, was not significantly related to the protein function, in our study, majority of the aa substitutions were observed ( / ), and most of them were located on the surface of its d structure. even e (t → i) and e (t → s) were found in the binding interface between genotype i and grp but not genotype v, further suggesting that the substitution of e (t → s) may be playing a key role in the binding of the e protein of genotypes i and v to the receptor. moraes et al. found that, with changes in the ph, the specific interaction between di and diii of the denv e protein is destroyed, resulting in its conformational change during entry into the cell, whereas nayak et al. also observed the presence of a bundle structure consisting of four polar aa residues at the interface between di and diii, of which his- and his were unique to denv , implying that the change of di conformation will also affect the realization of diii function. domain iii has an immunoglobulin-like structure and a functional region where denv binds to a cellular receptor. drumond et al. predicted the structure of the denv e protein in brazil and observed that the substitution of e (s → f) can reduce its interaction with several residues (ser , lys , lys , val , val , ile , tyr , and gly ), resulting in a change in the folding of the area, whereas genotypes i and v of denv in guangdong have a unified mutation in e (i → v). the residue qhg at position e -e of the e protein of denv and is highly conserved, and docking by the zdock method showed that it possibly interacts with the membrane receptor protein tim- , although we found the same region conserved at e -e . meanwhile, three conserved regions ( a- p, t- d, and e- k) were concurrently marked in the binding interface of the two genotypes found in guangdong. chen et al. localized the neutralizing determinants of the inhibitory mabs demonstrating strong effects to a sequence-unique epitope on diii of the denv e protein, centered near residues t and d ( tqngrlitanpivtd ) which were highly conserved among different genotypes of denv but different from those of the denv , denv , and denv serotypes and other flaviviruses. currently, we believe that vaccination and vector control are the fundamental measures to control df. however, research on vaccines and mosquito-control measures have not made significant breakthroughs. we need additional information to understand the epidemic situation of df and evolution of its virus in guangdong to develop effective preventive and control measures. epidemiological analysis reveals information on the cities and months during which df was substantially prevalent and helps to develop mosquito-surveillance and killing strategies. the phylogenetic tree revealed that denv , which is the main serotype of the virus, has been prevalent in guangdong since a long time. the strains isolated from epidemic cases occurring during the same period are homologous, and genotype i has formed a stable evolutionary lineage in recent years. these results suggested that denv may be lurking and circulating in guangdong, although it cannot be stated with certainty. however, it is highly recommended that we detect the denv in local mosquito vectors urgently. at the same time, the phylogenetic tree of the input source suggests the possible countries and regions from which importation of df in guangdong can occur. this information is of great significance for the development of a plan to monitor the departure and entry of populations from regions with a high incidence of dengue. e-mails: chienhai@ .com, murong @outlook. com, hexiaoen @ .com, maiblume@ .com, xiyuxie @ .com, zhuli @ .com, @ .com, milyx @ .com, harpul@ sina.com, wenying@gdciq.gov.cn, and zhangb@smu.edu.cn. zhicheng zhong, and danjuan ma dengue fever: new paradigms for a changing epidemiology a local outbreak of dengue caused by an imported case in dongguan china dengue viruses in brazil guidelines for diagnosis, treatment, prevention and control world health organization refining the global spatial limits of dengue virus transmission by evidencebased consensus the global distribution and burden of dengue flavivirus genome organization, expression, and replication the origin, emergence and evolutionary genetics of dengue virus insights of the genetic diversity of denv- detected in brazil in years: analysis of the envelope domain iii allows lineages characterization microevolution and virulence of dengue viruses the role of models in reconstructing evolutionary trees molecular evolution and phylogeny of dengue- viruses phylogeny 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protein in the postfusion conformation and its implications for membrane fusion phylogenetic analysis of dengue virus isolated from south minas gerais, brazil epitopes based drug design for dengue virus envelope protein: a computational approach characterization and epitope mapping of dengue virus type specific monoclonal antibodies this is an open-access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. key: cord- -o d aa authors: yu, xi; zhang, liming; tong, liangqin; zhang, nana; wang, han; yang, yun; shi, mingyu; xiao, xiaoping; zhu, yibin; wang, penghua; ding, qiang; zhang, linqi; qin, chengfeng; cheng, gong title: broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, sars-cov- and other enveloped viruses date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: o d aa viruses are the major aetiological agents of acute and chronic severe human diseases that place a tremendous burden on global public health and economy; however, for most viruses, effective prophylactics and therapeutics are lacking, in particular, broad-spectrum antiviral agents. herein, we identified secreted bacterial lipases from a chromobacterium bacterium, named chromobacterium antiviral effector- (cbae- ) and cbae- , with a broad-spectrum virucidal activity against dengue virus (denv), zika virus (zikv), severe acute respiratory syndrome coronavirus (sars-cov- ), human immunodeficiency virus (hiv) and herpes simplex virus (hsv). the cbaes potently blocked viral infection in the extracellular milieu through their lipase activity. mechanistic studies showed that this lipase activity directly disrupted the viral envelope structure, thus inactivating infectivity. a mutation of cbae- in its lipase motif fully abrogated the virucidal ability. furthermore, cbae- presented low toxicity in vivo and in vitro, highlighting its potential as a broad-spectrum antiviral drug. genomic comparison, csp_bj shares either . % identity to chromobacterium haemolyticum ch -bl or . % identity to chromobacterium rhizoryzae jp - strain. oral supplementation of this bacteria in a. aegypti largely impaired mosquito permissiveness to denv (supplementary figure a and b) and zikv (supplementary figure c and d) , suggesting a close relationship between the identified csp_bj and csp_p strains. we next aimed to understand how csp_bj resists viral infection in mosquitoes. bacteria usually exploit many effectors, such as cellular components, metabolites or secreted proteins, to regulate their host immune or physiological status for effective colonization. we therefore identified the bacterial effector(s) that modulate infection of denv and zikv through differential fragmentation. in this experiment, we cultured csp_bj for hr at °c. the cell-free culture supernatant was collected by centrifugation and filtration through a . µm filter unit, whereas the cell lysates were generated by sonication. either the bacterial cell lysate or the culture supernatant was mixed with plaque-forming units (pfu) of denv or zikv and incubated for hr, and then the infectious viral particles were determined by a plaque forming assay ( figure a ). incubation of the culture supernatant but not the bacterial lysates resulted in significant suppression of denv ( figure b ) and zikv ( figure c ) infectivity in vero cells, indicating that an extracellular effector(s) secreted by csp_bj was responsible for viral inhibition. next, we investigated whether the effector(s) was a secreted protein, small peptide, lipid, polysaccharide or other metabolite. therefore, the culture supernatant was separated using a kda-cutoff filter (wu et al., ) . either the upper retentate (proteins and large peptides) or the lower liquid filtrate (small molecule compounds and short peptides) was mixed with the viruses for incubation in vero cells ( figure d ). intriguingly, inoculation of the retentate rather than the filtrate inhibited the infectivity of denv and zikv ( figure e and f), suggesting that the effector(s) might be a protein(s) secreted by csp_bj. subsequently, the protein components in the upper retentate were separated by sds-page and then identified by mass spectrometry (figure a ). the highly abundant proteins with secretable properties were selected, expressed and purified in an escherichia coli expression system ( figure b) . of all the proteins tested, the bacterial protein encoded by gene (accession: mt ) significantly impaired denv and zikv infection in vero cells ( figure c and d). we named this protein chromobacterium antiviral effector- (cbae- ). intriguingly, a cbae- homologue with . % amino acid identity named cbae- (accession: mt ) was further identified from csp_bj based on sequence comparison (supplementary figure a and b) . both effectors encoded a lipase domain with a typical gdsl motif (casas-godoy et al., ) . to further confirm the virucidal activity, both proteins were expressed and purified in e. coli ( figure e) . a serial concentration of recombinant proteins was mixed with pfu of denv or zikv for plaque assay in vero cells. the half-inhibitory concentration (ic ) of cbae- was . µg/ml for denv and . µg/ml for zikv ( figure f and g). however, the ic of cbae- for both flaviviruses was - times higher than that of cbae- , indicating a much more robust virucidal activity of cbae- against flaviviruses ( figure f and g). thus, we identified bacterial effectors with a high virucidal activity from a csp_bj bacterium. we next investigated the mechanisms by which these bacterial effectors resist viral infection. according to sequence analysis, cbaes contain a conserved lipase domain. lipases are a group of enzymes that catalyse the hydrolysis of the ester bond of glycerides into fatty acids and glycerol (casas-godoy et al., ) . we therefore assessed whether cbaes have lipase activity. in a plate degradation assay, both cbaes directly digested egg yolk lipoproteins and formed lytic halos whose diameters correlated with lipase activity ( figure a ). the sequence gdsl is the core motif of lipase activity (casas-godoy et al., ) . consistently, a s g mutation in this motif of cbae- fully disrupted its lipase activity ( figure a ), validating cbaes as secreted lipases of csp_bj. given their lipase activity, we hypothesized that cbaes might use their enzymatic activity to degrade viral lipid envelope, which may result in exposure of the viral rna (muller et al., ) . to address this hypothesis, serial concentrations of cbaes were incubated with × pfu of denv or zikv for hr at °c. the mixture was then treated with rnase a to evaluate the degradation of exposed viral genomic rna. compared to mock treatments in which the viruses were incubated with bsa, a significant reduction in viral rna was recorded by rt-qpcr when the viruses were treated with cbaes ( figure b and c), indicating that the lipase activity of cbaes directly disrupted the virion structure, thus resulting in viral genome release. consistent with these results, the s g mutant of cbae- that had no lipase activity completely failed to suppress both denv and zikv infection ( figure d and e), further indicating that the virucidal activity of cbaes is lipase-dependent. to validate that cbaes disrupt viral lipidic membranes, we incubated cbae- with purified zikv virions and processed the samples for transmission electron microscopy ( figure f ). the zikv particle typically has a diameter of - nm (hasan et al., ) and, in consistent with that, we observed intact viral particles in our control sample treated with µg/ml bsa ( figure f ). however, upon treatment with µg/ml cbae- in the same buffer as bsa solution, the integrity of zikv particles was fully disrupted ( figure f ). this is in agreement with our previous finding that treatment of cbaes resulted in viral genome release. since cbaes blocked viral infection in the extracellular milieu, we further assessed whether treatment of cbaes prior to viral inoculation could effectively block viral infection. we pre-treated vero cells with µg/ml of purified cbaes. subsequently, . moi of denv or zikv was used to challenge the cbaes-treated cells. pre-incubation with cbae- fully blocked the infectivity of both flaviviruses, whereas treatment with cbae- exhibited %- % inhibition ( figure f and g). since the cbaes showed a direct catalytic action on the viral lipid bilayer, we assessed their virucidal activity against other enveloped viruses. sars-cov- is a newly emerging coronavirus that causes the severe acute respiratory disease covid- . to date, no specific therapeutics are available against sars-cov- infection. we therefore assessed the virucidal effect of the cbaes on sars-cov- . in contrast to the large difference in virucidal activity against flavivirus infection, both cbaes presented a similar antiviral effect against a sars-cov- pseudovirus in hek- t-ace cells ( figure a ). consistently, infection with lowpassage sars-cov- in vero cells was also significantly suppressed by treatment with the cbaes ( figure b ). additionally, both cbaes showed broad-spectrum antiviral effects on hiv- pseudoviruses ( figure c , d and e), as well as hsv- ( figure f ). notably, compared to a hiv-neutralizing antibody (n ) with a near-pan neutralization breadth (huang et al., ) , cbaes were very potent with a much lower ic for hiv pseudovirus strains ( figure c, d and e). nonetheless, it is puzzling that the cbaes did not show any effect against infection with influenza a virus (iav) ( figure g ). the lipidic envelope of a viral particle is generally derived from the plasma membrane or the endoplasmic reticulum (er) membrane of a host cell. thus, cbaes can also act on host cell membranes and their cytotoxicity to host cells may be a major concern for developing a cbae-based broad-spectrum antiviral drug. generally, cbae- showed much higher toxicity figure d ). altogether, these data suggest that cbae- is much safer than cbae- to hosts, and thus could be a broad antiviral drug candidate. in this study, we identified two virucidal effectors with lipase activity, cbae- and cbae- , from the chromobacterium sp. csp_bj. both cbaes showed a potent virucidal activity against a variety of enveloped viruses including denv, zikv, sars-cov- , hiv- , and hsv- . notably, neither cbaes exhibited any effect against iav. the toxicity assessment showed that cbae- was much safer than cbae- to both human cells and mice. indeed, accumulating evidence indicates that certain lipases present a potent antiviral activity. either lipoprotein lipase or hepatic triglyceride lipase impaired hepatitis c virus (hcv) infection in human huh . cells through degrading virus-associated lipoproteins (shimizu et al., ) . a secreted phospholipase a (pla ) isolated from naja mossambica snake venom showed a potent virucidal activity against hcv, denv, and japanese encephalitis virus (jev); while the protein did not exhibit significant antiviral activity against sindbis virus (sinv), iav, middle east respiratory syndrome coronavirus (mers-cov) or hsv- (chen et al., ) . a secreted human pla has been shown to neutralize hiv- by degrading the viral membrane (kim et al., ) or by blocking viral entry into host cells rather than a lipase-mediated virucidal effect (fenard et al., ) , suggesting diverse virucidal mechanisms by pla . cbaes may inactivate viruses by their lipase activity that also damage cellular membranes; nonetheless, their specificity and affinity for the viral envelope could be significantly improved, thus further reducing their cytotoxicity and increasing virucidal efficacy. for example, the sars-cov- surface protein spike binds to human ace with high affinity (wang et al., ) , and thus soluble ace could be engineered with cbaes to greatly enhance its affinity and specificity for the viral envelope. given that human recombinant soluble ace can also inhibit sars-cov- infection in organoids (monteil et al., ) , it is possible that a soluble ace -cbae construction may have improved efficacy in blocking sars-cov- infections. viruses are the major aetiological agents of acute severe human diseases that impose a tremendous burden on the global public health and economy (ghosn et al., ; girard et al., ; wilder-smith et al., ) . given the fact that development of virus-specific vaccines and antiviral drugs is usually lengthy, broad-spectrum antiviral drugs could be crucial to prevent wide spread of new viral disease in a timely manner (schein, ) . cbae- , with a broad antiviral effect and low toxicity to hosts, could be a potential choice. our study provides a future avenue for the development of broad-spectrum antiviral drugs that might reduce the clinical burden caused by emerging viral diseases. human blood was collected from healthy donors who provided written informed consents. the collection of human blood samples and their use for mosquito feeding was approved by the local ethics committee at tsinghua university. eight-week-old female icr mice purchased from vital river laboratories in china were used for toxicity assay. the mice were maintained in a specific pathogen-free barrier facility at tsinghua university. the animal protocol used in this study was approved by the institutional animal care and use committee of tsinghua university and performed in accordance with their guidelines. aedes aegypti (the rockefeller strain) was maintained on a sugar solution in a low-temperature, illuminated incubator (model , thermo electron corporation) at °c and % humidity, according to standard rearing procedures (cheng et al., ) . vero cells, hek- t cells and a cells were maintained in dulbecco's modified eagle's medium ( - , gibco) supplemented with % heat-inactivated foetal bovine serum ( - , gibco) and % antibiotic-antimycotic ( - , invitrogen) in a humidified % (v/v) co incubator at °c. the vero, hek- t and a cell lines were purchased from the atcc (ccl- , crl- and ccl- , respectively). denv- (new guinea c strain), zikv (prvabc strain), hsv- , iav (h n pr strain) and sars-cov- were grown in vero cells with vp-sfm medium ( - , gibco). denv, zikv, hsv and sars-cov- were titrated by a standard plaque formation assay on vero cells (bai et al., ) . iav were titrated using a standard % tissue culture infection dose (tcid ) assay (teferedegne et al., ; varada et al., ) . all experiments involving infectious sars-cov- were performed in a biosafety level (bsl ) containment laboratory. chromobacterium sp. beijing was grown in lb broth at °c for hr at rpm. culture supernatants were obtained by centrifugation and filtering the supernatant through a . µm filter unit (slgp rs, millipore). genscript) before the protein concentration was measured using a bradford assay ( - , bio-rad), and the protein purity was checked with sds-page. vero cells were seeded at ~ × cells per well in -well plates and then incubated at °c overnight before reaching - % confluence. denv, zikv, hsv- and sars-cov- virus stocks were diluted to plaque-forming units (pfu) per ml and incubated untreated or with a serial dilution of the cbaes in five-fold steps at °c for hr before being added onto vero cell monolayers for hr of infection. cell monolayers were washed once with pbs and covered with % agarose overlay dmem with % fbs. after - dpi (denv, zikv and hsv- ) or - dpi (sars-cov- ), vero cell monolayers were fixed and stained with . % crystal violet, and the number of pfu per ml was determined. the concentration of each protein necessary to inhibit virus infection by % (ic ) was calculated by comparison with the untreated cells using the dose-response-inhibition model in graphpad prism . (graphpad software, usa). for pre-infection treatment, vero cells were seeded in -well plates and allowed to form monolayers. ten micrograms/ml cbae- or cbae- was added to vero cell monolayers at °c for hr, and then denv or zikv ( . moi) was added and incubated for another hour at °c. after infection, cell monolayers were washed once with pbs buffer, fresh vp-sfm medium was added, and the cells were incubated at °c for hr before the supernatant was collected for rt-qpcr quantitation of the viral genome. total rna was isolated either from homogenized mosquitoes or infected cell supernatant using supplementary table . the lipase activity of cbae- , cbae- and cbae- -s g was measured with a plate assay as previously described (liu et al., ) . briefly, µg of cbae- , cbae- or cbae- -s g was spot inoculated onto a % agar plate with % egg yolk and incubated for hr at °c. phospholipase activity was indicated by the diameter of the lytic halo around each well. viral rna exposure assay. cbaes or pbs in a total volume of ml for hr at °c. then, the mixtures were treated with µl of rnase a (ge - , transgen) and incubated for hr at °c. viral rna was extracted, and rna degradation was evaluated by rt-qpcr as mentioned above. zikv viral particles were purified as described previously (tan and lok, ) . briefly, virus stocks were pelleted in % w/v peg at , ×g for hr, then purified by % w/v sucrose cushion for hr at , ×g (beckman sw ti rotor) and separated in potassium tartrate-glycerol gradient - % for hrs at , ×g (beckman sw ti rotor). purified viral particles were suspended in µg/ml bsa solution or µg/ml cbae- solution and incubated for hr at room temperature. the samples were then applied to a carbon grid, washed times with water and negatively stained with % w/v uranyl acetate. the images were acquired in a hitachi h- b tem microscope at . kv. the in vitro antiviral efficacy of the cbaes on iav was tested in a cells. briefly, a cells were seeded into a -well plate and incubated at °c for - h. iav ( tcid ) were incubated untreated or with a serial dilution of the cbaes in five-fold steps at °c for hr before being added onto a cell monolayers to allow infection to proceed for hr. then, the virus-protein mixture was removed, and the cells were further cultured with fresh vp-sfm medium. at hr p.i., relative viral rna copy numbers in the infected cells were quantified by rt-qpcr assays with specific primers. the neutralization titre was defined as the concentration of each protein necessary to inhibit the pcr signal by % (i.e., below the threshold of % of the mean value observed in virus control wells). hiv- pseudoviruses were generated by co-transfecting hek- t cells with env expression vectors and the pnl - r-eluciferase viral backbone plasmid, and a neutralization assay was performed as described previously (zhou et al., ) . briefly, pseudovirus titres were measured by luciferase activity in relative light units (rlus) (bright-glo luciferase assay system, promega biosciences, california, usa). neutralization assays were performed by adding tcid (median tissue culture infectious dose) of pseudovirus into serial : dilutions of cbae- or cbae- starting from µg/ml, following incubation at °c for hr and addition of ghostx /r cells. neutralizing activity was measured by the reduction in luciferase activity compared to that in the controls. the fifty percent inhibitory concentration (ic ) was calculated using the dose-response-inhibition model with the -parameter hill slope equation in graphpad prism . (graphpad software, usa). vesicular stomatitis virus g protein (vsv-g) pseudotyped lentiviruses expressing human ace were produced by transient co-transfection of pmd g (addgene # ) and pspax (addgene # ) plasmids and the transfer vector plvx-ace flag-ires-puro with vigofect dna transfection reagent (vigorous) into hek- t cells to generate the hek- t-ace cells for sars-cov- pseudovirus infection. sars-cov- pseudoviruses were purchased from genscript, and neutralization activity was measured using the hek- t-ace cell line with the same procedures as mentioned above. fresh human blood from healthy donors was placed in heparin-coated tubes ( , bd vacutainer) and centrifuged at , ×g and °c for min to separate plasma from blood cells. the plasma was heat-inactivated at °c for min. the separated blood cells were washed three times with pbs to remove the anticoagulant. the blood cells were then resuspended in heat-inactivated plasma. bacterial suspension ( . od) was mixed with viruses and treated blood for mosquito oral feeding via a hemotek system ( w , hemotek). fully engorged female mosquitoes were transferred into new containers and maintained under standard conditions for an additional days. the mosquitoes were subsequently euthanized for further analysis. the mosquitoes used in this experiment were previously antibiotic treated. briefly, mosquitoes were provided with cotton balls moistened with a % sucrose solution including units of penicillin and mg of streptomycin per ml ( - , thermo fisher scientific) for days to remove gut bacteria. the mosquitoes were starved for hr to allow the antibiotics to be metabolized prior to in vitro membrane blood feeding. removal of gut bacteria was confirmed by a colony-forming unit assay. the cytotoxicity of the cbaes was evaluated in vero cells and a cells. cell viability was measured by the mtt [ -( , -dimethylthiazol- -yl)- , -diphenyl tetrazolium bromide] (m , solarbio) method. confluent cell monolayers contained in -well plates were exposed to different concentrations of the cbaes for hr at °c. then, a final concentration of . mg/ml mtt was added to each well. after hr of incubation at °c, the supernatant was removed, and µl of dimethyl sulfoxide (dmso) was added to each well to solubilize the formazan crystals. after shaking for min, absorbance was measured at nm. the concentration of each protein necessary to reduce cell viability by % (cc ) was calculated by comparison with the untreated cells using a sigmoidal nonlinear regression function to fit the dose-response curve in graphpad prism . (graphpad software, usa). icr mice were used to test the in vivo safety of the cbaes. a number of icr -week old female mice were divided into groups (n= ) at random. cbae- or cbae- was dissolved in pbs and administered either intravenously once at doses of , , , and µg/kg or intranasally once at doses of , , and µg/kg. pbs and corresponding concentrations of bsa served as negative controls. the general behaviour, signs of toxicity, body weights and mortality of the mice were recorded after the administration of the cbaes. the half-lethal dose (ld ) was calculated using a sigmoidal nonlinear regression function to fit the dose-response curve in graphpad prism . (graphpad software, usa). animals were randomly allocated into different groups. mosquitoes that died before sample collection were excluded from the analysis. the investigators were not blinded to the allocation during the experiments or to the outcome assessment. no statistical methods were used to predetermine the sample size. descriptive statistics are provided in the figure legends. all analyses were performed using graphpad prism statistical software. (d, e) the s g mutant of cbae- fully lost its ability to suppress denv (d) and zikv (e) infection: inhibition curves of cbae- and cbae- -s g against denv (d) or zikv(e). serial concentrations of cbae- or cbae- -s g were mixed with pfu of denv or zikv in vp-sfm medium to perform standard plaque reduction neutralization tests (prnts). (f) representative negative stained transmission electron microscopy images of zikv particles treated with µg/ml bsa (arrow head) and those treated with µg/ml cbae- (empty arrow head); high magnification: , ×, low magnification: , ×. (g, h) rate of denv (g) or zikv (h) replication inhibition following exposure to cbaes before viral infection of vero cell monolayers. the viral genome was quantified by rt-qpcr. (b, c, g, h) significance was determined using unpaired t-tests. data are presented as the mean ± sem. aegypti. a mixture containing human blood ( % v/v), csp_bj bacterial suspension ( % v/v), and supernatant from denv-or zikv-infected vero cells ( % v/v) was used to feed antibiotic-treated a. aegypti rockefeller strain via an in vitro blood feeding system. mosquito infectivity was determined by rt-qpcr at days post blood meal. the final denv or zikv titre was × pfu/ml for oral infection. (a, c) the number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. a nonparametric mann-whitney test was used for the statistical analysis. (b, d) differences in the infectivity ratio were compared using fisher's exact test. (a) conserved domains of cbae- and cbae- protein sequences was analysed using a simple modular architecture research tool (smart) (letunic and bork, ; letunic et al., ) . (b) sequence comparison of cbae- and cbae- was performed using basic local alignment search tool (blast) on ncbi website with the program "needleman-wunsch alignment of two sequences" (altschul et al., ) . gapped blast and psi-blast: a new generation of protein database search programs antiviral peptides targeting the west nile virus envelope protein broad-spectrum agents for flaviviral infections: dengue, zika and beyond a non-live preparation of chromobacterium sp. panama (csp_p) is a highly effective larval mosquito biopesticide lipases: an overview broad-spectrum antiviral agents: secreted phospholipase a targets viral envelope lipid bilayers derived from the endoplasmic reticulum membrane a c-type lectin collaborates with a cd phosphatase homolog to facilitate west nile virus infection of mosquitoes the pandemic in mexico: experience and lessons regarding national preparedness policies for seasonal and epidemic influenza secreted phospholipases a( ), a new class of hiv inhibitors that block virus entry into host cells the a (h n ) influenza virus pandemic: a review structural biology of zika virus and other flaviviruses west nile virus spreads in europe identification of a cd -binding-site antibody to hiv that evolved near-pan neutralization breadth lysis of human immunodeficiency virus type by a specific secreted human phospholipase a years of the smart protein domain annotation resource smart: recent updates, new developments and status in pathogenicity of different isolates of vibrio harveyi in tiger prawn, penaeus monodon infections in engineered human tissues using clinical-grade soluble human ace phospholipase a isolated from the venom of crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope respiratory syncytial virus seasonality: a global overview inter-annual variation in seasonal dengue epidemics driven by multiple interacting factors in guangzhou, china human influenza virus infections chromobacterium csp_p reduces malaria and dengue infection in vector mosquitoes and has entomopathogenic and in vitro anti-pathogen activities the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak chromobacterium spp. mediate their anti-plasmodium activity through secretion of the histone deacetylase inhibitor romidepsin repurposing approved drugs on the pathway to novel therapies west nile virus infection lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis c virus (hcv) through their catalytic activities on hcvassociated lipoproteins zika virus: history, epidemiology, transmission, and clinical presentation respiratory syncytial virus hospitalization and mortality: systematic review and meta-analysis dengue virus purification and sample preparation for cryoelectron microscopy development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription pcr broad-spectrum coronavirus antiviral drug discovery a neutralization assay for respiratory syncytial virus using a quantitative pcr-based endpoint assessment structural and functional basis of sars-cov- entry by using human ace coronavirus disease (covid- ) situation report - epidemic arboviral diseases: priorities for research and public health a gut commensal bacterium promotes mosquito permissiveness to arboviruses sars-cov- : an emerging coronavirus that causes a global threat broadly resistant hiv- against cd -binding site neutralizing antibodies broad-spectrum antiviral agents key: cord- -rkher ly authors: pong, lian yih; yew, peng nian; lee, wai leng; lim, yau yan; sharifah, syed hassan title: anti-dengue virus serotype activity of tannins from porcupine dates date: - - journal: chin med doi: . /s - - - sha: doc_id: cord_uid: rkher ly background: dengue fever is currently endemic in tropical and subtropical countries worldwide and effective drug against denv infection is still unavailable. porcupine dates, which are traditionally used to treat dengue fever, might contain potential anti-dengue compounds. two porcupine dates, black date (bd) and powdery date (pd) from himalayan porcupine (hystrix brachyura), were investigated for their antiviral activities against denv- in vitro. methods: the methanol crude extracts (mbd and mpd) were prepared from the raw material of porcupine dates. the tannin-rich fractions (bdtf and pdtf) were isolated from their methanol crude extracts using column chromatography. the presence of tannins in bdtf and pdtf extracts was determined by fourier-transform infrared spectroscopy (ftir) and nuclear magnetic resonance (nmr) analyses. the cytotoxicity and anti-denv- activities including virus yield inhibition, virucidal, virus attachment and pre-treatment assays of the extracts were examined in vero cells. results: our findings revealed that all the extracts of porcupine dates exhibited antiviral activity against denv- in vero cells. the ic( ) of bdtf and pdtf were µg/ml and µg/ml respectively, while their methanol crude extracts demonstrated lower antiviral efficacy (ic( ) ≈ – µg/ml). bdtf and pdtf also exerted a similar higher virucidal effect (ic( ) of µg/ml) than methanol crude extracts (ic( ) ≈ – µg/ml). furthermore, all the extracts inhibited the attachment of denv- by at least %. pre-treatments of cells with bdtf and pdtf markedly prevented denv- infection when compared to methanol crude extracts. conclusion: this study suggests that porcupine dates possess antiviral properties against denv- , which is attributed to its tannin compounds. dengue fever is the most prevalent mosquito-borne viral disease with high morbidity and mortality that is currently endemic in more than countries within tropical and subtropical regions worldwide [ , ] . it is caused by dengue virus (denv) which is circulated as four distinct serotypes that are antigenic closely related, denv- , - , - and - . denv is an enveloped, positivesense single-stranded rna virus that has been classified into genus of flavivirus in the flaviviridae family. the genome of denv is approximately kb in size and it encodes for three structural proteins, envelope (e), capsid (c) and premembrane (prm) proteins and seven non-structural proteins, ns , ns a, ns b, ns , ns a, ns b and ns [ ] . denv is transmitted to human by mosquito species of aedes aegypti mainly as well as aedes albopictus [ ] . nowadays, dengue is still a public health concern globally as it spreads rapidly worldwide and the incidence of dengue keeps increasing dramatically in recent decades. world health organization (who) has estimated , people would be diagnosed as severe dengue annually with a fatality rate of . %. to date, there is no specific antiviral treatment for dengue illnesses. there is therefore, a compelling need to find and discover new drugs or compounds for dengue treatment. traditional medicine has been used occasionally as a primary treatment for dengue fever, especially in asian and african countries [ , ] . porcupine dates are one of the traditional medicines used by people in southeast asia to treat dengue fever by consuming orally the fine powder form of porcupine dates, and sometimes it has been used as multilateral treatment against various diseases including cancer and inflammation [ ] [ ] [ ] . porcupine dates are also known as porcupine bezoar, a phytobezoar that is formed from the partly digested plant materials or herbs in the stomach of himalayan porcupine (hystrix brachyura) [ , ] . many testimonies have claimed that porcupine dates can effectively cure dengue illness [ ] ; however, these claims lack scientific evidence and its relative mechanism of actions is still unknown. in our previous study, two types of porcupine dates, the black date (bd) and powdery date (pd) were found to have a significant high content of total phenolics with % of tannins as major compounds [ ] . in subsequent study, hydrolysable polygalloyl glucoses similar to commercial tannic acid ranging from to galloyl groups were identified in the tannins-fraction of bd and pd [ ] . tannin is an astringent large biomolecule that tends to bind and precipitate proteins, and it is known to be the main component in phytobezoar due to its agglomerating properties. in previous studies, tannin was found to possess antiviral activities against influenza a virus, human papillomavirus type , denv, human cytomegalovirus (hcmv), hepatitis c virus (hcv), measles virus (mv), respiratory syncytial virus (rsv) and herpes simplex virus type (hsv- ), in which these tannins effectively inhibited those infections by interfering the interaction between viral glycoprotein and cell surface glycosaminoglycans including heparan sulfate [ ] [ ] [ ] . apart from that, the oligomeric procyanidins derived from unripe apple peel, a condensed form of tannins, was found to induce innate antiviral immune responses against denv infection in human peripheral blood mononuclear cell (pbmcs) [ ] . in this study, we have hypothesized that the phytochemicals, particularly the hydrolysable tannins in porcupine dates have effective antiviral properties against denv- by interfering the denv- infectivity. the bd and pd from himalayan porcupine (hystrix brachyura) were investigated in this study in order to determine their antiviral activity against denv- . this study, for the first time, provides the scientific evidence on the anti-denv property of the porcupine dates as well as its mode of action in inhibiting the denv- in vitro. in addition, more in-depth nuclear magnetic resonance (nmr) analysis of the chemical content of porcupine dates was also reported for the first time. raw material of porcupine dates, bd and pd obtained from a local chinese medicinal shop were finely ground using pestle and mortar. one gram of samples were then extracted using ml ( : w/v) of % methanol and shaken at rpm using orbital shaker (protech model ) for h at room temperature. extraction process was repeated three times using the same method, and the extracts were then pooled and subjected to rotary evaporation and lyophilized. the yields of the methanol crude extract were . % for bd and . % for pd. two commercial tannic acids purchased from sigma-aldrich, st and friendemann schmidt, ft were included as reference compounds in the screening of antiviral activity. methanol crude extracts were subfractioned into ethanol soluble fraction using non-denatured absolute ethanol to remove the undesired compounds such as protein and non-phenolic phytochemicals. the ethanol fraction was subjected to column chromatography on sephadex lh- (sigma lh , - µm; × cm) using two mobile phases (solvent a: % etoh: % h o and solvent b: % acetone: % h o). samples were wet packed at the top of the column, and run through with three column volumes (≈ ml) of solvent a. eluent was checked with , -diphenyl- -picrylhydrazyl (dpph) until no phenolic compound is eluted. then, three column volumes (≈ ml) of solvent b was used to elute the tannins bound to sephadex lh- beads which were then collected as tannins fraction (sample of bdtf or pdtf). eluted samples were subjected to rotary evaporation and lyophilized. ftir analysis of bdtf, pdtf and st tannic acid samples were done using ft-ir spectrometer (perkin elmer spectrum two). approximately mg of finely ground sample powder or standards was placed on the diamond atr crystal of the spectrometer. pressure was applied to the sample until the force gauge reached a value of . triplicate of scans were obtained for each sample. h-nmr spectra of bdtf, pdtf and st tannic acid standard were obtained using bruker high resolution nmr fourier hd spectrometer with h nmr ( mhz). compounds were dissolved in dmso-d solvent at mg/ml for nmr analysis. solvent peak by dmso-d was identified at . ppm. vero cells (african green monkey kidney) were maintained in minimum essential media (mem) supplemented with % fbs, % hepes and % penicillin-streptomycin antibiotic (gibco) at °c in a humidified incubator with % co . dengue virus serotype (denv- ), which was isolated from dengue-infected patient serum, was used in this study. the serotype of this clinical isolate was confirmed by whole genome sequencing (genbank accession no. mh ). dengue virus stock was prepared by propagation in vero cells. the virus titer was determined via focus formation assay in vero cells. vero cells grown in -well plates were incubated with µl of growth medium containing various concentrations of extracts or tannic acids for h at °c in a humidified incubator with % co . thereafter, µl of mtt reagent was added at final concentration of . mg/ ml and cells were further incubated for h. an equal amount of solubilization solution ( % sds in . n hcl) was added and plate was further incubated overnight. the optical density was read at nm with a background absorbance subtracted at nm. the cell viability rate was plotted against various concentrations of compounds to determine the cytotoxic concentration % (cc ) that causes % reduction in cell viability. vero cells grown in -well plates were infected with denv- at multiplicity of infection, moi of . for h at °c. thereafter, the inoculums were removed and cells were rinsed once with pbs. infected cells were then incubated with various concentrations of extracts or tannic acids for h at °c in a humidified incubator with % co . after h of post-infection, supernatants of infected cells were harvested and subjected to focus formation assay in duplicate. the inhibitory concentration % (ic ) was determined by plotting the percentage of virus yield reduction against various concentrations of extracts. ffa was performed as previously described [ ] and according to the who guidelines [ ] with some modifications. the supernatants containing virus particles were serially diluted in tenfold. vero cells ( × cells) grown in -well plates were inoculated with µl of inoculum followed by infection for h at °c. after h of virus adsorption, inoculums were removed completely and % cmc (carboxymethyl cellulose) overlay medium containing mem, % fbs, % penicillin-streptomycin antibiotic and % hepes was overlaid. the plates were incubated at °c in a humidified incubator with % co for days. on day of post-infection, immunostaining against dengue virus was performed. the overlay medium was removed and cell monolayers were washed twice with wash buffer (tris-buffered saline containing . % tween- , tbst). after fixation with cold % acetone followed by washing, the cells were incubated with blocking buffer (wash buffer containing % bsa and . % triton x- ) at °c for min. cells were then incubated with mouse monoclonal dengue virus type , , and antibody [d - ( )] (genetex) at °c for h. after washing, alkaline phosphatase-conjugated rabbit anti-mouse igg (santa cruz) or alkaline phosphatase-conjugated goat antimouse igg (genetex) was added for h at °c followed by washing. the foci were visualized by adding a mixture of nbt (nitrotetrazolium blue chloride) and bcip ( -bromo- -chloro- ′-indolyphosphate p-toluidine salt) substrates (bio basic). the number of foci was counted and percentage of focus reduction was calculated. an equal volume of various concentrations of extracts or tannic acids serially diluted in fourfold starting at nontoxic concentration were incubated with foci-forming units (ffu) of denv- for h at °c. thereafter, µl of extract-virus mixture was inoculated on vero cells grown in -well plates for h at °c. after incubation, the inoculums were replaced by % cmc overlay medium. the plates were incubated at °c in a humidified incubator with % co for days. the immunostaining against dengue virus was performed as described in ffa. the ic values were determined as mentioned above. the virus attachment assay was performed as previously described with some modifications [ ] . vero cells grown in -well plates were infected with - ffu of denv- in the presence or absence of bdtf, pdtf, methanol crude extracts, tannic acids of ft and st at maximum non-toxic concentration (mntc) of µg/ ml, µg/ml, µg/ml, µg/ml and µg/ml respectively for h at °c. heparin (sigma-aldrich) was tested at µg/ml as a control in this assay. after h of virus infection, the inoculum was removed and cells were washed twice with cold pbs. cells were then covered with % cmc overlay medium and incubated at °c in a humidified incubator with % co for days. the immunostaining against dengue virus was performed as described in ffa. vero cells grown in -well plates were incubated with the extracts or tannic acids at mntc for h at °c. a control, heparin (sigma-aldrich) at µg/ml was included. thereafter, the compounds were removed completely followed by infection with ffu of denv- for h at °c. after h of virus infection, the inoculum was replaced by % cmc overlay medium. the plates were incubated at °c in a humidified incubator with % co for days. the immunostaining against dengue virus was performed as described in ffa. the data were subjected to statistical analysis using ordinary one-way anova followed by dunnett's multiple comparison test, unless otherwise stated. the nonlinear regression with a model of sigmoidal dose-response was used to determine all the cc and ic values except the ic value of mbd. the ic value of mbd was determined from the graph that was plotted by the percentage of virus yield reduction against the extract's concentrations of and μg/ml by using linear regression, as there was a lack of dose-response effect for mbd. the analyses were performed with graphpad prism software (version . ). the results are expressed as the mean ± sem of two independent experiments. the p values less than . were defined as statistically significant. ftir was used to identify the organic functional groups present in fractionated samples. the ftir spectra of both bdtf and pdtf highly matched to that of commercial tannic acid, which all have major bands at wavenumbers around , - , - , , , , , , , , and cm − (fig. ) . in view of the tannins as reported in literature [ ] , the functional group attributed to different positions of the galloyl group of tannic acid are depicted and summarized ( fig. and table ). based on the ftir results, it is suggested that the tannin compounds present in bdtf and pdtf are essentially tannic acid or its derivatives. however, some minor differences in the spectra such as and cm − bands in bdtf were not observed in commercial tannic acid standard. this suggests that bdtf and pdtf are slightly structurally different from the commercial tannic acid, possibly with minor substitution of the side group. based on the comparison of proton nmr spectra, the major chemical shifts in bdtf and pdtf are similar to that of tannic acid standard ( fig. and table ). the similarity of major proton chemical shifts further suggests the presence of tannic acids in bdtf and pdtf fractions. in a recent study, both bdtf and pdtf have been structurally determined using high-performance liquid chromatography (hplc) and esi tandem mass spectroscopy (esi-ms/ms) [ ] . the fragmentation pattern of respective mass spectrum suggests the presence of several hydrolyzed forms of tannins with varying number of galloyl groups. collectively, this is the first time compound analysis of porcupine dates has ever been reported. cytotoxicity of methanol crude extracts (mbd and mpd) and tannin fractions of black date (bdtf) and powdery date (pdtf) were first analysed in vero cells to establish appropriate concentrations for further studies. to evaluate the inhibitory effect of porcupine dates on denv- , the denv- -infected vero cells were treated with various types of porcupine dates extracts at various non-toxic concentrations. as illustrated in fig. , both methanol crude extracts, bdtf and pdtf were not toxic to vero cells at all concentrations up to µg/ml, µg/ml and µg/ml, respectively. mbd and mpd exhibited similar lower antiviral activity against denv- with an ic of and µg/ml respectively, and a selectivity index (si) of more than when compared to both bdtf and pdtf (fig. and table ). however, the inhibition of virus yield by mbd was not in a dose-dependent manner, thus the ic of mbd may not be accurate. in contrast, bdtf exhibited its antiviral activity against denv- in a dose-dependent manner with an ic of µg/ml and an si of more than (fig. c and table ). pdtf also showed similar inhibition toward denv- but with a greater antiviral activity; as little as of µg/ml of pdtf caused a % inhibition in denv- yield, resulting in a higher si of more than when compared to bdtf (fig. d and table ). these results suggest that pdtf exhibited greater antiviral activity against denv- than bdtf. to determine whether the antiviral activity observed in the porcupine dates is attributed to the tannins in those porcupine dates, two commercial tannic acids, ft and st were included as reference compounds. both ft and st were non-cytotoxic at all concentrations up to µg/ml (fig. ) . ft inhibited the denv- replication with an ic of and an si of more than , whilst st exhibited its antiviral activity with an ic of µg/ ml and an si of ( fig. and table ). the formation of denv- focus on vero cells treated with porcupine dates extracts or tannic acids is shown in fig. . to evaluate the virucidal effect of porcupine dates on denv- , the porcupine dates extracts at various nontoxic concentrations were incubated with the denv- directly and the ability of virus to replicate and infect was assessed via viral focus formation assay. mbd and mpd exhibited their dose-dependent virucidal activity against denv- with an ic of and µg/ml respectively, and these extracts inactivated the denv- significantly by . % and . % at concentration of µg/ ml, respectively (fig. a) . in contrast to mbd and mpd, bdtf and pdtf demonstrated an identical higher virucidal effect toward denv- with an ic of µg/ml ( fig. b) . based on the ic values, the virucidal effects of bdtf and pdtf were approximately sixfold and fivefold higher than the mbd and mpd, respectively. in addition, both bdtf and pdtf exterminated the denv- replication at concentration of µg/ml significantly and a % of significant virus inhibition was observed in both extracts at . µg/ml. intriguingly, both ft and st also exerted comparable virucidal effect on denv- replication in a dose-dependent manner with a similar ic of µg/ml, in which the virus yield was decreased significantly by at least % at concentration of µg/ml (fig. c) . to determine whether the porcupine dates extracts able to block the early step of denv replication, which is the virus attachment, the extracts were evaluated at mntc by incubating together with denv- on vero cells at °c. all the porcupine dates extracts significantly reduced the formation of viral foci by at least % (fig. ) . remarkably, bdtf was found to % inhibit the denv- infection at µg/ml, while pdtf-treated denv- infection was reduced by . % at µg/ml. similar results were observed in ft and st tannic acids with an inhibition of % and . %, respectively. furthermore, the antiviral activity of tannin fractions of bd and pd were found significantly higher than the methanol crude extracts, mbd and mpd by . % and . %, respectively. these results demonstrated that these porcupine dates extracts are active inhibitors against the adsorption step of denv- and this antiviral activity most probably attributed to the tannin compounds in porcupine dates. heparin, a known active inhibitor against virus entry of denv [ , [ ] [ ] [ ] , was also examined as a control in this assay. heparin was found to be less potent in inhibiting the attachment of denv- to vero cells, in which % of virus inhibition was observed table . tannic acid and its derivatives are putatively identified in bdtf and pdtf a the data of reported tannins are obtained from a previous study done by pantoja-castro and gonzález-rodríguez [ ] no. functional group wavenumber (cm − ) at the highest concentration been tested in this study ( µg/ml). nonetheless, this result shows that glycosaminoglycans on cell surface are important for the denv- attachment to host cells [ , ] . the pre-treatment antiviral effect of porcupine dates extracts was examined by incubating the vero cells with lyophilized samples were dissolved in deuterated dmso at mg/ml. the proton shift of each sample was obtained using bruker high resolution nmr fourier hd spectrometer ( mhz). similarity of major chemical shifts was observed in all three spectra the extracts at mntc for h at °c prior to denv- infection, in order to determine if there is any direct interaction between the extracts and host cells that contribute to the antiviral action. pre-treating the cells with bdtf was found to inhibit the virus infection by . % significantly and similar result was observed in ft tannic acid, which caused inhibition of . % significantly (fig. ) . however, pre-treatment with pdtf exhibited lower antiviral activity when compared to bdtf, which resulted in a significant virus inhibition of . %. in contrast, the antiviral activity of the pre-treatment with mpd or st showed only . - . % of inhibition and there was no virus inhibition observed when pre-treats the cells with mbd (fig. ). pre-treating the cells with heparin at µg/ml had no significant inhibitory effect on the infectivity of denv- , which is similar to the previous result attained by hung et al. [ ] in bhk cells. these results suggest that the tannins in bdtf and pdtf could be the key compounds that promote the antiviral effect. the pre-treatment of cells with bdtf evidently demonstrated the highest antiviral activity among the other extracts, indicating that this extract could efficiently block the virus attachment to the host cells. apart from the development of dengue vaccine, one of the alternative ways to fight against dengue infection is through the discovery of active compounds or natural products that are specifically directed at killing the virus or induce host innate immune responses against dengue infection without enhancing humoral responses which might cause complications in patient. currently, there is no active compound or alternative medicine available for treating dengue illnesses. therefore, it is of utmost importance that research in discovering anti-denv treatments for human is expedited. some natural products like marine seaweeds and approved drugs have been shown to inhibit denv- replication in vitro [ , [ ] [ ] [ ] [ ] [ ] ; however, their effectiveness in combating dengue infections in human or in vivo has not been determined. unlike these compounds, another natural product, the porcupine dates have long been used as a traditional chinese medicine to treat dengue illnesses in human in some of the southeast asian countries [ ] . in this study, methanol crude extract of porcupine dates (mbd and mpd) were shown as active denv- inhibitors that can inhibit the infectivity of internalized denv- in vero cells and extracellular denv- particles, providing evidence that porcupine dates could be used to treat denv- infection. both mbd and mpd the data were subjected to statistical analysis using ordinary one-way anova followed by dunnett's multiple comparison test. values represent the mean ± sem of two independent experiments performed in duplicate. *p < . , **p < . , ***p < . were non-toxic to vero cells up to μg/ml, which was similar to a previous study done on normal human colon cells [ ] . denv- was selected as the main target for the antiviral activity of porcupine dates in this study because most cases of secondary infection by denv- manifested a more severe form of dengue due to the antibody-dependent enhancement (ade) of infection exerted by the host immune system responses, when compared to the other serotypes [ ] [ ] [ ] [ ] . moreover, denv- has been shown to be the predominant strain among the other serotypes (denv- , denv- and denv- ) in causing dengue in many endemic countries [ ] [ ] [ ] . furthermore, to date, the search for an effective and efficacious vaccine is continuing. the efficacy of the first licensed dengue vaccine i.e. dengvaxia (cyd-tdv) against denv- is still at its lowest with an efficacy of . % among the other serotypes [ , ] . the porcupine dates, bd and pd have been reported to have promising in vitro antioxidant and intracellular reactive oxygen species (ros)/reactive nitrogen species (rns) scavenging properties, and this might be ascribed to the high content of tannins in those porcupine dates [ ] . furthermore, these tannins in porcupine dates were believed to inhibit the proliferation of the colon cancer cells in vitro by inducing apoptosis and cell cycle arrest in the cells [ ] . to study whether tannins in the porcupine dates are bioactive against dengue infection, we have isolated the tannin fractions from mbd and mpd for besides that, both bdtf and pdtf demonstrated similar and higher virucidal activity against denv- when compared to their methanol crude extracts. in virucidal assay, which is determining the viral activity after direct binding of virus to porcupine dates extract, the ic of all extracts except pdtf were found to be at least . -fold lower than the one observed in virus yield inhibition assay, indicating that the anti-dengue activity exhibited by those compounds was mainly due to its virucidal effect on the virus particles. apparently, the antiviral effectiveness of these porcupine dates was higher when they interacted directly with the denv- particles, thus suppressing the infectivity of denv- by inactivating the extracellular denv- particles. in denv, the envelope glycoprotein (e protein) on the viral surface is crucial for virus attachment and entry [ ] , and earlier study by chen et al. [ ] has proved that dengue virus infectivity is highly dependent on the binding of e protein onto heparan sulfate on host cell. based on these findings, it is highly deemed that the extracts bind to the e protein on denv- particles and thus inhibit the attachment of virus to the host cells, preventing virus infection. to ascertain this, virus attachment assay was conducted and confirmed that the porcupine dates extracts inhibited the denv- infection by blocking virus attachment to the host cells. moreover, the results of the virucidal and attachment assays correlated well with each other in term of antiviral effect, in which the porcupine dates extracts particularly the tannin fractions exerted the greatest antiviral activity against denv- . in both assays, the bdtf and pdtf showed similar the compound-virus mixture was then incubated with vero cells and the mixture was removed after h of infection. cells were covered with overlay medium and incubated for days. the number of foci was determined and counted via focus-formation assay. data were subjected to statistical analysis using ordinary one-way anova followed by dunnett's multiple comparison test. values represent the mean ± sem of two independent experiments performed in duplicate. *p < . , **p < . , ***p < . antiviral effect as observed in reference compounds, the ft and st tannic acids. high inhibition on the attachment of denv- by heparin at low concentration ( - µg/ml) in vero and bhk cells has been reported [ , ] . however, in this study, heparin appears to be less effective in abrogating denv- infection at µg/ ml with respect to virus attachment. this could be due to the different strain of denv- being used; the degree of denv- inhibition by heparin might be varied when tested on different strains of denv- . as the denv- heparin was assayed at µg/ml as a control. the mixture containing unadsorbed virus was replaced with overlay medium after washing. after days of incubation, the cells were subjected to immunostaining against denv. data were statistically analysed using ordinary one-way anova followed by dunnett's and sidak's multiple comparison tests. values represent the mean ± sem of two independent experiments performed in duplicate. *p < . , ****p = . , "n.s. " indicates not significant fig. effect of pre-treatment with porcupine dates extracts and tannic acids on denv- infection in vero cells. the individual extracts or tannic acids were incubated with vero cells at mntc for h at °c prior to infection with ffu of denv- for h. heparin was assayed at µg/ ml as a control. the inoculum was then replaced with overlay medium. the cells were subjected to immunostaining against denv after days of incubation. data were statistically analysed using ordinary one-way anova followed by dunnett's multiple comparison test. values represent the mean ± sem of two independent experiments. ***p < . , ****p = . used in this study was isolated from a patient with severe dengue, it is highly likely that the denv- strain was less susceptible to the heparin treatment. the antiviral activity ascribed to the interaction between the extracts and host cells was also investigated; pre-treating the cells with bdtf prevented the denv- infection by at least % and similar result was observed for ft tannic acid. this observation might be due to the presence of specific chemical structure in the ft tannic acid that is similar to the one in bdtf. it could be implied that the tannins in porcupine dates might interfere the host cell's receptors, which are essential for denv- attachment to the host cells, preventing the initiation of virus infection [ ] . a difference in the effect of pre-treatment was observed in the reference compounds, st and ft tannic acids, in which only . % of virus inhibition was observed in st in contrast to the . % of virus inhibition by ft. this might be attributed to the differences in the chemical composition between st and ft tannic acids viz. the absence of c-h functional group at wavenumbers of - cm − in st tannic acid (table ) , which is found in bdtf or pdtf. taken together, these results suggest that tannins in porcupine dates could be the key compounds that contribute to the antiviral effect of porcupine dates extracts against denv- infection. it appears likely that the tannins in porcupine dates exerted its antiviral activity by acting on the virus particles and host cells at the early stage of denv- replication, thereby suppressing the initiation of virus infection. in conclusion, porcupine dates are proven to be potentially effective in abrogating denv- infection in vitro. this anti-dengue property of porcupine dates is most likely attributed to its tannin compounds. the findings in this study signify the potential of porcupine dates as an alternative natural antiviral agent against denv. nonetheless, further investigation on the antiviral activity of porcupine dates dengue infection in vivo and against other dengue serotypes is warranted. besides that, the relationship and kinetics of the drugs in interacting with denv replication is worthwhile to be further elucidated in future. dengue diagnosis, advances and challenges dengue: guidelines for diagnosis, treatment, prevention and control. new ed. geneva: world health organization a structural perspective of the flavivirus life cycle human to mosquito transmission of dengue viruses clinical implications and treatment of dengue papaya, dengue fever and ayurveda the trade, forgery and medicinal use of porcupine bezoars in the early modern period (c. - ) a traditional folk medicine in malaysia: porcupine bezoar health beliefs and practices related to dengue fever: a focus group study • 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 m website views per year • at bmc antioxidant and intracellular reactive oxygen species/reactive nitrogen species scavenging activities of three porcupine bezoars from hystrix brachyura tannic acid-rich porcupine bezoars induce apoptosis and cell cycle arrest in human colon cancer cells hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus entry and cell-to-cell spread broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry tannins from hamamelis virginiana bark extract: characterization and improvement of the antiviral efficacy against influenza a virus and human papillomavirus oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human pbmcs optimization and validation of a plaque reduction neutralization test for the detection of neutralizing antibodies to four serotypes of dengue virus used in support of dengue vaccine development guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses the antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell study by infrared spectroscopy and thermogravimetric analysis of tannins and tannic acid dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate analysis of the steps involved in dengue virus entry into host cells heparin inhibits dengue- virus infection of five human liver cell lines antiviral activity of lanatoside c against dengue virus infection evaluation of antiviral activities of houttuynia cordata thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection screening of dengue virus antiviral activity of marine seaweeds by an in situ enzyme-linked immunosorbent assay antiviral activity of carbenoxolone disodium against dengue virus infection antiviral natural products and herbal medicines dengue hemorrhagic fever: two infections and antibody dependent enhancement, a brief history and personal memoir clinical manifestations of dengue in relation to dengue serotype and genotype in malaysia: a retrospective observational study dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity serotype influences on dengue severity: a cross-sectional study on confirmed dengue cases in vitória, brazil phylogenetic analysis revealed the cocirculation of four dengue virus serotypes in southern thailand co-circulation of all the four dengue virus serotypes and detection of a novel clade of denv- (genotype i) virus in pune, india during season dengue serotype-specific differences in clinical manifestation, laboratory parameters and risk of severe disease in adults clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in asia: a phase , randomised, observer-masked, placebo-controlled trial cyd study groupcyd study group. efficacy of a tetravalent dengue vaccine in children in latin america demonstration of binding of dengue virus envelope protein to target cells publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank soon hing cheong ginseng sdn. bhd. (malaysia) for providing financial support and the raw materials for this research. ssh and yyl conceived the study. ssh acquired the funding for this study. ssh, yyl and wll supervised this study. pny designed and performed the experiments related to extracts preparation. lyp designed and performed the experiments related to antiviral assays. lyp and pny analyzed and interpreted the data. lyp and pny drafted the manuscript. ssh, yyl, lyp, pny and wll revised the manuscript critically for important intellectual content. all authors read and approved the final manuscript. this research was funded by the infectious diseases and health cluster of the tropical medicine and biology platform, monash university malaysia, grant number . the funders had no role in the design of the study and collection, analysis, and interpretation of data and in the writing the manuscript. all data generated or analysed during this study are included in this published article. not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -a vt kso authors: ren, linzhu; peng, zhiyuan; chen, xinrong; ouyang, hongsheng title: live cell reporter systems for positive-sense single strand rna viruses date: - - journal: appl biochem biotechnol doi: . /s - - - sha: doc_id: cord_uid: a vt kso cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. there are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand rna virus infections. the first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. during infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. using subgenomic replicon, which are genetically engineered viral rna molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. the second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. the third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. this review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades. classical reporter systems for (+) ssrna virus the first type of classical reporter systems was based on live recombinant virus carrying a reporter protein, such as enhanced green fluorescent protein (egfp), firefly luciferase (fluc), or secreted alkaline phosphatase ( fig. a and table ). to generate a recombinant virus, a recombinant plasmid was constructed by fusing the reporter protein with the viral structural protein or by inserting the coding sequence of reporter protein behind the ′ terminus of the viral structural protein gene [ ] . subsequently, the recombinant virus was rescued in cells transfected with transcripts produced from the recombinant plasmid [ ] . similarly, based on an infectious complementary dna (cdna) clone of dengue virus (denv), a recombinant denv generating luciferase was developed by engineering the luciferase gene into the capsidcoding region of an infectious cdna clone [ ] . further studies indicated that the reporter system can be used to measure neutralization and antibody-dependent enhancement activity [ ] . a classical swine fever virus (csfv) stably expressing luciferase was developed by inserting the luciferase gene into n pro gene of csfv [ ] . the reporter virus enabled more sensitive and convenient detection of n pro protein expression and viral replication by a luciferase assay than by traditional methods [ ] . the csfv n pro was detectable as early as . h post infection [ ] . additionally, two semliki forest virus (sfv) constructs were developed by inserting egfp gene or renilla luciferase (rluc) gene into the virus replicase open reading frame between nsp and nsp flanked by nsp protease-recognition sites [ , ] . subsequently, infectious sfv expressing detectable egfp or rluc upon infection were obtained by electroporation of in vitro transcripts of the constructs [ , ] . another strategy of classical reporter systems utilizes a reporter virus particle (rvp) (fig. b) . an rvp is a type of virus-like particle (vlp) composed of viral structural proteins and a self-replicating replicon rna containing a reporter gene [ , ] . to obtain an rvp of the west nile virus (wnv), a red fluorescent protein gene was inserted into the wnv rna genome by replacing the c, prm, and e protein genes to generate a wnv replicon rna [ , , ] . subsequently, the replicon rna was transcribed in vitro and co-transfected into cells with two dna expression plasmids for viral c and prm/e proteins, and finally the rvp was generated [ , ] . in a new protocol for the preparation of rvps, the replicon rna was stably transfected and expressed in the cells [ ] . two plasmid dna expression plasmids for viral c and prm/e proteins were then co-transfected into the cells to package the replicon rnas into rvps [ ] . in these rvp systems, rvps were capable of only one round of infection in susceptible cells due to the lack of structural protein genes within the replicon rnas [ , , ] . however, rvps are useful tools for the study of viral genome packaging and cellular factors [ ] . although recombinant viruses can be infectious, the viruses expressing fluorescent proteins tend to be attenuated due to the modification of their genomes [ ] . furthermore, many viruses reporter system based on virus-like particle (vlp). the reporter gene was inserted into the viral genome by replacing the structural genes to generate a replicon. the replicon rna was transcribed in vitro and cotransfected into the cells with expression plasmids for viral structural protein, and finally the rvp was generated. then, the target cells were infected with rvp and the reporter protein can be detected in the cells. c reporter systems based on a viral subgenomic sequence. the reporter gene cassette controlled by viral promoter is constructed. the replicon rna is transcribed in vitro and transfected in to the cells. then, the reporter protein can be expressed and detected in the cells. d protease-sensor reporter systems for (+) ssrna virus. sequence of viral protease cleavage site is fused with reporter gene. the expression cassette is transfected into the cells and stably expressed in the cells. during the virus infection, the cleavage site is specifically cleaved by particular virus, and the reporter protein can be detected in cells or in cells culture medium. e replicase-sensor reporter systems for (+) ssrna virus. the reporter gene was regulated by specific viral rdrp in the defective replicon. without the virus infection, the replicon failed to express the reporter gene efficiently. however, when the cells were infected with the specific virus, the viral rdrp was provided by the virus and the defective replicon was activated, resulting in high-level expression of the reporter gene, which could be easily examined. r reporter protein, s structural protein, ns non-structural protein, rv recombinant virus, rvp reporter virus particle, pro protease, rep replicase, q quenching peptide [ ] are extremely virulent pathogens, such as the middle east respiratory syndrome (mers), severe acute respiratory syndrome coronavirus (sars-cov), and csfv, which can only be handled in specialized laboratories of the highest biosafety level. to overcome these limitations, viral subgenomic replicons were developed and have been proven valuable for measuring the replication kinetics of replicon ( fig. c and table ). self-replicating subgenomic replicons are genetically engineered viral rna molecules that are shorter than full-length viral genomes and are capable of replication but incapable of producing virions [ , , , ] . however, they can be packaged into viral particles if viral coat proteins are provided [ ] . for flaviviridae viruses, such as csfv, two self-replicating rnas (replicons) were produced by replacing the coding region for c to e of csfv with the rluc sequence or the coding region for c to e with the rluc- a sequence [ ] . subsequently, the translation and replication of the replicon rnas could be followed by the accumulation of luciferase protein as well as by detection of csfv non-structural protein (ns) production within the cells [ ] . similarly, two kinds of replicons were constructed for wnv and denv [ , ] . one replicon encoded an rluc followed by an internal ribosome entry site (ires) element for cap-independent translation of the open reading frame encompassing the carboxy-terminal sequence of the e protein to the ns protein. the second replicon contained a luciferase gene, foot and mouth disease virus a, and a neomycin phosphotransferase gene that allows establishment of a stable mammalian cell line expressing the rluc protein in the presence of the neomycin analog, g [ , ] . the stable replicon-expressing cell line has been used for cell-based screening and determination of % effective concentration (ec ) values for antiviral compounds that inhibited wnv replication [ , ] . for hepatitis c virus (hcv), lohmann and his colleague established efficient cell culture systems, which were based on the self replication of engineered hcv replicons [ , , ] . the first one was developed by co-transfecting in vitro-transcribed hcv bicistronic replicon rnas with the analogous mutants carrying the in-frame deletion of the ns b polymerase active site [ ] . the bicistronic replicons were composed of the hcv-ires, the neomycin phosphotransferase (neo) gene, the ires of the encephalomyocarditis virus, which directed translation of hcv sequences from ns or ns up to ns b, and the ′-untranslated regions (utrs) [ ] . however, most of these replicons focused on hcv genotype b, until efficient rna replication systems for genotype a [ , ] and genotype a [ ] were established. furthermore, to construct a tricistronic hcv replicon, two sequential iress were used to initiate translation of humanized rluc and hcv nonstructural genes along with an authentic hcv ires that initiated translation of the neomycin resistance gene [ ] . the results demonstrated that the tricistronic replicon was efficient to evaluate hcv infection [ ] . for the chikungunya virus (chikv), the replicon was constructed by replacing the structural gene of the virus with gaussia luciferase (gluc) gene [ ] . and two helper plasmids, which contain the ′ and ′ chikv replication signals and a subgenomic promoter followed by either the capsid gene or the remaining structural proteins, were constructed expressing either the chikv capsid protein or envelope proteins, respectively [ ] . by co-transfection of the replicon and the helper plasmids, the virus replicon particles (vrps) could be efficiently produced [ ] . positive-sense single strand rna virus genome encodes a large polyprotein precursor, which will be cleaved into structural and non-structural proteins by the action of cellular and viralencoded proteases [ , , , ] . each virus with such a genome has a specific protease that along with an authentic hcv ires that initiated translation of the neomycin resistance gene. cheng et al. [ ] flaviviridae pestivirus classical swine fever virus (csfv) the coding region for c to e of csfv was replaced with the renilla luciferase (rluc) sequence or rluc- a sequence. the translation and replication of the replicon rnas could be followed by the accumulation of luciferase protein expression as well as by detection of csfv ns protein production within the cells. risager et al. [ ] recognizes a specific cleavage site of the polyprotein. moreover, the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase [ ] , indicating that cleavage of the polyprotein depends on the particular virus infection and that the viral protease cleavage site sequence is an ideal element to report the viral infection. during the past few decades, a large number of virus-specific proteases and their cleavage sites have been identified and used to design and engineer reporter cells for specific virus infections ( fig. d and table ). of these viral proteases, the ns / a protease of hcv became an attractive target for the development of reporter systems for hcv infection. one strategy involved the fusion of egfp to secreted alkaline phosphatase (seap) through the ns / a protease recognition sequence, delta ab [ , , , ] . during hcv infection, the fused protein can be cleaved by the protease, and seap will be secreted into the extracellular culture medium. this strategy made it possible to monitor ns / a activity and replication of hcv genomic rna inside mammalian cells by measuring seap levels in the culture medium [ , , , ] . in another strategy, the recombinant caspase (rcasp ) was used as the specific substrate of the ns / a protease; the endogenous cleavage sites in the procaspase molecule were substituted with decapeptides specific for the ns / a protease cleavage [ ] . after hcv infection, the activation of rcasp depended on its specific cleavage by the ns / a protease and resulted in apoptosis of the hcv-infected cells [ ] . additionally, tanaka and his colleagues established an indicator cell system in which the transcriptional factors gal -tbp and human immunodeficiency virus type (hiv- ) tat protein were utilized [ ] . the chimeric tat and gal -tbp transcription factors, both containing the hcv ns / a cleavage sequence of a mitochondria-resident ips- , were manipulated to be localized in the endoplasmic reticulum. upon infection with hcv, the transcription factors were efficiently cleaved by hcv protease, migrated into the nucleus and activated the reporter gene under the tandem promoter [ ] . another protease is the ns protease of csfv. we previously developed a dark-tobright reporter cell for csfv infection [ ] . this assay was based on a novel reporter cell stably expressing egfp fused in-frame to a quenching peptide via a special recognition sequence of the csfv ns protease [ ] . without the csfv infection, although egfp can be expressed in the reporter cell, chromophore maturation of egfp in the reporter cells was inhibited by the c-terminal quenching peptide of egfp [ ] . however, when the cells were infected with csfv, the recognition sequence of the csfv ns protease was specifically cleaved by the protease, and the quenching peptide was released from the protein. subsequently, the egfp can undergo conformational rearrangement allowing maturation of the chromophore and gain of fluorescence, making the cell a dark-to-bright reporter of csfv infection [ , ] . similarly, a denv-specific reporter system was created that utilized the viral protease cleavage site resulting in nuclear localization of gfp in denv-infected cells [ ] . the reporter system was based on a plasmid-containing protease cleavage site of the denv- genome tagged with the sv nls (nuclear localization sequence) and egfp [ ] . during denv or rvps infection, egfp is transferred from the cytoplasm to the nucleus. the reporter system was shown to be effective in detecting infection of cells by all four denv serotypes as well as by low-passaged viral strains [ ] . furthermore, these systems are also valid for the detection of vlps, vrps, or rvps, and even constructs that can produce the protease rnas. positive-sense single strand rna virus genome also encodes a rna-dependent rna polymerase (rdrp), which catalyzes synthesis of viral rna and is considered as another target for virus detection and eradication [ , , ] . generally, the reporter gene was regulated by specific viral rdrp in the defective replicon (fig. e) . without the virus infection, the replicon failed to express the reporter gene efficiently. however, when the cells were infected with the specific virus, the viral rdrp was provided by the virus and the defective replicon was activated, resulting in high-level expression of the reporter gene, which could be easily examined [ ] . for alphaviruses, such as sindbis virus (sinv), a stable cell line, bhksinluc , containing a luciferase gene under the control of the sinv subgenomic rna promoter, was established [ ] . this cell line expressed high levels of luciferase activity during infection with sinv and provided a sensitive assay for titering the virus [ ] . moreover, a species-specific insect cellbased system for detecting wild-type sinv infection was developed, which produced sinv minigenome containing a virus inducible promoter and a fluorescent gene [ ] . during the virus infection, the fluorescent gene was specially induced and expressed in the cells [ ] . in another system, replicon-defective plasmids derived from the sinv were constructed by replacing the structural genes of sinv with reporter genes and deleting bp in the ns gene of the virus [ ] . upon infection with the virus, the viral components induced expression of the reporter genes in the cells transfected with the replicon-defective plasmid [ ] . another replicase-sensor reporter system based on a bicistronic reporter plasmid, designated as (+)fluc-(−)utr-rluc, was constructed by comprising the fluc gene and the rluc gene in reverse orientation flanked by both negative strands of the hcv ′-and ′-utrs, in which fluc and rluc proteins are regulated by host polymerase and functional ns b polymerase, respectively [ ] . then, the bicistronic plasmid was stably transfected into baby hamster kidney- (bhk- ) cells to generate the bhk-ns b-frluc reporter cell line, which can be used to simultaneously measure cellular toxicity and intracellular rdrp activity [ ] . this system is specific for the hcv rdrp activity assay and the inhibitor evaluation. cell based reporter systems, combined with other genetic and proteomic approaches including rna interference, microarray, and two-dimensional gel electrophoresis coupled with maldi-tof/tof identification, have shown promise as a strategy for research on virus infections and pathogenesis. in general, the recombinant virus or rvp can mimic the virus life cycle and the reporter protein can serve as an indirect readout, thus the functions of the viral proteins and interactions of the proteins can be elucidated by measuring the reporter protein activity. furthermore, the translation and replication of subgenomic replicon rnas could be followed by the accumulation of luciferase protein in the cells. using the reporter systems, it was demonstrated that the csfv core protein can regulate csfv rna synthesis by enhancing csfv rna-dependent rna polymerase (ns b) activity in a virus species-specific manner [ ] . further studies indicated that residues - of the core protein were required for enhancement of ns b activity [ ] . ns , ns a, and ns b of csfv are replication-associated proteins. however, these proteins also play an important role in internal ribosome entry site (ires)-mediated translation of csfv. csfv ns is a multifunctional protein possessing serine protease, rna helicase, and nucleoside triphosphatase (ntpase) activities [ ] . the rna helicase activity of ns could promote viral and cellular translation, whereas the protease domain of ns interacted with ns b to enhance viral translation [ ] . in contrast, the ns a protein had an inhibitory effect on ires-mediated translation, while the ns b proteins suppressed the inhibitory effect of ns a on viral translation by binding residues - located in the cterminal half of ns a [ ] . amino acids k , t , e , and l of ns a and aa - , aa - , and the highly conserved gdd motif of ns b were necessary for the interaction between ns a and ns b [ , ] . furthermore, the ′ terminal sequence harbored the positive and negative regulatory elements to control the ires-mediated translation of csfv [ ] . the negative cis-acting element was the ′-end hexamer cggccc sequence [ ] . moreover, mutations within ires affected the translation initiation efficiency of csfv [ ] . n pro of csfv was not only involved with virus rna translation in the cytoplasm [ ] but also counteracted double-stranded rnamediated apoptosis and ifn-α/β induction [ ] . further studies elucidated that interferon synthesis can be prevented by inhibiting transcription and promoting degradation of interferon regulatory factor (irf ) via the zinc-binding ability of viral n pro [ , ] . n pro also redistributed to mitochondria and peroxisomes to inactivate irf and inhibit apoptosis [ ] . in addition, n pro influenced the innate immune response at local sites of virus replication in pigs and contributed to the pathogenicity and viral replication of csfv [ ] . using the reporter system for denv, it was demonstrated that the ′ utr, ns , ns , and ns were essential for viral rna replication and translation. rna replication of denv required an rna-rna-mediated circularization of the viral genome, and the ′ and ′ utrs formed several additional rna elements that were involved in the regulation of translation and rna replication [ ] . the first nt in the ′ utr comprised a variable region (vr), which could be further divided into a ′-terminal hypervariable region (hvr) and a ′-terminal semi-variable region (svr) [ ] . the vr was important for denv replication and was associated with the accumulation of denv rna in mammalian cells [ ] . the core region of the ′-utr of denv rna can form two dumbbell structures ( ′and ′-dbs) and four pseudoknots, which were required not only for rna replication but also for optimal translation [ ] . the ns protein contained an n-terminal serine protease region joined to an rna helicase by an -amino acid linker [ ] . the linker region conferred flexibility to the ns protein that was required both for polyprotein processing and rna replication [ , ] . furthermore, the host factor p can interact with the ′ utr to facilitate viral rna replication [ ] . two nlss within the central region of ns ('anls' and 'bnls') can be recognized by the importin α/β and importin β nuclear transporters, respectively, leading to ns nuclear accumulation [ ] . moreover, the heterogeneous ribonucleoprotein (hnrnp) c /c interacted with ns to participate in viral rna synthesis [ , ] . during the viral assemblying, the mature capsid protein of denv accumulated on the surface of er-derived lipid droplets in the cytoplasm [ ] . in addition, denv can induce low levels of interferon regulatory factor and nf-κb activation by blocking the tlr-triggered erk-nf-κb activation, thus leading to reduced cytokine production [ , ] . additionally, the dynamics of denv infection in mice revealed that the virus localized predominantly to lymphoid and gut-associated tissues [ ] . cell-based reporter systems also represent rapid, low cost, and high throughput assays for antiviral agent and antibody evaluation. replicon systems were useful for anti-viral drug development. many anti-hcv compounds targeting ns / a, ns a, or ns b have been screened by using the hcv replicon systems, which are now available with sustained virological response up to % in the majority of patients [ ] . recently, pan and his colleagues developed a ns / a protease-based reporter assay, which was not only suitable for efficiently assessing hcv infection, but was also useful for high throughput screening of anti-hcv agents [ ] . in this system, virus replication/ infection in the cells could be quantitatively indicated by measuring the seap activity in the cell culture medium [ ] . similarly, a dual reporter system for wnv based on a subgenomic replicon encoding rluc and neomycin phosphotransferase was generated [ ] . incubation of the reporting cells with a known wnv inhibitor decreased luciferase activity as well as the replicon rna level. the efficacies of the inhibitors, as measured by the depression of luciferase activity in the reporting cells, were comparable to those derived from authentic viral infection assays, indicating that the reporting cell line can be used as a high throughput assay for anti-wnv drugs [ ] . for denv, another severe mosquito-borne viral pathogen, neither a vaccine nor an antiviral therapy is currently available to treat the disease [ ] . by using cell-based reporter systems, several compounds were identified as potential antiviral agents. two novel flavones, pmf and tmf, were discovered to have denv-inhibitory properties [ , ] . one effective compound of , small-molecule chemicals was found to suppress viral rna replication [ ] . a doxorubicin derivative, sa- , can inhibit denv replication in the very early stages of the viral replication cycle with an ec of . ± . μm [ ] . furthermore, nucleoside inhibitors targeting viral polymerases have proved promising for the development of drugs against viruses [ ] . it has been reported that a nucleoside analog, ′-c-methylcytidine ( cmc), exerted specific anti-denv rna polymerase activity in denv subgenomic rna replicon and infectious systems as well as in suckling mice models, with a % inhibitory concentration (ic ) value of . ± . μm [ ] . moreover, bp and bp were identified as small-molecule inhibitors of the denv ns b/ns protease using a stable cell line harboring an efficient luciferase replicon of denv [ ] [ ] [ ] . further studies elucidated that bp inhibited replication and viral rna synthesis by interacting with the central hydrophilic portion of the ns b cofactor with an ec of . ± . μm, while bp targeted the ns protease with an ec of . ± . μm [ , ] . in addition, new compounds were discovered as ns b-ns protease inhibitors with ic values ranging from . ± . to . ± . μm, and compounds belonging to two different scaffolds were active to some extent against denv based on luciferase reporter replicon-based assays [ ] . additionally, amodiaquine (aq) inhibited denv infectivity with ec and ec values of . ± . μm and . ± . μm, respectively, and inhibited viral rna replication with an ec value of . ± . μm in the replicon-expressing cells [ ] . both p-hydroxyanilino and diethylaminomethyl moieties were important for aq to inhibit denv replication and infectivity [ ] . in the context of virus disease control, a reliable, high throughput tool for antibody or vaccine evaluation is also important to allow for an understanding of the impact of neutralizing antibodies on disease progression and vaccine efficacy [ ] . a virus reporter system has emerged as a promising strategy for antibody and vaccine evaluation. a reporter system using denv rvps was used to measure neutralizing antibodies in human serum samples against all • fluorescent proteins tend to be attenuated. • lower than wild-type virus yield of viral progeny • genetic modification of a viral genome may be labor intensive. • higher risk of mutation • expression vectors of structural proteins need to be co-transfected into the cells. • lower than wild-type virus yield of viral progeny • genetic modification of a viral genome may be labor intensive. four denv serotypes [ ] . the results showed that denv rvps yielded serotypespecific responses and reproducible neutralization titers that were in statistical agreement with plaque reduction neutralization test (prnt) results [ ] . similarly, a stable rluc reporter system for denv (luc-denv) was developed for measuring neutralization and antibody-dependent enhancement activity [ ] . a luciferase value analysis using various known denv-specific monoclonal antibodies and clinical samples from infected animals and individuals showed good repeatability and a linear correlation with conventional plaque-based assays [ ] . additionally, a rapid system to produce chimeric single-round infectious particles (srips) of flaviviruses was developed using a japanese encephalitis virus subgenomic replicon plasmid [ ] . the srips of denv- were evaluated as antigens for functional antibody assays [ ] . as a result, a significant correlation was shown between antibody titers obtained using srips and those obtained using denv- antigens, indicating that srips can be used as an alternative antigen in functional antibody assays [ ] . a vrp-based neutralization assay for chikv infection was established using gluc as readout [ ] . the vrps could be produced efficiently in the bhk- cells by co-transfecting of the chikv replicon expressing gluc and the helper rnas [ ] . subsequently, the infection with vrps was measured via gluc secreted into the supernatant, and the chikv-neutralizing antibodies could be determined within a day by the assay without the need of using infectious chikv [ ] . cell-based reporter systems are also well suited for use in clinical examinations and epidemiological surveillance [ ] . the subgenomic reporter rna systems developed by steel and his colleagues were relatively species specific and allowed for rapid and simple visual detection of the wild-type viruses in mosquito cells [ ] . moreover, the replicon-defective reporter gene assay could detect a variety of alphaviruses from tissue cultures with a limit of detection between one and ten (plaque-forming unit) pfu for sinv while other rna viruses, such as the japanese encephalitis virus and tahyna virus, displayed negative results with this system [ ] . cell-based reporter systems have enhanced our understanding of the molecular mechanisms of virus replication and pathogenesis as well as virus interactions with host cells. they also provide platforms for virus detection and drug susceptibility testing (table ). however, there are still several issues with these systems that need to be further explored. first, not all positive-sense single strand viruses have a successful cell-based reporter system. one future challenge will be the generation of a sensitive cell-based reporter system for detecting or tracking emerging viruses and pathogenic viruses that are currently not cultivable. second, viruses are highly variable and undergo frequent mutation, so new reporter systems for these viruses need to be developed that can identify mutations and serotypes more efficiently and accurately. additionally, future challenges will lie in optimizing cell-based reporter systems for high throughput screening of antiviral agents. rna-dependent rna polymerases, viruses, and rna silencing inhibition of rna binding to hepatitis c virus rna-dependent rna polymerase: a new mechanism for antiviral intervention construction of self-replicating subgenomic dengue virus (denv ) replicon construction of self-replicating subgenomic west nile virus replicons for screening antiviral compounds the hepatitis c virus replicon system: from basic research to clinical application novel cell culture systems for the hepatitis c virus efficient replication of hepatitis c virus genotype a rnas in cell culture amodiaquine, an antimalarial drug, inhibits dengue virus type replication and infectivity dengue virus serotype blocks extracellular signal-regulated kinase and nuclear factor-kappab activation to downregulate cytokine production flavivirus induces interferon-beta gene expression through a pathway involving rig-i-dependent irf- and pi k-dependent nf-kappab activation a dark-to-bright reporter cell for classical swine fever virus infection tricistronic hepatitis c virus subgenomic replicon expressing double transgenes structural analysis of foot-and-mouth disease virus c protease: a viable target for antiviral drugs? foot-and-mouth disease virus c protease: recent structural and functional insights into an antiviral target role of human heterogeneous nuclear ribonucleoprotein c /c in dengue virus replication discovery of novel small molecule inhibitors of dengue viral ns b-ns protease using virtual screening and scaffold hopping development of a novel protocol for generating flavivirus reporter particles composition of the sequence downstream of the dengue virus ′ cyclization sequence (dcs) affects viral rna replication modulation of translation initiation efficiency in classical swine fever virus virus replicon particle based chikungunya virus neutralization assay using gaussia luciferase as readout diagnostic methods for detection of classical swine fever virus-status quo and new developments minigenomes, transcription and replication competent virus-like particles and beyond: reverse genetics systems for filoviruses and other negative stranded hemorrhagic fever viruses replicon cell culture system as a valuable tool in antiviral drug discovery against hepatitis c virus identification of a small-molecule inhibitor of dengue virus using a replicon system the ′-terminal hexamer sequence of classical swine fever virus rna plays a role in negatively regulating the ires-mediated translation a reporter cell line for rapid and sensitive evaluation of hepatitis c virus infectivity and replication host factors that interact with the pestivirus n-terminal protease, npro, are components of the ribonucleoprotein complex the pestivirus n terminal protease n(pro) redistributes to mitochondria and peroxisomes suggesting new sites for regulation of irf by n(pro construction and applications of yellow fever virus replicons a derivate of the antibiotic doxorubicin is a selective inhibitor of dengue and yellow fever virus replication in vitro efficient replication of the genotype a hepatitis c virus subgenomic replicon hepatitis c virus ns / a protease loss of interferon regulatory factor in cells infected with classical swine fever virus involves the n-terminal protease construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors establishment of a stable cell line coexpressing dengue virus- and green fluorescent protein for screening of antiviral compounds development of dengue type- virus replicons expressing gfp reporter gene in study of viral rna replication a reporter-based assay for identifying hepatitis c virus inhibitors based on subgenomic replicon cells development of a cell-based assay for monitoring specific hepatitis c virus ns / a protease activity in mammalian cells a cell-based reporter assay for inhibitor screening of hepatitis c virus rna-dependent rna polymerase characterization of the activity of ′-c-methylcytidine against dengue virus replication high-throughput cell-based screening for hepatitis c virus ns / a protease inhibitors functional interaction between cellular p and the dengue virus ′ utr development of a cell-based assay for monitoring hepatitis c virus ns / a protease activity rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay the classic swine fever virus (csfv) core protein can enhance de novo-initiated rna synthesis by the csfv polymerase ns b hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity potential high-throughput assay for screening inhibitors of west nile virus replication replication of subgenomic hepatitis c virus rnas in a hepatoma cell line flexibility between the protease and helicase domains of the dengue virus ns protein conferred by the linker region and its functional implications crystal structure of the ns protease-helicase from dengue virus a pcr-based protocol for generating west nile virus replicons identification of cis-acting elements in the ′-untranslated region of the dengue virus type rna that modulate translation and replication dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes a plasmid-based reporter system for live cell imaging of dengue virus infected cells development and application of west nile virus subgenomic replicon rna expressing secreted alkaline phosphatase structural basis of fluorescence quenching in caspase activatable identification of human hnrnp c /c as a dengue virus ns -interacting protein a cell line that expresses a reporter gene in response to infection by sindbis virus: a prototype for detection of positive strand rna viruses development of ns / a proteasebased reporter assay suitable for efficiently assessing hepatitis c virus infection a luciferase-based screening method for inhibitors of alphavirus replication applied to nucleoside analogues nuclear localization of dengue virus nonstructural protein through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection construction of yellow fever virus subgenomic replicons by yeast-based homologous recombination cloning technique analysis of classical swine fever virus rna replication determinants using replicons novel hepatitis c virus reporter replicon cell lines enable efficient antiviral screening against genotype a n(pro) of classical swine fever virus is an antagonist of double-stranded rna-mediated apoptosis and ifn-alpha/beta induction dengue virus capsid protein usurps lipid droplets for viral particle formation dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro generation of a recombinant classical swine fever virus stably expressing the firefly luciferase gene for quantitative antiviral assay classical swine fever virus ns b protein suppresses the inhibitory effect of ns a on viral translation by binding to ns a construction and characterization of subgenomic replicons of new york strain of west nile virus a novel reporter system for neutralizing and enhancing antibody assay against dengue virus subgenomic reporter rna system for detection of alphavirus infection in mosquitoes zinc binding in pestivirus n(pro) is required for interferon regulatory factor interaction and degradation characterization of the variable region in the ′ nontranslated region of dengue type virus insertion of egfp into the replicase gene of semliki forest virus results in a novel, genetically stable marker virus npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type i interferon induction at local replication sites establishment of an indicator cell system for hepatitis c virus an in vitro coupled transcription/ translation reporter system for hepatitis c virus rna-dependent rna polymerase comparison of six detection methods for classical swine fever virus development of egfp/gluc-tagged sindbis-like virus xj- effect of ns and ns b proteins on classical swine fever virus internal ribosome entry site-mediated translation and its host cellular translation influence of ns a protein of classical swine fever virus (csfv) on csfv internal ribosome entry site-dependent translation evaluation of single-round infectious, chimeric dengue type virus as an antigen for dengue functional antibody assays novel dengue virus-specific ns b/ns protease inhibitor, bp , discovered by a high-throughput screening assay a novel dengue virus inhibitor, bp , discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral ns b/ns protease characterization of an efficient dengue virus replicon for development of assays of discovery of small molecules against dengue virus construction and application of chimeric virus-like particles of tick-borne encephalitis virus and mosquito-borne japanese encephalitis virus packaging the replicon rna of the far-eastern subtype of tick-borne encephalitis virus into single-round infectious particles: development of a heterologous gene delivery system development and characterization of a stable luciferase dengue virus for high-throughput screening development of egfp/gluc-tagged sindbis-like virus xj- acknowledgments this work is supported by the jilin province science and technology development projects conflict of interest the authors declare that they have no competing interests. key: cord- -ityhcak authors: zhu, hanliang; podesva, pavel; liu, xiaocheng; zhang, haoqing; teply, tomas; xu, ying; chang, honglong; qian, airong; lei, yingfeng; li, yu; niculescu, andreea; iliescu, ciprian; neuzil, pavel title: iot pcr for pandemic disease detection and its spread monitoring date: - - journal: sens actuators b chem doi: . /j.snb. . sha: doc_id: cord_uid: ityhcak during infectious disease outbreaks, the centers for disease control need to monitor particular areas. considerable effort has been invested in the development of portable, user-friendly, and cost-effective systems for point-of-care (poc) diagnostics, which could also create an internet of things (iot) for healthcare via a global network. however, at present iot based on a functional poc instrument is not available. here we show a fast, user-friendly, and affordable iot system based on a miniaturized polymerase chain reaction device. we demonstrated the system’s capability by amplification of complementary deoxyribonucleic acid (cdna) of the dengue fever virus. the resulting data were then automatically uploaded via a bluetooth interface to an android-based smartphone and then wirelessly sent to a global network, instantly making the test results available anywhere in the world. the iot system presented here could become an essential tool for healthcare centers to tackle infectious disease outbreaks identified either by dna or ribonucleic acid. dengue fever, ranked as the most important mosquito-borne viral disease with pandemic potential, emerged as a serious public health concern. the world health organization (who) reported that in the past fifty years, the number of cases has increased times, causing huge human and economic cost which has yet to be tackled [ ] . according to studies [ , ] , there were an estimated million annual cases of dengue fever and an estimated . billion people in countries are at risk of being infected by dengue fever virus (denv) with different serotypes. the denv is affecting numerous areas in eastern and southeast asia, such as china [ ] ; the lowlands of nepal [ ] ; the slums in delhi, india [ ] ; hanoi, vietnam [ ] ; and iran [ ] as well as other parts of the world, such as the middle east, north africa [ ] , and south america [ ] . due to the lack of effective treatment of severe dengue fever [ ] , accurate and early detection methods to identify the denv serotype [ , , ] are required in order to provide careful patient monitoring and to prevent the disease's progression to a more severe stage. mobile laboratories suitable for tackling highly contagious diseases such as ebola, have been developed [ ] , but they are too bulky and expensive for non-contagious denv, malaria or similar diseases. an easy-to-use, affordable, truthful and reliable point of care (poc) device to perform rapid and economical denv diagnostic tests in high-risk areas is badly needed. moreover, it is of the utmost importance to immediately report the results of the poc, including its time and location, to authorities in a centralized location to take required measures. these days, a vast amount of the human population has possession of a cell phone with an embedded global positioning system (gps) connected to mobile networks via base stations using different generations of cellular mobile communications, such as second ( telecommunications system) or fourth (long term evolution (lte)/ worldwide interoperability for microwave access). connecting the easy to use and cost-effective poc devices providing the denv diagnoses via a mobile network would create an internet of things (iot) [ ] for healthcare [ , ] , an essential tool to tackle any infectious disease outbreak. the iot would speed up the information transfer from poc devices to a centralized location and then based on models using big data analysis [ , , ] , suggest targeting specific sites. the iot would be able to communicate with the poc systems, inform its operator where to go next to perform testing, remotely change the test protocols or provide other important information to the poc device user. this year, the fifth generation of cellular mobile communications will be released with a massively increased data rate compared to those of previous generations. nevertheless, the iot for poc applications such as denv testing can rely on the oldest generation of g, as the poc communication with the centralized location requires a low data rate, an important feature for cash strapped countries of the developing world. denv is often detected by a paper-based immunochromatographic test either alone [ ] or in combination with malaria [ ] , enzymelinked immunosorbent assay (elisa) [ ] , and/or reverse transcription polymerase chain reaction (rt-pcr) [ ] (allowing identification of the denv serotype) [ ] . the paper-based lateral flow immunechromatographic test is fast and easy to usetherefore is often used in remote areas. however, due to its lower sensitivity as well as specificity, a confirmation using a second detection method (elisa and/or rt-pcr) is often required. the elisa method relies on antibody production by the human immune system, the method being more suitable for advanced stages of the disease. the rt-pcr is the preferred method as, in principle, it can detect a single copy of specific ribonucleic acid (rna) [ , ] having both sensitivity and specificity required for early disease detection (lowering the cost of treatment). unfortunately, rt-pcr tests are typically only carried out in hospitals or certified diagnostic laboratories after the onset of denv symptoms. the rt-pcr method is highly specific and reliable; the problem is that not every infected patient makes it to the hospital to be positively diagnosed. as an outcome, the diagnostic results are not available as quickly and comprehensively as required for disease outbreak control. recently, a lot of effort was invested in the development of a portable system for poc diagnostics [ ] [ ] [ ] [ ] . numerous methods were developed using a smartphone for data communications as well as fully integrated systems [ ] such as a smartphone-based optical system for high throughput [ ] . we previously reported a hand-held polymerase chain reaction (pcr) system capable of four simultaneous reactions, demonstrating its performance by amplification of complementary deoxyribonucleic acid (cdna) of avian influenza a (h n ) rna, proving its principle of operation [ ] . later on, a reverse transcription step was added and rna of an ebola virus was detected with rt-pcr while using human transcript glyceraldehyde- -phosphate dehydrogenase rna as a positive control [ ] . here, we report an iot pcr device that can be used for pandemic disease detection and its spread monitoring. it is a portable system with a weight of ≈ g and physical dimensions of (≈ × ≈ × ≈ ) mm equipped with bluetooth (bt) communication between the pcr and the android wireless tool, such as a smartphone or a tablet. we utilized it to test cdna of a denv, which could also be used for rna detection. once the denv was detected, the unit sent this information, including the gps coordinates to a centralized location via the lte network, thus helping the authorities to map the disease spread (as suggested in fig. ). the core of the system consists of four virtual reaction chambers (vrc) on a hydrophobically coated glass (fig. c) which were successfully tested earlier for ultrafast real-time pcr with external optics [ ] , and a fully integrated real-time pcr [ ] . the glass was placed on a silicon chip made by micro-electro-mechanical system (mems) technology developed earlier [ ] . we improved the layout of the mems chip containing heaters and sensors using a nanolithography toolbox [ ] by adding an electrically grounded guard ring between the thin film sensor and the heater. we also increased the chip size to (≈ × ≈ mm) to assure easier handling. the chip was placed on its own printed circuit board (pcb) and was connected to the motherboard of the pcr unit by a connector for simple replacement and calibration ( fig. b and d) . the electronics as well as the optics ( fig. a) was also improved but it is not a subject of this contribution. the pcr systems were then assembled and calibration were ready for deployment. we tested the pcr device performance using cdna of denv prepared by synthesis. real field testing of the device would have to start with sample collection and preparation followed by reverse transcription before the pcr can be conducted [ , ] . also, reagents for single step qrt-pcr are needed. more details are in the discussion section. we selected an amplicon with a total length of base pairs (bp) from bp to bp, as follows: acaagtagaacaacctggtccatacacgccaaacatgaatggatg acaacggaagacatgctgacagtctggaacagggtgtggattcaaga aaacccatggatggaagacaaaactccagtggaatcatgggaggaaa tcccatacttgggaaaaagagaagaccaatggtgcggc using forward and reverse primers with sequences acaagtagaacaacctggtccat and gccgcaccattggtcttctc, respectively. we prepared a solution to conduct the pcr by mixing ≈ . μl of a commercial polymerase with contents of units μl − , ≈ μl of a pcr buffer consisting of mm tris (hydroxymethyl) aminomethane-hcl (ph ≈ . ), mm kcl, ≈ mm mgcl , ≈ . μl of a deoxyribonucleotide triphosphate mixture consisting of adenine, thymine, cytosine, and guanine each with a concentration of ≈ . mm, ≈ . μl of forward as well as reverse primer, ≈ . μl of eva green intercalating dye with original concentration × (diluted as per manufacturer instruction), ≈ μl of bovine serum albumin with contents of ≈ mg·ml − , and ≈ μl of cdna template with varied contents depending on the experiment. furthermore, the total volume was adjusted by adding ≈ . μl of sterilized water to a total volume of ≈ μl. prior to testing on an iot pcr device, we verified the master mix performance and its values of critical threshold (c t ) and the melting temperature (t m ) using a commercial real-time pcr system (supplementary section a) beginning with a hot start at °c for s followed by cycles of pcr amplification consisting of dna denaturation at °c for s, primer annealing at °c for s, and dna sequence elongation at °c for s, then followed by melting curve analysis (mca) from °c to °c. once the master mix performance was verified, we ran the same protocol on the iot pcr system. wireless communication between mobile platforms, such as a mobile phone or tablet, and the pcr system was provided via a bt module, which comprised a commercial unit supporting bt version . . this unit has its own dedicated v power supply with a universal serial bus (usb) connector, including ac-dc converter and voltage stabilizer, making it compatible with a standard usb power supply or power bank. the independent power supply for the bt module eliminates its influence on pcr system stability. data transfer between the bt unit and pcr system is conducted bi-directionally via a universal asynchronous receiver-transmitter interface. based on a request from a mobile system, such as a smartphone or tablet, the last pcr measurement data are sent to the mobile system. we developed an application (app) for android devices that allows them to receive data via the bt communication module from the pcr system, save them into a text file, and represent them in graphical format. the smartphone display is split into two parts: the top is used to show the pcr amplification curve and the bottom for mca. the system has the option of sending the data to a dedicated place with gps coordinates to inform authorities about the presence of the infectious disease. after pairing the android device with the bt unit of the pcr system, data from the pcr system are downloaded into the device and plotted on its display. they can also be automatically sent via lte network to a dedicated place monitoring the disease outbreak. for convenience, we also created a personal computer (pc) app for pcr system programming (fig. a) either via usb or bt interface. we performed two basic real-time pcr experiments. the first test was conducted with four samples having a master mix with identical contents of the cdna of denv template to verify real-time pcr system uniformity and repeatability as well as temperature settings by performing mca. the second test mimicked the actual field test having three samples and a no template control (ntc) (supplementary section b) . the captured analog-to-digital converter output values of pcr fig. . the schematic diagram demonstrates the iot system that could be applied for denv spread monitoring. once the sample potentially containing the denv is processed at point of interest location, the results of diagnoses as well as gps coordinates of the location are automatically transferred via the user's mobile phone interface through a global network to a control center. all results can be collected as cloud data by a network to create a disease outbreak map showing the disease outbreak area and carry out continuous monitoring. amplification data, as well as mcas, were displayed on the integrated thin film transistor (tft) screen of the pcr unit. then they were transferred to the smartphone via bt to be displayed by the mobile system and also further processed by subtracting the background noise and normalization. the data were then averaged from four consecutive runs and plotted in a linear (fig. b) and logarithmic (fig. c) scale. we then calculated the c t as the value of the cycle number with % of relative fluorescence amplitude as ( . ± . ) cycle (mean ± standard deviation from four measurements), showing an excellent point to spot result uniformity. we also extracted the mca (fig. d ) and performed the curve fitting using a modified boltzmann curve obtaining a t m value of ( . ± . )°c (mean ± standard deviation from four measurements). we subsequently performed a numerical differentiation of the fitted curves for better visualization (fig. e) by showing the values of t m as peaks of the -df/dt curve. concurrently, we performed identical testing using a commercial real-time pcr system getting comparable results for both, c t and t m (supplementary section a) . then, the data containing the time of experiment and gps location could be sent via lte to the centralized location for further analysis of the disease spreading. we developed an iot pcr system equipped with bt communication and a supporting android app for smartphones as well as a pc app for convenient pcr programming with a projected manufacturing cost between - usd. this real-time pcr is a robust device with no moving parts and uses four standard light emitting diodes (leds) with a diameter of mm as a light source with a lifetime of more than , h. the most fragile part is the micromachined silicon chip, here mounted on its own replaceable pcb. we performed a set of experiments using a cdna of denv with cycles of a three-step pcr protocol followed by an mca taking ≈ min in total. the testing time can be shortened either by system redesign [ ] or using probe-based pcr and performing a single pcr cycle as fast as s [ ] , thus having a total required time for the cycle pcr protocol as short as ≈ min. the system can be further expanded using multiplexing just by reprogramming and effectively doubling the throughput [ ] , reprogramming the pcr readout, or using multicolor leds with a dual bandpass filter. once the pcr was completed, the data were transferred via bt to a portable android platform and then they could be sent via lte network together with the pcr unit number, time of test completion and gps coordinates of its location to a centralized location. the positive result could indicate the spread of the infectious disease on a map in a similar form as the hypothetical results from xi'an, shaanxi province, p. r. china (fig. ) . we performed the first test in our laboratory capturing pcr data as well as the gps location, and then we emulated the tests performed through the city by capturing the locations only (supplementary section c). this pcr with bt and android-based wireless communication could be a key part of an iot system for healthcare ready for deployment to tackle infectious disease outbreaks. an actual approach to tackle infectious disease spreading would require obtaining samples from patients, processing them depending on the body fluid type, then mixing them with the reagents and pre-concentrating them into volume compatible with the miniaturized pcr, which is . μl or less. samples collected from cheek swabs or from saliva have very low contents of dna, thus hindering the pcr analysis. there are readily available single-step kits for real-time pcr systems capable of processing samples as they are collected [ , ] . the only requirement is mixing the sample with these reagents. after the mixing step, the cells are enzymatically lysed, so that dna/rna from cells is released, followed by heating to stop the lysis enzymes [ ] and activation of the polymerase, which is then followed by pcr thermal cycling. the last two steps would be performed on the hand-held pcr. samples that originate from blood contain albumin and other pcr inhibitors; these have to be first removed by a method compatible with hand-held pcr as shown before [ , ] using functionalized paramagnetic beads. this sample preparation also includes sample preconcentration to process small sample volumes by hand-held pcr. any part of the system getting into contact with the actual sample has to be disposable due to extremely high contagion of sample to be tested. both systems, potential sample preparation as well as the pcr reaction should be conducted on a disposable microscope cover slip glass coated with suitable fluorosilanes or teflon making their surface hydrophobic [ ] . all other parts of the system can be non-disposable since they do not come into contact with either the reagents or the sample. once the test is completed, the used glass objects are disposed of and are then replaced with fresh ones. the device is now ready making the entire process of diagnoses economical. here we tested a denv as a typical representative of an infectious disease spread by mosquitos. the same system can also be applied to monitor the spreading of other diseases, such as malaria. in principle, its deployment could also be possible for highly contagious diseases such as ebola, severe acute respiratory syndrome (sars), and other viruses, but in such circumstances, the system would have to be handled by highly trained professionals due to the highly infectious nature of the viruses. also, for practical reasons, the hand-held pcr system would have to be redesigned for autoclaving to prevent any contamination of the operator. we tested an iot real-time pcr system and demonstrated that the device sensitivity and reproducibility were improved. the pcr device performance is comparable with that of a commercial system. the protocol consisting of cycles to detect the cdna of a denv required ≈ min. the system was connected via bt communication with an android-based smartphone and then with the whole world via lte. this could become a basic block of the iot, thus helping to tackle infectious disease outbreaks. the device can be used for other diseases such as any type of influenza, malaria, and human immunodeficiency virus. it can be used as a "first-defender" tool to perform rapid early diagnosis and, more importantly, the data collected can be used to prevent pandemic outbreaks. pavel neuzil, ciprian iliescu and hanliang zhu wrote the manuscript, hanliang zhu performed the experiments and data analyses, and pavel podesva improved and assembled the pcr systems. xiaocheng liu and haoqing zhang fabricated the pcr chip, tomas teply programmed the internal pcr software as well as the android app; honglong chang participated in the organizing and writing of the manuscript; ying xu conceived the pcr as an iot device and participated in writing the manuscript; airong qian, yingfeng lei, and yu li chose the cdna sequence and designed the primers; andreea niculescu wrote the iot part of the manuscript; and pavel neuzil designed the portable pcr concept as well as the micromachined chip. authors declare no competing interests. global brief on vector-borne diseases, who the global distribution and burden of dengue refining the global spatial limits of dengue virus transmission by evidence-based consensus characterizing a large outbreak of dengue fever in guangdong province cost-effective real-time reverse transcriptase pcr (rt-pcr) to screen for dengue virus followed by rapid single-tube multiplex rt-pcr for serotyping of the virus widespread fear of dengue 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polymerase chain reaction in microfluidic drops disposable real-time micropcr device: lab-on-achip at a low cost he is currently a ph.d. student of school of mechanical engineering in npu. his research interests include microfluidics and miniaturization and application of pcr systems he was a postdoctoral research fellow at the npu for over two years and he is currently a researcher in but. his research interests include analytical chemistry, material chemistry and mems degrees from npu in and , respectively. he is currently a ph.d. student of school of mechanical engineering in npu. his research interests include adhesion force of gecko-inspired nanostructure and its application haoqing zhang received the b.s. and m.s. degrees from the xi'an university of technology in and , respectively. he is currently a ph.d. student of school of mechanical engineering in npu. his research interests include droplet pcr and digital pcr systems currently he is a lecturer and member of microsystems group in the department of microelectronics, fee ctu in prague. his interests include programming of microcontrollers, sensors application and electronic circuits design she had worked in r&d in medical device industry. she also obtained emba from wharton school of business, university of pennsylvania. she worked as a scientist at fraunhofer-ibmt in germany prior to joining npu as a research associate in the microfluidic systems lab respectively. currently, he is a faculty member as associate professor and vice director of department of microbiology in fourth military medical university. he has published over peer-reviewed journal and conference papers, and about chinese patents as principle investigator from fourth military medical university in , , and , respectively. currently, he is currently an associate professor with faculty of life sciences, npu. he has published over sci papers, and one international pct patent and six national invention patents. he is a member of the china cancer society's cancer mark professional committee, the shaanxi provincial cell biology society, and the shaanxi provincial cancer mark professional committee. his interests include in antibody drugs and tumor cell biology her research interests include user experience and interaction design. currently, she is working on several projects focusing on user interfaces for ai and iot applications he is member of the academy of romanian scientists. and now work in national institute for research and development in microtechnologies he is currently a professor with the ministry of education key laboratory of micro and nano systems for aerospace supplementary material related to this article can be found, in the online version, at doi: https://doi.org/ . /j.snb. . . key: cord- -okvvajms authors: lazzarini, luca; barzon, luisa; foglia, felice; manfrin, vinicio; pacenti, monia; pavan, giacomina; rassu, mario; capelli, gioia; montarsi, fabrizio; martini, simone; zanella, francesca; padovan, maria teresa; russo, francesca; gobbi, federico title: first autochthonous dengue outbreak in italy, august date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: okvvajms in august , during the coronavirus disease (covid- ) pandemic, five locally acquired cases of dengue virus type were detected in a family cluster in vicenza province, north-east italy where aedes albopictus mosquitoes are endemic. the primary case was an importation from west sumatra, indonesia. this is the first outbreak of autochthonous dengue reported in italy. during the covid- pandemic, screening of febrile travelers from endemic countries is crucial in areas where competent vectors are present. in august , during the coronavirus disease (covid- ) pandemic, five locally acquired cases of dengue virus type were detected in a family cluster in vicenza province, north-east italy where aedes albopictus mosquitoes are endemic. the primary case was an importation from west sumatra, indonesia. this is the first outbreak of autochthonous dengue reported in italy. during the covid- pandemic, screening of febrile travelers from endemic countries is crucial in areas where competent vectors are present. in europe, dengue is almost always imported from endemic countries, and only very few autochthonous cases and limited outbreaks have been reported. we present the first autochthonous outbreak of dengue in italy, due to an imported case from indonesia. a woman in her thirties from vicenza province, veneto region, italy, (case ) stayed in pulau weh, a tropical island in west sumatra, indonesia, for months and returned to her home town in vicenza province on july . she had a four-day stopover in djakarta before flying back to italy. after her return, she stayed at home for days to complete the coronavirus disease (covid- ) quarantine, mandated by the italian government for travellers outside european union (eu) countries at the time. since july, she experienced fever ( ° c), malaise, back pain and upper limb itching. on july, she was tested with oral and nasal swab for severe acute respiratory syndrome coronavirus (sars-cov- ) rna and found negative. clinical symptoms resolved within days. between and august, five of her seven household contacts started having symptoms of fever (> ° c), malaise, headache, and upper limb itching. the contacts included a female and male in their fifties (case and case , respectively), two males in their twenties, and a preschool child (cases , and , respectively). clinical symptoms resolved within days in all of them. case had stayed in in pulau weh in january for days. she was asymptomatic during and after travel. cases , , and never travelled abroad. case presented to the infectious diseases unit of our hospital on august, when clinical symptoms had already disappeared. she was investigated for west nile virus, usutu virus, dengue virus (denv), chikungunya virus (chikv) and zika virus (zikv) by molecular and serology testing, because she reported that a family member (case ) had had similar symptoms after a recent travel in indonesia. she was pcr tested for sars-cov- and found negative. the results were available on august, and she was consecutively diagnosed with denv type (denv- ) infection based on positive realtime reverse transcriptase (rt)-pcr on plasma, urine, and saliva, and positive denv ns antigen in plasma, while serology testing was negative. following the dengue diagnosis of case , cases , , , and were invited to present to the same unit, where they were tested on august and also found positive for denv- (table) . all serological tests for chikv, zikv, west nile and usutu vires were negative. all patients recovered fully without complications, and none may be classified as severe dengue. following the notification to the public health authority of veneto region on august, public health technicians inspected the house where the affected family lives, which is in a rural, underpopulated area. on the same day, and for three consecutive days, mosquito control was conducted in the area comprised in a radius of m around the house, and extended to sensible places nearby such as a hospital and a touristic castle, as indicated by the national plan against arbovirus infections [ ] . disinfestations included larvicides, adulticides and removal of the breeding sites. on august, an entomological monitoring was set up using bg-sentinel traps, ovitraps and manual aspiration in order to control the effectiveness of the disinfestations, to define the mosquito density outside the area and to search for the virus. a retrospective laboratory investigation was performed in all the patients from vicenza province who were referred for suspected arbovirus infection during the previous month, which excluded denv- infection in all of them. general practitioners in the district where the outbreak occurred were alerted to refer cases of unexplained fever for investigation to the local infectious diseases department. during and soon after disinfestation activities, local health professionals, entomologists and representatives of the local police went from door-todoor in the radius of m to interview the inhabitants on symptoms, and raise awareness for the need to avoid mosquito bites and the need to remove possible larval breeding sites. the population of the city where the family resides is about , inhabitants, and was provided with informative brochures about dengue. in addition, on august, the national transplant centre activated denv nat screening in organ, tissue and haematopoietic stem cell donors, as well as in blood donors, who reside in vicenza province or had stayed for at least one night in vicenza province in the days before donation. blood collections from donors in the town where the outbreak occurred have been suspended. this analysis was conducted as part of public health usual practice, and was not conducted for research. patients gave informed consent to anonymously publish their clinical and laboratory data for the purpose of this paper. [ ] . real-time rt-pcr assays were carried out using the one-step, real-time kit (thermo fisher scientific, waltham, massachusetts, us) and run on abi ht sequence detection systems (thermo fisher scientific). b denv ns antigen was detected in plasma by using a rapid immuno-chromatographic assay (dengue ns ag strips, bio-rad, hercules, california, us). c denv igm and igg antibodies were detected by a chemiluminescence immunoassay (virclia, vircell, granada, spain) in a thunderbolt instrument (vircell). between and , autochthonous cases of dengue fever in europe were reported in from france [ ] and croatia [ ] , in from madeira, portugal [ ] , from france in , , , , [ ] [ ] [ ] and from spain in and [ , ] . these cases were caused by denv- , as in cases described here, and denv- [ ] . previously, in italy no autochthonous cases of dengue had been reported, while two outbreaks of chikv disease were detected in emilia-romagna region and in central-southern italy in and in , respectively [ , ] . the veneto region started an integrated surveillance for imported fevers in [ ] . the surveillance, active between june and november, aims to increase the detection rate of denv and chikv (since , also zikv) infection in travellers from endemic areas and to promptly identify potential autochthonous cases. patients with fever > ° c in the last days and recent (less than days) return from endemic countries, after ruling out malaria, are referred to an infectious diseases department of the region to perform a rapid test for denv and to collect samples for the regional reference laboratory in padua, for second line testing. entomologic investigations are also performed in the areas surrounding ( m) the residence of human cases of denv, chikv and zikv infection, if notified as viraemic [ ] . in ten years of surveillance ( - ), ca , patients were tested and were positive for denv, for chikv and for zikv. each year, a variable proportion of febrile cases returning from endemic areas, ranging from . % in to . % in , are diagnosed with an arbovirus infection. all these cases were imported. a mathematical model was applied to epidemiological and entomological data to assess the risk of a denv outbreak in northern italy [ ] . the risk for denv outbreaks was estimated lower than for chikv, because of the lower competence for transmitting denv of ae. albopictus, the dominant vector in our area. we cannot exclude that unnoticed autochthonous cases of denv may have occurred in italy, especially if asymptomatic. however, we believe that any cluster of unexplained summer fever should be detected by existing clinical surveillance plans. the index patient in the outbreak described here was not promptly referred to an infectious disease department, but the mandatory covid- restriction measures also limited the risk of denv spread from the index case during the viraemic phase. at the same time, the ongoing covid- pandemic caused a diagnostic delay, as the first clinical suspicion was covid- . however, the high density of mosquitoes in the area where the source case (case ) was living, as well as her permanent presence at home due to quarantine, led to a high attack rate among household contacts. active clinical surveillance in the control area, aimed at diagnosing other possible secondary cases is still ongoing, as well as entomological surveillance, including installation of ovitraps, larval search and manual aspiration of adult mosquitos within and outside the control area. we suggest that risk of further local spread may be possible, depending on the number and movement of potential viraemic people outside the control area before clinical diagnosis and on the local mosquito density. clinical surveillance of summer fever and surveillance of febrile travellers returning from endemic areas should not be limited to rule out sars-cov- infection, and should include arbovirus screening in all countries where competent vectors are present. west nile virus and usutu, the surveillance and response plan. rome: ministry of health first two autochthonous dengue virus infections in metropolitan france autochthonous dengue fever in croatia clinical presentation and laboratory findings for the first autochthonous cases of dengue fever in madeira island autochthonous dengue outbreak in nîmes european centre for disease prevention and control (ecdc) european centre for disease prevention and control (ecdc) infection with chikungunya virus in italy: an outbreak in a temperate region detection of a chikungunya outbreak in central italy surveillance for west nile, dengue, and chikungunya virus infections human and entomological surveillance of west nile fever, dengue and chikungunya in veneto region potential risk of dengue and chikungunya outbreaks in northern italy based on a population model of aedes albopictus (diptera: culicidae) analytical and clinical performance of the cdc real time rt-pcr assay for detection and typing of dengue virus none declared. luca lazzarini and vinicio manfrin managed the patients. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- - xy s authors: hu, dan; zhu, zhongyu; li, shun; deng, yongqiang; wu, yanling; zhang, nana; puri, vinita; wang, chunyu; zou, peng; lei, cheng; tian, xiaolong; wang, yulu; zhao, qi; li, wei; prabakaran, ponraj; feng, yang; cardosa, jane; qin, chengfeng; zhou, xiaohui; dimitrov, dimiter s.; ying, tianlei title: a broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain iii date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: xy s dengue is the most widespread vector-borne viral disease caused by dengue virus (denv) for which there are no safe, effective drugs approved for clinical use. here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the denv envelop protein domain iii (diii) combined with depletion by an entry defective diii mutant, we identified a cross-reactive human monoclonal antibody (mab), m . , which bound with high affinity to denv diii from all four denv serotypes. immunogenetic analysis indicated that m . is a germline-like mab with very few somatic mutations from the closest vh and vλ germline genes. importantly, we demonstrated that it potently neutralized denv both in vitro and in the mouse models of denv infection without detectable antibody-dependent enhancement (ade) effect. the epitope of m . was mapped to the highly conserved regions on diii, which may guide the design of effective dengue vaccine immunogens. furthermore, as the first germline-like mab derived from a naïve antibody library that could neutralize all four denv serotypes, the m . can be a tool for exploring mechanisms of denv infection, and is a promising therapeutic candidate. a a a a a dengue virus (denv) causes the most prevalent mosquito-borne viral disease. over . billion people are at risk for infection in over countries, - million are infected with symptoms, and up to , die from dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) each year [ , ] . no specific antiviral drug has been available against denv infection; the only approved vaccine, dengvaxia, has caused considerable controversy regarding its safety and potential benefits [ ] [ ] [ ] [ ] . for decades, anti-denv vaccine and biological drugs development has been hampered by the high sequence divergence ( - %) among the four denv serotypes [ , ] . such divergence leads to the fact that one antibody may not be sufficient to neutralize all denv infection. instead, the induced humoral immune response to one denv infection can enhance the infection and disease processes brought by a subsequent infection with another denv serotype [ ] [ ] [ ] . these findings suggest that the development of new and broadly neutralization antibodies against all the serotypes of denv could be promising candidate anti-denv agents, and may also guide the design of effective and safe vaccine immunogens. the denv envelope glycoprotein (e protein), which mediates virus entry into cells, is the major neutralizing target of antibodies [ ] [ ] [ ] [ ] [ ] . e protein is a type ii fusion protein and consists of three domains: di, dii, and diii of which diii has been proposed to contain a receptor binding domain [ ] [ ] [ ] [ ] . recent studies revealed that cross-reactive conserved epitopes exist on dii as well as diii of the denv e protein [ , [ ] [ ] [ ] . during the naturally-occurring primary denv infection, a large fraction of the antibody repertoire consists of dii-specific antibodies which are, unfortunately, typically poor in neutralization and may increase the likelihood of severe disease upon subsequent infection through a mechanism known as antibody-dependent enhancement (ade) [ ] [ ] [ ] . in contrast, antibodies targeting diii have proven to be the most potent neutralizing antibodies, but very few could be elicited in naturally infected individuals [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite this, previous studies indicated that anti-denv diii serotype-specific and cross-reactive antibodies could be elicited using denv diii as vaccine immunogen [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and in infected humans [ ] [ ] [ ] [ ] . it has also been demonstrated that the lysine at position on diii is the critical residue in the cross-reactive epitope [ ] . therefore, the conserved epitope on diii represents an attractive target for the development of broadly neutralizing denv antibodies. here, we report the isolation of a potent denv diii-specific human monoclonal antibody (mab), designated as m . , from a large naïve antibody library constructed by the blood of healthy adult donors. a competitive sorting strategy using a diii mutant as competitor was applied to identify antibodies precisely targeting the conserved neutralizing epitope. to our knowledge, m . is the first human mab isolated from a naïve antibody library which could neutralize all the four serotypes denv viruses. importantly, both heavy and light chain genes of m . are very close to their putative germline predecessors. its fully human origin, the germline-like nature, combined with high-affinity and broad neutralizing activity toward all denv serotypes, suggest that m . is a promising candidate antiviral agent and may also provide a unique template for designing effective dengue vaccine immunogens. we previously prepared some large naïve antibody libraries using peripheral blood b lymphocytes of non-immunized healthy donors and used them for panning/screening against viral and cancer targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, we used a competitive library sorting strategy to isolate broadly neutralizing antibodies against denv - ( fig a and b) . the yeast-displayed naïve single chain antibody fragment (scfv) library was used to screen against the biotinylated denv diii, and, importantly, ten times concentration unbiotinylated diii k e mutant was used as the competitor. the yeast cells were selected to present the antibody-expressing cells that could bind well to the wild-type diii instead of the diii mutant, resulting in the isolation of antibodies that can target the cross-reactive neutralizing epitopes covering the residue lysine [ ] . potent enrichment was achieved after four rounds of sorting, and a panel of antibodies were identified ( fig b) . two antibodies, designated as m and m , bound potently to denv diiis. their scfv gene were fused with human igg fc for protein expression, and surface plasmon resonance (spr) experiments were used to evaluate the antigens binding. the equilibrium dissociation constant (k d ) of m for the denv - diiis were . nm, . nm, . nm and . nm, respectively. the mab m displayed a broader binding profile compared with that of m , with the k d of . nm, . nm, . nm and nm to denv - , respectively (table , s and s figs). to further improve the affinity of m and m with the four denv serotypes, we constructed a mutant library using the error-prone pcr technologies. following three cycles of mutagenesis and selection, two clones were identified from the enriched pool of yeast sorting, designated as m . and m . . biacore analysis showed that the cross-reactive binding activities of m . and m . to all diiis were preserved after the affinity maturation process. the k d of m . for the denv - diiis were . nm, pm, . pm and nm, respectively (s fig). although the binding to denv - diiis was improved, the m . had only slightly increased binding affinity to denv diii compared to its parental mab m . notably, the m . exhibited high affinity to all the denv diiis. the k d of m . for the denv - were . nm, . nm, . nm, and . nm respectively, which demonstrated that m . could bind to all the four serotype denv viruses with high avidity (table , fig ) . we also assessed the binding specificity of m . by elisa, and the results showed that m . had weak cross-reactivity with zika virus (zikv) diii and no binding with other irrelevant antigens (s fig). next, we assessed the neutralization capacity of m . and m . against the four denv serotypes using a denv luciferase reporter viral particle (rvp) neutralization assay. we used denv rvps against the four dengue serotypes that are common strains in denv research: denv- (westpac ), denv- (s ), denv- (ch ), and denv- (tvp ). the luminescent reporter expression was proportional to the number of rvps added to bhk dc-sign cells, confirming the linear correlation between the extent of rvp infection and reporter gene expression. in consistent with the biacore binding results, both m . and m . could neutralize all the four serotype denv, and m . displayed better neutralization than m . , with the % neutralization titers (ic to further evaluate the neutralization breadth of m . igg against the four denv serotypes, a standard plaque reduction neutralization assay (prnt) on bhk- cells was performed using denv - live viruses, including denv- (genbank fj ), denv- gz / (s fig, isolated from a denv- infected patient in guangzhou, china), denv- (genbank af ), denv- - (genbank af ), and denv- b (genbank af ). an irrelevant human mab g was used as the negative control [ ] , and a g , a broadly neutralizing mab against all the four denv serotypes, was used as the positive control [ , ] . as shown in fig , m . igg could neutralize all the four denv serotypes. the % neutralization titers (ic ) of m . against denv - was . , . , . , and . μg/ml respectively ( table ). we next used a well-established ade assay to detect the in vitro ade effect of m . igg. a mutated form of m . igg was also generated containing the leucine to alanine substitutions at positions and (m . igg-lala), which lacked binding to fcγ receptors. the ade effects of denv- or denv- by m . igg, m . igg-lala, as well as a g were measured. interestingly, neither m . igg nor m . igg-lala presented any ade effect against different serotypes of denv ( fig f, s fig) . in contrast, potent ade effects were observed for the dii-specific mab a g . these results showed that m . igg is a denv diii-specific mab without detectable ade effect. we further analyzed the sequences of mabs using the imgt tool to identify their closest vh and vλ germline genes. the results indicated that m . and m . originated from different b-cell lineages ( table ). the m . vh gene was derived from the ighv - and the vλ gene was from iglv - . in contrast, the m . vh gene was derived from the ighv - and the vλ gene was from iglv - . interestingly, we found that the encoding genes of both m . and m . closely resembled their corresponding germline gene segments. notably, m . vh and vλ gene shared . % and . % sequence identities with the ighv - � and iglv - � germlines respectively (fig a and b ). these results indicated that the mab m . is a germline-like antibody, which, in general, could show better drug properties and lower immunogenicity compared to somatically hypermutated antibodies [ ] . to further investigate the immunogenetic characteristics of m . -like antibodies, we analyzed in detail the ighv - recombination frequencies with specific ighd and ighj genes families from naïve immunoglobulin m (igm) repertoires of health adult donors and neonatal igm repertoires of newborn babies, using next-generation sequencing data previously generated from our antibodyome studies [ ] . by querying the m . sequence from the igm repertoires, sequences were found to display m . -like v(d)j recombination from the genes ighv- - , ighd , and ighj out of a total of , , sequences from healthy adult igm repertoires. in igm repertoires of newborn babies, a similar recombination frequency was also observed, in which sequences with m . -like v(d)j recombination were found from , , sequences. our analysis showed that ighv - is one of the most frequently used ighv genes, and identified that many of those sequences sharing a significant degree of resemblance to m . ( fig c) . in brief, analysis of these data showed the potential of eliciting robust immune responses with the m . -like germline antibodies by vaccination. to determine whether m . can protect denv infections in vivo, we firstly used a lethal denv - infection suckling mouse model. the mice were challenged with denv - at pfu/mouse via intracranial injection. four hours later, the mice were treated intracranial with a single dose ( μg) of m . igg, m . igg-lala mutant and g (unrelated antibody control). these animals were monitored for morbidity and mortality daily. as shown in fig , all the mice in control groups died from denv infection, and most of them died within the first two weeks of viral challenge. interestingly, there was no significant difference in therapeutic efficacy against denv - infection between m . and the lala-mutated m . . the m . igg protected % denv- , denv- , denv- and % denv- infection whereas (c) germline-rooted circular phylogenetic tree of m . -like antibody sequences found in igm libraries derived from healthy human adults and neonates. the m . and a sequence showed highest similarity to m . were shown in red. sequence id started with cb represents sequence derived from the neonates, and that started with hh represents sequence derived from the healthy adults. the phylogenetic tree was constructed by the neighbor-joining method. https://doi.org/ . /journal.ppat. .g lala-mutated m . protected % denv- , denv- and % denv- , denv- respectively. therefore, m . has no detectable ade as confirmed in both in vitro and in vivo experiments. we also used the ag (types-i and -ii ifn receptor deficient) mice to test the therapeutic effect of m . against denv- (s fig). the results showed that all the mice in the control antibody treatment group died while the survival rate of mice can reach % in m . treatment group, indicating that the antibody can also protect the lethal infection of denv- in ag mice. taken together, these results indicated that m . can protect denv - infections in vivo. to map the epitope of the germline-like mab m . and identify in greater detail the structural basis of denv neutralization, we employed multiple approaches (fig ) . sequence alignment of different denv genotypes and mapping of the conserved amino acid residues of denv diii showed that four serotypes denv diiis amino acid residues were different from one another between amino acids - ( fig a) . subsequently, serotype derived diii consensus gene was randomly mutated to construct a yeast-displayed mutant library. two rounds of sorting of those yeast cells showing expression on surface but lacking the binding to m . was performed. a total of binding escape mutants were aligned with the serotype consensus protein sequence. mutation frequency at each position was plotted against the residue position number. similarly, unique diii sequences derived from naturally isolated serotype dengue viruses from genebank were also aligned with the consensus sequence (fig b and c) . the superimposed profiles of the two set of sequences showed that many of the escaped mutations located in the well-conserved area, indicating the broad crossreactivity of m . to naturally isolated dengue viruses. besides, the epitope mapping shows that the m . epitope is at close to or partially overlaps the dimerization interface between domains ii and iii. these results may explain why m . is a potent cross-reactivity antibody to all the four denv serotypes. furthermore, computational docking of denv diii-m . antibody complex was performed using zdock method. we selected the three top scored docked complexes that contained the key residues identified from an experimental epitope mapping approach. one of the top scored docked models exhibited minimum clashes with appropriate protein interface parameters and was used to demonstrate the lactation the potential epitopes and their interactions with m . antibody, which might shed light on the molecular mechanisms of broadly cross-reactive neutralization. fig d showed the docking model of the diii-m . antibody complex in which these epitopes are highlighted in green surfaces. the docking model revealed a different orientation of antibody binding as compared to the diii complex structure with fab a d- that was previously determined [ ] . the epitope comprised of three structurally proximal regions, residues - in green, , and in dark green, and - at the c-terminal in lime. one of the key residues, k , contacts the cdr-l of m . which has a germline mutation. in env-diii-fab- a d- complex structure, the residue k contacts the cdr-h . the hydrophobic residues, ile and trp, of cdr-h contact the center part of the epitope, and other loops h , h , l and l also involve in the binding. the surface area of the interface between diii and m . antibody in the model complex is Å , a typical of antibody-antigen interactions. there are six hydrogen bonds likely to form and no salt bridges at the interface. in brief, the binding regions of the m . may be close to or partially overlaps the dimerization interface between domains ii and iii, which might indicate the broad crossreactivity of m . to the four serotypes of denv. dengue is a disease with a complex immune response orchestrated by host cells partially due to the presence of four serotypes of denv. importantly, after a primary denv infection, one can be protected against or aggravate of a secondary infection with a different serotype, which bring many difficulties to develop an effective vaccine. thus, it is very urgent to develop an effective and cross-reactive antiviral therapy against denv infection. monoclonal antibodies (mabs) are of growing importance for protective and pathogenic immune responses to viruses. at present, there are many therapeutic antibodies to treat viral infections under development, such as antibodies for hiv- , sars-cov, mers-cov, nipah and hendra viruses, and h n influenza virus [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . fortunately, screening antibodies from the large naïve libraries has enabled the rapid development of high-affinity human mabs, especially for the rapid response to the outbreak of emerging viruses and diseases. we recently successfully identified two human germline-like mabs against mers-cov and h n influenza virus from the naïve library, named m and m , respectively [ , ] . they both can naturally exist with very low level of somatic hypermutation in the naïve library with which they have potent binding activity against the envelop proteins of mers-cov and h n influenza virus. most importantly, m and m all showed highly therapeutic effective in the animal models. therefore, the naïve library screening can be quickly used to isolate germline-like antibodies that effectively bind to complex protein targets like those in denv viruses. how to increase the neutralization breadth is a key issue in developing anti-denv antibodies. previous studies revealed two classes of broadly neutralizing antibodies to flaviviruses, including antibodies targeting the conserved epitopes in dii or diii [ ] [ ] [ ] ]. while the conserved fusion loop epitope (fle) in dii is the immunodominant epitope in e protein, unfortunately, this epitope frequently induced poorly neutralizing and strongly infectionenhancing antibodies via ade [ ] [ ] [ ] . therefore, diii represents the ideal target for neutralizing antibodies. in this study, we applied a highly efficient yeast-display-based sorting strategy by using the highly diverse denv diiis as antigen and the competitive sorting technique. by applying this method, we quickly and efficiently identified two human germline-like broadspectrum anti-denv mabs (m and m ) from the naïve scfv yeast library using the diii antigen that make them as promising candidate therapeutics as well as the template for vaccine development. another class of highly efficient broadly neutralizing antibodies that target the envelope dimer epitopes (ede) from the secondary acute denv infection plasmablasts has been identified by dejnirattisai et al. [ ] . these antibodies may especially get through with high somatic mutations from the secondary virus infection. compared with the highly somatically mutated antibodies, germline-like antibodies typically have better safety and drug-related property [ ] . importantly, the hendra and nipah antibody m . is a near germline antibody and exhibited a very good drugability, which was from a similar library that was also used to isolate our m and m -like antibodies. m . was successful as a candidate therapeutic mab in animal models and was also completed the phase i clinical trial (actrn ) without side effects [ ] . to further improve the affinity of m with the four serotypes denv diiis, we subjected m to affinity maturation process, and named it as m . . subsequently, we analyzed m . sequence using the imgt tool to identify its closest vh and vλ germline genes. interestingly, we found that m . is still a germline-like antibody although it went through the mutation process in vitro, with over % identities of its vh and vλ genes to the ighv - � and iglv - � germline respectively. in order to evaluate the neutralization effect of the m . igg, we used a standard plaque reduction neutralization with bhk cells to measure denv infection and neutralization. the m . igg showed broadly neutralization towards the four serotypes denv as well as a recent denv isolate from clinical samples. more importantly, m . did not present any ade effects in different serotypes of denv. the in vivo study results demonstrated the therapeutic potential of m . against severe denv - infections. in brief, the m . could neutralize the four serotypes denv in vitro and protect the denv infection mouse model in vivo without detectable ade effects. we therefore expect that m . has a likeness drugability of m . and could be developed as a candidate therapeutic in the future. we have also localized the m . epitope by using a combination of computational structural modeling, display-based antigen mutagenesis, and sequence-based analysis of mutants. the epitope appears to overlap with the epitope previously explored as targets for cross-reactive murine mabs and close to or partially overlaps the dimerization interface between domains ii and iii. this further indicates that this epitope could be an important component of vaccine immunogens intended to elicit cross-reactive neutralizing antibodies. in progress are our experiments to crystallize the complex of m . with denv diii that would allow precise determination of the m . epitope. the major result of this study is the identification of a germline-like human mab, m . , from a naïve yeast antibody library which binds with high (picomolar) affinity to diiis from all serotypes and neutralizes the four denv serotypes. there are two major implications from this finding: ) m . is a potential candidate therapeutic which could be further developed in preclinical and clinical settings. ) the epitope of the germline-like mab m . could guide the design of effective candidate vaccine immunogens capable of eliciting m . and/or m . -like antibodies. bhk cells were cultivated in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) (biowest). mosquito cells c / were cultured in rpmi- medium supplemented with % fbs. all cells were maintained in a humidified atmosphere of % co at ˚c incubator, except for c / cells, which were cultivated at ˚c. denv- (genbank fj ), denv- gz / (isolated from denv- infected patient in guangzhou), denv- (genbank af ), denv- - (genbank af ), and denv- b (genbank af ) were propagated in c / cells by using rpmi medium and the titers were measured by standard plaque forming assay in bhk cells. denv diii genes from all serotypes were synthesized by genescript, inc (nanjing, china), fused with igg fc and a c-terminal avi-tag, and cloned into psectag expression vector. the diii. (serotype ) k e mutant was generated through overlapping pcr. for the conversion of igg from scfv, the heavy and light chains of scfv were amplified and recloned into the ptt-igg vector. the plasmids were transfected into expi cells (thermo fisher) for transient expression, and purified using protein g column (ge healthcare, piscataway, nj) according to the manufacturer's instructions. the purified protein was biotinylated by mixing with biotinylation reagents in pbs for min on ice, according to the manufacturer's instructions (pierce). a large yeast-displayed scfv library was used for antibody screening, and the screening protocols were essentially carried out as described previously [ ] . briefly, μg of binotinylated diii. -fc and cells of the initial naïve library were mixed and washed by pbsa, and incubated with μl streptavidin conjugated microbeads (miltenyi biotec, auburn, ca) before loading onto the automacs system (miltenyi biotec) for sorting. after three rounds of sorting, the downsized library was further sorted against binotinylated diii. -fc ( μg/ml) but also using unbiotinylated k e mutant ( μg/ml) as the competitor. to generate the m scfv and m scfv mutant libraries, random mutagenesis of the scfv genes were performed through error-prone pcr by using a genemorph ii kit (stratagene) following the manufacturer's instructions with minor modifications. to further diversify the mutation profile, um of each of the two nucleotide analogues ( -oxo-deoxyguanosine triphosphate and '-deoxy-p-nucleoside- '-triphosphate) was mixed in the pcr reaction mixture. for the second and third cycle library constructions, an extra step of dna shuffling pcr was inserted into the regular pcr cycles to combine the beneficial mutations obtained from previous maturation process. dna shuffling pcr step was performed as following: cycles of denature at ˚c for seconds followed by annealing/extension at ˚c for second on the bio-rad mycycler. binding affinities of m scfv, m scfv, m . scfv, and m . scfv to the denv diiis were analyzed by surface plasmon resonance technology using a biacore x instrument (ge healthcare). the antibodies were covalently immobilized onto a sensor chip (cm ) using carbodiimide coupling chemistry. a control reference surface was prepared for nonspecific binding and refractive index changes. for analysis of the kinetics of interactions, varying concentrations of antigens were injected at flow rate of μl/min using running buffer containing mm hepes, mm nacl, mm edta, and . % surfactant p- (ph . ). the association and dissociation phase data were fitted simultaneously to a : langumir global model by using the nonlinear data analysis program biaevaluation . . all the experiments were done at ˚c. neutralizing activity of mabs was measured using a standard plaque reduction neutralization with bhk cells as previously described [ ] . briefly, -fold serial dilutions of mabs were added to approximately pfu of a variety of dengue virus strains and incubated for h at ˚c. then, the mixture was added to bhk cell monolayers in a -well plate in duplicate and incubated for h at ˚c. the mixture was removed, and ml of . % (w/v) lmp agarose (promega) in dmem plus % (v/v) fbs was layered onto the infected cells. after further incubation at ˚c for days, the wells were stained with % (w/v) crystal violet dissolved in % (v/v) formaldehyde to visualize the plaques. prnt values were determined using non-linear regression analysis. prnt data were calculated by doing a non-linear regression analysis using sigmaplot (version . , systat software, inc., ca) as previously described [ ] . denv rvps from all four serotypes were pre-incubated with an equal volume of serially diluted antibodies ( μg/ml to . μg/ml pre-dilution or . μg/ml to . μg/ml predilution, as measured based on the dilution of antibody prior to combining with rvps) in dmem infection media for h at room temperature and transferred to wells of a -well plate. an equal volume of denv rvps were added to each well followed by slow agitation for h at room temperature. bhk dc-sign cells were added to each well at a density of , cells per well followed by incubation at ˚c in % co for h. cells were subsequently fixed in lysed and analyzed for luminescent reporter expression using the wallac victor. the percent infection for each concentration of mab or serum was calculated, and the raw data was expressed as percent infection versus log of the mab concentration or the reciprocal serum dilution. the data were fit to a sigmoidal dose-response curve using prism (graphpad software, la jolla, ca) to determine the titer of antibody that achieved a % reduction in infection. maximum infection was determined in the absence of antibodies. the in vitro ade assay was performed using k cells [ ] . briefly, serial -fold dilutions of antibodies under concentrations ranging from to . μg/ml were mixed with denv- or denv- , and incubated for h at ˚c. mixtures were then added to × k cells at multiplicity of infection of . ~ . for h in -well plates. the cells were subsequently washed times with serum free rpmi- medium. after collecting cells by centrifugation, the cell pellets were re-suspended with rpmi- medium containing % fbs and added to -well plates, then incubated for days at ˚c with % co . the titer of viruses in the supernatant was then measured using a plaque assay. the ade effect was calculated as different viral yields in the supernatant after infection in the presence of the added antibodies. the epitope mapping of m . was performed using previously described protocols [ ] . briefly, random mutagenesis of the denv diii. gene was performed using a genemorph ii kit (stratagene). as described above, the yeast-displayed mutant library was mixed with biotinylated m . scfv-fc, washed, and stained by mouse anti-c-myc antibody (roche), alexa- conjugated goat-anti-mouse antibody (invitrogen), and pe-conjugated streptavidin (invitrogen). after two rounds of sorting on a facsaria ii cell sorter (bd biocsiences, san jose, ca), the sorted cells were amplified and their plasmids were prepared and sequenced. homology modeling of variable regions of heavy (v h ) and light (v l ) chains for m . scfv antibody was carried out using the swiss-model workspace [ ] by selecting the closest template structures (pdb codes: qos for heavy chain and dd for light chain), whose sequence similarities were % and % respectively. the v h -v l orientation of m . scfv structure was assigned similar with one of the templates (pdb code: dd ) that showed minimum steric clash for creating the final m . scfv model. the crystal structure of denv diii serotype (pdb code: r ) was used for docking with the modeled scfv antibody m . . docking of scfv m . to the dengue env-iii was performed by zdock server (http://zdock.bu.edu) that uses a fast fourier transform (fft)-based rigid-body protein docking algorithm with scoring functions combining pairwise shape complementarity, desolvation and electrostatic energies. based on the escape mutants that led to the loss of epitopes and available crystal structure of denv diii, we selected a list of residues as biological constrains, , , , , , and , on the surface of env-diii as potential contacting residues for docking constraints. similarly, one or two residues from each of cdr-h , h and l loops were chosen at the docking interface. cdr-h and h loops had dominant hydrophobic residues whereas cdr-l had a germline mutation, and they all had high antigen-contacting propensities [ ] . results from the top zdock predictions were filtered using the userdefined residues and a angstrom distance cutoff. three predicted complexes were only kept as all residues selected come together at the interface and were further examined by pdbepisa (protein interfaces, surfaces and assemblies). pymol was used for the analysis of docked model and graphical illustration [ ] . the suckling mice were purchased from b&k universal group limited (shanghai, china) and housed under specific pathogen-free conditions at the animal facilities of the shanghai public health clinical center, fudan university (shanghai, china). before infection, the mice were transferred to the animal biosafety level (bsl- ) laboratory (shanghai, china). one day mice were used for all experiments. all mice were intracerebrally injected with pfu of denv - . at h post infection, mice were passively transferred a single dose of μg antibody m . igg, m . igg lala mutant or g igg as the negative control via intracerebrally injection. survival rates and disease sings were monitored daily. the ag mice (type i and type ii interferon receptor-deficient) were purchased from b&k universal group limited (shanghai, china) and housed under specific pathogen-free conditions at the animal facilities of the shanghai public health clinical center, fudan university (shanghai, china). before infection, the mice were transferred to the bsl- laboratory (shanghai, china). groups of mixed-sex -to -week-old mice were used for all experiments. all mice were intraperitoneally injected with x pfu of denv- in a volume of μl. at h post infection, mice were passively transferred a single dose of μg antibody m . igg-lala, or g antibody as the control via i.p. injection. survival rates, weight loss, and disease sings were monitored daily. specific-pathogen-free ag mice ( - weeks old) and suckling mice were used for all experiments. all experimental protocols were reviewed and approved by the institutional committee of the who dengue classification and case definitions: time for a reassessment global spread and persistence of dengue the risks behind dengvaxia recommendation dengvaxia: age as surrogate for serostatus. the lancet infectious diseases : dengvaxia controversy: impact on vaccine hesitancy ade and dengue vaccination cross-reacting antibodies enhance dengue virus infection in humans the growth and potential of human antiviral monoclonal antibody 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predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain ii antibodies induced by dengue virus type and envelope domain iii recombinant proteins in monkeys neutralize strains with different genotypes vaccines and immunization strategies for dengue prevention serotype-specificity of recombinant fusion proteins containing domain iii of dengue virus evaluation of the protective efficacy of a recombinant dengue envelope b domain fusion protein against dengue virus infection in mice short report: antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine mice immunized with a dengue type virus e and ns fusion protein made in escherichia coli are protected against lethal dengue virus infection an in-depth analysis of original antigenic sin in dengue virus infection in-depth analysis of the antibody response of individuals exposed to primary dengue virus infection dengue: defining protective versus pathologic immunity dengue virus neutralization by human immune sera: role of envelope protein domain iii-reactive antibody a potent germline-like human monoclonal antibody targets a ph-sensitive epitope on h n influenza hemagglutinin exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody eradication of tumors through simultaneous ablation of cd /b -h -positive tumor cells and tumor vasculature cd -targeted car t cells induce remission in b-all that is naive or resistant to cd -targeted car immunotherapy human monoclonal antibodies as candidate therapeutics against emerging viruses highly efficient selection of epitope specific antibody through competitive yeast display library sorting a human monoclonal antibody against small envelope protein of hepatitis b virus with potent neutralization effect a bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion a broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of e protein therapeutic antibodies, vaccines and antibodyomes broad and potent hiv- neutralization by a human antibody that binds the gp -gp interface broad and potent neutralization of hiv- by a gp -specific human antibody structural basis for broad and potent neutralization of hiv- by antibody vrc human infection with a novel avian-origin influenza a (h n ) virus therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies nonneutralizing antibodies induced by the hiv- gp nhr domain gain neutralizing activity in the presence of the hiv fusion inhibitor enfuvirtide: a potential therapeutic vaccine strategy a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus swiss-model: modelling protein tertiary and quaternary structure using evolutionary information antibody-antigen interactions: contact analysis and binding site topography simplifying and enhancing the use of pymol with horizontal scripts we thank professor dane wittrup from mit for providing the yeast display vector and yeast strain. key: cord- -qjyooq authors: king, chwan-chuen; chao, day-yu; chien, li-jung; chang, gwong-jen j; lin, ting-hsiang; wu, yin-chang; huang, jyh-hsiung title: comparative analysis of full genomic sequences among different genotypes of dengue virus type date: - - journal: virol j doi: . / - x- - sha: doc_id: cord_uid: qjyooq background: although the previous study demonstrated the envelope protein of dengue viruses is under purifying selection pressure, little is known about the genetic differences of full-length viral genomes of denv- . in our study, complete genomic sequencing of denv- strains collected from different geographical locations and isolation years were determined and the sequence diversity as well as selection pressure sites in the denv genome other than within the e gene were also analyzed. results: using maximum likelihood and bayesian approaches, our phylogenetic analysis revealed that the taiwan's indigenous denv- isolated from and dengue/dhf epidemics and one sporadic case were of the three different genotypes – i, ii, and iii, each associated with denv- circulating in indonesia, thailand and sri lanka, respectively. sequence diversity and selection pressure of different genomic regions among denv- different genotypes was further examined to understand the global denv- evolution. the highest nucleotide sequence diversity among the fully sequenced denv- strains was found in the nonstructural protein a (mean ± sd: . ± . ) and envelope protein gene regions (mean ± sd: . ± . ). further analysis found that positive selection pressure of denv- may occur in the non-structural protein gene region and the positive selection site was detected at position of the ns gene. conclusion: our study confirmed that the envelope protein is under purifying selection pressure although it presented higher sequence diversity. the detection of positive selection pressure in the non-structural protein along genotype ii indicated that denv- originated from southeast asia needs to monitor the emergence of denv strains with epidemic potential for better epidemic prevention and vaccine development. dengue fever (df) and its more severe forms, dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss), have emerged as major public health problems in tropical and subtropical areas [ , ] . infection with dengue viruses (denv), which are maintained in a human-mosquito transmission cycle involving primarily aedes aegypti and aedes albopictus, can result in various clinical manifestations ranging from asymptomatic to df, dhf, dss and death [ ] . the occurrences of dengue epidemics in the past years have been characterized by the rising incidence rates of infection and continuous expansion in geographic distribution of dhf epidemics [ ] . importantly, the epidemics of dhf have become progressively larger in the last years in many dengue endemic countries [ ] . the increasingly widespread distribution and the rising incidence of df and dhf are related to increased distribution of a. aegypti, global urbanization and rapid and frequent international travel. epidemiological analysis reveals that some denv strains are associated with mild epidemics with low occurrences of dhf cases and inefficient virus transmission, whereas others are more likely to cause severe epidemics with high incidence of dhf/dss and rapid virus transmission [ , ] . the large dhf epidemics in indonesia in the s and sri lanka after provided evidence supporting this phenomenon [ , ] . dengue virus serotype (denv- ) re-appeared in latin americain after its absence for seventeen years. the virus was detected initially in panama and soon dispersed throughout central and south america during the following years [ , ] . this introduction coincided with an increased number of dhf cases in this region. although the genotype originating in southeast asia has been postulated as the major cause of the increased virulence, the molecular marker associated with a difference in virulence among genotypes at the fullgenomic level is still largely unknown. dengue is caused by four antigenically related but genetically distinct viruses (denv- , - , - and - ) belonging to the genus flavivirus, family flaviviridae [ ] . denv is a single stranded, positive-sense rna virus, approximately , nucleotides in length. the genome contains a single open reading frame (orf) that encodes a polyprotein, which is co-and post-translationally processed to produce three structural proteins, including capsid (c), pre-membrane (prm) and envelope (e), and seven nonstructural (ns) proteins (ns , ns a, ns b, ns , ns a, ns b and ns ) [ , ] . a considerable number of studies have revealed that each serotype of denv is composed of phylogenetically distinct clusters that have been classified into "genotypes" or "subtypes," and each genotype is also composed of phylogenetically distinct "groups" or "clades." a previous study has classified denv- strains into four genotypes based on limited numbers of nucleic acid sequences from the prm and e protein genes [ ] ; denv- strains have also been re-classified into five genotypes [ ] . growing evidence suggests the existence of denv strains with different epidemic potentials. this evidence is supported by the following observations: ( ) the differences in fitness among various genotypes of denv- reflect their different replication capabilities in human monocytes and dendritic cells [ ] ; ( ) around , clade replacement among denv- genotype ii containing isolates from thailand was associated with changing serotype prevalence and incidence of dhf epidemics [ ] ; and ( ) sudden changes in the genotype of denv at a single locality have been observed that appeared to originate from the genetic bottleneck of a large viral population [ , ] . this sudden genotype replacement has been associated with more severe dhf epidemics in indonesia and sri lanka [ , ] . however, most of these studies involved the e gene alone. this raises an important question: is the introduction of different denv genotypes in disparate geographical locations a result of sequence differences outside of the e gene altering their epidemic potential, or it is simply a stochastic event in viral evolution? dengue epidemics in taiwan are usually initiated by imported index cases (king et al., ) . the re-emergence of dengue outbreaks in taiwan started when denv- was re-introduced into the off-islet of hsiao-liu-chiu in . in - , another large-scale denv- outbreak occurred in kaohsiung and pingtung in southern taiwan [ ] . although denv- was detected sporadically from imported index cases, no denv- -related epidemic occurred until dhf cases were confirmed in kaohsiung in and dhf cases in tainan in [ ] . taiwan neighbors many southeast asian countries and more than , travelers visit these adjacent countries annually. the surveillance system implemented by the center for disease control in taiwan (taiwan-cdc) routinely detects many imported dengue cases each year. thus, taiwan is an ideal place to study the evolution and dispersion of denv that may have different epidemic potential, particularly in the and dhf epidemics in taiwan that coincided with the dhf epidemics in southeast asian countries [ ] . complete genomic sequencing of denv- strains collected from different geographical locations and isolation years offers the opportunity to understand the genetic stasis and possible selection pressure sites in the denv genome other than within the e gene. the blood samples of suspected dengue patients, obtained from the sentinel hospitals/clinics located in tainan, kaohsiung and pingtung in southern taiwan, were sent to the infectious disease epidemiology laboratory at national taiwan university (ntu) and taiwan-cdc for laboratory confirmation. the study protocol was approved by the college of public health research human subject ethics review committee at ntu. a suspected and confirmed dengue case was defined as previously described and confirmed by both laboratories [ , ] . imported and indigenous dengue cases were defined based on the patients' travel history to dengueendemic or -epidemic countries within - days before the onset of the disease. due to few denv- epidemics and limited denv- isolates identified before in taiwan, we focused our study on comparing the sequences of different denv- isolates in and considering various epidemiological characteristics, including temporal, geographical and host factors. six denv- isolates were selected for full-length sequencing: ( ) an isolate from the imported denv- infected case in ; ( ) an isolate from the indigenous df and dhf cases during the epidemic in tainan, taiwan; ( ) the isolate from a geographical location in tainan other than the epidemic area; ( ) an isolate from the same geographical location as the tainan's epidemic but in ; and ( ) an isolate from indoor mosquitoes during the dengue/dhf epidemic in tainan. the epidemiological characteristics of these six denv- isolates are summarized in table , and their genebank accession numbers are dq -dq . in addition to the - denv- strains, four local isolates obtained from taiwan during previous years, kindly provided by taiwan-cdc, were also used for comparison, including four strains isolated from indigenous df patients during the - epidemic in kaohsiung [ twkh (accession no.: dq ), twkh (accession no.: dq ), twkh (accession no.: dq ), tw (accession no.: dq )]. isolate tw with low passage history (two passages in c / cells) was subjected to full-length genomic sequencing together with the above six isolates from - , constituting seven full-length denv- sequences from taiwan. the remaining three denv- isolates were sequenced only from the ' ncr to the cooh-terminus of the e gene region for phylogenetic analysis. acute-phase serum or plasma samples collected from the dengue patients within seven days after the onset of fever were used for both virus isolation and molecular diagnosis [ , ] . molecular diagnosis by reverse transcriptase polymerase chain reaction (rt-pcr) amplification and subsequent nucleic acid sequencing was performed as previously described, and a complete list of the pcr and sequencing primers utilized is available upon request [ ] . the rna genomic ' and ' terminal nucleotide sequences were not confirmed independently and were assumed to be of the same length and sequence as the prototype strain h in this study. a total of complete genomic sequences of denv- strains and one denv- strain a (genbank accession number ab ) were aligned using the multiple sequences alignment clustalx [ ] . these sequences were further combined with all available sequences of the complete e gene or the complete prm and partial e genes (to nucleotide position of the e gene) of denv- deposited in the genbank database at the national center for biotechnology information (ncbi). therefore, the complete e gene ( nt) dataset consisting of a total of sequences and the prm and partial e gene ( nt) dataset of a total of sequences were used for phylogenetic analysis. a complete list of the sequences along with associated epidemiological information is available upon request. the percentage of sequence similarities and differences were calculated using bioedit v . program [ ] . pairwise comparisons of both nucleotide and amino acid sequences of denv- isolates were performed using the program mega v . (molecular evolutionary genetics analysis, pennsylvania state university, pa) to determine the mean and range of the proportional difference (p-distance) [ ] . the model of nucleotide substitution that best described denv- sequence evolution was identified using the program modeltest . [ ] . the resulting most complex gtr+i+Γ substitution model (general time reversible model, gtr, a proportion of sites modeled as invariant, i, variation in rates among sites modeled using the gamma distribution, Ã) was selected to be the best fit to the data using the hierarchical likelihood ratio tests (hlrts) and akaine information criterion (aic). the estimated parameter values from this model were as follows: relative substitution rates among nucleotides were a ↔ c = . , a ↔ g = . , a ↔ t = . , c ↔ g = . , c ↔ t = . , g ↔ t = . ; proportion of invariable sites (i) was . ; gamma distribution of among-site rate variation (Ã) was . ; and estimated base composition of a = . , c = . , g = . , and t = . . a maximum likelihood (ml) tree using these parameter settings was estimated using the dnaml in phylip v . package [ ] . bootstrap analysis with , re-samplings was used to determine confidence values for groupings within the phylogenetic tree. in addition, a posterior probability distribution tree, generated by implementing the recently developed bayesian hierarchical phylogenetic model utilizing a metropolis-coupled monte carlo markov chains (mc) algorithm in the mrbayes program (version . , [ ] ) was compared with the evolutionary tree of denv- generated by the ml method. indeed, the bayesian approaches for constructing phylogenetics have several advantages. first, the primary analysis often provides faster estimates of the tree and measurements than the estimates obtained using ml bootstrapping techniques. secondly, bayesian model selection offers advantages over likelihood methods in that the competing evolutionary hypotheses need not to be nested, and it does not rely on standard likelihood assumptions. in other words, the starting trees in bayesian method are randomly chosen, and multiple runs of the same dataset are generally made with different starting trees to check convergence of the process. the programs' default settings for prior probability were used in our analysis. bayesian markov chain monte carlo (bmcmc) processes, considering the heterogeneity in the evolutionary process and thus incorporating a discrete gamma distribution of four classes of substitution rates across mutation sites, were run for , generations. output trees were sampled every generations but the first , trees were discarded before the process reached the convergence state. the resulting trees were rooted using a denv- strain a isolate as described. to analyze the selection pressure in denv- , the codeml program from the paml package was employed by implementing a maximum-likelihood method. this method presents major advantages over simpler pairwise comparisons in considering the transition/transversion rate bias, non-uniform codon usage, and phylogenetic relationships among the sequences [ ] . positive selection at a small number of codons can be detected by comparing various models of codon evolution which differ in how the rates of synonymous (ds) and nonsynonymous (dn) substitutions (denoted as ω) are treated among codons or within lineages using likelihood ratio tests. to analyze selection pressures at individual codons, we compared the m and m model. in the m model, categories were assigned and estimated from the data, which specified only neutral evolution; however, the m model allowed positive selection by add-ing an th codon category at which dn/ds can exceed . . to examine selection pressures along the lineages, the free ratio model, which allows certain lineages to have ω ratios different from the background, was implemented in the m model. additionally, parameters involving the incorporation of classes of codons where ω > were used by comparing the value of the likelihood from m , in which the specified neutral evolution of ω is constrained to be equal to or less than at all codons among all lineages. the comparison was again assessed using the likelihood ratio test. if positive selection was found, the bayesian method was applied to identify the specific codon that may have been subjected to positive selection pressure. we have determined the complete nucleotide sequences ( , nucleotides in length with an orf of , amino acids) of the seven different denv- strains from taiwan ( table ). the percentages of nucleotide and amino acid identities of the entire orf among these strains, compared with the prototype denv- strain h isolated in the philippines in , are shown in table . the indigenous denv- isolates from the epidemic area in tainan city ( tw and tw ) and from the sporadic case in pingtung ( tw ) displayed the highest similarity, with . % sequence identity in both nucleotide and amino acid sequences. the imported tw strain showed slightly lower nucleotide and amino acid sequence identity ( %) relative to these indigenous taiwanese denv- isolates. the denv- isolates of taiwan from years other than , including the kaoshiung tw and the tainan tw strains, showed higher sequence diversity compared with the denv- taiwan isolates ( % nucleotide and amino acid sequence identity), which suggested that they might have originated from different countries. further phylogenetic analysis revealed that these viruses belong to different genotypes (genotype i and iii; see the section ''phylogenetic analysis of denv- '' for details). compared to the prototype strain h , several unique amino acid substitutions that serve as unique signature sites for each genotype were found within the full genomic sequences of the selected denv- isolates from taiwan or other countries and are listed by the order of the gene in table . among those, several substitutions changed the polarity, charges, or hydrophobicity of these amino acids, which were present only in genotype iii of denv- , including the change from threonine (t) to alanine (a) at position of the c region, leucine (l) to histidine (h) at position of the prm region, l to t at position of the e region, isoleucine (i) to t at position of the ns region, and lysine (k) to t and aspartic acid (d) to asparagine (n) at positions and of the ns region. similar signature sites experiencing amino acid property alterations in genotype ii included a change from t to a at position of the prm region, l to serine (s) at position of the ns region, and a to t at position of the ns a region. thus, our data suggested that different genotypes of denv- experience different amino acid changes at both structural and non-structural genes, and the sites of these substitutions could serve as signature sites for genotype identification. the phylogenetic trees of denv- were constructed from the two different nucleic acid dataset alignments: ( ) partial sequences of the prm and e gene region (prm/e) from isolates obtained from taiwan and sequences available from genbank; ( ) complete e gene sequences including isolates from both taiwan and genbank. the trees derived from the maximum likelihood method the upper-right matrix corresponds to nucleotide sequences and the lower-left matrix to the amino acid sequences. geno v v i i i i ii ii ii ii ii iii iii iii t e v i i i i v i i i i l f f f f ns b v i i i i l and the bayesian method based on both datasets were very similar to each other. thus, only the posterior probability tree derived from the bayesian method based on the complete e gene sequences is shown (fig ) . the denv- strains isolated in taiwan with the lack of full-length sequences of viruses belonging to old american genotype iv, only four denv- genotypes, including representatives of genotype i ( tw ), genotype ii ( tw ), genotype iii ( tw ) and genotype v (h ) were compared. the sequence divergences in nucleotide and amino acid were calculated as the p-distance by adjusting the lengths of different genes [ , ] . the highest nucleotide diversity was found in the ns a gene (mean ± sd: . ± . ), followed by the e gene (mean ± sd: . ± . ). similar results were observed for amino acid diversity, which was also the highest in the capsid gene (mean ± sd: . ± . ), followed by the ns a gene (mean ± sd: . ± . ) ( table ) . to determine whether higher sequence diversity in certain genes could be the result of natural selection pressures, we implemented the m and m selection models to determine whether positive selection pressure among all codons from the full-length denv- sequences could be detected by using the codeml program from paml [ ] . the results suggested that both structural and non-structural genes of denv- were under neutral selection. although the e gene showed positive selection (ω = . ) with statistical significance (p = . ) when using the larger dataset with sequences, no specific site with positive selection could be detected. to further examine the selection pressure along the lineage, genotype i, ii, iii and v, based on the phylogenetic tree of the full-length sequences (fig ) , were examined separately using the m model. the results are summarized in table . although there were positive selection pressures detected in the c and ns b genes of genotype i, and in the e, ns and ns genes of genotype ii, only the ns gene of genotype ii showed statistically significant positive selection pressure. furthermore, positive selection was detected at position of the ns gene (substitution of s for l). changes occurring in the ' ncr and ' ncr were examined among the denv- viruses isolated in taiwan and other countries. in the ' ncr, positions , , and had nucleotide changes that were distinguishable for the specific genotype. among them, a g to a change at position was frequently seen in genotype i, a c to t change at position and a g to a change at position were observed only in genotype ii, and an a to g change at position was present in genotype iii. interestingly, the bayesian hierarchical consensus tree showing the phylogenetic relationships between denv- genotypes is based on the complete e gene sequences ( bp) from the denv- strains sampled globally figure the there was consistently an additional -nucleotide sequence, agtgaaaaaga, inserted in the ' ncr close to the end of the open-reading frame (orf) of the denv- strains isolated in recent years, compared to the prototype strain h . in the ' ncr, nucleotide changes at position , , and (nucleotide numbering beginning at '-terminus of ' ncr after the stop codon) were observed from the strains circulating recently, which differed from the strain h . however, none of these changes had any effect on the predicted secondary structure of the ' ncr rna (data not shown). the putative genome cyclization sequence ucaauaug, located between nucleotides and of the c gene, was conserved in all denv- viruses. viral sequence comparisons among isolates from dengue epidemics of different disease severities may provide valuable information regarding the molecular basis of the epidemic potential of the virus. denv- re-appeared in in taiwan and caused the df/dhf epidemic in tainan city after its first introduction in [ ] . this stimulates a great interest in understanding the molecular relationship of denv- isolates in taiwan during interepidemic periods and in comparing them with the strains circulating globally to understand evolutionary trends and geographical expansions. here, we confirmed that the dengue epidemics in taiwan were strongly associated with the globally circulating denv- due to constant introduction of viruses from southeast asia by taiwanese travelers. our data demonstrates the sequence diversity among the full-genomic sequences of denv- and the positive selection pressures exerted in different lineages (i.e. genotypes) at sites in denv- non-structural genes. since most taiwan dengue epidemics were initiated by the introduction of virus from imported cases [ ] , phylogenetic analysis provides essential information to understand the history and origin of all taiwan denv- isolates originating in other countries (fig. ) . the high nucleotide sequence identity (> . %) among the strains isolated in indicates that they were from a single origin and further spread to different townships, such as pingtung (id# tw ). the only imported denv- isolated from a traveler who had recently visited indonesia was more closely associated with the genotype ii isolates from myanmar and older isolates from thailand. this virus differed from the virus isolated during the tainan outbreak, which might suggest that multiple genotypes of denv- circulated in indonesia. this observation is consistent with a previous study indicating that at least two subtypes of denv- were present in indonesia [ ] . the phylogenetic analysis also suggested that a single isolate (id# tw ) from the same location as the epidemic was grouped together with the genotype iii sri lanka isolates. additional denv- isolates from the first denv- -caused dhf outbreak in taiwan ( ) ( ) were grouped into genotype i. all these results implicated that repeated introductions of different genotypes of denv- into taiwan since were important causes of dengue epidemics, and that denv- was not endemic in taiwan. this situation may be similar in the subtropical region of china. our country initiated airport fever screening during the severe acute respiratory syndrome (sars) outbreak in - , and it successfully identified confirmed, imported dengue cases [ ] . airport fever screening can thus quickly identify imported dengue cases, and may prevent a significant number of dengue outbreaks that would have been initiated by imported index cases. however, its cost-effectiveness in preventing any dengue epidemics in taiwan will need to be evaluated in the future. with different denv- genotypes imported into taiwan from southeast asia and other parts of the world, this virus collection provides an excellent opportunity to examine the sequence diversity of different genes of the full-length denv- viral rna genome for genotypes other than genotype iv. the highest p-distance of nucleotide diversity of the full-length genomes occurred for the ns a gene ( . % ± . %), followed by the e gene ( . % ± . %). in contrast, the highest p-distance of amino acid diversity of the full-length genomes occurred for the cap- the maximum likelihood phylogenetic tree shown here is based on the complete genomic sequences of denv- strains available from genbank figure the maximum likelihood phylogenetic tree shown here is based on the complete genomic sequences of denv- strains available from genbank. the tree was rooted using denv- strain a (genbank accession number: ab ) as the outgroup. the major amino acid changes along lineages within genotype i and ii are also labeled. taiwan denv- isolates are marked with a star. sid gene ( . % ± . %), followed by the ns a gene ( . % ± . %). this observation is consistent with the previous analysis in denv- , denv- and denv- [ , ] , although the precise cause of the increased rate of amino acid change in the ns a gene is unknown. a similar observation could also be made while analyzing the full genomic sequences of west nile virus (wnv) isolated from different animal species [ ] . the flavivirus ns a, a protein important for viral replication and particle formation [ ] , is cleaved by viral serine protease. a mutation at the basic p cleavage site residue in ns a blocks this processing event and is lethal for virus production while still allowing rna replication [ , ] . furthermore, this basic residue in ns a and an acidic residue in ns are important determinants for virus assembly and/or release [ ] . although the relative high sequence diversity of the ns a gene of denv- may be due to the lesser structural constraint required for ns a, it is possible that positive selection pressures may be exerted on this gene. especially in light of recent studies, ns a together with ns b and ns a were identified as dengue virus-encoded proteins that could antagonize the interferon (ifn) response during viral infection [ , ] . our analysis didn't detect any selection pressure exerted on the ns a gene probably due to the small sample size; future studies will be needed to focus the selection pressure analysis on non-structural proteins and denv evolution. several evolutionally conserved amino acid changes are preserved, which are unique in different denv- genotypes (table ) . these substitutions resulted in changes of its polarity, hydrophobicity or charge. especially notable was the change from l to s at position of the ns region, which is an amino acid substitution unique to genotype ii. this might be the result of positive selection within the lineage of genotype ii but not other genotypes. all denv- isolates from thailand belong to genotype ii, and interestingly, based on a previous publication [ ] , strains of denv- isolated prior to in thailand may have been replaced by two new locally evolving strains. this could be a sign of a new genotype evolving in thailand; however, most of the mutations or substitutions occurring were deleterious and a purifying selection of denv- was suggested [ ] . it is very likely that the previous analysis focused on only the e protein gene. determining the possibility of a positive natural selection site in the non-structural genes of the new thailand lineage will require further study. a number of t-and b-cell epitopes are present on the non-structural proteins, especially the ns gene [ ] [ ] [ ] . even though the biological significance of the l to s change at position of the ns region is unclear, growing evidence supported by in vitro and in vivo studies suggest that there are certain evolutionary forces acting on the ns gene shaping the gene flow of the dengue viral population, which might differ during viral replication in mammalian and mosquito cells [ , ] . this is the first time that a positive selection pressure site was detected in a non-structural protein in denv- and its importance together with its functional relevance to epidemic severity will need to be examined with a larger sample size. the global distribution of different genotypes of denv- indicates that they originated in southeast asia; these genotypes demonstrated higher epidemic potential with regards to severe dhf epidemics in sri lanka, central and south america [ , ] . genotype iii, once its transmission cycle was established locally, soon resulted in dhf epidemics regardless of an increase in virus transmission or a change in circulating serotypes [ , ] , supporting the hypothesis that virus strain is an important risk factor for dhf [ , ] . two sub-lineages (isolated before and after ) existed within the denv- genotype iii strains from sri lanka, and the viruses isolated after were associated with the dhf epidemic [ ] . we found that the strain isolated in from a indigenous dengue patient ( tw ) that did not lead to a large-scale epidemic of df or dhf was more closely related to the lineage of the denv- genotype iii sri lankan strain isolated before . similarly, in indonesia two sub-lineages of denv- were present (isolated before and after ), and a greater dhf epidemic, especially in adult cases, was caused by the denv- strains isolated after [ ] . the denv- strain isolated in taiwan during the dhf outbreak in was actually more closely related to the old indonesian strain of genotype i from - . while it is currently unknown how the different sub-lineages within each genotype are associated with different dhf epidemic potential, a recent publication suggested that changing serotype prevalence could lead to differential susceptibility to cross-reactive immune responses [ ] . furthermore, wearing et al suggested that both vector and short-termed host cross-immunity are two factors responsible for dengue epidemics [ ] . it would be necessary to strengthen comprehensive dengue virological surveillance, especially in those endemic and hyper-endemic areas/countries, to monitor the emergence of denv strains with epidemic potential for better epidemic prevention and vaccine 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type after amplification in tissue culture strategically examining the full-genome of dengue virus type in clinical isolates reveals its mutation spectra clustal: a package for performing multiple sequence alignment on a microcomputer biological sequence aligment editor for winxp mega : integrated software for molecular evolutionary genetics analysis and sequence alignment modeltest: testing the model of dna substitution phylogeny inference package mrbayes: bayesian inference of phylogenetic trees paml: a program package for phylogenetic analysis by maximum likelihood dengue type virus in plasma is a population of closely related genomes:quasispecies the molecular epidemiology of dengue virus serotype in bangkok complete genome sequences and phylogenetic analysis of west nile virus strains isolated from the united states flaviviridae: the viruses and their replication cleavage of dengue virus ns -ns a requires an octapeptide sequence at the c terminus of ns in vitro processing of dengue virus type nonstructural proteins ns a, ns b, and ns inhibition of interferon signaling by dengue virus inhibition of alpha/beta interferon signaling by the ns b protein of flaviviruses precise location of sequential dengue virus subcomplex and complex b cell epitopes on the nonstructural- glycoprotein recognition of snthetic oligopeptides from nonstructural proteins ns and ns of dengue- virus by sera from dengue virus-infected children development and evaluation of serotype-and group-specific fluorogenic reverse transcriptase pcr (taqman) assays for dengue virus temperature sensitive mutations in the genes encoding the ns , ns a, ns , and ns nonstructural proteins of dengue virus type restrict replication in the brains of mice e/ns modifications of dengue virus after serial passages in mammalian and/or mosquito cells phylogenetic analysis of dengue- viruses prevalent in guatemala during - internaltional notes dengue type infection -nicaragua and panama cities spawn epidemic dengue viruses disease exacerbation caused by sequential dengue infections: myth or reality? evolution of the dengue- virus in indonesia and throughout se asia may contribute to recent changes in outbreak dynamics ecological and immunological determinants of dengue epidemics we sincerely thank shih-ting ho at the sin-lau christian hospital, chien-ming li at the chi-mei foundation medical center and shih-chung lin at the kuo general hospital for cooperation in kindly providing the clinical samples. the study was supported by the grants from the national health research institute (nhri), taipei, taiwan (grant number: nhri#dd - x-cr- p and nhri#cn-cl p) and the national science council (nsc# - -b- - , nsc# - -b- - ) in taiwan. the authors declare that they have no competing interests. dyc and cck designed and performed all the experiments and drafted this manuscript together. dyc participated in the sequence alignment and statistical analysis. jhh and ycw helped with collecting field human isolates and ljc helped with sequencing experiments, together. thl helped for the field mosquito collection and gjc for- key: cord- -yeaw nr authors: nedjadi, taoufik; el-kafrawy, sherif; sohrab, sayed s.; desprès, philippe; damanhouri, ghazi; azhar, esam title: tackling dengue fever: current status and challenges date: - - journal: virol j doi: . /s - - - sha: doc_id: cord_uid: yeaw nr according to recent statistics, million apparent dengue infections were estimated worldwide in . this figure is by far greater than the who prediction which indicates the rapid spread of this disease posing a growing threat to the economy and a major challenge to clinicians and health care services across the globe particularly in the affected areas. this article aims at bringing to light the current epidemiological and clinical status of the dengue fever. the relationship between genetic mutations, single nucleotide polymorphism (snp) and the pathophysiology of disease progression will be put into perspective. it will also highlight the recent advances in dengue vaccine development. thus far, a significant progress has been made in unraveling the risk factors and understanding the molecular pathogenesis associated with the disease. however, further insights in molecular features of the disease and the development of animal models will enormously help improving the therapeutic interventions and potentially contribute to finding new preventive measures for population at risk. dengue fever is a major cause of illness and death worldwide. the disease is caused by dengue virus which gets transmitted to humans by the bites of infected mosquitoes, aedes (ae.) aegypti and ae. albopictus [ ] . the disease represents a global health issue as it is endemic in around countries, most of which are in tropical and sub-tropical areas. over the last decades, the incidence rate and the geographic distribution of dengue have rapidly increased (almost -fold). data from the world health organization (who) estimates up to million cases of dengue fever each year [ ] . however, a recent published work by bhatt et al. ( ) suggested that the burden of dengue is far more than the who estimation and indicated that million infections of dengue virus could have happened every year [ ] . changes in dengue epidemiology and the increase in incidence rates (with and without co-morbidities) have led the who to propose a new dengue classification system according to disease severity ( fig. ) [ ] . dengue fever is caused by infection with dengue virus (denv). the denv is a vector-borne virus transmitted to humans primarily by bites from two mosquito species, ae. aegypti or ae. albopictus. denv is a single positivestranded rna virus belonging to flavivirus genus of the flaviviridae family and has major serotypes (denv - ) that are antigenically distinct from each other. each denv serotype is phylogenetically distinct suggesting that each serotype could be considered a separate virus [ ] . three dengue serotypes out of four (denv - ) have been found in middle eastern countries including saudi arabia and yemen. interestingly, denv- strain isolated in saudi arabia exhibited a high genetic similarity with denv- strain isolated from asian population, suggesting a widespread of the asian genotype, probably through asian pilgrims [ , ] . a recently published article has unveiled a new serotype , to be added to the existing ones [ ] . this discovery is still controversial and little-known enough to conclude how the th dengue serotype might add to the burden associated with dengue infection. mosquitoes transmit the virus by feeding on blood of infected persons. at first, the virus infects and replicates in the mid-gut epithelium of the mosquito and then spreads to other organs until it reaches the salivary glands after - days where it can be inoculated to another person during subsequent blood meal. vertical transmission of denv in mosquitoes, i.e. from mosquito to larvae has been reported by a number of research groups. in india, angel & joshi ( ) reported the detection of dengue virus by indirect fluorescence antibody test (ifat) in laboratory reared mosquitoes originating from larvae collected from urban and rural areas [ ] . a similar study was conducted in brazil by martins et al. ( ) and confirmed the isolation of denv-type in ae. albopictus larvae and denv-type in ae. aegypti larvae [ ] . similar findings were also reported in mexico [ ] and indonesia [ ] . on the other hand, mother-to-infant transmission of dengue virus via cord blood or breast milk remains controversial [ ] [ ] [ ] . based on the results from several studies, the who has launched a new dengue classification. this classification divides dengue cases into a) cases with/without warning signs and b) severe dengue cases [ ] . however, it is important to note that numerous research groups have debated the rational of this classification as it does not fit their unique local settings. the criteria for dengue case classification are presented in fig. . clinically, dengue infection has a broad spectrum of features. the vast majority of cases are asymptomatic and passes unnoticed. typically, the symptoms start to be prominent after an incubation period of - days [ ] . the severity of the clinical manifestations varies from mild symptoms to severe life threatening symptoms in the case of dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) [ ] . predicting the progression of the mild signs to a severe dhf/dss remains a challenge due to non-specificity of clinical presentation and the incomplete understanding of pathophysiology of the disease and its underlying molecular mechanisms. the early signs of the disease are non-specific. according to the who classification ( ), df is characterized by febrile episode (≥ °c for - days) frequently associated with rash, nausea, vomiting, and headache. although the disease affects people of all ages from infancy through to adulthood [ ] , epidemiological data showed that children tend to tolerate this phase of illness better than adults [ ] . the persistence of the aforementioned symptoms and appearance of other symptoms, such as abdominal pain, mucosal bleed, and lethargy and restlessness can be seen - days later. laboratory analysis of mild dengue fever cases usually shows abnormal leukocyte counts and moderate elevation of the hepatic amino-transferase enzyme activity [ ] . the emergence of these symptoms is a warning sign for disease progression to severe form (dhf/dss) if therapeutic intervention is not undertaken. at this stage clinical intervention and continuous surveillance are imperative to prevent vascular leakage, especially in an endemic area. this form of dengue infection can be attributed to any of the four known serotypes denv - . the likelihood of developing dhf/dss is high in patients who have experienced dengue infection in the past with heterogeneous serotype [ ] . about - % of patients progress to develop a severe dhf/dss which can be fatal unless treated promptly [ ] . this form develops at a late stage of df, where patients may go through defervescence phase characterized by a sudden drop of body's temperature. this phase is also distinguished by severe bleeding, particularly bleeding from the gastrointestinal tract (black, tarry stool), and thrombocytopenia (< , /mm ), which may affect up to % of dhf cases [ ] . interestingly, there was an observed negative correlation between the severity of dhf and the level of platelets in the blood. the exact mechanism of this correlation has yet to be delineated. the drop of platelet counts and the loss of their functionality lead to a vascular fragility increasing the risk of hemorrhage and plasma leakage [ ] . it has been suggested that during acute phase of the infection denv replicates quickly in platelets, as this is very critical for virus survival and dissemination [ , ] . the existence of other symptoms such as retro-orbital pain, maculopapular rash, petechiae, or bleeding from the nose or gums will help making definitive diagnosis for df [ ] . evidence of plasma leakage in various body cavities such as the pleural cavity and the peritoneal cavity, associated with profuse perspiration, adynamia, and sometimes fainting are signs of rapid progression to shock. subsidence in systolic pressure and hypotension may result in profound shock, known as dengue shock syndrome (dss). the duration of dss for a long time might predispose to further complications such as massive bleeding, disseminated intravascular coagulopathy (dic), respiratory failure, multi-organ failure, and infrequently encephalopathy leading to death [ , ] . it has been proposed that case fatality related to dhf may reach % of all cases, however, proper medical care and symptomatic management can reduce mortality rate to less than % [ ] . an early and accurate laboratory diagnosis of dengue infection is of paramount importance in the management of the disease. it has been estimated that the number of misdiagnosed dengue cases could reach a record ratio of % of all cases, mainly due to a large disparity of dengue signs and symptoms which overlap with the symptoms of other viral infections, especially for persons living in or traveling to endemic areas of tropical infectious diseases. dengue fever should be distinguished from other illnesses which share similar symptoms such as chikungunya, mayaro fever, ross river fever, west nile fever, zika fever, yellow fever and viral hemorrhagic fevers [ ] . until the antiviral vaccine becomes available, the prevention of severe cases and cut-down of the economic burden of the disease rely enormously on early and accurate diagnosis. the latter is made possible through the availability of several diagnostic laboratory and virological tests. the onset of later stage symptoms of the illness can be overwhelming and more pathognomonic. nonetheless, based on who classification schemes, the appearance of leukopenia in patients with febrile illness is a major consideration in making diagnosis of dengue infection [ ] . overall, there is an urgent need to reduce dengue morbidity and mortality by improving the diagnosis and molecular analysis of emerging dengue virus. thus far, two diagnostic modalities have been applied to detect the disease at an early stage. the first one is a direct method targeting the acute phase of dengue disease, which is based upon detection of genomic rna by rt-qpcr or soluble ns by antigen capture in blood samples from viremic patients. the second is the indirect method that relies on serological tests to detect denguerelated immunoglobulins par mac-elisa for the capture of specific igm or indirect elisa for the capture of anti-den iggs [ ] [ ] [ ] [ ] [ ] . several risk factors have been associated with dengue infection and its progression to severe dhf/dss forms. recent advances in molecular biology have revealed that the genetic makeup of the three elements of dengue infection (the virus, the vector, and the host) plays a primordial role in the pathogenesis of the disease and could potentially contribute to the dhf progression [ , , ] . hence, an in-depth analysis of genetic variability including polymorphism and mutations could be beneficial in identifying the possible factors and mechanisms of disease development [ ] . the list of host's genetic factors that confer susceptibility or resistance to dengue infection is summarized in table . like most arboviruses, denv infect different organs of the mosquito, including the salivary glands and the central nervous system. mosquito infection elicit behavioral changes including increase of the probing time which lead to host interruption that might lead to wider spread of the virus [ ] . it has been demonstrated that denv infection induced the expression of cathepsin-b, a putative cystatin, and a hypothetical ankyrin repeat-containing protein genes [ ] . the latter could alter the efficiency of virus replication in the salivary gland. this study has shown that modulation of obp and obp genes expression as well as denv infection-responsive odorant-binding protein genes increase the time length for initiation of probing before a successful blood meal, resulting in changes in the host seeking behavior of the mosquito. comparative analysis of the salivary gland transcriptomes of native and denv-infected ae. aegypti identified a number of differentially expressed genes related to sugar/protein digestion enzymes, immunity related genes and blood meal acquisition enzymes that might have an impact on the efficiency of viral replication or mosquito feeding behavior. this study showed that denv infection alter the expression of key host-seeking genes in the mosquito's main olfactory organs and the antennae [ ] . recent updates have indicated that resistance of ae. aegypti to conventional insecticides is related to different mechanisms, one of which is associated with genetic abnormalities within the vector's genome. single point mutation in the voltage-gated sodium channel gene at position (f c) resulting in phenylalanine to cysteine substitution in ae. aegypti confers resistance to permethrin. this mutation is widespread in this vector in southeast asia and latin america [ , ] . it has also been reported that a single amino acid substitution valine to glycine at position in domain ii, segment of the voltage-gated sodium channel gene was associated with less sensibility of ae. aegypti to deltamethrin in thailand [ ] . numerous multi-disciplinary studies confirmed that race, young age, virus strain, female sex and high body-mass index correlate well with increased burden of dengue [ , ] . thus, a closer consideration of human genes regulating the severity of dengue infection, especially genes associated with the immune response, might help in controlling disease spread and improve the acute symptoms of the infection. a number of studies have investigated the relationship between the host genetic polymorphisms and denv infection ( table ) . a single nucleotide polymorphism (snp) in the promoter of cd /dc-sign was associated to increased risk of developing dengue fever [ ] . association studies have successfully identified a link between polymorphisms in the human major-histocompatibility-complex (hla) class i/ii genes and non-hla host genetic factors and severity of dengue disease [ ] [ ] [ ] . polymorphisms of the tap and tap genes could be directly associated with the risk of developing dengue disease among the primary-infected individuals [ ] . both tap and tap are located within the mhc class ii region and homozygosity of the tap at position and and for tap at position , respectively, was found to protect against developing severe forms of dengue [ ] . in an independent study [ ] , the authors showed that single nucleotide polymorphism of the oligoadenylate synthetase genes (oas , and ), of the oas/rnase l antiviral immune system, enhance susceptibility to clinical outcomes of dengue infection. an association between the severity of the disease and other genes including human leukocyte antigen class i and class ii genes, tumor necrosis factor-alpha, fcgriia, vitamin d receptor, transporters associated with antigen presentation, and jak has also been proposed [ ] . the importance of vit-d in denv pathogenesis was concluded from newly-gathered data showing that vit-d impairs denv replication and polymorphism of vit-d gene increases the expression of both cd /dc-sign and fcgriia receptors that enhance denv entry in the target cells [ , ] . in another study [ ] , the authors have successfully applied genome-wide association study (gwas) approach to identify loci that confer susceptibility to severe forms of dengue disease. the investigators used samples from children affected with severe dengue infection against population control cases in vietnam. the data showed that snps at two loci, micb and plce , significantly increased the likelihood of developing dss in children. this finding was further validated in an independent cohort of cases and controls [ ] . a snp in the micb gene coding for the mhc class i polypeptide-related sequence b, an inducible activating ligand for the nkg d type ii receptor of immune cells could alter the protective role of natural killer and cd + t cells in the host responsiveness to denv at the early stage of infection [ , ] . on the other hand, plce plays a primordial role in maintaining intact vascular endothelial cell barrier function, hence, polymorphism of the plce gene may lead to blood vessels leakage and circulatory hypovolemia during dss [ ] . other host candidate genes have also been associated with early onset dengue disease. among these genes, there were receptors/attachment factors for denv linked to immune system and inflammatory response. the chemokines cxcl , cxcl and its respective chemokine receptor cxcr were reported as biomarkers for severe form of dengue infection [ ] . these results are in agreement with recent emerging data indicating strong association between cxcl , cxcl and cxcr and vascular permeability [ ] . the three genes are components of the nf-kb pathway and are involved in the pathogenesis of sars and west nile virus encephalitis [ , ] . [ ] . this process depends enormously on vector-derived salivary factors inoculated on the skin cells [ ] . till-date, there is no effective, commercially available, therapy/vaccine for dengue virus. numerous groups have already made intensive efforts and made good progress to develop a safe, affordable and effective vaccine against all serotypes for global public health [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vaccines which are being developed use various approaches such as live attenuated viruses, inactivated viruses, subunit vaccines, dna vaccines, and chimeric viruses using yellow fever vaccine and attenuated dengue viruses as backbones ( table ) . currently, only one tetravalent vaccine against dengue virus, developed by sanofi-pasteur (france) has reached phase iii clinical trial and is expected to be launched in . this vaccine is based on the production of four chimeric live dengue-yellow fever viruses in which the yellow fever (yf) d vaccine sequences encoding the envelope proteins prm and e genes were substituted by the prm and e genes from dv of serotype , , , or in a molecular clone of yf- d [ ] . this vaccine was produced and tested over people using four dengue virus isolates from indonesia and thailand. this candidate vaccine was found to be attenuated and stable in animal models with respect to plaque size and yellow fever virus neurotropism [ ] . results of the clinical trials showed no adverse effects except moderate injection site pain, headache, and myalgia. another randomized, controlled trial was launched using a total of thai school children to investigate the efficacy of a recombinant, tetravalent vaccine for dengue virus and only dengue cases were reported [ ] . phase i trial of the vaccine in the philippines showed that the seropositivity increased gradually ( , & %) after - vaccinations against all four serotypes as compared to control group. the most promising results were observed in children - years old who exhibited high levels of reactivity of , , , % for denv - ; respectively [ ] . another placebo-controlled trial was conducted on , children from vietnam (vaccine, n = vs placebo, n = ) to determine the clinical efficacy and safety of cyd-tdv. the results demonstrated virologically-confirmed cases in % of the vaccine group as compared to the control group ( %). the efficacy was achieved in up to . % ( % ci . - . ). these findings indicated that the vaccine is highly efficacious with good safety profile when three injections were given to children with age group - years at , and months intervals [ ] . the data emerging from another randomized phase ii trial in india indicated that the vaccine has no serious adverse events and the immunogenicity and safety of cyd-tdv were satisfactory [ ] . a pilot study carried out in five latin american countries where more than , children aged - were recruited to receive either the cyd-tdv vaccine or placebo. the results on efficacy ( . %) and safety profiles were consistent with the previous findings [ , ] . interestingly, the vaccine efficacy ( . %) against hospitalization for dengue was promising and represented a step forward to developing an effective dengue vaccine [ ] . other candidate dengue vaccines have been developed in usa by the johns hopkins university and national institute of allergy and infectious diseases (niaid) and have reached advanced clinical trials [ ] . four liveattenuated denv/delta- were generated each containing nucleotides deletion of the '-untranslated region of genomic rna (delta- ). these vaccines efficiently impaired viral growth in human liver carcinoma cells [ ] . to improve the attenuation of denv- /delta- and denv- /delta- , chimeric denv were developed by substitution of the prm-e gene region of denv- / delta- virus with the prm-e genes of denv- and denv- [ , ] . the results from phase i clinical trial showed that all four live-attenuated denv/ delta- are safe and immunogenic with minor side effects such as faint rash and transient leucopenia only after higher dose [ , ] . dengue virus serotype- antigen was expressed in a vector based on pediatric live-attenuated schwarz measles vaccine (mv) by using the envelope domain iii (ediii) fused with the ectodomain of the membrane protein (ectom). after immunization, long-term production of denv- serotype-specific neutralizing antibodies was observed in measles virus susceptible mice [ ] . a new strategy was evaluated based on single minimal tetravalent denv antigen expression using viral vector derived from pediatric live-attenuated measles vaccine (mv). a recombinant mv vaccine construct was developed using envelope domain iii (ediii) and ectodomain of the membrane protein. the neutralizing antibodies were induced against all four serotypes of dengue virus after two injections in mice susceptible to mv infection. a strong memory neutralizing response was observed against all four serotypes in immunized mice after inoculation with live denv from each serotype [ ] . a naked dna-based candidate vaccine against denv has been developed by the naval medical research center [ , , ] . the genes encoding prm and e of denv were cloned into a shuttle vector under the transcriptional control of human cytomegalovirus (cmv) promoter. the results of phase i clinical trial showed no adverse effects except mild injection site pain, swelling, and fatigue. after second dose, strong igm and igg antibody response was observed which favors the safety profile of this vaccine. to get a better immunogenicity profile, a vaccine based on lipid adjuvant vaxfectin (vical incorporated, san diego, usa), was developed and the results demonstrated good protection profile against denv compared to dna alone [ ] . based on this technology, different groups have developed other candidate vaccines and achieved good protection in mouse models using envelope glycoproteins prm and e, the non-structural protein ns and the helicase/protease ns as vaccine antigens [ ] [ ] [ ] . the first purified inactivated vaccine was developed with aluminum hydroxide (alum) adjuvant and tested in mice and rhesus macaques in the mid- s, by walter reed army institute of research against dengue serotype and good virus protection was reported after two doses [ , ] . using similar technology, second generation japanese encephalitis (je) piv vaccine was developed [ , ] . currently, a new je vaccine (ixiaro; novartis vaccines) has been approved for use in many countries, including the usa [ ] . another dengue vaccine (dengue piv), recombinant subunit dengue e glycoprotein antigen (r e) was also developed and has entered phase i clinical trial [ ] [ ] [ ] . the centers for disease control and prevention (usa) have also developed a live-attenuated vaccine named denvax, which was found to be highly immunogenic in both children and adults and has currently entered phase i clinical trial in the united states [ , ] . recently, a novel third generation approach is being used to develop a vaccine containing recombinant subunit e domain iii (ed ) and the results of laboratory tests have shown the development of potent neutralizing antibodies in a mouse model [ ] [ ] [ ] . using the same technology, a tetravalent vaccine was developed and expressed in pichia pastoris by splicing and using flexible pentaglycyl linkers of the four ediii. the observed results showed that this antigen elicit specific antibodies against all four denv serotypes in balb/c mice [ ] . animal models are very useful for vaccine test development. the lack of animal models significantly hampered the development and efficacy testing of dengue vaccine. currently only rhesus macaques and aotus monkeys are being used for testing the vaccine before clinical trials are initiated [ ] . the d me vaccine was evaluated in both aotus monkeys and rhesus monkeys, and found to be immunogenic with - % protection against dengue infection [ , ] . porter et al. ( ) demonstrated that injection of non-human primate with three doses on day , and , with tetravalent dengue dna vaccine vaxfectin-adjuvanted, was more efficient against live dengue- virus compared to control animals. this finding support initiation of vaxfectin-adjuvanted phase i clinical trial [ ] . successful induction of immune response was obtained in mice and rhesus monkeys to the vaccines developed using dengue prm-e, dengue prm-e-nonstructural (ns) , and dengue ns antigens, and piv adjuvanted with alum [ , ] . centers for disease control and prevention (fort collins, co), hawaii biotech, and simmons developed different vaccines that showed good immunogenicity in animal models [ ] . similarly, the psoralen/uv inactivation dengue vaccine was found to be more immunogenic and protective against dengue serotype virus in aotus monkeys [ ] . thus far, there are no antiviral drugs available to treat dengue fever; therefore the community will continue to depend on the control of the mosquito vector as the main route to prevent the spread of disease. alternative approaches have been utilized against flaviviruses by targeting and inhibiting virus entry and the essential elements used in virus replication, nonstructural proteins, rna polymerase, and proteases. the most important target elements include ns helicase nucleoside triphosphatase (ntpase/ rna ' triphosphatase (rtpase), ns methyl transferase/ rna-dependent rna polymerase, and ns /ns b protease [ ] [ ] [ ] . rna interference (rnai) technology is also being used to impair virus replication against respiratory syncytial virus, hepatitis viruses, influenza virus, poliovirus and hiv [ , ] . low molecular weight phenolic compounds such as flavonoids and phytochemicals isolated from plants were previously tested and are being used for anti-dengue therapy [ , ] . an anti-viral inhibitory effect ranging from - % against denv replication was observed when methanolic extracts of momordica charantia and andrographis paniculata were used in cultured primate cells [ ] . several attempts have been made in the past to tackle dengue through elimination of ae. aegypti. the most successful experiences were related to vector control programs adopted in cuba and singapore. the programs were based on intensive insecticidal treatment and reduction of the availability of aedes larval habitats [ , ] . unfortunately, lack of sustainability of these stringent measures led to reappearance of dengue outbreaks. recently, a novel form of biological control of dengue transmission has been developed and is currently being applied. this is based on the development of genetically modified (gm) mosquitoes infected with a bacterium known as wolbachia to combat dengue infection. this bacterium blocks replication of the virus inside the mosquito and prevents its transmission to humans [ ] . in , million gm male mosquitoes were released in the wild to decrease the number of aedes mosquitoes and reduce the rate of dengue transmission. a closer monitoring of the insects revealed that over % of the eggs were wolbachia-positive which indicated that gmmosquitoes were overriding wild-mosquitoes resulting in decreased virus transmission [ ] . in an initiative to eradicate dengue fever, scientists from australia, are leading eliminate dengue (ed) program which involves community engagement as a key component in this program. since the program kicked off in , millions of wolbachia mosquitoes were released across the north queensland city-australia. based on the promising results obtained from local trial, eliminate dengue became an international research program across countries affected by dengue including australia, vietnam, indonesia, brazil and colombia [ , ] . dengue infection can be prevented by alternative approaches. the first one includes blocking virus entry into cells which is mediated by the viral envelope glycoprotein e via receptor-mediated endocytosis [ ] . dendritic cells, monocytes, and macrophages are the main targets of denv infectious entry. the second approach involves blocking virus attachment to specific cellular receptors expressed on immune cells, liver cells, and endothelial cells. small molecules and peptides targeting the hydrophobic pocket of the envelope e glycoprotein are characterized as inhibitors of virus entry. nicholson et al. ( ) explored the inhibitory effects of dn and oan , peptide entry inhibitors. the authors demonstrated that dn and oan can effectively block antibody dependent enhancement (ade) in-vitro suggesting that entry inhibitors are potential candidates to prevent development of dhf/ dss [ ] . two other compounds have also been shown to qualify as potent inhibitors of dengue virus infection are imino-sugars deoxynojirimycin and castanospermine [ ] . these compounds are natural alkaloids derived from the black bean and act as inhibitors against all dengue serotypes by disrupting the folding pathways of the envelope glycoproteins prm and e [ ] . various types of carbohydrate-binding agents, isolated from different organisms, have been shown to have antiviral activities. three plant lectins, hippeastrum hybrid agglutinin, galanthus nivalis agglutinin and urtica dioica agglutinin isolated from amaryllis, snowdrop and stinging nettle respectively were found to be potent inhibitors of denv- infection by inhibiting viral replication [ ] . heparan sulfate (hs) is a putative receptor for denv which interacts with domain iii of the e-protein. virus entry can be blocked by targeting the e-protein-hs interaction with soluble gags and other highly charged hs [ ] . fucoidan was isolated from marine algae and showed antiviral activity against denv- in bhk cells [ ] . similarly, carrageenan and dl galactan, sulfated polysaccharides from red seaweeds, exhibited strong antiviral activity against denv- and denv- but a very weak activity against denv- and denv- . furthermore, two α-d-glucans were isolated from a chinese herb and demonstrated high anti-denv- activities in bhk cells [ , ] . dengue fever represents a real economic burden especially in affected countries. extensive efforts are needed to tackle disease spread and reduce the mortality rates and the associated healthcare cost. there is a need for more scientific research which we believe is a key route to provide further insight in the pathogenesis of dengue infection and help understanding the underlying molecular mechanisms associated with progression to the severe forms of the disease (dhf/dss). this will be a step forward to develop an adequate preventive vaccine and effective treatment. the authors disclose that there is no conflict of interest. authors' contributions tn participated in the review design, coordination and helped to draft the manuscript. sk and ss participated in the review design and helped to draft the manuscript. pd participated in the article revision. gd and ea participated in the review design. read and approved the final manuscript. this project is funded by the king abdulaziz city for science and technology (kacst) under grant number ( ‫ﺍ‬ ‫ﺕ‬ - - ). the authors are also grateful to the diagnosis of dengue: an update guidelines for diagnosis, treatment prevention and control the global distribution and burden of dengue dengue viral infections outbreak of viral hemorrhagic fever caused by 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α-dglucans from gastrodia elata bl. and their anti-dengue virus bioactivities submit your next manuscript to biomed central and we will help you at every step: key: cord- -z js mr authors: chen, huixin; parimelalagan, mariya; lai, yee ling; lee, kim sung; koay, evelyn siew-chuan; hapuarachchi, hapuarachchige c.; ng, lee ching; ho, phui san; chu, justin jang hann title: development and evaluation of a sybr green–based real-time multiplex rt-pcr assay for simultaneous detection and serotyping of dengue and chikungunya viruses date: - - journal: j mol diagn doi: . /j.jmoldx. . . sha: doc_id: cord_uid: z js mr chikungunya virus (chikv) and dengue virus (denv) have emerged as the two most important arbovirus diseases of global health significance. similar clinical manifestations, transmission vectors, geographical distribution, and seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases and vice versa. in this study, we developed a rapid and accurate laboratory confirmative method to simultaneously detect, quantify, and differentiate denv serotypes , , , and and chikv. this sybr green i–based one-step multiplex real-time rt-pcr assay is highly sensitive and specific for chikv and denv. melting temperature analysis of pcr amplicons was used to serotype denv and to differentiate from chikv. the detection limit of the assay was , , , , and rna copies/reaction for denv- , denv- , denv- , denv- , and chikv, respectively. our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, and human coronaviruses. the feasibility of using this assay for clinical diagnosis was evaluated in denv- and chikv-positive patient sera. accordingly, the assay sensitivity for denv- , denv- , denv- , denv- , and chikv was . %, . %, . %, . %, and . %, respectively, with % specificity. these findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection and serotyping of denv and chikv in clinical samples. chikungunya virus (chikv) and dengue virus (denv) are two of the arthropod-borne diseases that are affecting many countries worldwide, especially in the tropics and subtropics. belonging to the genus alphavirus of the togaviridae family, chikv has a genomic structure of cap-nsp -nsp -nsp -nsp -(junction region)-c-e -e - k-e -poly (a)- . denv is a flavivirus of flaviviridae family and has a genomic structure of utr-c-prm (m)-e-ns -ns a-ns b-ns -ns a-ns b-ns - utr. both denv and chikv infections have similar geographical distribution and seasonal correlation , because they share the same vectors for transmission: aedes albopictus and aedes aegypti mosquitoes. clinical manifestations of denv infections are often indistinguishable from chikv infections, which makes clinical diagnosis difficult. , although both denv and chikv are single-stranded, positive-sense rna viruses that have similar vectors, transmissibility, and clinical symptoms, denv infections can be more severe when compared with chikv infections. , denv is traditionally classified into four serotypes: denv- , denv- , denv- , and denv- . a primary infection with one serotype of denv is believed to confer lifelong immunity to the same serotype. on a secondary, heterologous denv infection, antibodies predominantly directed against the initial denv serotype are produced rapidly. , although most of the produced antibodies bind to the heterologous virus serotype, they are more likely to have nonneutralizing properties against the new serotype, and these have been found to enhance infection, a phenomenon called antibody-dependent enhancement. e in addition, the dominant serotype during recent large denv outbreaks is swapping. therefore, accurate clinical diagnosis is important in providing health care personnel the information required for proper case management and planning preventive measures. in addition, serotyping data could contribute to warn impending epidemics. thus, it is of great concern to develop a diagnostic kit for accurate identification of the chikv and denv and at the same time to serotype denv at the point of care. currently, the diagnosis of denv and chikv is determined by virus isolation, detection of virus-specific antibodies by enzyme-linked immunosorbent assay, genomic rna detection using rt-pcr, or real-time rt-pcr. , although virus isolation is the gold standard, the method is time-consuming. enzyme-linked immunosorbent assay, especially capture of igm, has been considered the standard for the diagnosis of arbovirus infection during the acute phase and allows diagnosis to days after the onset of illness but is hampered by cross-reactions between members of the flaviviruses and alphaviruses. shu et al developed a sybr green iebased real-time rt-pcr assay for denv serotyping, but at least four tubes of reactions are needed per sample. chien et al developed a taqman probeebased real-time rt-pcr assay for denv serotyping, but the method is unable to detect chikv simultaneously. in addition, a few taqman probeebased multiplex real-time rt-pcr assays for denv and chikv has been developed, but these are not capable of serotyping denv at the same time. in sybr green iebased real-time rt-pcr assays, the melting temperature (tm) of a pcr product depends on the concentration, length, and nucleotide composition of the amplicon so that tm analysis has the potential to differentiate denv serotypes. in the present study, we aimed to develop and evaluate a sybr green iebased multiplex real-time rt-pcr assay to detect, differentiate, and quantify denv and chikv and simultaneously to serotype denv based on tm analysis of the pcr amplicon. primers targeting the prm region of the denv genome and nsp region of the chikv genome were selected and evaluated. detection limit of the assay was determined using serially diluted in vitro transcribed viral rna samples. in addition, a panel of rna viruses, including members of togaviridae, flaviviridae, orthomyxoviridae, coronaviridae and picornaviridae, were also used to examine the cross-reactivity of our assay. lastly, the clinical sensitivity and specificity of the assay were evaluated with a panel of serum samples collected from denv-or chikv-infected and healthy individuals in singapore. viruses chikv (eu . , fj ), denv- (s strain), denv- (stp and new guinea c strain), denv- (eden / strain), and denv- (s strain) were propagated in c / cells, and viral titers were determined by the plaque-forming assay. viral rna of ross river virus (rrv); zika virus (zikv, mr strain); kunjin virus (kunv, mrm c strain); west nile virus (wnv, sarafend strain); human enterovirus (hev , af strain); enteric cytopathic human orphan virus ; enteric cytopathic human orphan virus ; coxsackie b virus (cb ) and a virus (ca , world health organization strain); poliovirus types , , and (sabin strain); human coronavirus (cov); and influenza a virus subtypes h n and h n were also used to examine the crossreactivity of this assay. the rrv, kunv, and wnv were provided by prof. mary mah-lee ng (national university of singapore, singapore). the human cov and influenza a viruses were provided by associate professor tan yee joo (national university of singapore, singapore). a set of serum samples from chikv-infected patients and from uninfected individuals were collected at the national university hospital, singapore, with informed consent, to evaluate the clinical sensitivity and specificity of the real-time rt-pcr assay. all the serum samples were validated using a real-time rt-pcr detection assay targeting the chikv envelope glycoprotein (e ) gene, and a confirmed case of chikv infection was defined as febrile illness associated with a positive result from the real-time rt-pcr. this part of the study was performed in accordance with a national university of singapore institutional review board approved protocol (no. - ). the environmental health institute of the national environmental agency of singapore provided a set of serum samples from chikv-infected patients and serum samples from denv-infected patients. besides suggestive clinical signs, multiplex rt-pcr for chikv and denv- - the journal of molecular diagnosticsjmd.amjpathol.org these samples were confirmed using a serotype-specific rt-pcr assay. viral rna was extracted from ml of infected cell culture supernatants or serum samples using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. the rna was eluted in a final volume of ml of nuclease free water and was stored at À c until use. the full genome nucleotide sequences of strains of each denv serotype and chikv from recent outbreaks were retrieved from genbank (http://www.ncbi.nlm.nih.gov/ genbank; accession numbers are provided in supplemental table s ) and aligned using the clustalx version . sequence alignment software. a number of oligonucleotide primers for specific denv and chikv were designed using national center for biotechnology information primer designing tool and modified manually (table ) . denv serotyping primer (dest) binding sites selected are well conserved across all four denv serotypes, whereas the nucleotide sequences between the forward and reverse primer binding sites are less conserved, resulting in different tms of the pcr amplicons for denv serotyping. standard rna templates containing primer-binding sites were obtained by in vitro synthesis from dna templates. in vitro transcription (ivt) was performed using the maxiscript ivt kit (catalog no. , ambion) on the rt-pcr products of denv and chikv. in brief, first, a standard rt-pcr was performed using a t promoter sequence incorporated forward primer. pcr product containing t promoter was subjected to ivt at c for hour. the ivt products were then treated with u of dnase i and incubated at c for minutes to remove the remaining dna. the dnase i activity was stopped by adding ml of . mol/l edta and heat deactivated further at c for minutes. the excess nucleotides and pyrophosphate were removed by nh oac/ethanol precipitation. the rna pellet was suspended in diethylpyrocarbonatetreated water. the amount of ivt-generated rna fragments was determined using the nanodrop nd spectrophotometer (nanodrop technologies, inc., wilmington, de) and converted to molecular copies by using the following formula: then -fold serially diluted ivt rna samples were subjected to real-time rt-pcr to generate a standard curve for the quantification of viral rna in terms of copy number. all rt-pcr reactions were performed in -ml reactions with ml of rna template. ivt rna of denv- , - , - , and - and chikv was included as a control in every rt-pcr run. sybr green iebased one-step real-time rt-pcr was performed on the applied biosystems ste-poneplus real-time pcr system (applied biosystems, bedford, ma). samples were assayed with nmol/l of each primer in a  final concentration of sybr green ready mastermix. the rt-pcr conditions for the real-time rt-pcr consist of a -minute rt step at c and minutes of taq polymerase activation at c, followed by cycles of pcr at c for seconds (denaturation) and c for minute (annealing and extension). the fluorescence emitted was captured at the end of the extension step of each cycle. amplification graphs were checked for the cross-point value of the pcr product. melting-curve analysis was performed after pcr amplification to verify that the correct product was amplified by examining its specific tm that was also used to serotype denv. (dest ) and the chikungunya virus (chk ) were tested in combination for simultaneous detection and differentiation of denv and chikv. the melting curve for each targeting virus is well separated, indicating that they not only are able to detect denv and chikv but also have good compatibility in terms of similar annealing temperature and low tendency of forming heterodimers. the results from the combination of dest and chk also reveal good consistency compared with use of dest alone in terms of specific melting temperatures (tms) of amplicon from denv- , - , - , and - . the consensus regions of the denv genome were carefully selected, and pairs of primers were designed for denv serotyping. among them, dest was selected based on its capability to detect and serotype denv and its low likelihood of forming primer dimer. the specific tm of pcr amplicons from denv- , - , - , and - was . c (coefficient of variation of . %), . c (coefficient of variation of . %), . c (coefficient of variation of . %) and . c (coefficient of variation of . %), respectively (figure ). dest was further tested in combination with each of pairs of primers for chikv detection for compatibility and capability of differentiating denv from chikv. the pair of dest and chk (figure ) (the specific tm of chikv pcr amplicon was . c, with coefficient of variation of . %) was selected because not only was it able to detect denv and chikv but also it had a similar annealing temperature and low tendency of forming heterodimers. the results for the combination of dest and chk revealed good consistency compared with use of dest alone, in terms of specific tms of amplicons from denv- , - , - , and - . figure lists the temperature ranges for the pcr amplicons specific to each denv serotype and chikv. the detection limit of the real-time rt-pcr assay was determined using -fold serially diluted standard ivt rna samples. figure gives the standard curves of denv- , - , - , and - and chikv using the dest and chk primers. the detection limits were calculated to be rna copies per reaction for denv- , rna copies per reaction for denv- , rna copies per reaction for denv- , rna copies per reaction for denv- , and rna copies per reaction for chikv. all standard curves revealed good correlation of coefficient to the data. the pcr reaction efficiency for denv- , - , - , and - and chikv was . %, . %, . %, . %, and . %, respectively. besides the use of standard rna samples of denv- , - , - , and - and chikv as positive controls, rna of rrv was used as a representative member of alphavirus; kunv, wnv, and zikv were used as representative members of flavivirus in the cross-reactivity study. in addition, a panel of other rna viruses, including h n , h n , polio , polio , polio , cov, enteric cytopathic human orphan virus , hev , cb , and ca , were also evaluated. all chikv and denv samples produced positive results, and none of the other viral rna samples produced false-positive results ( table ). since , major chikv outbreaks causing debilitating disease have been reported in africa, india, europe, the middle east, and southeast asia. the recent resurgence of chikungunya fever has drawn global attention because of its explosive onset, rapid spread, high morbidity, and myriad of clinical manifestations. denv that causes dengue fever, hemorrhagic fever, and shock syndrome has been defined as "the most important mosquito-borne viral disease in the world" ,p by the world health organization, with an estimated . billion people at risk for infection in tropical and subtropical regions. global estimates vary, but approximately million to million dengue infections, , episodes of severe dengue infections, and > , dengue-related deaths occur annually. , because of common transmission vectors and similar clinical manifestations, chikv and denv can be misdiagnosed in areas where both viruses are co-circulating. this may lead to poor patient management at point of care. in addition, serotyping information is important for the understanding of dengue epidemiology. for instance, serotype switch is used as an early warning of epidemics in singapore because data have revealed that such switches are associated with epidemics. in recent studies, several denv serotyping assays have been reported to require four tubes of tests or to be unable to detect chikv at the same time. , recently, a few probe-based multiplex real-time rt-pcr assays were published for simultaneous detection of chikv and denv, which required individual primer and probe sets to detect each targeted virus, but none of them can be used for denv serotyping, e probably because of the limitation in the number of channels required for discriminating the viruses using real-time pcr instruments. for instance, cecilia et al reported a single-step multiplex real-time rt-pcr assay for simultaneous detection and quantification of all dengue serotypes and chikv. the assay was developed using primers and taqman probes targeting denv- / / , denv- , chikv, and b-actin. the assay revealed high sensitivity for denv ( %) and chikv ( . %) with high specificity ( %) when compared with conventional rt-pcr assay. detection limit was set within the range of to pfu/ml ( pfu/ml for denv- and , pfu/ml for denv- and chikv, and pfu/ml for denv- ). the assay is used primarily for detection of all four denv serotypes and chikv but lacks serotyping capability. we report a rapid and accurate laboratory confirmation method to simultaneously detect, quantify, and differentiate denv multiplex quantitative rt-pcr denv- denv- denv- denv- chik denv- (s ) þ À À À À denv- (stp ) À þ À À À denv- (new guinea c) À þ À À À denv- (eden / ) À À þ À À denv- (s ) sybr green iebased assays are more cost-effective than probe-based ones, and only two detection channels (rox and sybr green) are needed, increasing adaptability of the assay to the whole range of real-time pcr instruments. however, the primary disadvantage of the sybr green i dye chemistry is that it binds to any double-stranded dna, including nonspecific pcr products and primer dimers, which may lead to false-positive results. to counter the problem, primer sets used in this study were carefully designed and examined based on heterodimer formation likelihood and thermal compatibility. in brief, denv serotyping primers were designed based on genome alignment of multiple strains of all four serotypes of denv. the primer-binding sites selected were well conserved across all four denv serotypes, whereas the nucleotide sequences in between, flanked by the forward and reverse primers, were less conserved to yield different tm of the pcr amplicons. dest (the targeting prm region of the denv genome) was selected among pairs of denv serotyping primers because of its outstanding performance in primer verification. for chikv, primers were designed to preferably target nonstructural protein-coding regions because they were usually well conserved. chk (the targeting nsp region of the chikv genome) was selected based on its exceptionally good compatibility with dest , reproducibility, sensitivity, and specificity in primer verification. in addition, a temperature range, instead of a fixed value, for pcr amplicons is given for each virus so that fluctuations of the tm from the respective pcr amplicons (ie, sample variance, strain variations) could be tolerated. we have modified the primer sequences and optimized the pcr thermal profile to achieve balanced sensitivity to all of the targeted viruses without sacrificing assay specificity. furthermore, sybr green i dye chemistry limits the use of internal controls because the fluorescent signal from internal controls would be nondifferentiable from targeting viruses due to same fluorescent channels (sybr green i) being used. to overcome this issue, external positive controls (ie, standard viral rna) were included in every rt-pcr run throughout this study. the detection limit of the assay was determined as rna copies/reaction for denv- , rna copies/reaction for denv- , rna copies/reaction for denv- , rna copies/reaction for denv- , and rna copies/reaction for chikv. in general, denv infection in human results in high viral load (approximately copies/ml), with even higher viral load in dhf cases. , however, the viral titer starts to decline rapidly after the acute phase, and more severe clinical symptoms start to appear. an assay that is able to detect low rna copy number with wide dynamic range for viral detection could benefit both the operators and patients at the point of care. in conclusion, this multiplex real-time rt-pcr assay for denv serotyping has superior analytical and clinical performance with high sensitivity and specificity. this enabled our assay to be a useful tool for epidemiologic surveillance and for the molecular diagnosis of large-scale analyses, such as during chikv and denv outbreaks. to our knowledge, this is the first sybr green iebased real-time rt-pcr assay 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and virus serotype correlate with disease severity the rrv, kunv, and wnv were provided by prof. mary mah-lee ng, and the human cov and influenza a viruses were provided by prof. tan yee joo (national university of singapore, singapore). supplemental material for this article can be found at http://dx.doi.org/ . /j.jmoldx. . . . key: cord- - poerd authors: vermeulen, tom d; reimerink, johan; reusken, chantal; giron, sandra; de vries, peter j title: autochthonous dengue in two dutch tourists visiting département var, southern france, july date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: poerd we report dengue virus (denv) infection in two dutch tourists who visited département var, southern france, in july and august . as some autochthonous dengue cases have occurred in europe in recent years, awareness among physicians and public health experts about possible intermittent presence of denv in southern europe is important to minimise delay in diagnosis and treatment. quick diagnosis can lead to timely action to contain the spread of vector-borne diseases and minimise transmission. we report dengue virus (denv) infection in two dutch tourists who visited département var, southern france, in july and august . as some autochthonous dengue cases have occurred in europe in recent years, awareness among physicians and public health experts about possible intermittent presence of denv in southern europe is important to minimise delay in diagnosis and treatment. quick diagnosis can lead to timely action to contain the spread of vector-borne diseases and minimise transmission. travel-related diseases may serve as sentinels of transmission of disease in the visited area. prompt diagnosis and notification of such diseases may assist in the detection and control of disease outbreaks. when we diagnosed dengue in a dutch tourist who visited southern france, we coordinated joint action between the patient and clinical and public health experts. this led to rapid international notification and consecutive outbreak control efforts by french authorities. a second, related, dutch patient with a recent fever was retrospectively also diagnosed with dengue. a previously healthy woman in her s (patient ) spent a -week holiday with relatives in la croix valmer, france, from to july . in the first week of august, patient stayed with other friends in another house nearby. on august, she developed fever accompanied by myalgia in her calves and neck, as well as a painful skin (day of the disease episode). on day post onset of symptoms (pos), she was nauseous and vomited once. on day pos, she returned to the netherlands. a test for severe acute respiratory syndrome coronavirus (sars-cov- ), on a nasopharyngeal swab, was negative. on day pos, the patient noticed an itchy erythematous rash on her hands and lower legs. on day pos, she consulted her general practitioner who, suspecting petechiae, referred her to our hospital. in addition to the reported signs and symptoms, she mentioned a blurred, colourful spot in the field of vision of her left eye. during childhood, the patient was vaccinated according to the dutch national vaccination scheme; she had never received any vaccinations for yellow fever, japanese encephalitis or tick-borne encephalitis. interestingly, one of her family members upon our clinical suspicion of dengue virus infection spontaneously acknowledged having seen tiger mosquitoes (aedes albopictus) around their holiday home. physical examination showed normal vital signs and revealed slight erythematous exanthema on her hands and upper limbs and a confluent petechiae-like exanthema on both legs. the presumptive diagnosis of dengue was made, common laboratory tests including dengue virus (denv) serology were ordered and she was referred to the ophthalmologist. fluorescein angiography of the eyes showed an inflammatory foveolitis in her left eye. laboratory results on day pos showed a mild thrombocytopenia and leukocytopenia with plasmocytosis, and moderately elevated serum levels of the liver enzymes (table ). on the same serum, comparative igm and igg serology (immunofluorescence arbovirus fever mosaic , euroimmun ag, lübeck, germany) against chikungunya virus (chikv), denv and japanese encephalitis virus (jev) was performed at the dutch national institute for public health and the environment (rivm) laboratory. high concentrations of denv-specific igm and igg antibodies were detected ( table ). there was a slight igg response, without igm response, against jev, interpreted as non-specific cross-reactivity. rt-pcr was not done. patient spent the holiday in la croix valmer, together with patient , from to july. he returned to the netherlands week before patient . on august, he noticed a mild pain in his right ear (day of the disease episode). on day pos he had a high fever ( . ° c). on day pos, suspecting bacterial otitis, he was prescribed amoxicillin. on day pos, he noticed a mild pain behind his eyes. on day pos, day after defervescence, he noticed a slight rash on his trunk and extremities and interpreted this as allergy to the amoxicillin. patient 's blood sample of september (day pos) tested positive for igm and igg antibodies against denv, with high titres ( table ). the igg response against jev was interpreted as non-specific cross-reactivity. also this patient had been vaccinated according to the dutch national vaccination scheme. on august , upon confirmation of the serological results, patient was reported by the rivm to the french authorities through the early warning and response system of the european union as an autochthonous denv infection probably acquired in france with cross-border implication. the french authorities contacted the patient and announced the case by press release on september [ ] . santé publique france advised that also other family members with symptoms should be tested. that identified patient . the other seven dutch family members visiting the holiday home between and july did not develop any disease symptoms and neither did the individuals with whom patient stayed during the first week of august in the other house nearby. none of the household members had recently travelled outside europe. notification to the french authorities led to a prompt local public health response including mosquito control around the holiday home (with deltametrine and bacillus thuringiensis israelensis on a m radius) and door-to-door investigations in order to identify other cases and raising awareness among local healthcare professionals and the public [ ] . here we describe two patients with dengue from the same family, who acquired the disease in department var, southern france. the signs and symptoms, as well as the plasmocytosis, of patient were typical for dengue [ ] . the list of differential diagnoses was therefore very short and the high igm and igg titres for denv were considered confirmative, even though definite confirmation would require demonstration of virus or serodiagnosis on paired samples [ ] . pcr was not conducted to detect denv in blood or urine because the chance for a positive test was considered low in this rather late stage of disease and it was not deemed necessary for confirmation of disease, clinical management, notification or public health measures. dengue is endemic in large parts of the world and a common illness among returning travellers from (sub) tropical regions [ ] . because of globalisation in travel and trade and under changing ecological conditions, the geographical distribution of the vector of denv, ae. albopictus, gradually expanded over the last decades and may continue to do so [ ] . imported infections can continue to cause autochthonous outbreaks. a lack of awareness and a long interval between the viraemic episode of the patient with imported dengue and the first registration in the public health system were identified as possible drivers of local outbreaks [ ] . aedes albopictus has been established in france since and currently, the mosquito species is endemic in large parts of the country including one area close to the belgian border [ , ] . the presence of ae. albopictus in multiple sites in europe means that also other diseases can be transmitted. upon introduction by returning viraemic travellers, european cases of chikv, denv and zika virus infection have been reported [ , ] . as recently as august , five patients in vicenza province, northern italy, were confirmed to have a denv infection weeks after a household member infected with denv returned from west sumatra [ ] . autochthonous denv infection was first reported in france in and has since been reported at an almost yearly basis [ , ] . in , by the end of september, six other autochthonous denv cases had been reported by french authorities, one in the department hérault and five in the department alpes-maritime [ , , ] . our case signalled the first evidence of local denv activity in département var in . in the recent past, between and , six cases of autochthonous transmission were confirmed in the départment var [ ] . however, our cases do not seem to have any connection with the other autochthonous cases identified in southern france this year. the cases reported here again illustrate that travel medicine can have a role as a sentinel for detection of silent circulation of infectious diseases [ ] . clinicians should be aware of the possibility of 'tropical' vectorborne diseases acquired by travellers within european areas where competent vectors are present, even when cases have not been reported (yet) by local authorities. rapid notification by clinicians and communication between national authorities is essential to ensure timely local risk management and disease control. none declared. tom d. vermeulen: clinical description of case. johan reimerink: serology and co-authoring manuscript. chantal reusken: international notification, epidemiological perspective, co-authoring manuscript. sandra giron: french public health perspective, co-authoring manuscript. peter j. de vries: clinical case management and description, corresponding author. marseille: agence régionale de santé high incidence of peripheral blood plasmacytosis in patients with dengue virus infection dengue guidelines for diagnosis, treatment, prevention and control: new edition. geneva: who travel-associated illness trends and clusters past and future spread of the arbovirus vectors aedes aegypti and aedes albopictus from importation to autochthonous transmission: drivers of chikungunya and dengue emergence in a temperate area chronology of the development of aedes albopictus in the alpes-maritimes department of france european centre for disease prevention and control (ecdc) vector-borne transmission of zika virus in europe, southern france ongoing and emerging arbovirus threats in europe first autochthonous dengue outbreak in italy Émergences de dengue et de chikungunya en france métropolitaine bilan de la surveillance des arboviroses en : transition vers une surveillance des cas confirmés de chikungunya, dengue et d'infection à virus zika en france métropolitaine. [review of arbovirus surveillance in : transition to surveillance for confirmed cases of chikungunya, dengue and zikavirus in metropolitan france an outbreak of indigenous cases of dengue detected in the alpes-maritimes cinq cas autochtones de dengue détectés à nice a case of dengue type virus infection imported from africa to italy license, supplementary material and copyright this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- -pbqjg hf authors: song, ke-yu; zhao, hui; jiang, zhen-you; li, xiao-feng; deng, yong-qiang; jiang, tao; zhu, shun-ya; shi, pei-yong; zhang, bo; zhang, fu-chun; qin, e-de; qin, cheng-feng title: a novel reporter system for neutralizing and enhancing antibody assay against dengue virus date: - - journal: bmc microbiol doi: . / - - - sha: doc_id: cord_uid: pbqjg hf background: dengue virus (denv) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. antibody plays distinct roles in controlling denv infections. neutralizing antibody is protective against denv infection, whereas sub-neutralizing concentration of antibody can increase denv infection, termed antibody-dependent enhancement (ade). plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. results: in this study, a novel reporter virus-based system was developed for measuring neutralization and ade activity. a stable renilla luciferase reporter denv (luc-denv) that can produce robust luciferase signals in bhk- and k cells were used to establish the assay and validated against traditional plaque-based assay. luciferase value analysis using various known denv-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. the newly developed assay was finally validated with clinical samples from infected animals and individuals. conclusions: this reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for denv antibodies. the four serotypes of dengue virus (denv) belong to the genus flavivirus within the family flaviviridae [ ] . the clinical manifestations of denv infections cover a wide range of symptoms, from mild dengue fever (df) to severe life threatening dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) [ ] . commonly, dhf/dss is associated with sequential denv infection by different serotypes [ , ] . annually, to million people in over countries are infected with denv and dhf/dss can be fatal in up to % of affected individuals. no vaccine or specific antiviral drugs is currently available. denv is a typical positive-sense, single-stranded rna virus. the genome is about kb in length and encodes three structural proteins (c, prm and e) and seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ). neutralizing antibody is predominantly induced against e protein, and laboratory and clinical studies have demonstrated that protection of animals or individuals from denv infection is best correlated to titer of neutralizing antibody (> : ). however, preexisting sub-neutralizing concentration of antibody or non-neutralizing antibody was also evidenced to enhance denv infection in fc gamma receptor (fcγr) -positive cells and appears to be a risk factor for severe diseases. this phenomenon is known as antibody-dependent enhancement (ade) infection [ , ] . thus, human antibodies are believed to play distinct roles in controlling denv infection. it is important to characterize antibody with neutralizing or enhancing activities against denv for both basic and applied research. currently, plaque-based analysis is the most widely accepted method measuring neutralizing or enhancing antibodies [ ] and has been recommended by the world health organization. however, this traditional method is time-consuming and labor intensive, and not suitable for large-scale samples analysis. further, plaque-based assay can only be performed in cells that permit plaque forming and quantified by an operator-error prone manual readout based on the number of plaques. there is a great need of novel technology for characterizing dnev neutralizing and enhancing antibodies in a simple, rapid, and highthroughput manner [ ] . recently, we have developed a stable luciferase reporter denv (luc-denv) for antiviral high-throughput screening [ ] . in this study, we aim to adapt the luc-denv for anti-dnev neutralizing and enhancing antibodies evaluation. this newly developed reporter virus-based assay is validated using various known monoclonal antibodies (mabs) and clinical samples from infected animal and patients, demonstrating well correlation with the traditional plaque-based assays. the luc-denv was developed by engineering the renilla luciferase gene into the capsid-coding region by reverse genetic technology [ ] . we have shown that luc-denv replicates efficiently in both mammalian and mosquito cells with high stability. as shown in additional file : figure s and additional file : figure s , increasing amounts of luciferase signal were observed from to h post-infection in luc-denv infected bhk- and k cells. to adapt luc-denv for neutralizing assay, we firstly assayed three identified neutralizing mabs g [ ] , b [ ] and a g [ ] by using plaque-based and luc-based assay, respectively. standard prnt was performed in -well plates using -fold dilution of each mab. the results showed that all three mabs significantly reduced the numbers of plaques in a dose-dependent manner (figure ,abc, right ordinate). the prnt of g , b and a g was . , . and . μg/ml, respectively. the rlu based assay was performed in the -well plate using the same dilutions of each mab. the results demonstrated that all three mabs significantly decreased rlu in a dose-dependent manner (figure , abc, left ordinate). lrnt of three mabs calculated from a fitting curve were . , . and . μg/ml, respectively, which was of the same order of magnitude with prnt . an unrelated mab against ev showed no neutralization for both plaque and luc-based assay (data not shown). data fitting was made between values above. as expected, a linear correlation (r > . ) was demonstrated between pfu and rlu assay, and the linear equation between rlu and pfu is calculated as rlu = . pfu + ( figure d ). our results supported the application of luc-based assay for neutralization antibodies against denv. to develop the luc-denv for ade assay, k cells were infected with luc-denv in the presence of serial -fold dilutions of a g . the viral titers in the supernatants were measured by standard plaque-based assay and rlu-based assay, respectively. the results showed that the viral yield was markedly enhanced in the presence of a g at dilutions ranging from μg/ml to . μg/ml, and the peak enhancing was . -fold at a concentration of . μg/ml (figure a , right ordinate). the rlu assay showed similar pattern of enhancing, and the peak enhancing was . -fold at a concentration of . μg/ml ( figure a , left ordinate), of the similar magnitude with plaque based assay. to get a linear equation between rlu and pfu, the results obtained with a g were plotted on a scatter graph ( figure b ). as expected, the enhancing antibody titer determined by rlu was linear correlated to pfu (r > . ), and the linear equation between rlu and pfu obtained was rlu = . pfu + , similar to the neutralizing equation. together, these results indicated that this novel reporter system using luc-denv is readily for antibody neutralizing and enhancing assay with equivalent reliability to the conventional pfu-based assays. finally, this rlu based assay was validated with clinical samples from immunized monkeys and patients. neutralization assays were performed using -fold serial dilution sera in bhk- cells. for enhancing assay, sera were -fold serial dilution and assay was performed in k cells. sera from rhesus monkeys (# , # ) immunized with a live attenuated denv- showed strong neutralizing activity, and lrnt was calculated to and , respectively ( figure ). negative control (#ns) from healthy monkey showed no neutralizing activity as expected. luc-based enhancement assay showed that both sera from immunized monkeys could significantly enhanced luc-denv replication at dilutions from × - to - (# ), and - to - (# ), respectively. the enhancing activity of # is higher than that of # . no enhancement was observed for #ns as expected ( figure ) . three convalescent sera from df patients (# - , # - , # - ) were validated with the newly developed assay in k cells. as shown in figure , all three samples were able to enhance denv infection at dilutions from × - to - (# - ), - to - (# - ), and - to - (# - ), respectively. negative control (#nc) from healthy adult in varying dilutions showed no impact on rlu as expected. meanwhile, serum # - and # - showed strong neutralizing activities at a dilution of - or even lower, and lrnt was calculated to and -fold dilution separately, whereas no neutralizing activity can be observed in serum # - at detected dilutions. together, these results indicate that the luc-based assay is suitable for detecting both neutralization and ade activity of immune sera from vaccinated or infected individuals. a reliable, rapid, and high-throughput assay for denv neutralization antibodies is critical for laboratory and clinical studies of denv infection and vaccine. considering the limitations of plaque based assay, some novel methods for neutralizing assays have been described [ [ ] [ ] [ ] [ ] [ ] [ ] [ ] . che and coworkers recently developed a novel elispot based neutralization test, demonstrating a well correlation with the conventional prnt assay [ ] . pseudo infectious denv reporter virus particles (rvp) carrying green fluorescent protein (gfp) reporter were also used to measure neutralization antibodies with rapidity, stability and reproducibility [ , , ] . infection with rvp could be monitored by the gfp signals using flow cytometry. however, gfp is not suitable for realtime quantification, and production of rvp requires special cell lines and replicon based plasmids. live reporter virus carrying luciferase reporter replicates almost the same as wild type virus, representing a more advanced tool. many reporter viruses, including sars-related corona virus, human hepatitis c virus, parainfluenza virus, hiv, adenovirus, have been described and well applied for antiviral screening, live imaging, or function studies [ ] [ ] [ ] [ ] [ ] . live reporter denv engineering a reporter gene at the capsid gene has been developed [ ] . recently, we described the stable luciferase denv reporter virus luc-denv and used it for high-throughput screening for antiviral drugs [ ] . in this study, we demonstrated the utility of luc-denv for measuring neutralization and enhancing antibodies. using three identified neutralizing mabs, luc-based assay showed well correlation with the prnt-based assay. g and b are both igg isotype mabs, and a g belongs to igg a isotype. b recognizes the domain iii of denv e protein and inhibit viral binding, while a g and g inhibit fusion. all three mabs were active in inhibiting plaque forming and luc expression in luc-dnev infected vero cells. the value of prnt and lrnt are well correlated (r > . ). the luc-based assay was readily applied in evaluation of clinical samples from vaccinated animals and infected patients. ade infection of denv has been well demonstrated in vitro and in vivo, and represents one of the major impediments against vaccine development. previously, different methods based on infection rate [ , ] , progeny viral yield [ ] , and number of infectious centers [ , ] have been reported to measure the ade activity in fcr expressing cells including k , u or thp- cells. the facs analysis has been commonly used to quantify the infection rate in c / cells, raji b, and human peripheral blood mononuclear cells [ , ] . progeny viral yield can be detected either by conventional plaque assay or ns -based elisa [ ] , elispot [ ] , and realtime rt-pcr [ ] . recently, moi et al. [ ] successfully established stable bhk- cell lines that express fcriia, which facilitate both neutralization and ade assay. the plaque based assay determined the infectious particles released from virus-infected cells, whereas the rlu based assay described in this study offered a simple method which detected viral protein expression in cells. linear correlation was established between the two assays for both neutralization and ade assays ( figure d and figure b ). the newly developed assay method is comparable to the traditional plaque assay, with some unique advantages. first, this luc-based assay is more substantial and time saving. the conventional plaque test used -well plates and - days observation for the plaque forming, the new test is compared performing the same protocol involved -well plates and cost no more than days. second, this new assay method has a more wide-range scope of application with high repetitiveness and reliability. luc-denv replicates well in multiple cells including bhk- , k , vero and thp- and a cells, and luciferase activity can also be detected stably in various cells. neutralization and ade assays can be performed in the same cells [ ] . third, this new assay method is easy to adapt for a highthroughput manner [ ] , which is of critical importance for large-scale clinical samples assays during clinical trials of dengue vaccine. together, we establish a novel reporter system for neutralizing and enhancing antibody assay against denv by using an engineered denv stably expressing renilla luciferase. the newly developed assay described here is rapid, low-cost, and time-saving, providing a useful tool for both basic research and epidemiological investigation. baby hamster kidney cells (bhk- ) and african green monkey kidney (vero) cells were cultured in dulbecco's modified essential medium (dmem; invitrogen) supplemented with % fetal bovine serum (fbs; hyclone) and % penicillin-streptomycin at °c in a % co . human erythroleukemic k cells were maintained in rpmi medium (invitrogen) supplemented with % fbs (gibco) at °c in a % co . the reporter luc-denv has been previously described [ ] and was prepared and tittered in vero cells. the following characterized monoclonal antibodies (mabs) against denv were used in this study: g , b and a g . serum samples were collected from rhesus monkeys (# , # ) immunized with a single dose of a live attenuated denv (unpublished data), and serum from the unimmunized animal was set as negative control (#ns). human convalescent sera from df patients (# - , # - , # - ) and control serum negative for denv (#nc) were from guangzhou no. people's hospital, guangzhou, china. all samples were inactivated at °c for min before assay. prnt were performed as previously described [ ] . briefly, × cells/well of bhk- cells were seeded into -well plates and incubated overnight. μl serially diluted antibody samples were mixed with an equal volume of luc-denv containing pfu. after h incubation, μl of antibody-virus mixture was added to bhk- cell monolayer in -well plates for another h. next, the supernatant was removed, and cells were overlaid with ml of . % (w/v) agarose (promega) in dmem containing % fbs. after further incubation at °c for days, the overlay was removed, and cells were fixed with % formaldehyde for min, and stained with % (w/v) crystal violet. dmem served as negative control, and each sample was assayed in triplicate. plaques were counted and prnt is defined as the antibody dilution resulting in % plaque reduction referred to negative control. luc-based neutralization assay was performed in -well plates, and the procedure was similar to the conventional prnt assay. briefly, virus-antibody mixture was added to bhk- cells in -well plates and adsorbed for h at °c. supernatant was removed and ml dmem- % fbs was replenished onto cells. after h incubation at °c, the supernatant was removed, cells were lysed with μl lysates (promega) per well for minutes. μl lysed suspension was assayed for enzyme activities after adding μl substrate reagent. data was collected using a continuous-read luminometer (glomax microplate luminometer, promega) integrated over seconds with a second delay. medium served as negative control, each sample was assay in triplicate. the antibody dilution resulting in % reduction of rlu value referred to the negative control was defined as lrnt . the protocol for ade assay has been previously described [ ] . briefly, pre-formed antibody-dnev complex were prepared by incubating serially -fold diluted antibody with luc-denv at moi of . in °c before applying to × k cells in -well plates. cells were incubated for additional hours, and the virus titer in the supernatant was titrated by standard plaque assay on bhk- cells. the luc-based ade assay was operated similar with plaque-based enhancement assay as above described in -well plates. serial dilutions of antibodies mixed with luc-denv were incubated for hours on k cells, cell lysates were then subjected to luciferase activities assay as described above. the enhancing activity was evaluated by comparing the rlu value from cells harboring antibody-luc-denv complex and that from cells harboring luc-denv alone. all statistical analyses were performed using spss . . graphs were performed using the prism software (graphpadprism , san diego, ca). the data were presented as means plus standard deviations from there independent experiments. a p value < . was considered statistically significant. epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the st century cross-protective immunity can account for the alternating epidemic pattern of dengue virus serotypes circulating in bangkok neutralization and antibody-dependent enhancement of dengue viruses dengue viruses and mononuclear phagocytes. i. infection enhancement by non-neutralizing antibody dengue virus identification by the plaque reduction neutralization test dengue 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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 additional file : figure s . growth curve of luc-denv on bhk- cells expressed by luciferase activity. cells were infected with virus at moi of . , collected and lysed at the indicated time points to measure the luciferase activities. each data point represents the mean obtained in three separate assays with sd (indicated by bars).additional file : figure s . growth curve of luc-denv on k cells expressed by luciferase activity. cells were infected with virus at moi of . , collected and lysed at the indicated time points to measure the luciferase activities. each data point represents the mean obtained in three separate assays with sds (indicated by bars). the authors declare that they have no competing interests.authors' contributions cfq and kys conceived and designed the experiments. kys, hz, zyj, xfl and yqd performed the experiments. kys and hz analyzed the data. tj, syz, bz, edq, fcz and pys provided reagents and advice. cfq and kys wrote the paper. all authors read and approved the final manuscript. key: cord- -j slaefb authors: silva, josé v.j.; ludwig-begall, louisa f.; oliveira-filho, edmilson f. de; oliveira, renato a.s.; durães-carvalho, ricardo; lopes, thaísa r.r.; silva, daisy e.a.; gil, laura h.v.g. title: a scoping review of chikungunya virus infection: epidemiology, clinical characteristics, viral co-circulation complications, and control date: - - journal: acta tropica doi: . /j.actatropica. . . sha: doc_id: cord_uid: j slaefb abstract chikungunya fever is a mosquito-borne viral illness characterized by a sudden onset of fever associated with joint pains. it was first described in the s during a chikungunya virus (chikv) outbreak in southern tanzania and has since (re-) emerged and spread to several other geographical areas, reaching large populations and causing massive epidemics. in recent years, chikv has gained considerable attention due to its quick spread to the caribbean and then in the americas, with many cases reported between and . chikv has further garnered attention due to the clinical diagnostic difficulties when zika (zikv) and dengue (denv) viruses are simultaneously present. in this review, topical chikv-related issues, such as epidemiology and transmission, are examined. the different manifestations of infection (acute, chronic and atypical) are described and a particular focus is placed upon the diagnostic handling in the case of zikv and denv co-circulating. natural and synthetic compounds under evaluation for treatment of chikungunya disease, including drugs already licensed for other purposes, are also discussed. finally, previous and current vaccine strategies, as well as the control of the chikv transmission through an integrated vector management, are reviewed in some detail. chikungunya virus (chikv) is the etiological agent of chikungunya fever (chikf), an arthropod-borne disease transmitted mainly by aedes genus species (weaver, ) . first described in in present-day tanzania, the early chikf cases were treated as dengue virus (denv) infection (lumsden, ) . following isolation of chikv from infected patients' sera, as well as ae. (stegomyia) aegypti (linnaeus, ) and culex spp. mosquitoes in , the virus was placed in the arbovirus group a (ross, ; calisher and karabatsos, ) . currently, chikv is grouped within the alphavirus genus, togaviridae family, and identified as member of the semliki forest virus (sfv) antigenic complex (van duijl-richter et al., ; ictv, ) . shortly after identification in east africa ( ), chikv was described in both central and southern regions of africa (uganda and sub- both urban and sylvatic chikv transmission cycles have been described (caglioti et al., ) . the sylvatic cycle (especially in africa) may involve the participation of some aedes species, such as aedes furcifer (edwards, ) , aedes taylori (edwards, ) , aedes luteocephalus (newstead et al., ) , aedes vittatus (bigot, ) and aedes fulgens (edwards, ) , and different non-human primate (nhp) species, possible reservoirs or amplifiers hosts for chikv [e.g. african green monkeys (chlorocebus sabaeus) (linnaeus, ), patas monkeys (erythrocebus patas) (schreber, ), guinea baboons (papio papio) (desmarest, ), guenons (cercopithecus aethiops) (linnaeus, ), bushbabies (galago senegalensis) (geoffroy, ), mandrills (mandrillus sphinx) (linnaeus, ), red-tail monkeys (cercopithecus ascanius schmidti) (matschie, ) and chacma baboons (papio ursinus) (kerr, )] (mcintosh, ; mccrae et al., ; diallo et al., ; chevillon et al., ; pruetz et al., caglioti et al., kading et al., ; althouse et al., ) . a vector role has also been suggested for culex and anopheles mosquitoes, which have been found infected in senegal from to (diallo et al., ) . in the urban cycle, ae. aegypti and ae. albopictus are known as the main vectors (weaver, ) . currently, they probably remain as the main vectors for chikv transmission in the americas, africa, europe, asia and oceania (horwood et al., ; vega-rúa et al., zeller et al., ngoagouni et al., . significantly, ae. aegypti can also be the main vector of other viruses, such as zikv and denv, and co-transmission events may be observed (carrillo-hernández et al., ) . however, it has been demonstrated that simultaneous infections by denv/chikv/zikv or denv/chikv in ae. aegypti does not compromise the vector competence (le coupanec et al., ; rückert et al., ) . it is known that horizontal transmission of chikv in aedes mosquitoes can occur and act positively in the maintenance of infection cycles ( fig. a ) (mavale et al., ) . vertical transmission of chikv into aedes has also been observed under natural and experimental conditions and has been pinpointed as a possible reason for viral persistence under harsh environmental conditions (fig. b) (agarwal et al., ; chompoosri et al., ; jain et al., ) . once inside the arthropod vector, chikv must replicate and reach the mosquitoes' salivary glands within roughly seven to ten days for transmission to a susceptible human (lim et al., ) (fig. c) . in human hosts, the intrinsic incubation time can vary from one to twelve days and infected individuals may present viremia of up to ten days (kam et al., ; simon et al., ; azevedo et al., ) . maternal-fetal transmission has also been reported in humans (fig. e ). neonatal encephalitis, as consequence of vertical transmission, was observed, for instance, during the brazilian epidemic in (bandeira et al., a; lyra et al., ) . however, no breast milk transmission has been evidenced (patterson et al., ) . despite the fact that chikv rna has been detected in semen even after days post symptom onset, indicating possible transmission via sexual intercourse, horizontal transmission between humans has not yet been reported ( fig. d ) (bandeira et al., b; patterson et al., ) . approximately - % of individuals infected with chikv develop clinical disease with fever and arthralgia (staples et al., ; ayu et al., ; nakkhara et al., ) . chikv infection has been associated with sudden onset of febrile illness (> . °c) ( % of patients), arthralgia ( % of patients), back pain ( % of patients), headache ( % of patients) and fatigue (who, ; thiberville et al., ) . the most common symptom in chikf is polyarthralgia, typically of bilateral polyarticular nature, affecting mainly peripheral joints (ankles, wrists and phalanges) and some large joints (knees and elbows) (who, ; morrison, ) . cutaneous manifestations are reported in circa % of acute cases. the lesions are characterized by macular or transitory maculopapular eruption, which can be edematous or itchy, often occurring in the body extremities, palms, soles of the feet, torso and face (simon et al., ; thiberville et al., ) . gastrointestinal symptoms, such as diarrhea, vomiting, nausea and abdominal pain, occur in - % of cases during the acute phase (thiberville et al., ) . other possible symptoms include erythema, asthenia, conjunctival effusion, persistent conjunctivitis and cervical lymphadenopathy (staples et al., ; staples and fischer, ; madariaga et al., ) . different studies have demonstrated that chikv infection can reach high viral loads, ranging from to copies of viral rna/ml, which seem to be correlated with the presence and severity of clinical signs and symptoms (chow et al., ; appassakij et al., ) . polyarthralgia and/or polyarthritis are hallmark symptoms of chronic chikungunya, mostly affecting small joints, such as phalanges and wrists, as well as large joints (e.g., ankles, knees and shoulders). the condition is usually severe and leads to mobility limitations of afflicted patients (hoarau et al., ) . polyarthralgia has been described to persist for varying periods of time, lasting from weeks to several months and, in some cases, up to five years, depending on the populations evaluated (borgherini et al., ; sissoko et al., ; manimunda et al., ; simon et al., ) . the persistence of polyarthralgia in some alphaviruses, such as sfv and sinv, seems to be associated with persistence of viral antigens and immune responses (inflammation) in the joints (atkinson et al., ; perri et al., ; hoarau et al., ; labadie et al., ; simon et al., ; poo et al., ; silva and dermody, ) . about the viral antigens, there is still no consensus whether they have replication competence (perhaps with mutations to promote their persistence) or are only the result of a delayed clearance of non-replicating viral antigen (atkinson et al., ; perri et al., ; poo et al., ; weaver and lecuit, ) . precisely on chikv, a study described the presence of macrophages with chikv genetic material and viral proteins in the synovial tissue of an -month-long chronically infected patient (hoarau et al., ) . experimental studies have shown chikv persistence in lymphoid organs, liver, joints, muscles and macrophages from nhps (labadie et al., ) . in addition, the presence of infiltrating cells, mainly macrophages, monocytes and lymphocytes, and specific proinflammatory mediators, such as il- , il- , and mcp- , within the synovial fluid probably also contribute to the chronicity of the inflammation in chikungunya disease (silva and dermody, ) . moreover, severe cases of chikungunya may be related to age and diverse underlying medical conditions, such as hypertension, respiratory conditions and diabetes mellitus (borgherini et al., (borgherini et al., , sankari et al., ; economopoulou et al., ; sissoko et al., ; tandale et al., ) . the pathogenesis of rheumatoid arthritis in chikf is still under debate. while certain studies have suggested that viral infection may trigger initiation of this chronic inflammatory disorder, other studies did not find inflammatory markers in infected individuals with chronic symptoms (staples et al., ; schilte et al., ) . chikv infections can also lead to atypical clinical manifestations. guillain-barré syndrome (gbs), for instance, has been associated with chikv infection (lebrun et al., ; oehler et al., ) . gbs comprises an acute inflammatory demyelinating polyneuropathy of global incidence, in which about two-thirds of cases occur after bacterial (e.g. campylobacter jejuni) (heikema et al., ) or viral infection (oehler et al., ) , such as by dengue- (simon et al., ) , west nile- (leis and stokic, ) , influenza- (choi and yeon, ) , cytomegalo- (steger et al., ) , human immunodeficiency- (girgin et al., ) , epstein-barr (phillips, ; kim et al., ) and zika viruses (rozé et al., ) . during the more recent chikv outbreaks, total or partial alopecia on the head or body, predominately in female patients, and ophthalmological alterations, such as uveitis and retinitis, were described during the chronic phase of infection (martínez-pulgarín et al., ; cunha and trinta, ) . in newborns, congenital infections may be accompanied by varying clinical signs, such as fever, lack of appetite, apnea, skin manifestations, distal and cerebral edema, encephalitis and hemorrhage (gopakumar and ramachandran, ; bandeira et al., a; lyra et al., ) . heart-and gastrointestinal disorders and cutaneous lesions are reported to manifest up to two days after the onset of fever in chikv-infected newborns and children (ernould et al., ) . bullous lesions associated to chikv infection have also been reported in four-month-old babies, who had % of their body surface affected on the second day after the onset of fever (robin et al., ) . deaths from chikv infection were previously considered a rare event. this perception, however, has changed since the latest epidemics, which presented a considerably increased mortality rate, probably due to neurological affections, mainly in neonates, immunocompromised and elderly (rampal et al., ; kee et al., chusri et al., bandeira et al., a) . it is challenging to differentiate clinical signs and symptoms of chikv infection from other pathologies, especially when zikv and denv are co-circulating in the same geographical region (hua and combe, ) . individuals infected by these arboviruses can present a wide range of similar clinical manifestations, such as rash, myalgia, exanthema, arthralgia, joint pain, headache, lymph node hypertrophy, neurological impairment and fever (brito and cordeiro, ) . in addition, it is difficult to determine the frequency and intensity of the symptoms and correctly assess pain (mild, moderate and intense) of afflicted patients (table ). in this context, variations in the clinical presentation of cases can give hints as to the viral etiology; for instance, the salient and prolonged polyarthralgia, often accompanied by rash, is typically more indicative of chikungunya, while hemorrhagic manifestations and myalgia are more commonly observed in denv infections (lee et al., ) despite the patients co-infected with chikv/denv, chikv/zikv and chikv/denv/zikv often do not show exacerbation of clinical signs, the co-infection presents as additional obstacle during differential diagnosis (furuya-kanamori et al., ; villamil-gómez et al., ; carrillo-hernández et al., ) . tables and summarize clinical and laboratory features that should be evaluated for efficient differential diagnosis of chikv, denv and zikv infections. since the variety and intensity of symptoms associated to chikv, denv and zikv infections are so similar and make clinical diagnosis difficult in areas of co-circulation, laboratory analysis is necessary to confirm the respective viral etiology. hematology findings associated to chikv infection are commonly either unspecific, however, lymphopenia and hypocalcemia were the most frequent observation, and severe thrombocytopenia is rare (borgherini et al., ; brito and cordeiro, ; paho, ) . moreover, the c-reactive protein is generally elevated in the acute phase illness (table ) (venugopalan et al., ; brito and cordeiro, ; paho, ) . elevation of hepatic enzymes, as well as elevated creatinine and creatine phosphokinase levels, have also been reported (danis-lozano et al., ) . the different laboratory patterns observed during chikv, denv and zikv infections, added to clinical findings, may be incorporated to support a correct diagnosis (tables and ) . laboratory tests for specific diagnosis of chikv infection are based on virus isolation, viral rna detection and serology (johnson et al., ) . despite not usually employed in routine diagnosis, viral isolation can be performed from sera collected up to seven days after onset of illness and inoculated into mosquito-or mammalian cell lines, in which cytopathic effects can appear between one to three days after inoculation. confirmation of results is possible via immunofluorescence or rt-pcr assays (panning et al., ; staples et al., dash et al., . recently, an immunochromatographic assay used anti-chikv e monoclonal antibodies to detect different chikv genotypes in samples from acutely infected patients. this highly specific and sensitive assay may also be an alternative method for chikv infection diagnosis (okabayashi et al., ) . molecular methods of chikv diagnosis, such as rt-pcr, rt-lamp, qrt-pcr, have gained increasing importance. they are more sensitive and faster than viral isolation, and permit rna detection from all chikv lineages with high specificity. usually, serum samples collected up to seven days of symptom-onset are suitable for chikv detection by molecular diagnostic platforms (edwards et al., ; litzba et al., ; sharma et al., ) . in addition, novel multiplex assays are capable of differentiating chikv from other infectious agents with a similar clinical spectrum. among them, rt-lamp assay has been shown to be capable of differentiating between zikv, chikv and denv infections (yaren et al., ) . a rt-qpcr capable of successfully differentiating between zikv-chikv-denv and chikv-denv-leptospira infections was also recently described (pabbaraju et al., ; giry et al., ) . in later phases of infection, chikv detection is usually based on serological methods, such as elisa and plaque reduction neutralization testing (prnt). elisa techniques are useful to distinguish between acute or convalescent infections via detection of anti-chikv igm or igg antibodies. igm can be detected from two/four days up to three months after the onset of illness, while igg can be detected for several years (grivard et al., ; pialoux et al., ; reddy et al., ) . moreover, elisa for chikv diagnosis are highly specific and have a high accuracy (johnson et al., ) . igm antibody-capture elisa (mac-elisa), via which igm antibodies can be detected in serum samples collected from four days after onset of symptoms, are the most table signals and symptoms that may contribute in the differential diagnosis between dengue, chikungunya and zika illnesses. commonly used tests for laboratory-based diagnoses (reddy et al., ) . prnt, used as a parameter to measure circulating neutralizing antibodies, is useful to establish immunoprotection levels based on the determination of serum antibody titers required to neutralize a known amount of infectious virus particles (azami et al., ) . alternative techniques for anti-chikv antibody detection include immunofluorescence and hemagglutination inhibition (staples et al., ). in general, any suitable etiological diagnosis should be reached through combined epidemiological, clinical and laboratory approaches performed by experienced health professionals. an algorithm-guided infographic with specific laboratory key findings to be used for differential diagnosis of chikv, denv and/or zikv infections is summarized in fig. . chikungunya dengue zika fever > °c ( - d a ) > °c ( - d) ≤ °c ( - d) rash ++ (d -d b ) + (d c ) +++ (d -d there is no licensed specific antiviral available for the control of chikv replication, thus therapeutic strategies must be supportive and symptomatic, including fluid intake (jain et al., ; who, ; kaur and chu, ) . in this context, non-steroidal anti-inflammatory drugs (nsaids), such as paracetamol, are indicated to reduce the fever and relieve arthralgic pain (who, ) . however, nsaids that interfere (at secondary level) with platelet aggregation or with other mechanisms of blood clotting (e.g. aspirin) should be avoided (goupil and mores, ) . the co-administration of nsaids with low-dose systemic corticosteroids has been recommended to reduce pain and improve quality of life in treating acute chikungunya cases with arthralgia (padmakumar et al., ) . past concerns that corticosteroid treatment may exacerbate alphaviral arthritides appear unjustified, since serodiagnosis has demonstrated antiviral immunity (mylonas et al., ) . a succinct overview of current strategies for inhibition of chikv infection has recently been published (subudhi et al., ) . briefly, among the anti-chikv drugs under evaluation, there are preparations that target the viral adsorption and fusion, translation of viral protein and genome replication (mainly in viral non-structural protein , nsp ), maturation of viral glycoproteins and immunological molecules (brighton, ; briolant et al., once the molecular test is negative, serological test must be performed. *perform serological tests for samples collected ≥ days after the onset of clinical signs and symptoms; **prnt is required due to the cross-reaction between denv and zikv; ***test also for other flavivirus (e.g. west nile and saint louis encephalitis viruses). reference: cdc ( ) and paho ( ). drugs and compounds under evaluation for treatment of chikv infection. licensed for malaria treatment antiviral activity has been already demonstrated against hiv a , sars-cov b and sfv. a pilot study shown that chloroquine could be employed in the treatment of arthralgia in chronic chikungunya. however, its use in the acute phase is still debated and some studies have also shown an increase of viral replication of sfv and ecmv after treatment with chloroquine. (brighton, ; de lamballerie et al., ; maheshwari, srikanta, bhartiyaet, ; khan et al., ) arbidol inhibition of the fusion between virus particle and plasma membrane, and between virus particle and the membrane of endosome ( silymarin reduction of both chikv replication efficiency and down-regulating of viral proteins involved in replication. silymarin interferes with post-entry stages of chikv infection, reducing, in a dose dependent manner, the nsp , nsp and e proteins production. (lani et al., ) ribavirin probable inhibition of the viral mrna polymerase by binding to the nucleotide binding site of the enzyme. antiviral licensed for treatment of rsv f and hcv g . patients in the drug group reported improvement in the joint pains and the soft tissue swelling also reduced. together with ifn-alpha b was able to inhibit chikv and sfv replication in vero cells (ravichandran, manian; palumbo, ; turner et al., ; briolant et al., ) decanoyl-rvkr-chloromethyl ketone (dec-rvkr-cmk) inhibition of the maturation of e glycoprotein by inhibition of furin. wichit et al., ) . examples of drugs and compounds under evaluation are available on table . in the absence of therapeutic strategies and licensed vaccines, efficient vector control plays a crucial role in chikv prevention (huang et al., ) . unfortunately, uncontrolled urbanization, lack of proper basic sanitation and increasing resistance to various classes of insecticides challenge the true impact of vector control measures for the reduction of arbovirus incidence (resnik, ; liang et al., who, . to overcome these obstacles, integrated anti-virus control is required and should include: a) epidemiological surveillance; b) environmental management focusing on educative actions to eliminate potential mosquito breeding sites and reduce standing water sites; c) chemical control using repellents (mainly for travelers and pregnant women) and insecticides, respecting the vectors resistance; and d) biological control against eggs, larvae and mosquitoes (fig. ) (hemingway and ranson, ; dumont and chiroleu, ; benelli, ; islam et al., ; benelli, ) . in this last context, larvivorous fish belonging to the genus gambusia (e.g. gambusia affinis) (baird and girard, ) and poecilia (e.g. poecilia reticulata) (peter, ) have been suggested in several countries and regions for mosquito control, mainly for ae. aegypti (hoy, ; das and prasad, ; cavalcanti et al., ; walton, ; chandra et al., ; seng et al., ; dumont and chiroleu, ; kweka et al., ; kamareddine, ; shulse et al., ; pereira and oliveira, ; chobu et al., ; . more natural mosquito predators, including copepods [e.g. mesocyclops thermocyclopoides (harada, ) and mesocyclops longisetus (thiébaud, ) ] and other invertebrate aquatic organism, have also been implemented successfully to reduce ae. aegypti and other culicidae populations (rawlins et al., ; manrique-saide et al., ; vu et al., ; schaper, ; mahesh kumar et al., ; benelli, ; pavela, ) . plant-borne molecules have shown activity against aedes, anopheles and culex larval instars . more recent approaches, based on "green"-synthesized nanoparticles, have been suggested for use against denv and ae. aegypti vector (madhiyazhagan et al., ; sujitha et al., ; . microbiological interventions, such as the use of bacillus thuringiensis var. israelensis (bti) (berliner, ) and entomopathogenic fungi [e.g. metarhizium anisopliae (metschnikoff, ) sorokin, and beauveria bassiana (balsamo) vuillemin, )], have shown effects on malaria mosquitoes, as well as on ae. aegypti and ae. albopictus (novak et al., ; blanford et al., ; armengol et al., ; knols et al., ; lam et al., ; ritchie et al., ; paula et al., a, b) . recently, bti was used to prevent zikv transmission in the continental united states (stoddard, ) . in another promising approach to arbovirus control, including also zikv, the release of wolbachia-infected male mosquitoes, sterile mosquitoes or insects carrying a dominant lethal gene can be used to reduce ae. aegypti and ae. albopictus populations (fig. ) (ferreira et al., ; alphey et al., ; walker et al., ; boyer, ; alphey et al., ; massonnet-bruneel et al., ; lee et al., ; zhang et al., a, b; dickens et al., ) . despite the theoretical basis for their effectiveness, additional studies are needed to verify the true risks and benefits of programs based on altered mosquitoes. this concern appears to be greater in relation to transgenic mosquitoes, especially on their efficacy, sustainability and impact on the environment and target and non-target species (wilke et al., ) . although many of these strategies have been initially directed against denv, their application to transmission control of chikv should be considered, mainly within an integrated approach, which rationally combines different measures. control programs for other insect-borne diseases, such as malaria (benelli and beier, ) , can also serve as model for chikv prevention. in a synergistic way, mass immunization against chikv would be an important tool for viral control and prophylaxis. several distinct vaccine approaches are currently under development, however, no vaccine has been licensed. anti-chikv candidates that have been already tested in humans and/or animals include inactivated-, attenuated-, virus like particle-(vlp), dna-and chimeric vaccines (eckels et al., ; levitt et al., ; muthumani et al., ; wang et al., ; tiwari et al., ; sharma et al., akahata et al., plante et al., ; wang et al., ; gorchakov et al., ; brandler et al., ; chang et al., ; garcía-arriaza et al., ; tretyakova et al., ; van den doel et al., ; erasmus et al., ) . in the past, viral inactivation strategies were the first to be tested in the course of anti-chikv vaccine development. chikv replicated in cell culture and subsequently inactivated by formalin, ether or , iodonapthyl azide (ina) was able to stimulate the production of neutralizing antibodies (eckels et al., ; tiwari et al., ; sharma et al., ) . particularly, the use of ina resulted in reduced binding capacity of anti-e neutralizing antibodies (sharma et al., ) , while formalin inactivation stimulated the cellular immune response with the production of anti-and pro-inflammatory cytokines (tiwari et al., ) . with the same aim of outlining the virulence of chikv infection, brandler et al. ( ) reported a recombinant live-attenuated measles vaccine expressing a vlp composed of the capsid (c) and envelope (e) proteins from the la réunion - chikv strain (ecsa lineage). in mice susceptible to measles virus (mv), immunization with mv-chikv induced high titers of chikv antibodies, specific cellular immune responses and protected all animals from lethal chikv challenge (brandler et al., ) . in phase i clinical trial (european clinical trials database, - -fig. . management for an integrated aedes vector control. *the use of insecticides and repellents should be carried out taking into account the mosquitoes' resistance profiles; ** it is important to consider and evaluate the influence of these interventions on ecosystem balance. ), a second vaccination resulted in % seroconversion for all participants. the immunogenicity of the mv-chikv was not affected by preexisting anti-mv immunity and no vaccination-related serious adverse effects were recorded (ramsauer et al., ) . also using the vaccine platform vlp-based, akahata et al. ( ) proposed the use of a vaccinal vlp expressing chikv structural proteins. the vlp-based vaccine, vrc-chkvlp - -vp, was obtained by transfection of a plasmid expressing c and e proteins of the chikv strain (wa lineage). the vaccine stimulated the production of neutralizing antibodies against the e protein of different chikv strains and was able to protect challenged monkeys. further tests in humans showed vaccine efficacy and immunogenicity, without reports of arthralgia as side effect (chang et al., ) . the vrc-chkvlp - -vp is one of the most developed strategies and is currently undergoing phase ii clinical trials (national clinical trials, ) . another approach to chikv vaccine development is the use of dna vaccines. in this context, a plasmid expressing consensus sequences of c, e and e chikv proteins elicited a robust cellular immune response and the production of high antibody titers capable of recognizing wild virus antigens (muthumani et al., ) . in another study, mice immunized with a dna vaccine containing the complete chikv genome of the / strain developed neutralizing antibodies and were protected from neurovirulent chikv (tretyakova et al., ) . these strategies involving inactivated viruses, vlp and dna vaccines often stimulate the humoral immune response, one of the main mechanisms for control and prevention of chikv infection (lum et al., ) . empirically-or reverse-engineered attenuated live vaccines, however, have been shown to be capable of inducing both cellular and humoral immune responses and have also been suggested to prevent chikv infection (levitt et al., ; wang et al., wang et al., , plante et al., ; gorchakov et al., ; garcía-arriaza et al., ) . an important example of attenuated chikv vaccine is the tsi-gsd- , a vaccine based on an empirically attenuated chikv strain (asian lineage) isolated from patient sera from the chikv outbreak in thailand (levitt et al., ) . by the end of its phase ii clinical trial evaluation, % of the vaccinated individuals presented neutralizing antibodies, % remained seroconverted after one year and . % presented temporary arthralgia (edelman et al., ) . in addition to classical attenuation via serial viral passage in cells, reverse genetics strategies have been employed as platforms for construction of recombinant attenuated viruses or vaccine chimeras (wang et al., ; plante et al., ; wang et al., ; garcía-arriaza et al., ; van den doel et al., ; erasmus et al., ). an anti-chikv strategy involves the development of an attenuated chimeric vaccine using the internal ribosome entry site (ires) of the encephalomyocarditis virus (ecmv). in this approach, the chikv subgenomic promoter from the lr -opy strain (ecsa lineage) is knocked via insertion of synonymous mutations and the ecmv ires sequence is added as new promoter for subgenomic rna transcription (plante et al., ) . briefly, the addition of the ires sequence to the attenuated viral genome prevents viral propagation in mosquito cells, thus restricting the target population. this vaccine strategy has been shown to lead to the stimulation of tcd and tcd responses in mice (plante et al., ) . in nhp, chikv/ires vaccine was also showed to be safe and immunogenically effective (roy et al., ) . finally, the preclinical safety of the chikv/ires vaccine was ensured in interferonα/β receptor-incompetent mice (a mice) (plante et al., ) . other viral chimeras have been proposed in the context of anti-chikv vaccine strategies. notably, attenuated strains of the eastern equine encephalitis virus (veev) or eastern equine encephalitis virus (eeev) were used as backbones to express chikv structural proteins, creating immunogenic chimeras able to stimulate production of neutralizing antibodies and protect mice against disease and viremia after chikv challenge (wang et al., ) . another chimeric construction, the modified vaccinia ankara expressing e glycoprotein or e -e - h-e proteins was shown to stimulate production of neutralizing antibodies in ifn-i (ifn α/β) and ii (ifn-γ) receptor knockout mice (ag mice), protecting against lethal infection (van den doel et al., ) . a replication-incompetent adenovirus vector also has been used to express the orf coding chikv structural polyproteins. a single dose of the chimera induced high antibodies titers capable of neutralizing asian and iol chikv lineages, protecting mice against viremia and arthralgia . most recently, a promising vaccine based on a chimera of eilat virus (eilv) and chikv has been reported (erasmus et al., ) . the eliv/ chikv possesses non-structural proteins of eilv and structural and accessory proteins of chikv strain (asian lineage). since the eilv is an alphavirus specific for insects, the eilv/chikv was unable to replicate in vertebrate hosts, thereby providing a high degree of safety. in nhp, eliv/chikv stimulated immune response and guaranteed protection against viremia (erasmus et al., ) . in the last decade, there has been an increase in the dissemination and co-circulation of several arboviruses. currently, areas that were endemic just to denv, for instance, are with autochthonous cases of chikungunya and zika diseases. these arboviruses have similar clinical spectrum and require an efficient laboratory diagnosis, especially for a rigorous epidemiological surveillance. in addition, chikv infections represent a serious public health problem. the high morbidity of chikungunya often results in absenteeism of afflicted individuals, incurring both psychosocial and economic impacts. in this context, the development of a specific anti-chikv drug is certainly an important demand. on the other hand, the ideal control strategy for chikv should combine an integrated vector management with mass immunization. although the different vector biocontrol strategies are promising, mainly within an integrated use, it is necessary to think about their sustainable use, assessing their real impacts on ecosystem equilibrium. the absence of chikv serotypes is considered a facilitative aspect in anti-chikv vaccine development, since a formulation developed from one strain will likely result in immunity against all chikv (smalley et al., ) . despite recent advances in vaccine strategies, the major challenge regarding chikv vaccine development remains the establishment of an equilibrium between immunogenicity and safety, notably a reduction of side effects, such as secondary arthralgia following immunization with attenuated virus. finally, recognizing the competence of vector and arboviruses control measures, we believe that the prevention of chikv infections should be planned within a global and multifactorial approach. this interdisciplinary strategy, currently framed within the one health concept, should thus integrate all aspects of health care for humans, animals and the environment (benelli and duggan, ) . the authors have no conflicts of interest to disclose. acknowledgments e.f. oliveira-filho and r. durães-carvalho are supported by fundação de amparo à ciência e tecnologia do estado de pernambuco (facepe) and mct/cnpq dcr grants. j.v.j. silva júnior is supported by coordenação de aperfeiçoamento de pessoal de nível superior (capes). t.r.r. lopes is supported by conselho nacional de desenvolvimento científico e tecnológico (cnpq). evidence of experimental vertical transmission of emerging novel ecsa genotype of chikungunya virus in aedes aegypti a virus-like particle vaccine for epidemic chikungunya virus protects nonhuman primates against infection sterile-insect methods for control of mosquito-borne diseases: an analysis genetic control of aedes mosquitoes role of monkeys in the sylvatic cycle of chikungunya virus in senegal viremic profiles in asymptomatic and symptomatic chikungunya fever: a blood transfusion threat long-lasting effects of a bacillus thuringiensis serovar israelensis experimental tablet formulation for aedes aegypti (diptera: culicidae) control persistence of 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a chikungunya virus receptor protein assessment of flavaglines as potential chikungunya virus entry inhibitors point of sampling detection of zika virus within a multiplexed kit capable of detecting dengue and chikungunya chikungunya: its history in africa and asia and its spread to new regions in - combining the sterile insect technique with wolbachia-based approaches: ii-a safer approach to aedes albopictus population suppression programmes, designed to minimize the consequences of inadvertent female release combining the sterile insect technique with the incompatible insect technique: i-impact of wolbachia infection on the fitness of triple-and double-infected strains of aedes albopictus key: cord- -peqz okh authors: girard, marc; nelson, christopher b.; picot, valentina; gubler, duane j. title: arboviruses: a global public health threat date: - - journal: vaccine doi: . /j.vaccine. . . sha: doc_id: cord_uid: peqz okh a conference on «arboviruses, a global public health threat» was organized on june – , at the merieux foundation conference center in veyrier du lac, france, to review and raise awareness to the global public health threat of epidemic arboviruses, and to advance the discussion on the control and prevention of arboviral diseases. the presentations by scientists and public health officials from asia, the americas, europe and africa strengthened the notion that arboviral diseases of both humans and domestic animals are progressively becoming dominant public health problems in the world. the repeated occurrence of recent deadly epidemics strongly reinforces the call for action against these viral diseases, and the need for developing effective vaccines, drugs, vector control tools and strong prevention programs. declaring a dengue pandemic in the s was a sentinel call to action in the fight against a range of emerging arboviral diseases of humans [ , ] . the past years have seen a dramatic émergence/re-emergence of epidemic arboviral diseases [ , ] . the recent outbreak of neurological disorders and neonatal malformations associated with zika virus (zikv) infection in latin america { }, the yellow fever (yfv) epidemics in angola and brazil with importation to china [ ] , the ever-expanding west nile virus (wnv) epidemic in the americas [ ] , the recent emergence in east africa and global spread of chikungunya virus (chikv) [ ] , as well as the ongoing and expanding dengue virus (denv) pandemic in the tropics and subtropics [ ] have reinforced the call for action in the fight against emerging and re-emerging arboviral diseases. these epidemics underscore the urgency and need for integrated control and prevention of arboviral diseases, especially those transmitted by aedes mosquitoes in urban areas [ , ] . prevention and control strategies currently focused on vector control, including insecticide treatment, environmental management and social mobilization have not been effective in practice. it is widely recognized that no strategy alone can fully address the problem. however, some intervention tools have helped reduce the disease burden. for example, timely access to clinical services and appropriate care can reduce mortality dramatically [ ] , indoor residual spraying (irs) and indoor space spraying (iss) may be effective in reducing mosquito populations and exposure to arboviruses [ ] . in addition, personal protection, clinical diagnosis and management, laboratory-based surveillance and vaccination, can be effective [ ] . vaccines are available to protect against japanese encephalitis and yellow fever [ ] , and the first dengue vaccine, even though limited in its applications, was licensed in [ ] . dr duane gubler (duke-nus medical school, singapore) reminded the audience that the frequency and magnitude of the arboviral epidemics and the extent of their geographic spread have progressively increased over time, accelerating in the past years and now occurring globally in the tropics [ , ] . https://doi.org/ . /j.vaccine. . . - x/ as an illustration, denvs were found in the s in less than endemic countries and only a few thousand cases were reported each year. in contrast, in the virus had become endemic in countries, causing an estimated million yearly infections and million symptomatic cases [ ] . in the s, denv serotypes and could be found only in south-east asia. but in the early s, all four serotypes of denv had dramatically spread to to all regions of the tropics [ ] . similarly, a new strain of chikv emerged in east africa in , spreading to asia and then to the rest of the tropical world in years [ ] . and epidemic zikv emerged in the pacific and spread around the world in only years [ ] . all of these viruses are transmitted by the urban mosquito, aedes aegypti. west nile virus (wnv), transmitted primarily by bird feeding culex mosquitoes, was introduced to the western hemisphere for the first time in , rapidly spreading from the east coast of the usa to the rest of the country and to canada before invading the caribbean, central and south america [ ] . in , , cases of wnv encephalitis in horses and , cases in humans were reported in the usa. wnv is now enzootic in the region. dr joao bosco siqueiras (institute of tropical pathology and public health, goias, brazil) described another dramatic example, that of yellow fever, which is also transmitted by aedes aegypti. the mosquito is widely prevalent in the tropics including tropical america and most countries in subsaharan africa. in - yellow fever emerged and expanded into the south and southeastern parts of brazil, where yellow fever vaccination was not common. then, in - , it emerged in central brazil, infecting many travelers. cases of yellow fever were exported from brazil to europe, peru and the usa. the virus continued to spread in - into the areas of bahia, rio and sao paolo and was detected in municipalities, causing small urban epidemics [ ] . the death toll increased to persons in and in the first half of . in africa, yellow fever spread from angola to the democratic republic of congo in - , and emerged in nigeria and uganda in [ ] . more dramatically, cases were imported from angola to china, which is the first time in history that confirmed yellow fever was introduced to asia [ ]! as outlined by dr duane gubler, the new and worrisome aspect of emerging arbovirus epidemics is that they can occur in urban centers, as was observed with dengue fever, zika, chikungunya and yellow fever. the urban vectors are aedes mosquitoes, primarily aedes aegypti, which has spread around the world in past centuries, and secondarily aedes albopictus, whose spread began in the s [ ] . the emergence and spread of dengue, chikungunya and zika infections actually reflect the growing geographical spread of aedes spp across all continents. the fact that arboviral diseases have become a major threat to urban populations is the result of: ) population growth and urbanization, which results in crowding of humans, inadequate housing, waste management and accumulation of trash, including used automobile tires, plastics, tins, etc, creating ideal ecological conditions for urban aedes populations to thrive; ) the spread of aedes spp mosquitoes around the world and throughout the tropics and subtropics; ) the lack of effective vector control and infectious disease prevention; and ) the globalization of air transport, which facilitates the rapid spread of pathogens and the diseases they cause (more than billion passengers have travelled on air lines in the year ) [ , , , ] . as these trends will continue, epidemic arboviral diseases will increasingly threaten global, national and local political and economic security and could create a global public health emergency, similar to or even greater than the current sar-cov- pandemic. one should not forget that over . billion people live in areas exposed to urban aedes mosquitoes. dr duane gubler also noted that since arboviral diseases represent a major threat to urban populations, it is necessary to reassess surveillance strategies. currently, surveillance systems are based on passive reporting of loosely defined clinical syndromes with infrequent laboratory confirmation. he suggested that all at-risk countries should have an enhanced passive disease surveillance system based on well-defined case definitions supported by serological and virological laboratory testing. three major arbovirus pathologies should be monitored: systemic febrile syndrome, haemorrhagic disease syndrome, and meningo-encephalitis syndrome. an active laboratory-based syndromic surveillance network is also badly needed. weekly reports by local physicians, as done in brazil, where they have turned out to be most useful, should also be encouraged. however, as discussed by dr elizabeth hunsperger (cdc, atlanta, usa), differential diagnosis in the clinical setting may be difficult, especially outside of seasonal disease, as mild and asymptomatic infections are common. as an example, laboratory testing for dengue is important in order to distinguish the disease from other febrile illnesses such as malaria, leptospirosis or influenza, other arbovirus diseases such as chikungunya, zika, west nile, japanese encephalitis or yellow fever, and even from typhoid/paratyphoid (salmonella typhi or salmonella paratyphi spp bacteria). recently, who has recommended the serotesting of individuals in dengue vaccination settings to screen out those with no evidence of past infection. each of theses objectives requires different test characteristics. standard dengue diagnostics assays include rt-pcr, virus neutralization assays, immunoassays to detect denv ns antigen, and assays to detect anti-denv igms [ref] . the most sensitive and specific assays are rt-pcr and the ns elisa assay. low-cost point-ofcare (poc) rapid diagnostic tests (rdts) are currently not adequate for the clinical and vaccination settings, while elisa, rt-pcr and other strategies with much higher levels of performance are are too slow and costly. the latter tests are needed in public health surveillance and research settings. these diagnostic approaches are instrument-dependent and require appropriate facilities and highly trained technical staff to perform complex diagnostic tests, unfortunately none of which are available in resource-limited areas. dengue diagnostics will greatly benefit from a dengue poc test that meets the who assured criteria (affordable, sensitive, specific, rapid, user-friendly and delivered to those in need) [ ] . note that laboratory diagnosis of dengue can be achieved with a single serum specimen obtained during the febrile phase of the illness by testing for denv nucleic acid, non-structural protein (ns ) and anti-denv igms. the current poc rdts detect both anti-denv igms and iggs as well as the ns antigen. this has the potential to change the current situation in resource-limited areas and improve dengue clinical management. denv viremia occurs for up to days following the onset of fever, and anti-denv igms begin to appear around days after fever onset. detection of denv nucleic acid by rt-pcr is the most sensitive and specific means to confirm acute infection, but it may not necessarily reflect infectious virus as it only detects viral rna, which may persist in biologic fluids after infectious virus is cleared. immunoassays to detect denv ns antigen also provide acceptable levels of sensitivity and specificity. both these diagnostic approaches are instrument-dependent and require appropriate facilities to perform complex diagnostic tests. recently, it was found that apoprotein h (apo h) can be efficiently used to detect bacteria and viruses in blood samples. as described by dr francisco veas (french institute for dvelopment (ird), montpellier, france), beads coated with apo h can readily capture viruses with a glycoprotein-rich envelope such as influenza virus, ebola virus, denv and other arboviruses, or even hcv. the beads are easy to use, the process is very fast and shows high sensitivity: one can readily detect pfu of virus in ml of whole blood. dr rome buathong (bureau of epidemiology, dept of disease contrtol, thailand ministry of public health) reminded the audience that until the brazilian outbreak in - , zikv was a little known arbovirus presenting with a mild dengue-like illness without any major complications [ ] . it initially spread from sub-saharan africa to asia without detection. thus, although the virus was present in thailand, it was not detected until may , when a canadian traveler returning from thailand was diagnosed with the disease. a zikv surveillance program was launched in the country (from january to december ) revealing a yearly peak of zikv infection during the rainy season, occurring - weeks after the peak of dengue disease. a total of confirmed cases of zikv infection was recorded in thailand during this two year period. the age groups most affected were the - followed by the - year groups. a total of cases of zikv infection was recorded in pregnant women, leading to miscarriages and birth abnormalities, including cases of microcephaly. aedes mosquitoes in the country were found to be zikv positive, but other routes of transmission included blood transfusion and sexual intercourse. zikv rna could readily be detected from plasma, saliva and urine of infected patients. sexual transmission of zikv has been documented to occur up to three weeks after onset of illness in the male partner. shedding of the viral rna in semen has been well documented, but only % of semen samples that were pcr positive could be shown to contain infectious virus by cell culture. dr nikos vasilakis (university of texas medical branch) reviewed the explosive emergence of zikv in - in the south pacific and south america, with a focus on brazil where major clinical outcomes in pregnant women were common. the alarming numbers of zikv associated microcephaly and other gestational and neonatal complications brought the virus into the spotlight. increased reporting of guillain-barré syndrome (gbs) and other neurological manifestations was also associated with zikv infections. the first association between zikv infection and microcephaly was reported in in recife, brazil where three women infected with zikv during their pregnancy delivered babies with microcephaly. it was retrospectively discovered that neurological diseases, including microcephaly, in babies born to zikv-infected mothers, had been observed in french polynesia as early as . as reported by dr patricia brasil (oswaldo cruz foundation, rio do janeiro), a prospective cohort study for zikv infection in pregnant women and infants was initiated in rio de janeiro in early . zikv was recovered from the amniotic fluid and placenta of the pregnant women, and also from the cerebral spinal fluid of their babies. microcephaly was observed in %- % of the newborns, while structural birth defects such as hypertonicity, cardiac defects, ophtalmic abnormalities, elbow abnormality, and seizures were observed in up to . % of the newborns. a follow-up of brazilian babies born to mothers infected by zikv during pregnancy showed that up to % had abnormal hearing or other manifestations, and % showed signs of abnormal behavior. at months of age, only % of the babies displayed normal neurodevelopment. dr mauricio lacerda nogueira (faculty of medicine, sao jose do rio preto brazil) talked of the interplay between various flaviviruses. given that brazil is hyperendemic for arboviruses, there was concern that previous heterotypic flavivirus (e.g. dengue) exposure could exacerbate zika disease pathogenicity. denv antibodies (ab) bind to zikv. could they drive greater zikv replication through antibody-dependent enhancement (ade)? the phe-nomenom was initially described for denv, but it has also been found to exist for rabies virus, coxsackievirus b , coronaviruses, and even hiv. to answer the question, a cohort of healthy volunteers in vila toninho, brazil, was followed up from to , looking for evidence for a possible ade phenomenom. in spite of the fact that % of the cohort had denv ab at the start of the study, no significant clinical difference could be observed in zikv infections between denv ab positive and denv ab negative persons. thus, the epidemiological evidence in brazil did not support the hypothesis that previous exposure to denv infection could enhance zikv pathogenicity. by contrast, it has been reported that denv- and denv- ab might be protective against zikv infection. similarly, the presence of yfv ab has recently been associated with a better prognosis of zikv infection in pregnant women. other hypotheses, such as enhanced infectivity of zikv for aedes mosquitoes, have not been confirmed. interestingly, a a v mutation in the e gene of chikv has recently been described which is associated with enhanced infectivity of the virus for aedes albopictus mosquitoes. as discussed by dr nikos vasilakis, the most convincing explanation for the sudden aggressivity of zikv lies in the discovery by yuan et al [ ] in of a mutation in the prm gene of the brazilian strain of zikv, which could strongly contribute to the generation of fetal microcephaly. the mutation was also found in zikv strains from french polynesia, where numerous cases of microcephaly have been recorded, but not in virus strains from africa, where microcephaly has never been observed. dr marc lecuit (pasteur institute, paris), addressing the question of the cellular and molecular mechanisms of microcephaly, reminded the audience that, in contrast with chikv, which does not cross the placental barrier, zikv can readily penetrate and infect placental cells. inoculation of zikv in the brain of mouse embryos triggers an endoplasmic reticulum stress which perturbs the physiological unfolded protein response (upr) within apical cortical progenitor cells that control neurogenesis. sustained endoplasmic reticulum stress leads to apoptosis. thus, it is likely that, in pregnant women, zikv crosses the placental barrier, gets to the brain of the foetus and crosses the blood-brain barrier, then targets apical cortical progenitor cells, which leads to the blockade of upr and the arrest of neurogenesis, and, as an obvious consequence, to microcephaly. microcephaly has not been observed in africa; it appears to be a specific property of the brasilian zikv strain, and is most likely related to the prm mutation identified by yuan et al [ ] . dr michael gaunt (london school of hygiene and tropical medicine) reported a series of experiments done on zikv and denv to identify potential genetic determinants of ade. a amino acid long motif in the denv glycoprotein was identified which might be associated with ade. dr annelies wilder-smith (london school of hygiene and tropical medicine) reviewed the eu research program on zikv (the 'zikaplan'), which oversees institutional global partners with plans to set up a sustainable latin american research preparedness network for emerging diseases. a longitudinal cohort study of , subjects aged - is being followed for three years in different locations in brazil, to further refine the full spectrum and risk factors of congenital zika syndrome, including neurodevelopment milestones, mental retardation, etc, during the first years of life. also to be addressed is whether neurological problems associated with zikv infection, such as meningoencephalitis or acute zika myelitis, are the direct consequence of zikv infection, or are mediated by immune responses. novel zikv diagnostic tests will also be developed. dr louis lambrechts (pasteur institute, paris) reminded the audience that aedes aegypti, which is distributed throughout the tropical and sub-tropical world on all continents, serves as the major vector to transmit urban arboviruses, including the denvs, zikv, yfv, chikv, etc. whereas most female mosquitoes take only one blood meal a day, aedes aegypti females need multiple blood feedings every day, which makes them more efficient at transmitting epidemic disease. the comparison between aedes aegypti and ae albopictus also shows that, although ae albopictus is often more susceptible to infection by denv than ae aegypti, it appears to be significantly less efficient at transmitting the virus. in addition, factors such as the mosquito genotype, its geographical location, and the strain of arbovirus, influence the transmission dynamics of the disease. for example, zikv infection of ae aegypti mosquitoes seems to be influenced by the sequence of the ns protein of the virus. as reported by dr scott ritchie (james cook university, cairns, australia), the use of wolbachia infection of aedes mosquitoes has great potential to control the spread of the arboviruses they transmit. wolbachia-infected male aedes mosquitoes become sterile and, if sterile males are repeatedly introduced in a given area, the resident aedes populations can be suppressed. alternatively, wolbachia-infected male and female mosquitoes can be introduced together, which leads to a progressive replacement of the resident aedes population, which could then show a much reduced capacity at transmitting denv, zikv, chikv, yfv and possibly other viruses [ , ] . the use of wolbachia is recommended by the world mosquito program to eliminate dengue. a trial in australia has demonstrated that, shortly after release of wolbachia-infected aedes mosquitoes, over % of the mosquitoes population at the site became wolbachia-infected, and the transmission of denv was stopped [ ] . similar trials are being conducted in viet nam, indonesia and brazil. the fear that wolbachia infection of ae aegypti would lead to its replacement by ae albopictus seems unwarranted so far, but it requires further studies. dr joao bosco siqueira reported that brazil had suffered from a shortage of yellow fever vaccine. fortunately, the usual dose of the brazilian vaccine contains , pfu of attenuated yfv (strain d), when only , pfu is a sufficient protective dose [ ] . it has therefore, been possible to use fractional doses of vaccine with evidence of protection and relatively no difference in reported adverse events. faced with the spread of yellow fever throughout the country, the brazilian government has updated its vaccination policy and now recommends that the entire population should be vaccinated. as part of this effort, a change in schedule for children was implemented from a single dose at years of age to a dose schedule with the first dose administered at months and a second dose at years. this effort is complicated by inadequate vaccine supply and also false rumors that the vaccine would not be protective. fake news is unfortunately powerful and, as a result, vaccine refusal has been rising in the population! as reviewed by dr in-kyu yoon (international vaccine institute, south korea), dr christopher nelson (sanofipasteur, lyon, france) and dr annelies wilder-smith, major advances in dengue research have resulted in development of new vaccines that show promise for use in dengue prevention, such as sanofi pasteur's recently licensed cyd-tdv (dengvaxia Ò , sanofi pasteur, lyon, france) and six other dengue vaccine candidates in various phases of clinical trials. cyd-tdv is a tetravalent live attenuated chimeric vaccine consisting of a d yfv genome with the pre-membrane (pre-m) and envelope (e) genes from each of the four antigenically distinct denv serotypes. the vaccine has undergone large-scale phase clinical trials in asia and latin america [ , ] , demonstrating increasing efficacy with age and higher efficacy in baseline seropositives. efficacy was highest at preventing severe dengue and hospitalization, and moderate for overall dengue. it also varied with serotype, being higher ( - %) for denv- and - infection, and lower for denv- and - . notably, during the third year of the asian phase trial, an increased risk of dengue hospitalization was seen in children aged - years. efficacy of the vaccine was only % or less in - years old and % in - years old. the vaccine was licensed in with an age indication of - years (up to years of age in some countries). the cyd-tdv vaccine is now licensed in countries, and has been introduced into public immunization programs in endemic areas of the philippines and brazil. the vaccine showed high efficacy and good safety in seropositive persons in the - years age group, but a risk of severe dengue was observed in individuals who were naive for denv infection at the time they were vaccinated. it was hypothesized that cyd-tdv mimics primary infection among individuals with no prior dengue infection (previously dengue-unexposed, seronegatives) and a secondary-like infection among those with prior dengue infection (previously dengue-exposed, seropositives) [ , [ ] [ ] [ ] [ ] . a retrospective case-cohort study was undertakern to analyze vaccine safety among dengue seropositive and seronegative trial participants. this work used anti-ns results from month of follow-up that were obtained using an anti-ns igg elisa developed for this purpose. the data confirmed the substantial benefit of cyd-tdv vaccination in those who are dengue seropositive and aged years or older, reducing symptomatic dengue, hospitalized dengue and severe dengue by~ %. however, in individuals of any age without evidence of prior dengue infection, the vaccine elicited an increased risk of severe dengue, as announced by sanofi pasteur in november . the vaccine should therefore be administered only to dengue seropositive persons, which obviously implies the need for a pre-vaccination screening strategy [ ] . the who sage dengue working group reviewed these data and, in april , the sage committee recommended to vaccinate only those with evidence of past dengue infection (seropositives) or with medical documentation of past dengue infection. these results have implications for other dengue vaccine candidates in clinical development, and for immunization program policy and program implementation. two other dengue vaccine candidates are currently in phase clinical trials: tdv, developed by the us cdc and manufactured by takeda, and tv / , developed by the us nih. tdv is a tetravalent live attenuated chimeric vaccine that uses an attenuated denv- backbone with the pre-m and e genes from each of the other three denv serotypes. the tdv vaccine is undergoing phase trials in brazil, columbia, nicaragua, panama, sri lanka and thailand. preliminary phase data show the vaccine is safe and has good efficacy against denv- and- , but the results for denv- and - are uncertain because there were only a small number of cases of these serotypes [ ] . tv /tv is a tetravalent live attenuated vaccine which includes three denv serotypes (denv- , - , À ) that have undergone attenuation through direct mutagenesis (a base-pair deletion), while the denv- candidate consists of a denv- -denv- chimera. the butantan institute in brazil, which is the sponsor of butantan-dv (tv ), received regulatory approval in december to begin a phase trial in brasil. three other denv vaccines are in less advanced development. they are an inactivated vaccine, a dna vaccine, and a subunit (the e glycoprotein) vaccine. but the difficulties of developing effective and safe dengue vaccines are multiple, as seen in the cyd-tdv experience, and many issues still remain, including: . the vaccine should elicit equal protection against the four denv serotypes, which has been extremely difficult to achieve, in view of competition between strains and/or ade of some strains: the question has been raised of whether it really is a must to develop tetravalent vaccines? . whether antibodies are associated with protection or risk (ade) seems to depend on titer, homotypy versus heterotypy, etc. how can that be understood and controlled? . why does severity of the disease, especially that of the postvaccination adverse events, vary with age? and . what is the feasibility and what will be the cost of a necessary pre-vaccination screening? nevertheless, the substantial public health benefits of even a moderately effective dengue vaccine continue to drive all the efforts at developing dengue vaccines [ , ] , hoping that safety concerns may be adequately addressed. a large number of zika vaccine candidates are under development and have entered clinical trials, including: a mrna vaccine; a dna vaccine that encodes the zikv prm and e genes and is presently entering phase iib trials in the usa, brazil, and several countries in latin america (puerto rico, mexico, panama, peru, columbia..); a purified inactivated vaccine adjuvanted with alum, which was shown to be immunogenic after two doses by the im route in a phase clinical trial; and a chimeric measles virus vaccine that expresses the prm and e genes of zikv. as outlined by dr anna durbin (john hopkins bloomberg school of public health) many hurdles still remain before licensure of a safe and effective zikv vaccine can occur. the transmission of zikv has declined so much that it will be difficult to perform a phase efficacy study. in addition, clinical trial efficacy endpoints for a zikv vaccine are not well established: one can wonder if a vaccine will not have to fully prevent infection in order to prevent microcephaly, a very high bar for any vaccine indeed! dr georges thiry (sas senergues, france) reported the creation of the 'coalition for epidemic preparadness innovations' (cepi), which was launched in january in oslo, london and washington with the aim to prevent future epidemics by the development of new vaccines. three major targets have been selected for the manufacturing and testing of candidate vaccines: mers-cov, lassa fever virus and nipah virus. cepi is responsible for vaccine development from preclinical to phase a trial. several vaccines are being followed up: a lassa-vsv vaccine, developed by iavi; a lassa-dna vaccine; a lassa-measles (mv) vaccine, developed by themis; a mers-cov-mv vaccine, also developed by themis; and a nipah glycoprotein subunit vaccine with adjuvant. most phase studies have taken place in - and phase a trials are expected to follow in - . arboviruses are in the news and at the top of social, political and public health agendas. arbovirus epidemics will increasingly threaten global, national and local political and economic security and create a global public health emergency. not only is there an increased risk of epidemic arboviral diseases in rural areas, but there also is now an increased risk of urban epidemics. dengue epidemics have recently seen their amplitude increase dramatically: in , there were fold more reported cases of denv infection in brazil than during the preceding year, leading to deaths; during the same period, , dengue-infected persons had to be hospitalized in bangladesh, where some deaths occurred, and more than deaths from dengue were reported in the philippines! an estimated million dengue infections occur annually [ ] . the development of vaccines and vector control tools to prevent arbovirus diseases is actively being pursued [ , ] , and the possibility of developing a trivalent vaccine against zikv, chikv and wnv is even being entertained. many hurdles however, remain before licensure of a safe and effective zikv vaccine can occur, as outlined above. similarly, the difficulties at developing an effective and safe denv vaccine are multiple, and many questions still arise. what will be the cost of implementing pre-vaccination screening? whom to vaccinate in places where denv seropositivity in years of age turns out to be very low (only % in singapore for example)? the dramatic emergence and spread of epidemic arboviral diseases has made it necessary to reassess surveillance strategies. all at-risk countries should have an enhanced passive disease surveillance system based on well-defined case definitions and supported by serological and virological laboratory testing. an active laboratory syndromic surveillance system, such as was developed in the past by who for influenza or for poliomyelitis, is badly needed. it is hoped that such a system will be set up rapidly. but we also need more effective mosquito control tools to use in conjunction with vaccines, timely access to clinical serices and appropriate care. only by using an integrated approach to prevention and control will we be able to successfully reverse the trend of emergent epidemic arboviral diseases. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. the xxth century dengue pandemic dengue and dengue hemorrhagic fever the global threat of emergent/reemergent vector-borne diseases. in: vector-borne diseases: understanding the environmental, human health, and ecological connections epidemic arboviral diseases: priorities fror research and public health increase in reported prevalence of microcephaly in infants born to women living in areas with confirmed zika virus transmission during the first trimester of pregnancy -brazil importation of yellow fever into china: assessing travel patterns the continuing spread of west nile virus in the western hemisphere the emergence of arthropod-borne viral diseases: a global prospective on dengue, chikungunya and zika fevers the global distribution and burden of dengue global strategy for dengue prevention and control community effectiveness of indoor spraying as a dengue vector control method: a systematic review dengue haemorrhagic fever: diagnosis, treatment, prevention and control who immunization, vaccines and biologicals. vaccine position papers efficacy and longterm safety of a dengue vaccine in regions of endemic disease yellow fever epidemiological update identifying global vulnerabilities to urban transmission of yellow fever virus aedes albopictus and the world trade in used tires, - : the shape of things to come urbanization and globalization: the unholy trinity of the st century the global distribution of the arbovirus vectors aedes aegypti and ae albopictus policy and practice. a guide to aid the selection of diagnostic tests isolation of zika virus from aedes aegypti mosquitoes in malaysia a single mutation in the prm protein of zika virus contributes to fetal microcephaly assessing the epidemiological impact of wolbachia deployment for dengue control using wolbachia for dengue control: insights from modelling the awed trial (applying wolbachia to eliminate dengue) to assess the efficacy of wolbachia-infected mosquito deployments in yogyakarta, indonesia: study protocol for a cluster randomised controlled trial immunogenicity of fractional-dose vaccine during a yellow fever outbreak -final report benefits and risks of the sanofi-pasteur dengue vaccine: modeling optimal deployment dengue vaccine: hypotheses to understand cyd-tdvinduced protection the long-term safety, public health impact, and cost-effectiveness of routine vaccination with a recombinant, live-attenuated dengue vaccine (dengvaxia): a model comparison study effect of serostatus on dengue vaccine safety and efficacy efficacy of a tetravalent dengue vaccine in healthy children and adolescents beyond efficacy: the full public health impact of vaccines estimating the full public health value of vaccination the organizing committee of the meeting included the following scientists: drs duane j gubler, jacques louis, christopher b nelson, mauricio nogueira, valentina picot, usa thisiyakorn, in-kyu yoon, and mrs cindy grasso.moderators and presenters were drs duane gubler, usa thisiyakorn, joao bosco siqueira, mauricio lacerda nogueira, christopher nelson, rome buathong, nikos vasilakis, michael gaunt, patricia brasil, marc lecuit, anna durbin, georges thiry, annelies wilder-smith, louis lambrechts, scott ritchie, in-kyu yoon, and elizabeth hunsperger. key: cord- -wmfwl bh authors: jung, eunhye; nam, sangwoo; oh, hyeryeon; jun, sangmi; ro, hyun-joo; kim, baek; kim, meehyein; go, yun young title: neutralization of acidic intracellular vesicles by niclosamide inhibits multiple steps of the dengue virus life cycle in vitro date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: wmfwl bh dengue fever is one of the most important mosquito-borne viral infections in large parts of tropical and subtropical countries and is a significant public health concern and socioeconomic burden. there is an urgent need to develop antivirals that can effectively reduce dengue virus (denv) replication and decrease viral load. niclosamide, an antiparasitic drug approved for human use, has been recently identified as an effective antiviral agent against a number of ph-dependent viruses, including flaviviruses. here, we reveal that neutralization of low-ph intracellular compartments by niclosamide affects multiple steps of the denv infectious cycle. specifically, niclosamide-induced endosomal neutralization not only prevents viral rna replication but also affects the maturation of denv particles, rendering them non-infectious. we found that niclosamide-induced endosomal neutralization prevented e glycoprotein conformational changes on the virion surface of flaviviruses, resulting in the release of non-infectious immature virus particles with uncleaved pr peptide from host cells. collectively, our findings support the potential application of niclosamide as an antiviral agent against flavivirus infection and highlight a previously uncharacterized mechanism of action of the drug. in this study, the anti-dengue activity of niclosamide was evaluated using four denv serotypes. after h post-infection (p.i), viral titres from the supernatants and the number of infected cells were measured by fluorescence-activated cell sorter (facs) analysis and focus-forming assay, respectively. the proportion of cells positive for denv antigen decreased in a dose-dependent manner in cells treated with niclosamide compared to that in dmso-treated controls as determined by facs analysis (fig. a) . specifically, the percentage of denv-positive cells was significantly reduced when infected cells were treated with niclosamide at a concentration of . μm or higher for all four serotypes of denv (fig. a) . the ec values of niclosamide against denv- , denv- , denv- , and denv- were approximately . μm, . μm, . μm and . μm, respectively. to rule out the possibility that virus infected cells were more sensitive to niclosamide treatment resulting in synergistic cytotoxicity compared to mock-infected cells, the percentages of live and dead cells from mock-and denv- infected cultures with increasing concentrations of niclosamide were determined by facs analysis. the data showed no difference in cell viability between mock-and denv-infected cells treated with increasing concentrations of niclosamide ( supplementary fig. s ). next, the % cytotoxicity concentration (cc ) value of niclosamide was determined by measuring cell viability using the mtt assay. the estimated cc value of niclosamide was > μm; however, minor cytotoxic effects at all sub-lethal doses were observed in huh- cells (supplementary fig. s ). similarly, niclosamide inhibited the production of infectious denv particles of all four serotypes in a dose-dependent manner as quantified by the focus-forming assay. significantly, no infectious denv particles were detected when infected huh- cells were treated with niclosamide at a concentration of μm or higher (fig. b) . these results together confirm that niclosamide effectively inhibits denv infection independent of the virus serotype within a non-cytotoxic range in huh- cells. demonstrating that niclosamide has an antiviral effect against denv independent of its serotype, the denv- ngc strain was employed in all subsequent experiments. we performed a time-of-addition experiment to investigate the stage of the denv life cycle during which niclosamide exerts its antiviral activity. huh- cells were infected with denv- and subsequently treated with μm niclosamide starting at , , and h p.i. until the medium and cell lysates were harvested at h p.i. (fig. a) . analysis of the infectious progeny released in the supernatant revealed that niclosamide not only affected an early stage of the viral life cycle but also reduced progeny titres when added at later stages. specifically, there was a complete inhibition of infectious virus production when niclosamide was added at h p.i., while a -log reduction was observed when niclosamide treatment was initiated at , or as late as h p.i. (fig. b) . similarly, niclosamide treatment was maximally effective (~ %) in reducing intracellular viral rna when added at h p.i., while the inhibitory effect markedly decreased when the drug was added at later time points ( , and h p.i.), suggesting that niclosamide interferes with viral rna replication (fig. c) . likewise, there was a marked reduction of viral genome copies released in the supernatant from samples that received niclosamide treatment at h p.i. (~ -log reduction), while the effect diminished www.nature.com/scientificreports www.nature.com/scientificreports/ when the drug was added at later time points such as , or as late as h p.i. (~ -log reduction, fig. d ). thus, the data together indicated that niclosamide possibly inhibits viral rna replication when it is added at early step of viral life cycle but it also affects a late stage of virion biogenesis, such as maturation, since a strong antiviral effect was consistently observed in infected cells with niclosamide treatment starting as late as h p.i. (fig. bd) . in addition, viral ns and e protein levels were significantly reduced upon niclosamide treatment at early post-infection time points, whereas the antiviral effect diminished when the drug was added after h p.i. (fig. e) . the data represent the means (±sd) of at least two independent experiments performed in duplicate. n.d., not detected. *p < . , ***p < . and ****p < . compared to dmso control. www.nature.com/scientificreports www.nature.com/scientificreports/ taken together, these data suggest that niclosamide potently inhibits the early stages of the denv life cycle, which impacts viral rna accumulation and protein expression, as well as the production of infectious viruses. in addition, the data revealed that niclosamide affects not only an early stage but also a late stage of the denv life cycle independent of the effects on intracellular viral rna accumulation and protein synthesis. neutralization of endosomal ph by niclosamide inhibits denv rna genome replication and viral polyprotein processing. niclosamide is a proton carrier and blocks endosomal acidification . we tested whether niclosamide neutralizes the low-ph compartments in huh- cells by using acridine orange (ao), which is a fluorescent ph-sensitive dye. as shown in fig. a , the low ph of endosomes (red) present in mock-treated huh- cells was completely absent in cells treated with niclosamide (green), suggesting that it effectively blocked endosomal acidification in huh- cells. bafilomycin a (bafa ) and ammonium chloride (nh cl) were used as controls. to further confirm the correlation of the antiviral effect with the neutralization of endosomal ph, lysotracker (a marker for acidic compartments) analysis was performed together with immunofluorescence staining for the detection of double-stranded rna (dsrna) in denv-infected cells treated with niclosamide at early (pre, − h to h p.i.) or late stages (post, h p.i. to h p.i.) (fig. b) . the results showed that viral dsrna expression, an intermediate product in replication, was significantly inhibited in infected cells treated with niclosamide at early and late stages compared to that in dmso-treated controls, indicating that viral rna replication is severely affected (fig. c) . notably, there was no difference in the levels of endosomal acidification in cells treated with niclosamide during the early stage and subsequently removed from the culture, whereas the endosomal ph was effectively neutralized in cells that received the treatment at the late stage and the treatment remained until lysotracker staining, indicating the reversible effect of the drug on the inhibition of endosomal acidification (fig. c) . to further corroborate that suppression of viral genome replication is specifically due to niclosamide-induced ph neutralization, denv replicon reporter bhk cells encoding the renilla luciferase gene were treated with niclosamide at the time points indicated in fig. b . ribavirin, a broad-spectrum antiviral compound known to impede rna virus replication, was used as a positive control. cell viability of replicon cells in the presence of niclosamide and ribavirin treatment was determined by the mtt assay ( supplementary fig. s ). as shown in fig. d , treatment of denv replicon reporter cells with μm niclosamide at early time points (pre, − h to h) resulted in a significant reduction in luciferase activity, comparable to that of ribavirin, whereas the inhibitory effect was almost restored to the level of dmso-treated cells when the cells were treated with niclosamide after h of culture (post, h p.i. to h p.i). in contrast, the reduction in luciferase activity reached a maximum when ribavirin was added at h p.i. and remained in the culture until harvest. thus, the replicon-based assay suggested that niclosamide inhibits the steps involved in viral rna replication independent of its effect on entry, membrane fusion and genome release. next, we evaluated the impact of niclosamide on the proteolytic activity of denv ns b-ns , which plays an essential role in viral polyprotein processing required for the initiation of viral replication. the proteolytic activity of denv ns b-ns protease, using the fluorogenic substrate, was evaluated in the presence of various concentrations of niclosamide. as a result, the half-maximal inhibitory concentration (ic ) for niclosamide was determined to be . μm, showing only a modest inhibition of ns protease activity (fig. e ). lastly, the expression level of denv non-structural (ns ) protein after niclosamide treatment at early (pre, − h to h p.i.) or late stages (post, h p.i. to h p.i.) was examined by western blot analysis (fig. f) . consistent with the immunofluorescence assay results, the expression of ns protein was significantly reduced in cells treated at early and late stages, although the inhibition was more pronounced in cells treated at the early stage of virus replication than in those treated after h p.i. (fig. f) . overall, the data indicate that the antiviral activity of niclosamide during the early stage of the denv life cycle correlates with the neutralization profile of the low-ph compartments, suggesting that blocking endosomal acidification results in the inhibition of viral genome replication and polyprotein processing, which further impedes viral protein expression and virus production. neutralization of low-ph compartments by niclosamide also impairs the denv maturation process. to further corroborate the effect of niclosamide-induced neutralization of low-ph compartments on virion biogenesis, particularly maturation, which is a ph-dependent process, we analysed the composition of prm and e proteins in the virus particles produced by infected cells treated with niclosamide. briefly, denv- -infected huh- cells were treated with μm niclosamide at h p.i. and supernatants were collected at h p.i. subsequently, tissue culture medium obtained from niclosamide-treated and mock-treated cells was pelleted by ultracentrifugation. samples were adjusted to equal viral genomic copy numbers, separated by sds-page, and viral proteins were detected by western blot using monoclonal antibodies against e and prm proteins. the data showed that virus particles obtained from niclosamide-treated and untreated (dmso) samples had comparable levels of denv e protein (fig. a) . in contrast, a clear prm protein band (approximately kda) was detected in denv released from niclosamide-treated cells, in which the pr peptide was not cleaved during maturation, resulting in the release of immature and non-infectious virus particles in the medium. in contrast, the prm protein band was barely detected from virions released from dmso-treated cells, indicating that they were mature and fully infectious (fig. a) . next, the morphology of denv particles released from dmso-and niclosamide-treated cells was examined by transmission electron microscopy (tem). as shown in fig. , denv particles derived from dmso-treated cells had relatively smooth and spike-less outer surfaces, which are characteristics of mature particles (fig. b,c [upper panels]) compared to those obtained from niclosamide-treated cells, which had the expected spiky appearance of immature particles (fig. b,c [lower panels]). similar results were observed in human monocytic u -dendritic cell-specific icam-grabbing non-integrin (u -dc-sign) cells suggesting that the antiviral effect of niclosamide is not limited to huh- cells (supplementary fig. s ). taken together, these results indicate that inhibition of endosomal acidification by niclosamide interferes with the ph-dependent denv maturation process by preventing cleavage of the pr peptide from the m protein on surface of the virus. www.nature.com/scientificreports www.nature.com/scientificreports/ the zikv particle maturation process is affected. we hypothesized that niclosamide-induced neutralization of low-ph compartments also impacts zikv, a member of the flavivirus genus, virion biogenesis. we first corroborated that niclosamide reduced the number of zikv-positive cells and infectious viral production in a dose-dependent manner in huh- cells with an ec of . μm as evaluated by facs analysis and focus-forming www.nature.com/scientificreports www.nature.com/scientificreports/ assay, respectively (fig. a,b) . next, the composition of prm and e proteins in zikv virions released in the medium of niclosamide-treated cells was examined to confirm whether niclosamide impacts the maturation process. as shown in fig. c , zikv particles released in the medium of dmso-treated cells were completely processed with no detectable prm protein band, while a prominent m protein band at approximately kda was observed, indicating that the virions are mature and fully infectious. in contrast, zikv particles released in the medium of niclosamide-treated cells had a prominent prm protein band, and almost no m protein was detected, indicating that cleavage of the pr peptide during the maturation process had been hampered (fig. c) . consistent with these results, the electron microscopy images also demonstrated that virions from dmso-treated cells had a smoother surface (fig. d , upper panels), while those obtained from niclosamide-treated cells had a spiky outer surface (fig. d , lower panels), indicating that conformational surface protein changes and pr peptide cleavage were blocked. taken together, our data suggest that niclosamide-induced neutralization of low-ph compartments interferes with the denv and zikv maturation process, resulting in the release of prm-containing immature and non-infectious viral particles into the extracellular environment. niclosamide is a well-established drug that has been safely used for antihelmintic therapy against tapeworm infection for approximately years . its antiparasitic activity is attributed to its ability to inhibit mitochondrial oxidative phosphorylation and anaerobic atp production, which affect the ph homeostasis of parasites , . recently, niclosamide has also been identified as an effective antiviral agent against a number of ph-dependent viruses. its inhibitory effect has been attributed to the neutralization of endo-lysosomal ph, which interferes with ph-dependent membrane fusion that is critical for virus entry . in this study, we found that neutralization of low-ph intracellular compartments by niclosamide not only inhibited the early stage of the denv viral life cycle, such as viral rna replication, independent of the entry step but also the late stage, specifically, the maturation of virus particles into infectious virions. similar to previous reports, our data showed that niclosamide neutralized the low-ph intracellular compartments in a reversible manner and suggested that its antiviral effect correlates with neutralization of these acidic environments, which are critical for denv replication , . here, we confirmed that niclosamide effectively inhibited intracellular viral rna synthesis, protein expression and production of infectious denv particles when the drug was added at an early time point during infection. the immunofluorescence assay coupled with staining of intracellular acidic compartments in live denv-infected cells showed that niclosamide-induced ph neutralization during the first h of infection was sufficient to completely block viral dsrna replication and its subsequent steps, such as viral protein expression and production of infectious viral particles. indeed, niclosamide treatment www.nature.com/scientificreports www.nature.com/scientificreports/ during the first h of infection reduced denv replication in bhk- cells harbouring dengue replicons to a level comparable to that of ribavirin, suggesting that the drug affects viral rna replication and/or translation independent of its effect on entry, membrane fusion and genome release. furthermore, we provide evidence that niclosamide impairs the proteolytic activity of denv ns b-ns , which is essential for the initiation of www.nature.com/scientificreports www.nature.com/scientificreports/ viral replication and may explain its effects on denv genome replication and/or translation, strengthening the findings of li et al. . however, these findings are somewhat contradictory to the results reported by kao et al. , which, using a replicon-based assay, indicated that niclosamide had no effect on denv genome replication. we speculate that the variance in the efficacy of niclosamide against viral genome replication using replicon-based cells could be due to a difference in the times of drug-addition used in each study. in this study, we identified a new potentially interesting mode of action of niclosamide, which impacts a late stage of the virus life cycle in huh- cells. our time-of-addition data demonstrated that the quantity of infectious virus particles released into the media was significantly reduced even when niclosamide was added to the cells several hours after infection, whereas intracellular and extracellular viral rna levels were not affected to the same extent as the production of infectious virus particles. these data led us to the hypothesis that niclosamide-induced intracellular ph neutralization affects the maturation of denv particles. it has been extensively described that the low-ph-induced conformational rearrangement of the prm and e proteins is critical for denv maturation and infectivity , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . indeed, our data showed that denv, as well as zikv particles obtained from niclosamide-treated cells, contained high levels of uncleaved prm proteins compared to the untreated virions, suggesting that most of the virus particles were immature and non-infectious. similarly, transmission electron microscopy images showed that the denv and zikv particles secreted from niclosamide-treated cells were substantially larger than untreated control virions and had irregularities on the surface, indicating that the viral particles were immature and contained uncleaved prm on the surface. taken together, we believe that we have provided the first evidence that neutralization of the low-ph intracellular compartments by niclosamide prevents conformational changes to e glycoproteins on the virion surface during the flavivirus maturation process, which results in the release of immature non-infectious virus particles from host cells. in summary, we confirmed that the antiviral effect of niclosamide against flaviviruses is mostly associated with neutralization of the low-ph intracellular organelles. as a consequence, multiple ph-dependent steps of the flavivirus life cycle, including viral and host membrane fusion and uncoating, viral rna replication (by inhibition of viral polyprotein processing), and the maturation process of the progeny virions, are impaired, highlighting the complexity of the antiviral efficacy of niclosamide (fig. ) . collectively, the data presented in this study provide further evidence to support the repurposing of niclosamide as a potential therapeutic option against ph-dependent rna viruses, particularly flaviviruses. . all denvs were propagated in c / cells, and tissue culture fluid (tcf) supernatants were harvested at - days post-infection. tcf was clarified by centrifugation, and -ml aliquots were stored at − °c until further use. viral titres were quantified by focus-forming assay on vero cells as described previously . zika virus, an mr strain (atcc ® vr- tm ) purchased from atcc, was amplified, and viral titres were measured by plaque assay on vero cells. to determine the mechanism of action of niclosamide, huh- cells were inoculated with the denv- ngc strain at an moi of . at °c for h. unbound virus was removed by washing with ice-cold phosphate buffered saline (pbs), fresh medium was then added, and plates were shifted to °c to allow synchronous entry and infection. soon after the temperature shift, µm niclosamide was added at , , and h and maintained throughout the infection. at h p.i., cell culture supernatants were collected for virus titration and extracellular viral rna quantification by focus-forming assay and quantitative reverse-transcription polymerase chain reaction (rt-qpcr), whereas cell lysates were harvested and subjected to intracellular viral rna and viral protein analyses by rt-qpcr and western blot assays, respectively. rna quantification, total cellular rna was purified from cell lysates using an rneasy mini kit (qiagen, valencia, ca, usa) according to the manufacturer's instructions. viral rna in the tcf samples was extracted using a qiaamp ® viral rna mini kit (qiagen) according to the manufacturer's instructions. rt-qpcr was performed using a superscript iii one-step rt-pcr system with platinum taq polymerase (invitrogen), primers/probe sets targeting ns or e genes and a quantstudio real-time pcr system (applied biosystems, foster city, ca, usa) as described previously . the relative viral rna expression levels were calculated by the ΔΔc t method, and β-actin was used as an endogenous control. absolute viral rna genome copy number was calculated based on the in vitro-transcribed denv rna standard curve and reported as the absolute number of viral rna genome copies per ml of tcf . two biological replicates, each with technical duplicates, were used for quantification. western blot analysis. at h p.i., virus-infected cells were washed with pbs and lysed using m-per buffer (thermo fisher scientific, waltham, ma, usa) containing . % protease inhibitor cocktail (pierce, rockford, il, usa). the cell lysates were clarified by centrifugation, and the total protein content was determined by the bradford assay (bio-rad, hercules, ca, usa). equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and electro-transferred to a pvdf membrane. viral proteins were detected using primary antibodies specific for denv ns , denv e, denv prm, zikv e, and zikv prm followed by a horseradish peroxidase (hrp)-conjugated goat anti-mouse or anti-rabbit secondary antibody. as a loading control, cellular β-actin was detected with an anti-β-actin-specific primary antibody and hrp-conjugated goat anti-mouse secondary antibody. after the addition of a chemiluminescent hrp substrate (supersignal west pico chemiluminescent substrate; pierce), images were obtained using a las- luminescent image analyzer (fujifilm, tokyo, japan). denv replicon assay. bhk- cells encoding a luciferase-expressing denv- replicon (bhk-d -rluc) were used to determine denv rna replication efficiency. briefly, bhk- replicon cells were seeded in -well plates and incubated with different concentrations of niclosamide at the indicated time points at °c for h. after incubation, antiviral activity was measured using the renilla luciferase assay (promega, madison, wi, usa) in a microplate luminometer (tecan, männedorf, switzerland). in vitro protease activity assay. the denv- ns b-ns protease expression plasmid used in this study was kindly provided by dr. rolf hilgenfeld, university of lübeck, lübeck, germany. the in vitro denv- ns b-ns protease activity assay was performed as described elsewhere . the fluorogenic peptide substrate (boc-gly-arg-arg-amc) was purchased from bachem ag (bubendorf, switzerland). to measure ph changes in cytoplasmic membrane-enclosed vesicles, cells were stained with acridine orange (ao, thermo fisher scientific), as described previously . briefly, huh- cells ( × cells per well) were cultured at °c in mm glass-bottom dishes (greiner bio-one, frickenhausen, germany). on the following day, cells were treated for h with μm niclosamide, nm bafilomycin a (a v-atpase inhibitor), or mm ammonium chloride (an intralysosomal ph-neutralizing agent). acridine orange was added to the culture medium at a final concentration of μg/ml, and the cells were imaged with a confocal microscope zeiss lsm meta (carl zeiss, oberkochen, germany). the excitation wavelength was nm, and images were collected in two emission windows: - nm and - nm. for labelling and tracking of acidic organelles in niclosamide-treated denv-infected cells, a deep red-fluorescent dye, lysotracker dnd- (ltr, thermo fisher scientific), was used. briefly, huh- cells were grown on -well chamber slide and incubated overnight. the huh- cells were infected with the denv- ngc ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ strain at an moi of and treated with μm niclosamide at h prior to infection and left in culture until h p.i. (pre − to h) or treated at h p.i. and the treatment maintained throughout the course of the infection. after h of incubation, cells were washed and loaded with . μm lysotracker dnd- (invitrogen) for min at °c. subsequently, the cells were washed with pbs and fixed with % paraformaldehyde in pbs for min at room temperature. the fixed cells were incubated with antibodies against denv e protein or dsrna antibodies for h at room temperature. the cells were then incubated with af -conjugated goat anti-mouse igg antibodies for h at room temperature. nuclei were counterstained by incubation for min with μg/ml dapi, and the coverslips were mounted in prolong tm gold antifade reagent (invitrogen). image analysis was performed using a confocal microscope (zeiss lsm meta). electron microscopy. huh- cells were infected with denv- (ngc strain) or zikv (mr strain) at an moi of . for h at °c. at h p.i., μm niclosamide or dmso was added and infected cultures were further incubated for h. after a total of h of infection, virus particles were pelleted by ultracentrifugation at °c in a beckman type sw rotor at , × g for h. virion pellets were resuspended in nte buffer. five microliters of resuspended denv- and zikv were mounted on plasma-cleaned mesh, carbon-coated, copper grids (electron microscopy sciences, hatfield, pa). grids were washed once with distilled water and then negatively stained with % aqueous uranyl acetate (electron microscopy sciences, hatfield, pa) for s. the solution was blotted with filter paper, and the sample grids were rinsed briefly with distilled water three times. after drying in air, the grids were examined under a zeiss leo ab transmission electron microscope (carl zeiss) at an accelerating voltage of kv and an fei tecnai g t- s transmission electron microscope (fei company, hillsboro, or) at an accelerating voltage of kv. statistical analysis. all statistical analyses were performed using graphpad prism version . 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for point-of-need diagnosis of dengue virus infection by use of the pockit nucleic acid analyzer protegrin- inhibits dengue ns b-ns serine protease and viral replication in mk cells intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin the authors are grateful to dr. rolf hilgenfeld (university of lübeck, lübeck, germany) for kindly providing denv- ns b-ns protease expression plasmid used in this study. we would also like to acknowledge dr. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to 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