key: cord- - mi aop authors: haddad, rodrigo; kashima, simone; rodrigues, evandra strazza; azevedo, rochele; palma, patrícia vianna bonini; de magalhães, danielle aparecida rosa; zago, marco antonio; covas, dimas tadeu title: silencing of htlv- gag and env genes by small interfering rnas in hek cells date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: mi aop since the discovery of rnai technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. rnai-based antiviral therapies are being studied for the treatment of retroviruses such as hiv- . these studies include the silencing of regulatory, infectivity and structural genes. the htlv- structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. to examine the possibility of silencing htlv- genes gag and env by rna interference technology, these genes were cloned into reporter plasmids. these vectors expressed the target mrnas fused to egfp reporter genes. three small interference rnas (sirnas) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by rnai technology. the plasmids and sirnas were co-transfected into hek cells. the results demonstrated that the expression of the htlv- gag and env genes decreased significantly in vitro. thus, sirnas can be used to inhibit htlv- structural genes in transformed cells, which could provide a tool for clarifying the roles of htlv- structural genes, as well as a therapy for this infection. human t-lymphotropic virus (htlv- ) is a member of delta retroviruses (retroviridae family). it is the etiologic agent of adult tcell leukemia-lymphoma (atll) (ratner, ) and the neurologic disease htlv-associated myelopathy/tropical spastic paraparesis (ham/tsp) (nagai and osame, ) . the htlv- proviral genome is composed of pol, gag and env structural genes, and regulatory genes such as tax and rex, flanked by long terminal repeat (ltr) sequences at both ends (seiki et al., ) . the pol gene codes for the reverse transcriptase and integrase, and gag and env code for the structural proteins of htlv- . the mature matrix (ma), capsid (ca) and nucleocapsid (nc) proteins are produced after the cleavage of gag precursor polyproteins by the viral protease. the htlv- env protein is derived from an envelope polyprotein precursor that is cleaved by cellular proteases, yielding a mature protein. this protein is composed of two subunits, a surface subunit (su) and a transmembrane (tm) subunit (le blanc et al., ; manel et al., ) . gag precursor polyproteins direct membrane targeting, which is required for viral assembly and release (le blanc et al., ) . previous studies found that htlv- uses the glucose transporter glut- to infect the target cell, and this interaction involves also env proteins (manel et al., ) . these features of htlv- structural proteins make them important targets in studies directed toward the development of new therapeutic methods. observed initially in plants but described accurately in caenorhabditis elegans (fire et al., ) , rna interference (rnai) refers to a cellular mechanism by which a double-stranded rna (dsrna) inhibits the expression of a specific gene. the dsrnas are processed by the endonuclease dicer into small interference rnas (sirnas), which are composed of approximately nucleotides. these sirnas are incorporated into the rna-induced silencing complex (risc) by the risc-loading complex (rlc), and the sense strand of sirna is removed. the risc is guided by the sirna antisense strand to the complementary target mrna. then, the mrna is cleaved by the ago protein. if partial complementarity occurs between the sirna antisense strand and the target mrna, translation is inhibited and there is no mrna degradation (kurreck, ) . rnai is involved in the inhibition of viruses and in the silencing of identical elements in plants, insects, fungi and nematodes (waterhouse et al., ; voinnet, ; wilkins et al., ; wang et al., ; segers et al., ) . when this genetic technique is used table primer sequences used to amplify gag and env genes. gene sequence sequence to facilitate digestion hindiii or bglii sites gag or env gene sequence in mammals, dsrnas elicit the innate antiviral immune response by inducing interferon-linked pathways (rana, ) . furthermore, it has been found that the transfection of - -nt synthetic sirnas, with -nt overhangs, into mammalian cells effectively inhibits an endogenous gene in a sequence-specific manner (elbashir et al., ; caplen et al., ; holen et al., ) . since , when the first report on inhibition of human respiratory syncytial virus (rsv) using rnai technology was published (bitko and barik, ) , many other studies have been performed examining the suppression of viral infections (ma et al., ; haasnoot et al., ) . they include the following: human immunodeficiency virus type (hiv- ) (novina et al., ; hayafune et al., ; naito et al., ) , hepatitis c virus (hcv) (krönke et al., ; sakamoto et al., ) , hepatitis b virus (hbv) (wu et al., a; ying et al., ) , severe acute respiratory syndrome coronavirus (sars-cov) (shi et al., ; wu et al., b) and influenza a virus (iav) (zhou et al., ) . in the present study, sirnas were used for inhibition of the expression of the htlv- structural genes gag and env. reporter systems were constructed that express viral target genes fused to the enhanced green fluorescent protein gene (egfp). these genes were transformed into hek cells, and sirnas were then introduced into cells expressing the structural proteins. the specific sirnas corresponding to the htlv- gag and env genes knocked down specifically these mrnas, and inhibited the expression of gag and env proteins. these results may be important for the development of new gene therapy-based anti-htlv- drugs. total rna of htlv- -infected cells (mt- , ecacc, salisbury, wiltshire, uk) was extracted by the trizol tm method and treated with dnase i. reverse transcription was performed using a high capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa). the entire gag and env genes were amplified by pcr. the primer sequences used are shown in table . the amplified dna fragments were digested with hindiii and bglii and inserted into the multi-cloning site of the pegfp-c expression vector (clontech, mountain view, ca, usa) between the hindiii and bglii sites. these vectors express the egfp reporter gene under cytomegalovirus (cmv) promoter control. in the resulting plasmids, pegfp-gag and pegfp-env, the egfp gene was located upstream of the target genes ( fig. a ). block-it tm rnai designer software (invitrogen, carlsbad, ca, usa) was used to design the sirnas corresponding to the gag and env genes (genbank accession numbers x and x respectively). this algorithm performs a statistical analysis of the target sequence and correlates it with data collected from multiple validated sirna oligo sets tested on different gene targets. three sirnas (sirnas gag , gag and gag ) and one scrambled sirna (sirna scr gag) were designed for the gag gene, and three other sirnas (sirnas env- , env- and env- ) and one scrambled sirna (sirna scr env) were designed for the env gene ( fig. b and table ). one non-related sirna was designed for both targets. all sirna sequences were blast searched in the us national center for biotechnology information (ncbi) against all human sequences deposited in the genbank and refseq databases and no significant similarities with human genes were found. the sir-nas were resuspended in ml of nuclease-free water for further use. the human embryonic kidney cell line (hek) was cultured and maintained in dulbecco's modified eagle's medium (dmem) (gibco-brl, gaithersburg, md, usa) containing % heat-inactivated fetal bovine serum (gibco-brl), supplemented with l-glutamine ( mm), streptomycin ( g/ml) and penicillin ( u/ml) (gibco-brl) at • c in a % co incubator. forty-eight table sirnas sequences used to inhibit gag and env genes and negative controls. hours before transfection, × cells were seeded to % confluence in -well culture plates with dmem ( ml/well). for the transfection of adherent hek cells, a total of g of reporter plasmid and l of sirna ( m) mixed with superfect transfection reagent (qiagen, valencia, ca, usa) was used according to the manufacturer's instructions. the cells were incubated at • c in the presence of % co . forty-eight hours after transfection, the expression of egfp-target (gag or env) fusion proteins was observed directly under an inverted fluorescence microscope. this experiment was done in triplicate. immunocytochemistry was also conducted to observe the expression of proteins. immunocytochemistry was performed to confirm the expression of egfp-gag/env fusion proteins. cells ( × ) were seeded to % confluency in -well culture plates with medium ( ml/well), and transfections were carried out after h. fortyeight hours post-transfection, pegfp-gag/env-transfected cells and non-transfected cells (negative controls) were fixed in % paraformaldehyde (merck, whitehouse station, nj, usa) for min at room temperature, washed three times with pbs and then permeabilized with . % triton x- (sigma-aldrich, st. louis, mo, usa) for h at room temperature. the cells were blocked for h with % bovine serum albumin (bsa) and % goat serum in pbs. samples were incubated with primary antibodies anti-htlv-i p (for gag) and anti-htlv-i gp (for env) (abcam inc., cambridge, ma, usa) at room temperature overnight. the secondary antibody used was alexa fluor mouse antibody (cat# a- , invitrogen, carlsbad, ca, usa). nuclei were stained with dapi (vysis, des plaines, il, usa). cells were visualized using a confocal laser scanning microscope (clsm) (lsm ; carl zeiss, oberkochen, germany) with in × objective lens in immersion oil, with a numerical aperture of . . an argon laser of nm was used to excite gag or env labeled with secondary antibodies, and emission was measured at nm. egfp fluorescence was excited with an argon laser at nm, and the emission was measured at nm. visualization of the light scattering for each excitation wavelength was recorded in the multitracking mode using separate detection channels. image analysis was carried out using carl zeiss zen software (carl zeiss). control and test images were captured using identical settings. the fluorescence of egfp-target in transfected hek cells was observed h after transfection under an inverted fluorescence microscope (olympus ix , tokyo, japan) exciting the cells at nm. light microscope and fluorescence images in the same field were captured. after analysis by fluorescence microscopy, the cells were trypsinized, harvested and washed twice with phosphate-buffered saline, ph. . (pbs). after centrifugation at rpm for min at • c, the cell pellet collected was resuspended in pbs to measure fluorescence using a bd facscalibur tm flow cytometer (bd biosciences, san jose, ca, usa) with filters (emission, nm; excitation, nm). non-transfected hek cells were used as a control. the data collected by flow cytometry were analyzed with the cellquest software (bd biosciences). the mean fluorescence intensity of the cell population that exceeded the fluorescence intensity of control cells was calculated. forty-eight hours post-transfection, total rna from transfected and non-transfected cells was extracted using trizol tm reagent (invitrogen), analyzed for integrity by % agarose gel electrophoresis and treated with dnase i amplification grade following manufacturer instructions (invitrogen). one microgram of treated rna was then reverse-transcribed into cdna using the high capacity cdna reverse transcription kit (applied biosystems) according to manufacturer's recommendations. pcr amplification was carried out in -well plates with optical adhesives. the final reaction volume for the target reaction was l, consisting of . l taqman universal pcr master mix (applied biosystems), . l specific probe (fam and mgb labeled, m), . l of each specific primer ( m), l of cdna template and l of nuclease-free water. the final reaction volume for the endogenous gene (gapdh) reaction was l, consisting of l of taqman universal pcr master mix (applied biosystems), . l of human gapd (gapdh) endogenous control (vic/tamra probe, primer limited, part number e) from applied biosystems, l of cdna template and . l of nuclease-free water. for each run, standard cdna, sample cdna and no template con- nuclei were stained with dapi. the cells transfected with pegfp-env presented the same profile (data not shown). (b) fluorescence microscopy of hek cells co-transfected with pegfp-gag + negative control sirna, pegfp-gag alone (without sirna), pegfp-gag + sirnas scr gag and sirna gag , gag or gag . (c) fluorescence microscopy of hek cells co-transfected with pegfp-env + negative control sirna, pegfp-env alone (without sirna), pegfp-env + sirnas scr env and sirnas env , env or env . the upper panels represent the cell fluorescence images recorded h post-transfection and the lower ones represent the light microscopic view of cells in the same field ( ×). (d) mean fluorescence intensity of cells co-transfected with pegfp-gag and sirnas, and transfected with pegfp-gag alone (without sirna) by flow cytometry. mean fluorescence was reduced significantly (*p < . ) in sirnas gag , gag and gag compared to pegfp-gag and sirna scr gag co-transfected cells. (e) mean fluorescence intensity of cells co-transfected with pegfp-env + sirnas, and transfected with pegfp-env alone (without sirna) by flow cytometry. a significant reduction (*p < . ) was observed in sirna env , env and env co-transfected cells compared to pegfp-env and sirna scr env co-transfected cells. means and standard deviations from three independent experiments are shown in (d) and (e). trol were all assayed in duplicate. the reaction conditions were: • c for min, • c for min, followed by cycles of s at • c and min at • c. the sense and antisense primers for the env gene were -tctagtcgacgctccaggatatg- and -cagttggctgggttcggtat- , respectively. the sense and antisense primers for the gag gene were -cccccaagttcttccagtca- and -tgccatgggcgatggt- . both probes were marked with the reporter dye fam and the quencher mgb, which were -ccccatctggttcct- for the env and -ccacatggtgcccc- 'for gag, respectively. data were reported as means ± sds. statistical analysis was performed by one-way analysis of variance (anova), and the dunnet multiple comparison test was applied to determine which means were significantly different (p < . ) from the control mean. all analyses were carried out using the graphpad prism software package (graphpad software, san diego, ca, usa). before using the vectors constructed for the inhibition experiments, the expression of egfp-gag and of egfp-env was analyzed in transfected cells under a fluorescence microscope at various time points, post-transfection. immunocytochemistry was also preformed to confirm the expression of fusion proteins. both vectors expressed the fusion gene efficiently ( fig. a-c) . the fluorescence at h post-transfection was chosen for experiments with both constructs. pegfp-gag + sirnas and transfected with pegfp-gag alone (without sirna) compared to pegfp-gag + sirna scr gag co-transfected cells. the differences in gene expression were not statistically significant. (b) mean relative expression of the env gene in cells co-transfected with pegfp-env + sirnas and transfected with pegfp-env alone (without sirna), compared to pegfp-env + sirna scr env co-transfected cells. significant reduction (*p < . ) of gene expression was obtained in cells co-transfected with sirnas env or env and pegfp-env compared to sirna scr env co-transfected cells. means and standard deviations from three independent experiments are shown in (a) and (b). the silencing of gfp was confirmed by fluorescence microscopy and flow cytometry in at least three independent experiments for the gag and env genes. hek cells were observed h posttransfection under a fluorescence microscope to determine the silencing effect of sirnas on gag and env protein expression in cultured cells (fig. b and c) . the data showed no difference between cells transfected with pegfp-gag or pegfp-env alone and cells co-transfected with pegfp-gag or pegfp-env and scrambled sir-nas (sirna scr gag or sirna scr env). on the other hand, the cells co-transfected with sirnas gag , gag and gag and pegfp-gag demonstrated less fluorescence than those co-transfected with pegfp-gag and scrambled sirna (fig. b) . for the env gene, the fluorescence intensity of hek cells co-transfected with pegfp-env and negative control or scrambled was stronger than that cells co-transfected with pegfp-env and sirnas env , env or env (fig. c) . the inhibitory effects of sirnas on the expression of egfp were validated quantitatively by flow cytometry, as shown in fig. d and e. egfp down-regulation was quantified by assessing the mean fluorescence of egfp-positive cells. no significant differences in scrambled sirna co-transfected cells were detected when compared to cells transfected with pegfp-gag or pegfp-env alone. mean fluorescence was reduced significantly by %, % and % in sirnas gag , gag and gag , respectively (p < . ), compared to pegfp-gag and sirna scr gag co-transfected cells. significant reductions (p < . ) of %, % and % were observed in sirnas env , env and env co-transfected cells respectively, compared to pegfp-env and sirna scr env co-transfected cells. . . gag and env mrna quantification gag and env mrna was quantified h post-transfection by real-time quantitative pcr using specific primers and probes and gapdh expression as endogenous control. although no significant differences were observed, the mean results demonstrated that sirnas gag and gag reduced gag gene expression by % and %, respectively, compared to sirna scr gag co-transfected cells. no silencing effect was observed for sirna gag (fig. a) . when env gene expression was analyzed, the results showed a significant reduction (p < . ) of gene expression by % or % in cells co-transfected with sirnas env or env and pegfp-env, respec-tively, compared to sirna scr env co-transfected cells (fig. b) . the results demonstrated that sirna env reduced env gene expression by %, but this did not reach statistical significance. rnai technology is accepted by the scientific community as a potential clinical tool against infectious diseases (ma et al., ; haasnoot et al., ) . to test htlv- gag and env gene silencing by rnai, these genes were cloned downstream of the egfp gene in a plasmid report system. reproducible expression of fusion protein was observed after h by fluorescence microscopy, providing an efficient reporter system for further experiments. this reporter system was co-transfected with sirnas in hek cells. based on the fluorescence data, flow cytometry and real time quantitative pcr, two sirnas targeted to the htlv- gag gene reduced efficiently gene expression by different amounts compared to the negative sirna, scrambled sirnas and controls without sirna. the three sirnas targeted to the env gene were effective, inhibiting gene expression compared to the control. different levels of inhibition can be justified since the efficiency of different sirnas against the same target rna can vary significantly. the characteristics of the target rna and intrinsic mechanisms of sirna itself play a role in silencing (kurreck, ). one of the two strands can be assembled preferentially into the risc depending on the stability of the two sirna strands; if the complementary strand is not assembled into risc, silencing would thus not be observed. furthermore, the sequence of the target rna could be inaccessible to the complementary strand, affecting the silencing (schubert et al., ) . these factors may partly explain the variation of gene suppression observed in this study. in addition, sirna efficiency is strongly related to complementarity. sirna may recognize and bind to the target sequence with incomplete homology, resulting in merely repression of translation without mrna target degradation. this fact may explain the difference between the results obtained by flow cytometry and real-time pcr. despite the global spread of htlv- , and because of the scarce number of studies conducted on infected patients, it is difficult to estimate its prevalence, but approximately - million people are infected worldwide (cooper et al., ) . most htlv- -infected individuals remain asymptomatic, but the virus is associated with severe diseases subdivided into neoplastic (atll), inflammatory syndromes (ham/tsp and uveitis) and opportunis-tic infections (strongyloidiasis, scabies, etc.). current treatment of htlv- -related diseases is aimed primarily at reducing symptoms. combination chemotherapy, allogeneic stem cell transplantation, molecular-targeted agents, nucleoside analogues and interferons have been reported as therapeutic strategies for these diseases, but with only short-term benefits and, in some cases, a short median survival (verdonck et al., ; yasunaga and matsuoka, ; ishitsuka and tamura, ; oh and jacobson, ) . the identification of new targets is an important part of drug development. many investigators are using rnai to combat viral infections. the first study reporting the use of sirna for the treatment of viral infection showed an effective and specific degradation of viral mrna, as well as a resultant ablation of the specific viral protein (bitko and barik, ) . the use of sirnas for the treatment of hiv- infection, specifically directed against the gag gene (novina et al., ) , was reported subsequently. at the same time, studies have demonstrated the inhibition of the regulatory hiv- rev gene and infectivity factors such as the vif and nef genes (jacque et al., ) . the structural htlv- gene gag is related to virion assembly and release (le blanc et al., ) , whereas the env protein is involved in the interaction between the surface receptor of the host cell and the virus (manel et al., ) . considering previous studies showing efficient inhibition of hiv- structural proteins gag and env (novina et al., ; park et al., park et al., , , and the importance of the corresponding genes in htlv- for its biology, in the present study, rnai technology was utilized to determine its efficacy for the inhibition of htlv- structural genes. in addition, few studies have used rnai during htlv- infection. this technique has been employed previously to demonstrate the importance of glut- in htlv- cell entrance (manel et al., ) , some regulatory tax gene functions (nomura et al., ; qu et al., ; hara et al., ; jung et al., ; hieshima et al., ) , virus release (blot et al., ) and host cell gene functions (tomita et al., ) . most of these studies used sirnas against genes of the host cells or the regulatory tax gene. no other report used sirnas for htlv- structural genes. in conclusion, this is the first report that demonstrates the specific silencing of htlv- gag and env genes by sirna. the present findings could be useful for studies of gag and/or env gene function and provide a tool for the development of new therapeutic strategies for the treatment and prevention of development htlv- -related symptoms in infected individuals. 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effective small interfering rnas targeting matrix and nucleocapsid protein gene inhibit influenza a virus replication in cells and mice this work was financially supported by fundaç ão de amparo a pesquisa do estado de são paulo (fapesp), centro de terapia celular da fundaç ão hemocentro de ribeirão preto (ctc/fundherp) and conselho nacional de desenvolvimento científico e tecnológico (cnpq). the authors would like to thank sandra navarro for the artwork and svetoslav n. slavov for revising the english text. key: cord- - fwkf fn authors: nan title: subject index, volumes - date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: fwkf fn nan hiv; ltr circles; new marker ( ) hiv antigen/antibody combined assay; hiv; serology; hiv- p antigen assay ( ) ( ) ( ) ( ) yeast expression; pichia pastoris; sars-cov; n protein ( ) enzyme analysis; flaviviruses; rt-pcr key: cord- - bsixsgd authors: chirnside, e.d.; francis, p.m.; de vries, a.a.f.; sinclaira, r.; mumford, j.a. title: development and evaluation of an elisa using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus date: - - journal: j virol methods doi: . / - ( ) -u sha: doc_id: cord_uid: bsixsgd a recombinant glutathione-s-transferase fusion protein expressing amino acids – of equine arteritis virus (eav) g(l) (rg(l) – ) was tested in an elisa for its ability to detect serum antibodies to eav. host antibodies induced following eav infection bound the recombinant antigen by elisa. the elisa specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. a good correlation existed between eav neutralizing antibody titers and elisa absorbance values (r = . ). the sensitivity and specificity of the elisa were . and . %, respectively, compared with the eav neutralization test and the recombinant antigen did not crossreact in elisa with equine sera directed against other common equine respiratory viruses. three post-eav infection equine sera raised against different eav isolates reacted strongly in the elisa, as did two equine sera raised against eav vaccines, indicating that the viral epitope was conserved between the viruses tested. following vaccination with an inactivated whole virus vaccine, antibody detected with the recombinant antigen elisa preceded the development of a virus-neutralizing response. the study demonstrates the potential application of rg(l) – as a diagnostic antigen. of virologicul methodc ( ) equine viral arteritis (eva) has to date only been reported to infect horses (doll et al., ; chirnside, ) although seropositive donkeys have been found (paweska and barnard, ) . the disease has been known for some years and manifests itself with widely varying clinical signs. in its most severe form equine arteritis virus (eav) infection causes abortion (doll et al., ) and foal death (golnik et al., ; vaala et al., , although it is more usual for this virus to cause mild respiratory disease (chirnside, ) . disease outbreaks are identified infrequently due to clinically inapparent infection and field isolates of the virus are rare. the causative agent, eav, is a small positive single-stranded rna virus with a genome organization, mode of replication and gene expression strategy similar to that of corona-and toroviruses (de vries et al., (de vries et al., , den boon et al., ; snijder et al., ) . eav has recently been classified in the genus arterivirus (cavanagh et al., ) along with lactate dehydrogenase-elevating virus of mice (ldv) (godeny et al., ; kuo et al., ) , simian hemorrhagic fever virus (shfv) (plagemann and moennig, ) , and the virus causing porcine respiratory and reproductive syndrome (prrsv) (conzelmann et al., ) lelystad virus (lv) (meulenberg et al., ) . eav is transmitted by the respiratory and venereal routes, with a % carrier state existing in seropositive stallions (timoney et al., ) making the latter route a particular cause for concern, as these stallions shed virus in their semen and may consequently infect broodmares neu et al., ) . eva is geographically widespread (chirnside, ) and most european and north american countries have eav-seropositive animals. some outbreaks have occurred following the importation of persistently infected animals causing primary transmission of eav by the venereal route, followed by secondary respiratory spread (wood et al., ) . in the light of the potential economic importance of this disease to the horse breeding and racing industry, a requirement exists for both prophylactic treatment and reliable serodiagnosis of infection. laboratory-based serological tests include eav virus neutralization test (nt), complement fixation (cf) and enzyme-linked immunosorbent assay (elisa) (senne et al., ; fukunaga and mccollum, ; cook et al, ) . the elisa has a relatively low specificity when applied to sera from horses previously vaccinated against equine influenza and herpesviruses; this is primarily due to host antibody induced by tissue culture contaminants of these vaccines reacting with cell culture-derived antigen present in the whole-virus elisa antigen. the cf test has limited temporal sensitivity and is consequently only useful for serodiagnosis up to weeks postinfection. at present, nt results from designated laboratories are internationally accepted for import/export testing. infection with eav elicits a virus-neutralizing antibody response within - days. stallions which have previously been exposed to eav, and therefore may be shedders of infectious virus, can be detected by the nt as a discernible level of circulating neutralizing antibody persists for many years after recovery (gerber et al., ) . the nt is sensitive, but suffers from the disadvantage that it requires laboratory testing to maintain cell cultures and stocks of infectious virus. in addition, the nt takes several days to complete. laboratories use varied test protocols and different reagents which can lead to disparate results. moreover, the nt does not differentiate between the host serological response induced by vaccination and that resulting from natural infection. it has recently been demonstrated that neutralizing monoclonal antibodies recognize a determinant on the large envelope glycoprotein, g, (balasuriya et al., ; deregt et al., ) . this paper describes an indirect elisa using a recombinant glutathione-stransferase fusion protein (smith and johnson, ) as an antigen to screen equine sera for the presence of antibodies to eav, and its evaluation as a diagnostic test with large numbers of equine serum samples. plasmid dna manipulations were carried out as described previously (sambrook et al., ) . transformations with plasmid pgex- x (smith and johnson, ) were carried out in escherichia coli tgl and recombinant clones selected by antibiotic resistance. clones were subsequently screened for fusion protein expression after iptg induction by analysis in sds- . % polyacrylamide gels, and the eav-specific insert size, orientation and in-frame insertion confirmed by restriction endonuclease digestion analysis and dsdna sequencing of purified plasmid dna (chirnside et al., ) . the expression construct srsal, expressing rg, - , comprised nucleotides , - , of eav open reading frame (orf) fused in-frame to the carboxyl-terminus of the glutathione-s-transferase gene in the expression vector pgex- x. fusion protein was affinity purified using glutathione sepharose b (pharmacia). a -ml culture of bacterial cells expressing recombinant antigen was harvested by centrifugation for min at g and °c resuspended in ml mm tris-hcl (ph . , mm edta, mm nacl (ste) and recentrifuged to pellet the cells. the supernatant was discarded and the cells resuspended in ml ste, ~ lysozyme ( mg/ml) added and the tube incubated at °c for min. the tube was then frozen at - °c overnight followed by a short immersion in a boiling water bath to thaw the frozen suspension. dnase ( pg) and ~ of m mgcl, were then added to the thawed cells and the tube incubated at °c for min. triton x- was then added to a final concentration of l%, the mixture kept on ice for min and centrifuged at , g and °c for min. glutathione sepharose b gel was added to the supernatant and the solution mixed by gentle agitation on a rotary mixer for min. the gel was washed times with pbs and the recombinant antigen eluted from the gel with reduced glutathione ( mm glutathione, mm tris-hcl (ph . )). eluate was collected in l-ml fractions and analyzed by sds-polyacrylamide gel electrophoresis prior to pooling fractions containing purified protein. pooled protein was dispensed into -~ aliquots and frozen at - °c until used. protein concentration was determined with a modified lowry reagent solution from a protein assay kit (sigma). optimal concentrations of reactants were determined by checkerboard titration. immulon microtiter plates (dynatech) were coated with gst or fusion protein diluted in mm carbonate buffer (ph . ) and kept overnight at °c. plates were then washed times with pbs containing . % tween (pbst) and ,ul of pbst containing % goat serum (pbstg) added to each well. following incubation at °c for h and washes with pbst, ~ of horse sera diluted lo-* in pbstg was added to each well and the plates incubated for min at °c. plates were then washed times with pbst, ~ of a lo-" dilution of affinity-purified biotin-labeled goat anti-y chain specific horse igg (kpl) was added to each well and the plates incubated for min at °c. the plates were then washed times with pbst, and ~ of a -j dilution of streptavidin-peroxidase (kpl) in pbstg added to each well and the plate incubated at room temperature for min. after a final washes with pbst, substrate solution ( . mg/ ml o-phenylenediamine dihydrochloride dissolved in mm phosphate-citrate buffer (ph . ), containing . % sodium perborate) was added to each well and the plate incubated at room temperature for min, after which the reaction was stopped by the addition of ~ m h,so, to each well and the absorbance read at nm. each serum sample was assayed in duplicate wells against both purified gst and fusion protein and the mean absorbance value taken as the a,,,, reading to each antigen. each elisa was validated by the inclusion of nt-/elisam control sera. the mean absorbance value for these control sera was < . in each individual elisa test, with a grand assay mean over all tests of . . in addition, nt+/elisa+ control sera were also run as standards in each test to confirm the sensitivity of the assay. immunoblots were carried out with equine sera diluted o-'. using a modified version of the elisa protocol after electrophoretic transfer of proteins from sds-polyacrylamide gels onto nitrocellulose membrane. non-specific binding of equine antibody to the nitrocellulose membrane was first blocked by overnight incubation at °c in pbstg. the membrane was then washed times in pbst and the elisa protocol for binding and washing the components of the antibody sandwich performed as above with washes, each of min between steps. to develop a signal from the immunoblots the ecl detection system (amersham) was employed using the manufacturer's protocol. the eav neutralization test was carried out according to the method of senne et al. ( ) with minor modifications. all sera were initially screened at a : dilution in replicate wells of a microtiter plate. virus neutralizing sera were subsequently titrated in replicate wells from an initial dilution of : to : . each individual nt included positive control sera, a virus control to ensure that lootcid,, of the bucyrus strain of eav was added to each well, and a series of rk- cell controls. individual wells were scored for > % cytopathic effect (cpe) after h incubation ( °c % co,) and titers calculated according to the formula of karber ( ) . a serum was considered seropositive when it had an nt titer > : (> log,, . ) and a -fold rise in titer regarded as a seroconversion following virus infection or vaccination. throughout the study, the same batch of the bucyrus strain of eav and of guinea pig complement was used, and rk- cells were used at passage numbers - . this ensured excellent comparability between eav neutralization tests. the field sera used for this study were submitted to the diagnostic service of the animal health trust. the virus type-specific sera were provided by dr. yoshio fukunaga of the equine research institute, japan, and the panels of european and american sera obtained from dr. margaret lucas, central veterinary laboratories, uk. fusion proteins derived from eav g, have been shown to react with postinfection, eav neutralizing horse sera (chirnside et al., ) . the glutathione-s-transferase of fusion protein expressed by plasmid construct rsal contains amino acid residues - of eav g, derived from the bucyrus isolate. this fusion protein (rg,, - ) was expressed at high levels in iptg-induced cells and purified readily by affinity chromatography on glutathione sepharose b; the purified rg, - contained few contaminating bacterial proteins (fig. la) . in immunoblots to equine sera (fig. lb-d) rg, - was very strongly recognized by eav neutralizing sera (fig. lb) , faintly by the antiserum against gst (fig. id) and only very slightly by the non-neutralizing equine serum tested (fig. lc) . purified gst was recognized by equine sera raised specifically to gst and also by both virus neutralizing and non-neutralizing equine sera. the optimal antigen and antibody concentrations in elisa were determined by checkerboard titration. a serum dilution of l/ and antigen concentration of . pg per well were selected to give maximum discrimination between nt+ and nt-sera. the majority of the equine sera had some reactivity to gst, in the absorbance range o- . , at these reagent concentrations. consequently the absorbance value to gst was subtracted from the absorbance value to the recombinant protein to give an eav-specific figure for each test sample. equine sera raised specifically against equine herpesvirus types - , equine rhinovirus types and and equine adenovirus did not recognize rg, - in elisa; the recombinant antigen was only bound by eav neutralizing equine sera. in order to evaluate the potential of rg, - as antigen for a diagnostic elisa, and to compare results directly with the eav neutralization test, the elisa absorbances of equine sera were compared with their nt titers (fig. ) . from the data plotted in fig. an a, ,,] > . (the y intercept plus two standard deviations as determined by linear regression analysis in fig. ) was taken as the cut-off point determining an elisa seropositive value. table shows a numerical break-down of the results using these seropositive/seronegative cut-off values. virus neutralizing sera were clearly distinguishable in elisa from samples seronegative in the nt. the elisa sensitivity in detecting nt seropositives was . % ( detected from ). only one virus-neutralizing sample, with a low antibody titer (log,, . , was elisa-(a,,, = . ). the specificity of the elisa to detect nt seronegative samples correctly was . % ( detected from ). this was due to the detection of samples which had absorbance readings > . but which were negative by nt (log,, < . ). this nt-/elisa+ group included blood samples from horses held on equine premises affected during the eav outbreak in england (wood et al., , diagnostic samples from assorted horses submitted for serological screening which included an eav nt, and blood samples from horses which had previously been vaccinated with either one or two doses of an inactivated eav vaccine. the anomalous equine sera were subjected to further investigation by: ( repeating the nt; ( ) repeating the elisa with a fresh batch of rg, - ; ( ) elisa nt-samples were elisa' to all eav-specific antigens; this number comprised serum samples from vaccinated horses, from outbreak-associated animals and two from sera submitted for general serodiagnosis. paired sera originating from horses during the course of the uk eav outbreak (wood et al., ) seroconverted in both the nt and elisa tests from nt-/ elisato nt+/elisa+. a further paired sera from horses associated with the eav outbreak did not seroconvert in either diagnostic test. paired sera from horses were tested by nt and elisa both prior to, and - weeks after administration of two doses of an inactivated eav vaccine. among the vaccinees l/ ( %) was nt-/elisa-, / ( %) were nt+/elisa+ and / ( %) had an elisa antibody response detectable in the absence of any vaccine-induced neutralizing antibody. the possible effect of variation in g, between viruses was investigated by comparing elisa absorbance readings and nt titers of two post vaccination sera raised against a live and killed vaccine derived from the bucyrus strain, and postinfection eav isolate specific equine sera (fig. ) . although differences between absorbance values were evident between the sera, all were positive by elisa. the homologous sera to rg, - , derived from infection with the bucyrus isolate (bucyrus) or vaccination with inactivated virus (kill bucyrus) had higher absorbance values than equine sera raised to the heterologous virus isolates ( -ky-al, wroclaw- ) and the live attenuated vaccine (arvac; fort dodge laboratories) although little variation in nt antibody titer to the bucyrus isolate of eav (from which the recombinant antigen is derived) was detectable between isolates. to evaluate the utility of rg, - two further panels of equine sera were assayed by elisa. the panels, one of european and one of american origin, comprised a mixture of postinfection and eav-negative sera and have been used as reference sera to monitor the standard of virus neutralization testing between laboratories (unpublished). with these two serum panels (tables and ) the elisa sensitivity was % and the specificity %; / nt+ sera tested elisa', / nt-sera tested elisa-and / nt-sera tested elba+. the serological reactivity of a recombinant eav g, fusion protein is reported and its use is described for the detection of equine antibodies to eav in elisa. the assay proved to be highly sensitive and specific for the detection of eav antibodies; it correlated well with results from the eav microneutralization test and detected postinfection seroconversions in horses following natural infection and vaccination. in the elisa, field sera diluted : reacted with gst causing a variable background absorbance of o-os. it should be possible to reduce this background absorbance by cleaving the gst moiety from the g, fusion protein, or by cloning eav g, - into a different expression vector. however in its present format, the correlation between the rg, - elisa and nt is high (r = . ) with the elba detecting additional seropositives to the nt. the elisa+/ nt results could be due to differences in sensitivity between the two tests or because the elisa detects equine antibodies that bind eav g,, of which those capable of neutralizing virus in a nt are a subpopulation. alternatively, the detection of elisa+/nt-samples, including vaccinated horses, poses questions about the specificity of the elisa and its biological relevance when compared with the nt. however, following one or two doses of an inactivated vaccine, it was possible to demonstrate elisa+/nt-results in % of vaccinees. if a similar situation exists following natural infection with eav, then the additional seropositive field samples detected by elisa may have originated from horses previously infected with eav, but in which a virus neutralizing antibody response was not induced or in which the response has dropped below detectable levels. this observation requires further investigation since the presence of circulating virus neutralizing antibody has to date been accepted as evidence of prior exposure to eav, either through infection or vaccination. the rg, . - elisa results suggest this may not be the case. the membrane topology of eav g, remains to be established. however, the hydropathy profile (de vries et al., ) and position of the sole n-glycosylation site (den boon et al., ) both determine that the protein ectodomain is likely to encompass residues - . by testing > equine sera in elisa to g, - , we have demonstrated that amino acid residues - of the bucyrus strain of eav g,_ encompass a highly immunoreactive antigen which correlates closely with the host virus neutralizing response. eav g, has been shown to contain a major antigenic site (chirnside et al., ) and virus neutralizing murine monoclonal antibodies react with a protein of a molecular size ( kda) equivalent to g,, by western blots (balasuriya et al., ; deregt et al., ) . eav strain variation has been demonstrated at the nucleotide level by rnase tl fingerprinting (murphy et al., (murphy et al., , and by direct sequencing of the eav n-and m-genes (chirnside et al., ) and antigenic variation inferred by cross-neutralization studies (fukunaga et al., ) . the equine immune response to eav induced by different eav isolates was detectable in elisa with rg, - derived from the bucyrus isolate, implying a high degree of conservation of this epitope. to substantiate this finding sequencing studies of eav g, from different isolates is currently being undertaken. changing from reliance solely on the nt, to an initial rapid screen by elisa followed by neutralizing antibody testing of elisa+ samples would improve the speed of diagnosis for eav seronegative horses, lower the cost of initial testing, and potentially eradicate differences in test results between laboratories. additionally, the enhanced detection of seropositive animals allied to a vaccination policy and selective breeding practices would allow the better control and possible eradication of eav from the breeding population. the expression and purification of g, - is simple and the elisa is easily standardized between different testing laboratories. since the recombinant protein is not infectious it affords the opportunity to undertake eav serodiagnostic assays rapidly and safely without virus containment facilities. in addition, the use of a purified protein expressed in a bacterial system removes the problem of non-specific antibody crossreactivity to cell culture-derived antigen which has plagued previous eav elisa tests (cook et al., ) . a k envelope glycoprotein of equine arteritis virus expresses neutralisation determinants recognised by murine monoclonal antibodies revision of the taxonomy of the coronavirus, torovirus and arterivirus genera equine arteritis virus: an overview comparison of m and n gene sequences distinguishes variation amongst equine artcritis virus isolates equine arteritis virus neutralizing antibody in the horse is induced by a determinant on the large envelope glycoprotein g molecular characterisation of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group the effects of vaccination with tissue culture-derived viral vaccines on detection of antibodies to equine arteritis virus by enzyme-linked immunosorbent assay all subgenomic rnas of equine arteritis virus contain a common leader sequence the structural proteins of equine arteritis virus monoclonal antibodies to equine arteritis virus proteins identify the g, protein as a target for virus neutralisation an outbreak of abortion caused by the equine arteritis virus complement fixation reactions in equine viral artcritis induction of immune response and protection from equine viral artcritis (eva) by formalin inactivated-virus vaccine for eva in horses an attempt to protect against persistent infection of equine viral arteritis in the reproductive tract of stallions using formalin inactivated-virus vaccine y ) use of the serum neutralisation test for equine viral arteritis with different virus strains serological investigations on equine viral arteritis complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (ldv) yxl) natural equine viral arteritis in foals beitrag zur kollektiven behandlung pharmakologischer reihenversuche. naunyn-schmiedebergs arch lactate dehydrogenaseelevating virus (ldv): subgenomic mrnas, mrna leader and comparison of 'terminal sequences of two ldv isolates the recovery of virus from horses with experimental cases of equine arteritis using monolayer cell cultures of equine kidney responses of vaccinated and non-vaccinated mares to artificial insemination with semen from stallions persistently infected with equine arteritis virus lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav analysis of genetic variation among strains of equine arteritis virus genomic variability among globally distributed isolates of equine arteritis virus persistent infection of the reproductive tract of stallions persistently infected with equine arteritis virus serological evidence of equine arteritis virus in donkeys in south africa lactate dehydrogenase-elevating virus, equine arteritis virus and simian hemorrhagic fever virus: a new group of positive-strand rna viruses molecular cloning: a laboratory manual equine viral arteritis: a standard procedure for the virus neutralisation test and comparison of results of a proficiency test performed at five laboratories single-step purification of polypeptides expressed in escherichia coli as fusion proteins with glutathiones-transferase the coronaviruslike superfamily demonstration of the carrier state in naturally acquired equine arteritis virus infection in the stallion fatal, congenitally acquired infection with equine arteritis virus in a neonatal thoroughbred the first recorded outbreak of equine viral arteritis virus in the united kingdom this work was funded by the animal health trust and maff contract research award csa . patent no. gb . covers the use of rg,. key: cord- -nvilxnzl authors: adachi, d.; johnson, g.; draker, r.; ayers, m.; mazzulli, t.; talbot, p.j.; tellier, r. title: comprehensive detection and identification of human coronaviruses, including the sars-associated coronavirus, with a single rt-pcr assay date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: nvilxnzl the sars-associated human coronavirus (sars-hcov) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (sars). this study presents a single-tube rt-pcr assay that can detect with high analytical sensitivity the sars-hcov, as well as several other coronaviruses including other known human respiratory coronaviruses (hcov-oc and hcov- e). species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the sars outbreak in toronto, canada. severe acute respiratory syndrome (sars) is a newly described, emerging infectious disease presenting with high fever and atypical pneumonia. the disease first appeared in the guangdong province of china and quickly spread to other countries; outbreaks were reported in mainland china, vietnam, singapore, hong kong, taiwan and canada (drazen, ; lee et al., ; poutanen et al., ; tsang et al., ; vu et al., ; who, a) . a new, previously unknown coronavirus was first isolated from patients with sars at the university of hong-kong (peiris et al., a; who, a) ; this was quickly followed by reports from several other laboratories of the demonstration of a new coronavirus in samples from patients with sars (drosten et al., ; ksiazek et al., ; poutanen et al., ; who, a) . the complete genomic sequence of the virus has been determined (marra et al., ; rota et al., ) and confirmed the classification of the sars-associated agent as a new coronavirus, distinct from the previously known groups of coronavirus (marra et al., ; rota et al., ) but with some relationship with group (snijder et al., ) . experimental infection of cynomolgous monkeys permitted the fulfillment of koch's postulates for this pathogen and firmly established the sars-associated human coronavirus (sars-hcov) as the etiologic agent of sars . this study presents a rt-pcr protocol that allows for the detection of the sars-hcov in clinical samples. this single-tube rt-pcr is based on consensus primers targeting conserved regions of coronavirus genome sequences and allows for the detection and species identification of several coronaviruses including sars-hcov, with high analytical sensitivity. this assay is expected to be helpful in fulfilling the world health organization laboratory case - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.jviromet. . . definition of sars, which recommends the use of two different pcr assays (who, b) . in addition, even if the sars-hcov does not reappear in the human population, this assay will still be useful, since it provides a diagnostic test for the respiratory coronaviruses hcov- e and hcov-oc . rna was extracted with trizol reagent (life technologies), as per manufacturer's recommendations, from a lung biopsy from a patient with sars. the rna pellet was resuspended in l of mm of dithiotreitol with % (v/v) of rnasin ( - u/l, promega), serially diluted, aliquoted and frozen at − • c. quantification of the rna serial dilution was achieved by measuring the amount of sars-hcov rna in the aliquots with the realart hpa coronavirus rt-pcr (artus gmbh, hamburg, germany). human coronaviruses oc and e strains were initially obtained from atcc and passaged in cell culture as described (sizun et al., ) . stocks of viruses were titrated as described (sizun et al., ) . rna was extracted from the titrated stocks using trizol (life technologies) as per the manufacturer's recommendations and serially diluted. tissue culture-adapted infectious bronchitis virus (ibv) stocks, strain massachussets and strain baudette, were obtained from dr. e. nagy, ontario veterinary college, university of guelph, ontario. rna was extracted and serially diluted as for hcov-oc and hcov- e. during the sars outbreak in toronto ( ), several samples from patients with probable or suspected sars were referred to our laboratory for sars-hcov detection by rt-pcr. of these, were assayed using the new primers described here, including lung biopsies, bronchoalveolar lavages, nasopharyngeal swabs, throat swabs, pleural fluid samples, endotracheal aspirates, blood samples, stool samples and urine samples. rna was extracted from lung tissue using the rneasy kit (qiagen) as per the manufacturer's recommendations. for all other samples, rna was extracted using the guanidium thiocyanate buffer (gtc) extraction methods, as described (johnson et al., ) . primers were designed to target segments of the pol b coding region conserved among several species of coronavirus ( fig. ) . we used the sense primer coro ( -tga tgg gtt ggg act atc cta aat gtg a- ) and the antisense primer coro ( -gta gtt gca tca ccg gaa gtt gtg cca cc- ), homologous to the segments [ , ] and [ , ] of the sequence of the tor strain of sars-hcov (genbank accession number ay ), respectively. this was done using the qiagen one-step rt-pcr kit. each reaction was performed in a . ml tube (diamed pre ) in a total volume of l overlaid with l of mineral oil. each reaction mix contains l of × qiagen buffer, l of dntp mix (each dntp at mm concentration), l of each primer ( m stock), l of molecular grade double distilled water (ddh o) and l of the one-step enzyme mix (qiagen). the master mix was then aliquoted in tubes, to which l of template rna was added. the pcr thermal cycling was done on a stratagene robocycler , with an initial incubation at • c for min, followed by an incubation at • c for min, and cycles consisting of denaturation at • c for min, annealing at • c for min, and elongation at • c for min. extensive precautions against pcr contamination, as previously described (johnson et al., ) , were strictly observed. positive and negative controls, as well as extraction controls and controls for pcr inhibition, were set up essentially as described (johnson et al, ) . a l volume of each reaction was submitted to electrophoresis on % agarose or % nusieve : (biowhittaker molecular applications, rockland, usa) gels containing ethidium bromide. the gels were visualized on a uv transilluminator and photographed. each reaction mixture in which the expected bp amplicon was detected was digested with the restriction enzyme alui, which cuts within the sequence agct. the digestion reaction consisted of l of the rt-pcr mixture, . l of the × enzyme buffer, l of alui ( u/l, new england biolabs) and . l of ddh o for a total of l. the reaction was incubated at • c for h and analyzed by agarose gel electrophoresis. amplicons were submitted to automated sequencing, for both strands, using the pcr primers as sequencing primers. (coro rc). dots denote homology with the consensus sequence on top. the sequence alignment was calculated using clustalx for windows version . (thompson et al., ) and edited with genedoc version . for windows (nicholas k.b., ). sequencing was performed by the dna sequencing facility, centre for applied genomics, hospital for sick children. sequence editing and analysis were done using the programs generunner for windows version . (hasting software). sequence alignments were calculated using clustalx for windows version . (thompson et al., ) , with the default parameters for gap opening and gap extension. phylogenetic trees were inferred using treecon for windows version . .b ( van de peer and de wachter, ) , using a distance method; in brief, the distance was calculated without correction; the tree topology was inferred with the neighbor joining method and the trees re-rooted at the internode. bootstrap analyses were done with replicates. initially, an amino acid sequence alignment of the pol b open-reading frame (orf) was used to identify well-conserved regions across species of coronaviruses comprising the sars-hcov and members of the three previously described groups of coronaviruses. the corresponding nucleotides alignment was then used to design pcr primers targeting these well-conserved regions. fig. illustrates the alignment of the nt segments of these coronavirus sequences homologous to the segment [ , ] of the tor strain of sars-hcov, as well as the corresponding sequence of the newly described human coronavirus hcov-nl (fouchier et al., ; van der hoek et al., ) , along with the sequence of the primers. fig. illustrates the phylogenetic trees of the nucleotide sequences (fig. a) and amino acid sequences (fig. b) in that segment, demonstrating that there are enough sequence diversity in the region bracketed by the primers to allow for unambiguous species identification. using as templates the rna of sars-hcov, hcov- e and hcov-oc , as well as rna from two strains of ibv, we could readily and reproducibly obtain the expected bp amplicon. aliquots of a -fold serial rna dilution prepared from a lung biopsy sample of a patient with sars (see section ) were used to compare our assay with the realart hpa coronavirus rt-pcr (artus gmbh). the results are displayed in table and show that our assay has essentially the same sensitivity as that of the realart hpa coronavirus rt-pcr assay; it is estimated that the analytical sensitivity is between and genome copies. for hcov-oc , viral rna could be detected from as little as × − tcid (tissue culture infective dose ); for hcov- e, viral rna could be detected from as little as × − tcid . for all coronaviruses tested in this study the species was verified by sequencing; in all cases the sequence internal to the primers were identical to the expected sequences. based on the expected sequences of the amplicons a rapid screening test for species identification for the human coronaviruses was designed using the alui restriction enzyme. as illustrated on fig. , the sars-hcov amplicon is cut into -bp and -bp fragments, the hcov- e amplicon into -bp, -bp and -bp fragments, and the hcov-oc amplicon is not cut by alui; based on the reference sequence, the amplicon of ibv would not contain a alui site. nucleic acids from human tissue, or from other respiratory viruses whose presence was detected in various samples (including influenza a, respiratory syncytial virus, parainfluenza and adenoviruses), did not generate amplicons of bp. from the clinical samples listed above, the presence of the sars-hcov rna was demonstrated in one lung biopsy, two bronchoalveolar lavages, two nasopharyngeal swabs, two urine samples and one throat swab. table comparison between our rt-pcr assay and the realart hpa coronavirus rt-pcr (artus gmbh) dilution rt-pcr with coro and coro realart hpa coronavirus rt-pcr (genome copies) − + − + − + − + − + − − -column two shows the result (scored as positive or negative) obtained with our assay using l of the indicated rna dilution (see section ). for each dilution the assay was done on two different aliquots and the results were in agreement. column three shows the average (on two experiments) of the measured number of genome copies in l of the rna dilution with realart hpa coronavirus rt-pcr. this study presents a single-tube rt-pcr assay designed to detect several species of coronaviruses, including the sars coronaviruses, by using consensus primers designed from an alignment of the sequences of a region in the pol b orf of species of coronaviruses. a previous version of these primers, based on an alignment of four species of coronaviruses and of the berne torovirus, was used to demonstrate the presence of a new coronavirus in patients with sars in canada (kumar et al., ; poutanen et al., ) at a time when the complete sequence of the sars coronavirus was still unknown. the use of the revised primers and the optimization of the rt-pcr has led to an improvement in analytical sensitivity of three orders of magnitude compared to the previous assay. its sensitivity has proven sufficient for the detection of the sars-hcov in several clinical samples. the assay was designed to be broadly reactive with the genome of many coronavirus species; it is demonstrated here that it can detect coronaviruses from all four known groups of coronaviruses, including the hcov- e and hcov-oc , which are well-recognized human pathogens (holmes, ) . although not all the known coronavirus species were tested, it is expected that the primer pair can amplify with high analytical sensitivity the targeted genome segment from the species used in the design of the primers, with the possible exception of the porcine epidemic diarrhea virus (pedv) whose sequence has a mismatch at the third nt from the end of the primer coro (fig. ) . the current assay can detect with high sensitivity three coronaviruses known to infect humans, and sequencing of the amplicons would unambiguously identify the species (fig. ) . although there has not been yet an opportunity to test this assay on isolates of the newly described hcov-nl ( van der hoek et al., ) , it is predicted, based on the sequence alignment in fig. , that our rt-pcr assay would also detect hcov-nl , and sequencing of the amplicon followed by phylogenetic analysis (fig. ) would readily confirm the identity of the virus. based on the published sequence of hcov-nl , digestion of the amplicon by alui would yield a distinct pattern consisting of bands of bp and bp. since the sequences targeted by the primers coro and coro are conserved across several coronavirus species (and this is particularly clear at the amino acid level), this suggests strong selective pressure on that part of the genome. in turn, this suggests that the assay would still be able to detect mutant or variant strains of sars-hcov, as mutations would likely not occur in the regions targeted by the primers. to date, the various isolates of sars-hcov have all displayed a great genetic homogeneity (ruan et al., ) , which argues for the emergence of sars-hcov in the human population only very recently. however, should the virus emerge again and be allowed to propagate, its genetic diversity may well increase (although the genomes of many known coronaviruses appear relatively stable). of note, a blast search in the genbank database using the -nt segment of sars-hcov (fig. ) showed a % homology with sequences of sars-hcov deposited to date, including representative of all clusters (guan et al., ) , and an homology greater than % ( / ) with two other sequences of sars-hcov. as well, the recent sars outbreak suggests that other coronaviruses may emerge as new human pathogens. again, assuming that the conservation of the sequences targeted by the primers stems from strong evolutionary constraints, the rt-pcr assay described here would very likely detect such an emerging coronavirus. in this study a simple restriction enzyme analysis as a screening test was shown to be very helpful in identifying the sars-hcov. indeed, in all the samples that tested positive for sars-hcov in our laboratory during the toronto outbreak, the expected restriction pattern was demonstrated (and based on sequences deposited in genbank this pattern would have been demonstrated in all isolates of sars-hcov sequenced to date). however, because rna virus genomes are much more variable than dna virus genomes, one should not rely completely on such an analysis and the failure to obtain the expected digestion fragments (or if the amplicon is not cut, as is the case for hcov-oc ) should prompt the determination of the sequence of the amplicon. as an illustrative case in point, whereas the amplicon from the ibv strain baudette is not cleaved by alui, the amplicon from strain massachussetts is in fact cleaved by alui, as verified by both sequencing and digestion of the amplicon (data not shown). after sequencing the amplicon, a comparison of the sequence to that of several coronaviruses in a phylogenetic analysis, as in fig. , should permit a definitive identification. indeed, should a new coronavirus emerge, it appears likely that it could be detected and identified as a new virus by using our rt-pcr assay followed by sequencing and phylogenetic analysis. since the end of the global sars outbreak in , a total of three laboratory confirmed sporadic cases of sars in guangdong have occurred (who, ) , which however did not spread further, as well as three laboratory acquired incidents (normile, ) . while surveillance for sars is still recommended (who, b) there is great concern about the negative impact of a false positive laboratory test, and the world health organization has recommended protocols for the laboratory diagnosis of sars in the post outbreak period (who, b) , which includes the use of two different rt-pcr assays. the assay described here provides an alternative test that can be helpful in that regard. furthermore, because our assay detects several coronaviruses, it is quite feasible to use rna from a coronavirus other than sars-hcov as a positive control. in fact, our laboratory now uses as positive control rna transcribed from the cloned amplicon of the ibv; ibv has never been implicated in human infections. the use of ibv as a positive control removes a potential source of pcr contamination by sars-hcov genetic material in the laboratory. in this study, the presence of the sars-hcov was demonstrated in a variety of clinical samples, which illustrates the usefulness of the assay. the number of positive samples reported here is too small to draw firm conclusions but taking into account the distinction between probable and suspected cases of sars, as well as previous observations made earlier in our laboratory during the outbreak, it appears that the sars-hcov is more likely to be demonstrated in lower respiratory tract samples, such as broncho-alveolar lavages or lung biopsies rather than in a nasopharyngeal swab; this parallels the findings of other reports (peiris et al., b; tang et al., ) and it has been suggested that the sars-hcov appears at a high titer in the nasopharynx relatively late in the disease (peiris et al., b) . thus, the negative rt-pcr results on such samples do not reflect a poor analytical sensitivity of the assay but rather the biology of the virus. a systematic study of various samples obtained throughout the course of the disease in several patients will be required to determine the best testing algorithm. certainly in the control of the outbreak in toronto, carefully established epidemiological links between patients fitting the case definition seemed the most effective method, but laboratory confirmation of some cases in the chain also proved to be very helpful (wallington et al., ) . in summary, a single-tube rt-pcr assay for the detection of human coronaviruses, including the sars-hcov, has been developed. it is expected to remain effective with possible sars-hcov mutants and to be of value should other new coronaviruses emerge as human pathogens. identification of a novel coronavirus in patients with severe acute respiratory syndrome case cluster of the severe acute respiratory syndrome koch's postulates fulfilled for sars virus a previously undescribed coronavirus associated with respiratory disease in humans molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome coronaviruses comprehensive pcr-based assay for detection and species identification of human herpesviruses a novel coronavirus associated with severe acute respiratory syndrome severe acute respiratory syndrome (sars) in a liver transplant recipient and guidelines for donor sars screening a major outbreak of severe acute respiratory syndrome in hong kong mounting lab accidents raise sars fears coronavirus as a possible cause of severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus associated sars pneumonia: a prospective study national microbiology laboratory, canada, canadian severe acute respiratory syndrome study team comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infections comparison of immunofluorescence with monoclonal antibodies and rt-pcr for the detection of human coronaviruses e and oc in cell culture unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage interpretation of diagnostic laboratory tests for severe acute respiratory syndrome: the toronto experience the clustalx windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools a cluster of cases of severe acute respiratory syndrome in hong kong treecon for windows: a software package for the construction and drawing of evolutionary trees for the microsoft windows environment identification of a new human coronavirus sars in northern vietnam update: severe acute respiratory syndrome-toronto world health organization multicentre collaborative network for severe acute respiratory syndrome (sars) diagnosis, . a multicentre collaboration to investigate the cause of severe acute respiratory syndrome alert, verification and public health management of sars in the post outbreak period new case of laboratory confirmed sars in guangdong this work was supported by the canadian institutes for health research and by the department of paediatric laboratory medicine, hospital for sick children, toronto. key: cord- -muskjaw authors: black, elizabeth m; lowings, j.paul; smith, jemma; heaton, paul r; mcelhinney, lorraine m title: a rapid rt-pcr method to differentiate six established genotypes of rabies and rabies-related viruses using taqman™ technology date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: muskjaw a rapid and sensitive reverse transcriptase-polymerase chain reaction (rt-pcr) assay incorporating taqman™ probes has been developed that can distinguish among the six established rabies and rabies-related virus genotypes. taqman™ probes were designed and validated against rabies and rabies-related virus isolates, one isolate of the australian bat lyssaviruses (genotype ), and other non-rabies viruses important in the veterinary field. the n gene was used as the target for the probes as it is well conserved and has been intensively used to genotype rabies isolates. additionally, it was found to contain regions specific to each genotype conducive to probe design. the rt-pcr assay described amplifies a portion of the nucleoprotein gene of all rabies and rabies-related viruses, but none of the other viruses tested. inclusion of taqman™-genotype-specific probes in the rt-pcr assay permits rapid identification of the virus present. by combining rt-pcr with taqman™ genotyping probes suspect rabies virus isolates can be identified in a single closed tube system that prevents potential pcr-product carry over contamination. rabies viruses are in the order mononega irales (enveloped negative strand rna viruses with unsegmented genomes), family rhabdo iridae, and belong to the lyssa irus genus. based on the nucleotide sequence and deduced percentage of amino acid similarity in the nucleoprotein (n), the lyssa irus genus is divided into six established genotypes (bourhy et al., , . the genus is composed of classical rabies virus (genotype ) and the rabies-related viruses: lagos bat virus (genotype ), mokola virus (genotype ), duvenhage virus (genotype ), and european bat lyssaviruses (ebl) and (genotypes and , respectively) (familusi et al., ; crick et al., ; foggin, ; king and crick, ; bourhy et al., ) . the more recent australian bat lyssavirus, first reported in (fraser et al., ) , has been classified as a separate and distinct genotype (genotype ) within the lyssa irus genus (gould et al., ) (fig. ) . all mammals are susceptible to rabies, but the major hosts are wild and domestic canines and felines, viverrids, mustelids, racoons and microchiroptera (insectivorous and haematophagous bats). all genotypes, with the exception of genotype , are known to cause human disease. the illness caused by the rabies-related viruses is virtu-ally indistinguishable from classical rabies (smith et al., ) . classical rabies viruses have a worldwide distribution except for a few island nations such as great britain, ireland, new zealand and hawaii, the continents of australia and antarctica and an increasing number of western european countries. the lagos bat, mokola and duvenhage viruses of genotypes - are mainly restricted in distribution to the african continent. however, a genotype isolate has been isolated recently from an imported fruit bat in france (aubert, ) . the bat viruses of genotypes and have only been isolated in europe. european bat lyssa irus (ebl ) isolates are found primarily in eptesicus bats, whilst ebl isolates have only been identified in myotis bats (brass, ) . the ebls have been further classified into the phylogenetic lineages ebl a, ebl b, ebl a and ebl b (amengual et al., ) . fig. . phylogenetic tree to demonstrate the genetic distances between the bp region from the amino terminus of the nucleoprotein gene of selected rabies and rabies-related virus isolates. (multiple sequence alignments were created using the clustal w programme, the tree was generated using the dnadist (maximum likelihood option) and neighbour programmes of the phylip package using an estimated transition/transversion ratio derived from the puzzel programme.) the accepted diagnostic standard for rabies detection, the fluorescent antibody test (fat), detects virus antigen in brain smears using fluorescently-labelled anti-rabies antibodies (dean and abelseth, ) . the sensitivity of this detection method is good with classical rabies isolates, but may be reduced with isolates from the rabies-related viruses (perrin et al., ) . in addition, fat cannot be used to distinguish between the genotypes. pcr-based assays have the added advantage over the standard diagnostic assays that they produce a product that can be further analysed by sequencing. the resultant data can then be used for phylogenetic analyses that enable a highly accurate characterisation of virus isolates. a hemi-nested reverse transcriptase pcr (hnrt-pcr) assay that uses a cocktail of primers capable of detecting the six established genotypes of rabies and rabies-related viruses has been described (heaton et al., ) . the hnrt-pcr offers a higher level of sensitivity than the fat for normal and decomposed tissues (heaton et al., ) , but it does not distinguish between the genotypes. however, the resultant n-terminal bp sequence data is sufficient to distinguish between the various genotypes ( fig. ) . more recently, an assay using pcr-elisa was described which could amplify and distinguish genotype , and viruses (black et al., ) . a technique that gives rise to phylogenetic data much more rapidly than sequencing involves the use of taqman™ probes. taqman™ is an adaptation by perkin-elmer (livak et al., ) of the % nuclease pcr assay described by lee et al. ( ) . a result confirming the genetic lineage of an isolate can be achieved using taqman™ probes within a few minutes of completing a standard pcr or reverse transcriptase-polymerase chain reaction (rt-pcr) cycle. a closed tube system can be used for the pcr and to measure the fluorescence emitted by the taqman™ probes, resulting in minimal post-pcr manipulation and therefore, a reduction in potential cross contamination. since its development, taqman™ has found many applications in research, viral and bacteriological diagnostics and epidemiological studies (chen et al., ; sharma et al., ; mcgoldrick et al., ; kimura et al., ; laue et al., ) . using the sequence data from a number of isolates from each of the rabies and rabies-related virus genotypes, taqman™ probes were designed to distinguish the six established genotypes of rabies and rabies-related viruses. due to the genetic diversity of genotype isolates, a combination of three probes was found to be necessary to detect all of the classical isolates tested. the remaining five genotypes required only a single probe each. using these probes rapid identification and classification of suspect rabies virus isolates can be made within a few hours and provide additionally useful epidemiological data in general research. virus isolates (n= ) from the six established genotypes of rabies and rabies-related viruses and one australian bat lyssa irus isolate were selected for this study. the rabies virus isolates were propagated and passaged in mice or wherever possible in bhk- cells as described (king, ; heaton et al., ; black et al., ) . the first bases of the n gene had previously been sequenced to confirm the genotype of the strain. forty-three genotype viruses and all available genotype - viruses were tested. the original hosts and geographical sources are summarised in table . the rna from eighteen other non-rabies viruses important in the veterinary field was also tested to assess the specificity of the primers and probes. total rna was extracted directly from rabiesinfected bhk- monolayers or infected mouse brain tissue using trizol ® (gibco brl) according to manufacturer's instructions. complementary dna was produced using the rna extracted from each of the infected cultures or mouse brains and the messenger sense rt primer jw as described (heaton et al., ). the primer sets for pcr amplification were based on those of heaton et al. ( ) although a new messenger sense primer bb was found to be more efficient than jw for pcr amplification. the taqman™ probes were designed by eye using multiple alignments (megalign, dnastar) following the recommended criteria (applied biosystems, uk). they were based on regions of high homology that were specific to each genotype and were synthesised by applied biosystems. a list of primers and probes is given in table . the cdnas were amplified using the specific primer set bb (messenger sense) and a cocktail of jw (dpl)/jw (m)/jw (e) (genomic sense) ( table ) . for each probe the conditions were optimised with relation to mg + concentration and annealing temperature. amplification of ml of the cdna template was performed in a final volume of ml of × pcr buffer containing . - . mm magnesium chloride (applied biosystems) (mg + concentration for each probe given in table ), mm each dntp, . pmol of primer bb , . pmol of each primer jw (dpl), jw (m) and jw (e), . u of amplitaq gold (applied biosystems) and pmol of the probes for genotypes - or . pmol of each of the three genotype probes to be used in combination. the amplification was carried out in . ml taqman™ optical tubes on an applied biosystems thermal cycler . the cycling conditions were as described (black et al., ) with the exception of annealing temperatures (table ). fluorescence monitoring was performed according to the manufacturer's directions (perkin-elmer, ) . on completion of the pcr the samples were analysed in the closed pcr tubes using a applied biosystems ls b luminescence spectrometer to quantify the fluorescence emitted by the taqman™ probes. the tubes were scanned at nm (fam) and then nm (tamra), with an excitation wavelength of nm. data acquisition and analyses were carried out using the fluorescence data manager (applied biosystems) and microsoft excel spreadsheets. the increase in sample fluorescence was compared with the fluorescence of no template controls in triplicate. the magnitude of the generated signal (drq) represents the difference between the sample rq (rq+) and the mean value attained for the no template controls rq (rq−), where rq is the emission intensity of the reporter divided by the emission intensity of the quencher. in this equation, the quencher acts as an internal standard to normalise fluctuations in fluorescent intensity due to non-specific effects such as concentration changes due to volume fluctuations. any other fluctuations not due to pcr-related nuclease digestion are normalised by taking the rq + value for a tube that contains all the components including template and subtracting the rq− value for the no template control tubes. this final drq value reliably indicates the magnitude of the signal generated by the given set of pcr conditions. a final calculation, defined as the threshold drq, was carried out to obtain a numerical cut-off value above which a given drq should represent a positive result. this value is calculated at a % confidence interval using the standard deviation (s.d.) obtained from the three no template controls (perkin-elmer, ) . for the purposes of this assay a higher threshold drq ( . ) than that calculated using this method (usually b . ) was adopted. a confirmatory test for successful pcr amplification was carried out by ethidium bromide stained agarose gel electrophoresis during the development of the assay. using . mm magnesium at an annealing temperature of °c, the rt-pcr gave clear welldefined bands on agarose gels. however, these conditions produced poor drq readings in the taqman™ assay. the rt-pcr was therefore, re-optimised for each taqman™ probe with respect to magnesium concentration and annealing temperature to obtain the highest intensity of reporter fluorescence whilst maintaining the highest specificity possible. agarose gel electrophoretic analyses of the rt-pcr products in the taqman™ assays gave weaker specific and often secondary non-specific bands, but were useful for confirming negative taqman™ results during assay development. none of the eighteen non-rabies viruses tested gave any bands when examined by agarose gel electrophoresis. the drq values for each of the genotypes are summarised in fig. . fig. a-f displays the individual values returned for each isolate. the three genotype probes (tqm a, tqm b and tqm c) used in combination detected all of the genotype isolates and none of the isolates from the other genotypes or viruses tested (fig. a) . similarly, the probes used individually to detect genotypes - (tqm , tqm , tqm and tqm , respectively) specifically detected all isolates from their respective genotype panels and none of the other genotypes or viruses tested (fig. c-f ). the probe used to detect the genotype isolates also detected four of the genotype iso-lates (fig. b) . fortunately, the genotype probe detected only genotype isolates (fig. c) . thus, if an isolate tested positive with both the genotype and genotype probes it was classed as genotype . isolates detected with the genotype fig. . graphs demonstrating the drq values obtained for each virus isolate using the taqman probes (a) tqm a, b and c in combination for genotype (b) tqm for genotype (c) tqm for genotype (d) tqm for genotype (e) tqm for genotype and (f) tqm for genotype . probe only, were classed as genotype . similarly, isolates detected with the genotype probe only, were classed as genotype . the genotype isolates that gave positive results with the tqm probe were rv -rv . all four viruses were isolated from cats in zimbabwe and have identical sequences in the bp % region of the nucleoprotein gene (unpublished data). using the rt-pcr primers developed by heaton et al. ( ) as well as the rt-pcr primers used in the development of this taq-man™ assay these four isolates gave two bands on agarose gel electrophoresis. with respect to the tqm probe region, rv -rv have four base differences, whilst the remaining genotype isolates contain five base differences from the probe. the maximum number of base differences between the tqm probe and the genotype isolates is three for rv , rv and rv . these latter isolates gave relatively low drq results ( . , . and . , respectively) compared with the remaining isolates, which were all identical to the probe and, with the exception of rv ( . ), gave results in the range . - . . the drq results with tqm for the genotype isolates (rv -rv ) were in the range . - . . examination of the remainder of the available sequence data for these isolates as well as sequencing of dna from the second band on the agarose gels failed to reveal any further areas of possible homology to the probe. possible patterns in the drq values were sought to determine if the number of differences between the target sequence and probe within a genotype was reflected in the drq value. all the genotype isolates had from to differences between the tqm probes and target sequence but there was no apparent pattern between the number of differences and drq. all of the other genotypes had at least five differences between the target sequence and tqm probes. all of the genotype isolates had no or one base difference to the tqm probe and again no pattern was seen between the differences and drq. of the other genotypes, the closest to this probe was + differences with the genotype isolates and + differences with the remaining genotypes. all the genotype isolates were identical to the tqm probe, which had six or more base differences to the other genotypes. all the genotype and isolates had no or one base difference to their respective probes and again no patterns were seen between the differences and drq. the tqm probe had + base differences and the tqm probe had + base differences to the other genotypes. the only probe that seemed to show any pattern was the tqm probe as mentioned above, where differences in drq reflected the complementarity of probe to target sequences for all but one of the genotype isolates. it appears that the lower discriminatory limit of probe detection is four base differences between probe and target sequence. this was seen with the genotype probes and is the most likely explanation for the detection of the genotype isolates rv - by the tqm probe. no isolates with more than four base differences to the probe were falsely detected. the genotype isolate was tested with these probes and was negative with all of them. a genotype probe was not designed as only one isolate was available for testing. despite the availability of an effective vaccine, rabies is still a significant problem in many parts of the world. a rapid test that could distinguish between the rabies genotypes would be very advantageous for both epidemiological studies, for example in africa and europe where more than one genotype co-exist, and as a diagnostic test in the case of an outbreak. in the uk the most likely genotypes that could enter this country from mainland europe are classical rabies and the ebls. at present genotype differentiation relies on rt-pcr and subsequent sequencing or monoclonal antibody typing. rapid genotyping of any strain found in the uk would be particularly important, as the outcome may dictate the control measures implemented. classical rabies has the potential to establish within indigenous terrestrial animal populations and hence may require the implementation of extensive control measures, whereas, the ebls are likely to be limited to spread among the uk bat population and could prove difficult to control. this report describes the development and application of eight taqman™ probes to differentiate between six genotypes of rabies and rabies-related viruses. using this method a definitive result can be acquired within a few minutes of completion of a standard rt-pcr cycle. monoclonal antibody analysis usually necessitates the growth of virus in cell culture, which may be impossible or result in a delay of several days with decomposed material. an additional problem encountered with monoclonal antibody typing is that the results are subject to individual interpretation. sequence analysis gives a definitive answer and is thus more reliable, but can also take several days to achieve a result unless there is direct access to automated sequencing equipment. an additional advantage of taqman™ is that it is a closed tube system that significantly reduces the risk of cross contamination by pcr products and thus results in increased confidence in the results acquired. the n gene was selected as the target for this assay as it is well conserved and has been intensively used to classify rabies isolates into their respective genotypes (black et al., ; bourhy et al., , kissi et al., ; smith et al., ) . there is a large amount of n gene sequence data available, which, on examination, revealed conserved regions specific to each genotype that could be used to design genotype specific taqman™ probes. the classical rabies virus isolates have a greater representation in our archive and due to extensive geographical distribution and host range, demonstrate a much broader genetic diversity than the other genotypes. for this reason, three probes were required in combination to detect all of the classical isolates tested. all three probes cover the same region of the gene, but have one base change from the others in either the fifth or sixth position. the remaining five genotypes had sufficient similarities in specific regions to enable the use of a single probe for their discrimination. the taqman™ probes were optimised individually with relation to magnesium concentration and annealing temperature to yield the highest intensity of reporter fluorescent intensity without compromising specificity. unfortunately, when relative specificities were compared, the probes were found to have different optimum mg + concentrations. using the universal x master mix (applied biosystems), which became available more recently it may be possible to standardise the reaction mixes to ease panel preparation. magnesium chloride affects annealing and the melting temperatures of both the taqman™ probe and the pcr primers as it help stabilise the hybridisation complexes. it is also required for taq dna polymerase activity and affects fam quenching by tamra on the probe. increasing the magnesium concentration can enhance tamra quenching in the intact probe (livak et al., ) , which is important for calculation of rq −, that is, the lower the reporter emissions in rq −, the greater drq will be in the presence of specific template. however, too little or too much magnesium can result in reduced amplification efficiency or amplification of non-target sequences. the pcr primers were known to produce (heaton et al., ) specific product at a range of temperatures, although this was strongest and cleanest with respect to non-specific products at °c. the annealing temperature was therefore optimised, again to achieve the best possible drq values. we observed that when transferring the technology to the newer abi reader, the drq values were much higher than obtained previously using the ls b and the threshold value required re-evaluation. hence, the actual values represented in fig. may be significantly different when alternative readers are used. the assay appears to be highly specific and reproducible. all the rabies and rabies-related virus rna samples used in the development of this assay had been amplified previously using the pcr protocol described by heaton et al. ( ) and the products sequenced partially and genotyped. due to the large number of available isolates, a representative group of genotype isolates were carefully selected for this covering as wide a range of animal hosts, geographical isolation and sequence variation, as possible. all of the genotype - isolates available in our archive were tested and a selection of unrelated viruses were also tested to ensure the specificity of the assays for rabies and rabies-related viruses. seven of the eight probes were used directly to give a definitive result, whilst the remaining probe, that for detecting lagos bat isolates had to be used alongside the mokola probe to determine whether a positive result is a lagos bat or mokola isolate. detection by the mokola probe or both probes indicated a mokola isolate, while detection with the lagos bat probe and not the mokola probe indicated a lagos bat isolate. for complete confidence, automated sequencing may be used to confirm the genotype of an isolate. it was not deemed necessary to further discriminate between the ebl a and b and ebl a and b subgroups as generic ebl and ebl probes would be sufficient for the aims of this assay. in the case of an outbreak, the control measures introduced would be the same whether an 'a' or 'b' subgroup was responsible. the material used during the development of the assay was passaged in either bhk cells or mice to obtain large quantities of rna permitting uniform validation experiments. the assay has been used successfully on panels of original rabies infected material (data not included). the sensitivity of the assay with each probe was found to be similar to that reported by heaton et al. ( ) for the rt-pcr alone, approximately . tcid per ml (data not included). the assay described above is a rapid and sensitive method for distinguishing between the rabies and rabies-related viruses with several advantages over traditional genotyping techniques. it is more rapid than traditional typing methods and reduces the need for further processing of the rt-pcr product, thereby reducing the risk of cross-contamination. it has the potential to be used in both diagnostic and research laboratories in the identification and classification of suspect rabies isolates in the case of a potential outbreak situation, or for generating routine epidemiological information. the probes may be used in the development of quantitative real time pcr assays using either the light cycler (roche applied science) or abi (applied biosystems) technologies. evolution of european bat lyssa irus who: rabies bulletin europe. second quarter molecular methods to distinguish between classical rabies and the rabies-related european bat lyssa irus antigenic and molecular characterization of bat rabies virus in europe molecular diversity of the lyssa irus genus rabies in bats: natural history and public health implications the evaluation of a fluorogenic polymerase chain reaction assay for the detection of salmonella species in food commodities a new isolate of lagos bat virus from the republic of south africam the fluorescent antibody test a fatal human infection with mokola virus mokola virus infection in cats and a dog in zimbabwe encephalitis caused by a lyssa irus in fruit bats in australia characterisation of a novel lyssa irus isolated from pteropid bats in australia hemi-nested pcr assay for detection of six genotypes of rabies and rabies-related viruses quantitative analysis of epstein-barr virus load by using a real-time pcr assay cell culture of rabies virus rabies-related viruses genetic polymorphism in the rabies virus nucleoprotein gene detection of dengue virus rna in patients after primary or secondary dengue infection by using the taqman automated amplification system allelic discrimination by nick-translation pcr with fluorogenic probes oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting pcr product and nucleic acid hybridisation a novel approach to the detection of classical swine fever virus by rt-pcr with a fluorogenic probe (taqman) a modified rapid enzyme immunoassay for the detection of rabies and rabies-related viruses: rreid-lyssa semiautomated fluorogenic pcr assays (taqman) for rapid detection of escherichia coli :h and other shiga toxigenic coli epidemiologic and historical relationships among rabies virus isolates as determined by limited sequence analysis primary structure of leader rna and nucleoprotein genes of the rabies genome: segmented homology with vsv this work was supported by funding from the ministry of agriculture, fisheries and food and was carried out under roame project se . key: cord- - pjam em authors: stranieri, angelica; lauzi, stefania; giordano, alessia; paltrinieri, saverio title: reverse transcriptase loop-mediated isothermal amplification for the detection of feline coronavirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: pjam em the feline coronavirus (fcov) is the etiological agent of feline infectious peritonitis (fip), a lethal disease of felids. the role of molecular methods is controversial for the diagnosis of fip, while essential for the identification of the shedders. thus, a fast and inexpensive method for the detection of fcov could be beneficial, especially in multicat environments. a reverse transcription loop mediated isothermal amplification (rt-lamp) assay was developed. rna extraction and rt-npcr for fcov were performed on thirty-two samples ( faeces, blood, effusions, and lymph nodes) collected from cats. six rt-lamp primers were designed from the same conserved region of rt-npcr, and the assay was run at °c for one hour. results were evaluated through both agarose gel run and hydroxynapthol blue (hnb) dye and then compared with rt-npcr results for the assessment of sensitivity and specificity. the overall specificity was %, but the sensitivity was % and . % for agarose gel and hnb respectively. therefore, rt-lamp seems optimal to confirm the presence of the virus, but not applicable to exclude it. the feline coronavirus (fcov) is the etiological agent of feline infectious peritonitis (fip), a lethal disease of felids. the role of molecular methods is controversial for the diagnosis of fip, while essential for the identification of the shedders. thus, a fast and inexpensive method for the detection of fcov could be beneficial, especially in multicat environments. a reverse transcription loop mediated isothermal amplification (rt-lamp) assay was developed. rna extraction and rt-npcr for fcov were performed on thirty-two samples ( faeces, blood, effusions, and lymph nodes) collected from cats. six rt-lamp primers were designed from the same conserved region of rt-npcr, and the assay was run at • c for one hour. results were evaluated through both agarose gel run and hydroxynapthol blue (hnb) dye and then compared with rt-npcr results for the assessment of sensitivity and specificity. the overall specificity was %, but the sensitivity was % and . % for agarose gel and hnb respectively. therefore, rt-lamp seems optimal to confirm the presence of the virus, but not applicable to exclude it. © elsevier b.v. all rights reserved. feline coronaviruses (family coronaviridae, order nidovirales) are enveloped, single-stranded positive sense rna viruses belonging to the species alphacoronavirus , genus alphacoronavirus of the sub family coronavirinae (gonzalez et al., ) . the feline coronavirus (fcov) possesses a large genome (almost kb) organized in putative open reading frames (orfs) encoding nonstructural proteins involved in virus replication as well as four structural proteins (spike, s; membrane, m; nucleocapsid, n; envelope, e) and five accessory proteins ( a-c, a and b) (kipar and meli, ) . not yet well characterized viral mutations, along with an inadequate immune response of the host, lead to the inevitably deadly disease of felids called feline infectious peritonitis (fip) (pedersen, ; porter et al., ) . the diagnosis of fip is challenging in vivo and must rely on several clinico-pathological tests (e.g. serum protein electrophoresis, agp measurement, effusion analysis) and only the immunohistochemical demonstration of the viral antigen inside the typical pyogranulomatous lesions can be considered as a gold standard (pedersen, ) . the fcov is faecally-orally transmitted, worldwide distributed and highly prevalent in feline populations (addie et al., ) . since most of the molecular and serological tools available up to date are not able to distinguish between the not mutated and the mutated pathogenic form of the fcov, the use of reverse transcriptase polymerase chain reaction (rt-pcr) for the diagnosis of fip is still extensively discussed doenges et al., ; felten et al., ; longstaff et al., ) . on the other hand, pcr is an extremely useful tool for the diagnosis of fcov infection and shedding and consequently for the identification of shedders, when performed on faeces (pedersen, ) . since shedders can spread the virus in the environment for months, rt-pcr should be repeatedly performed to identify both shedders and cats free from the infection (addie and jarrett, ) . loop-mediated isothermal amplification (lamp) is an amplification gene technique developed some years ago (notomi et al., ) which allows to amplify nucleic acids in an hour and under isothermal conditions, and to evaluate the results by observation of the turbidity of the reaction or using different dyes. reverse transcriptase lamp (rt-lamp) has been used to amplify the genome of several coronaviruses of both humans and animals (amer et al., ; bhadra et al., ; cardoso et al., ; hanaki et al., ; nemoto et al., ; pyrc et al., ; thai et al., ; yu et al., ) . to our knowledge, rt-lamp has never been used for the identification of fcovs. the aim of this study was to develop a rt-lamp for the detection of feline coronavirus in the specimens most frequently used in clinical practice for both screening and diagnostic purposes. thirty-two samples from cats ( faeces, blood, effusions, and lymph nodes) submitted to our institution as part of a diagnostic panel for the clinical suspicion of fip or, regarding faeces, for screening purposes, were used. all the specimens were subjected to rna extraction using a nucleospin rna isolation kit (macherey-nagel bethlehem, pa). whole blood and effusion samples were centrifuged ( min at × g) and the obtained pellets were suspended in l of phosphate buffered saline (pbs) by vigorous vortexing and stored at − • c for further rna extraction. faecal samples were suspended at a final concentration of % (wt/vol) in pbs by vigorous vortexing. the supernatant was cleared by centrifugation for min at × g and stored at − • c for further rna extraction. for tissues, approximately mg of sample were thinly shredded on sterile plates using sterile scalpels, followed by vigorous vortexing in rna lysis buffer until complete disruption of the sample. all the further steps were performed according to the manufacturer's instruction. the extracted rna samples were tested for the presence of fcov using a reverse transcription nested pcr (rt-npcr) targeting a bp product of the highly conserved untranslated region ( utr) of the genome of both type i and type ii fcov (herrewegh et al., ) . rt-npcr positive fcov rna from a cat with fip was used as positive control and rnase-free water as negative control. pcr products were visualized under uv transilluminator on a % agarose gel stained with ethidium bromide. the rt-lamp primers targeting the utr of the fcovs were designed using the primer explorer v software (http:// primerexplorer.jp/elamp . . /index.html) based on a nucleotides sequence ( - bp) of the fcov c je strain (accession number: dq ) (table ) . a loopamp rna amplification kit (rt-lamp, new england biolabs, uk) was used to perform rt-pcr lamp and the reaction mixture was set up as follows: × isothermal amplification buffer, mm mgso , . mm dntps, u/ml of warm start dna, u/ml of warm start rtx reverse transcriptase, m of both forward inner primer (fip) and backward inner primer (bip), . m of both f and b primers, m of both loop f and loop b primers, m of hydroxynaphtol blue (hnb) dye (sigma-aldrich ® ) and l of rna template. the reaction mixture was then made up to l with rnase-free water and incubated in a thermal cycler (mycycler, bio-rad laboratories, hercules, ca, usa) for h at • c followed by min at • c for heat inactivation. the same positive control used for traditional rt-npcr, which tested positive also on the first rt-lamp assay, was then used as a positive control in the following rt-lamp assays, while rnase-free water was used as negative control. the products of the reaction were then inspected both by eye, in order to detect the color turning from violet to sky blue in case of positive results with hnb (goto et al., ) , and under uv transilluminator on a % agarose gel stained with ethidium bromide in order to detect a ladder-like pattern in case of positive result (parida et al., ) (fig. ) . results obtained with rt-lamp were then compared with those obtained with the rt-npcr and the sensitivity and specificity of rt-lamp obtained with both hnb and agarose gel were calculated. results are reported in table . all the negative samples using rt-npcr were also negative by rt-lamp, using both gel electrophoresis and hnb for the visualization of the results, leading to a % specificity. on the other hand, a conspicuous number of false negative results was recorded ( / for agarose gel and / for hnb) and the overall sensitivity was % and . % using gel electrophoresis and hnb, respectively. positive samples were characterized by a slight difference in the intensity of coloration, as shown in fig. . the sensitivity of rt-lamp was also different according to specimens: faeces showed a sensitivity of % and %, with gel electrophoresis and hnb respectively. on blood, the sensitivity was % and %, with gel electrophoresis and hnb respectively while on effusions the sensitivity was % with both the visualization methods. only on tissues, the sensitivity resulted to be absolute, but the number of tested samples was too low to be discussed in terms of diagnostic accuracy. based on the results of this pilot study, rt-lamp for fcov, due to its high specificity, appears to be a solid molecular test to confirm the diagnosis of fcov infection, and eventually to support a clinical diagnosis of fip, when performed on specimens from cats with an high pre-test probability of fip (e.g. cats with clinical signs and laboratory findings consistent with fip, like effusions with physico-chemical and cytological features consistent with fip) but its low sensitivity makes this test not reliable in case of negative results (pedersen, ) . the design of this study did not allow us to understand the possible mechanisms responsible for the low sensitivity recorded. technical problems are unlikely since the method described in this study has been developed after testing different primers, working conditions or temperatures (data not included in this short communication). possible explanations of the high rate of false negative results compared with conventional nested rt-pcr include the lower analytical sensitivity of rt-lamp, as previously reported for other coronaviruses when compared with real time rt-pcr (bhadra et al., ) , that allow to obtain positive results only in samples with a high viral burden. in the case the sensitivity of the test might be ameliorated through further studies, the rt-lamp could be extremely useful, due to its low costs and rapidity, in those situations where the table results obtained on the samples tested with rt-npcr (pcr) and lamp and evaluated with agarose gel electrophoresis (lamp gel) and hydroxynaphtol blue dye (lamp hnb detection of fcov must be repeated over time and on a high number of cats (e.g. breeding catteries). an additional future perspective would be the optimization of the test to obtain quantitative results, possibly by establishing a standard curve of color intensity using rna samples with known viral load (e.g. quantification of rna copies by quantitative pcr techniques). moreover, the development of an internal control to assess the integrity of rna in each sample would be advisable before the use of this test in field conditions. the authors declared no conflicts of interest regarding the research, authorship, and/or publication of this article. the authors received no financial support for the research, authorship, and/or publication of this article. use of a 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coronavirus spike gene mutations as a tool to diagnose feline infectious peritonitis a comparative sequence analysis to revise the current taxonomy of the family coronaviridae short technical reports reverse transcription-loop-mediated isothermal amplification for the detection of rodent coronaviruses detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr feline infectious peritonitis: still an enigma? feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification loop-mediated isothermal amplification of dna loop mediated isothermal amplification (lamp): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases a review of feline infectious peritonitis virus infection: - an update on feline infectious peritonitis: diagnostics and therapeutics amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis development of loop-mediated isothermal amplification assay for detection of human coronavirus-nl development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus development of a real-time reverse transcription loop-mediated isothermal amplification method for the rapid detection of porcine epidemic diarrhea virus none. key: cord- -ajzk rq authors: van weezep, erik; kooi, engbert a.; van rijn, piet a. title: pcr diagnostics: in silico validation by an automated tool using freely available software programs date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: ajzk rq pcr diagnostics are often the first line of laboratory diagnostics and are regularly designed to either differentiate between or detect all pathogen variants of a family, genus or species. the ideal pcr test detects all variants of the target pathogen, including newly discovered and emerging variants, while closely related pathogens and their variants should not be detected. this is challenging as pathogens show a high degree of genetic variation due to genetic drift, adaptation and evolution. therefore, frequent re-evaluation of pcr diagnostics is needed to monitor its usefulness. validation of pcr diagnostics recognizes three stages, in silico, in vitro and in vivo validation. in vitro and in vivo testing are usually costly, labour intensive and imply a risk of handling dangerous pathogens. in silico validation reduces this burden. in silico validation checks primers and probes by comparing their sequences with available nucleotide sequences. in recent years the amount of available sequences has dramatically increased by high throughput and deep sequencing projects. this makes in silico validation more informative, but also more computing intensive. to facilitate validation of pcr tests, a software tool named pcrv was developed. pcrv consists of a user friendly graphical user interface and coordinates the use of the software programs clustalw and ssearch in order to perform in silico validation of pcr tests of different formats. use of internal control sequences makes the analysis compliant to laboratory quality control systems. finally, pcrv generates a validation report that includes an overview as well as a list of detailed results. in-house developed, published and oie-recommended pcr tests were easily (re-) evaluated by use of pcrv. to demonstrate the power of pcrv, in silico validation of several pcr tests are shown and discussed. pathogens exhibit genetic variation as a result of genetic drift, adaptation and evolution, but also by random variation. since the late nineties of the th century, due to the improved sequencing techniques and high throughput sequencing machines, the number of sequences submitted to databases like genbank ® has increased exponentially. this results in an enormous increase of identified variants and quasi-species as well as sequences of newly discovered pathogens from all over the world. a few examples are the discovery of coronaviruses causing severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), nipah and hendra viruses, atypical pestiviruses, atypical and new serotypes of bluetongue virus, schmallenberg virus and new variants of avian influenza viruses (chua et al., ; demmler and ligon, ; drosten et al., ; hoffmann et al., ; hofmann et al., ; maan et al., ; marcacci et al., ; schirrmeier et al., ; van boheemen et al., ; wang, ; zientara et al., ) . currently, in many countries, the first line of pathogen detection is real-time pcr diagnostics. favourably, pcr tests can be highly sensitive and specific, and are often designed to detect all variants of a defined family, genus or species, while not detecting closely related pathogens. in addition, pcrv can also be used to validate in silico pcr assays that differentiate between lineages, serotypes or variants. therefore, pcr targets must be unique, and highly conserved. nonetheless, false negative results can arise by genetic drifting or by emergence of new variants, while false positive results can be caused by new variants of closely related pathogens. it is therefore important to frequently reevaluate and, if necessary, redesign pcr tests taking sequences of newly discovered pathogen variants into account. validation of pcr diagnostics should be organized in three stages, in silico, in vitro and in vivo validation. in silico validation covers the study on inventory of matching and non-matching sequences of the pcr target sequence in a nucleotide database. matching sequences enable in silico sensitivity (detection of all variants), while non-matching sequences support in silico specificity (selective detection of variants of the respective group of pathogens). in vitro and in vivo validation include testing of cultured pathogens, and field samples of defined positive and negative status. in vitro and in vivo validation for all virus variants is practically impossible and extremely costly. even more, not every pathogen variant has been cultured or isolated, and transport and handling of pathogens could imply safety issues. in contrast, sequences are rapidly becoming available by high throughput and deep sequencing, even without culturing of pathogens. therefore, in silico re-evaluation of validated pcr diagnostics is and will be an attractive alternative to obtain detailed insight in detection of circulating and (re-) emerging virus variants, and should be frequently executed. it will however become an increasing task due to the rapid increase of available sequences and full genome sequences of numerous species. we developed a software tool named pcrv to facilitate in silico validation of pcr tests entirely based on freely available software programs. pcrv links freely available software programs to automate the whole process, reduces labour, and generates a validation report that includes a brief summary as well as a list of detailed results. the software tool pcrv is written in the python programming language. pcrv consists of a user friendly graphical user interface and coordinates the use of software programs clustalw . (larkin et al., ; thompson et al., ) and ssearch (brenner et al., ; pearson, ; pearson et al., ; to perform in silico validation. pcrv is suitable to determine the in silico sensitivity (conservation of sequences) and in silico specificity (selectivity) of different pcr formats. to monitor the performance of pcrv, a set of flagged internal control sequences (fics) are randomly added to the sequence database. pcrv processes data and analyses results, and generates a validation report that includes a summarizing table as well as a list of detailed results for an easy check of potential false positives and false negatives. an overview of all actions executed by pcrv is shown in fig. . the sequences of a target organism are downloaded from the national center for biotechnology information (ncbi) database (https://www.ncbi.nlm.nih.gov/nuccore/) by using the respective taxonomy id number as search query. this guarantees that all available sequences of the defined taxon in the database are downloaded. to generate a multiple sequence alignment (msa) of these sequences, a full genome sequence was selected as a reference sequence. genome segments of pathogens with a segmented genome were concatenated to serve as an artificial full length genome. if a full genome sequence was not available, a representative large sequence of the taxon was selected as a reference sequence. a prerequisite is that this partial sequence contained the full target of the pcr test being validated. in order to drastically reduce computing time, pairwise alignments were calculated for each downloaded sequence to the reference sequence by using software program clustalw . (larkin et al., ) . to correct for orientation errors in the database sequences, alignment in the reverse complement orientation was also attempted. a score was calculated using a scoring scheme as follows: match (+ ), mismatch (- ), point deletion or gap (- ), every next adjacent point deletion (- ). the aligned orientation with the highest score was selected. to enable efficient alignment of large sequences, these large sequences were segmented in fragments of , nucleotides in length and individually aligned to the reference sequence and subsequently combined into one pairwise alignment. pcrv combined all individual pairwise alignments into one multiple sequence alignment (msa), including the pairwise alignments of primers and probes. the calculation of the msa was performed by a computer with an intel ® xeon(r) cpu e - v @ . ghz processor and gb of internal computer memory. the regions corresponding to primers and probes were selected from the msa to construct a conservation plot sorted in decreasing total number of mismatches. the in silico sensitivity was expressed as the percentage of hits with a cut-off value of a maximum of one mismatch per primer or probe. the entire nucleotide sequence database (compressed gzip file: nt.gz) was downloaded from the ncbi ftp-website (ftp:// ftp.ncbi.nlm.nih.gov/blast/db/) using pcrv. the integrity of the download was confirmed by calculation of the md checksum and subsequent comparison with the checksum published on the ftp-website (file nt.gz.md ). pcrv processed the data stream during download by several optimizations to improve the analysis. nucleotide code 'n' was replaced by the meaningless code 'z', which prevents infinite number of hits by the alignment search. the data stream was unpacked and subdivided into multiple fasta formatted text files. fasta files with a maximum size of mb were sequentially numbered and stored because the ncbi nucleotide database is too large to be analysed all at once. to increase the accuracy of the alignment search (see discussion), large sequences were fragmented in sequences of maximal nucleotides with an overlap of nucleotides to prevent the loss of hits of primer or probe sequences spanning the split site. fragmented sequences were tagged with a unique code allowing reconstruction of the original sequence. any nucleotide database in fasta format is compatible and could be added. flagged internal control sequences (fics) were added to enable validation of the alignment search. fics consisted of randomly generated sequences of nucleotides in length containing primer and probe sequences of the pcr test being validated. primer and probe sequences were inserted in all possible combinations and orientations potentially initiating amplification ( fig. ). multiple copies of each combination were inserted with an increasing number of randomly introduced mismatches from - in each primer and probe sequence ( fig. ). in total, ten copies of each control sequence per number of mismatches were linearly spread in each mb fasta file. an alignment search was performed with the default expectancy threshold value on all fasta files using primers and probes of the pcr test as search queries and the program ssearch available in the fasta sequence analysis package (brenner et al., ; pearson, ; pearson et al., ; . pcrv produced a list of hits of the alignment search of all possible primer/probe combinations potentially leading to detectable amplicons. hits of fics were stored separately. the percentage of returned hits of control sequences with an increasing number of mismatches was indicative for the sensitivity and accuracy of the alignment search per mb fasta file. the maximum number of returned mismatches in the control sequences was determined by use of the spearman-kärber method and demonstrated the validity of the computing process (wulff et al., ). an aborted search caused by an unknown error was visible by the incompleteness of returned fics. if the accuracy of the alignment search was not acceptable, the alignment search was repeated with a higher expectancy threshold value, which usually resulted in a longer analysis time. the specificity check was limited to a maximum of nucleotides in amplicon length and up to four mismatches per primer or probe. this limitation was however not applied to the fics in order to fics consist of randomly generated sequences of nucleotides in length containing the primer and probe sequence of the pcr test being validated. multiple copies were inserted with an increasing number of randomly introduced mismatches from - in each primer and probe sequence. ten copies of each fics per number of mismatches were linearly spread in each mb fasta file. b) overview of all eight possible combinations of positional orientations of forward primer (fwd), reverse (rev) primer and probe used as fics which are all capable of initiating an (nonspecific) amplification reaction in combination with a detectable probe signal. combinations of primers and probes according to other pcr formats (e.g. nested pcr, pcr using hybridisation probes or hydrolysis probe) are also supported by pcrv but are not shown. fully ascertain the validity of the executed alignment search. hits were interpreted as specific or nonspecific according to the taxonomy classified sequences as used to generate the msa. the in silico specificity is expressed as the percentage of specific hits of taxonomy classified sequences with a maximum of one mismatch per primer or probe as these are considered to be detected with the respective pcr test. to demonstrate the suitability of our in-house developed software tool pcrv, we determined the in silico sensitivity and specificity of three pcr tests for west nile virus (wnv) recommended by the world organisation for animal health (oie) (eiden et al., ; johnson et al., ) . these wnv pcr tests represented three different formats; a real-time pcr test, a conventional pcr test and a nested pcr test (table ) . available west nile virus nucleotide sequences were downloaded from the ncbi website using taxonomy id , (search query ncbi:txid on january th , ). in total, the download contained , wnv sequences. a msa was calculated using the full genome sequence with accession number nc_ as a reference sequence (borisevich et al., ) . primer and probe sequences were included in the alignment. the calculation of the msa with pcrv was completed in about . h. a limited number of - % of the aligned sequences encompassed the locations of primers or probes of the selected oie-recommended wnv pcr tests. the regions corresponding to primers and probes were taken from the alignment in order to construct a conservation plot. detailed results were sorted according to the number of mismatches to easily select individual sequences with > mismatch in order to check their origin (supplemented data a). note, sequences incorrectly classified as wnv as well as synthetically derived sequences should be discarded as these are irrelevant. results of the conservation plot were summarized according to the number of mismatches to a maximum of four mismatches per primer or probe ( table ).the overall in silico sensitivity of each pcr test was calculated and expressed as the percentage of sequences with a maximum of one mismatch per primer or probe. the real time pcr test for wnv showed the highest in silico sensitivity of . % ( . %+ . %). the conventional and nested pcr tests showed an in silico sensitivity of . % and . %, respectively. the entire nucleotide sequence database from the ncbi ftp-website was downloaded as a compressed gzip file (nt.gz) of gb on january th , . the download was valid according to the calculated md (johnson et al., ) , and the real time pcr test have been described (eiden et al., ) . sequences. an alignment search with primer and probe sequences was performed with a cut-off expectation value e of . the search per pcr test was completed in less than two hours. about . - . million individual primer and probe alignment hits were found and processed by pcrv as described (fig. ) . fics were found homogeneously in all database files indicating that the alignment search was completed properly. fics for each pcr test were returned with a mean of . - . mismatches per primer or probe demonstrating completeness and acceptable accuracy of the alignment search (table ) . potential amplicons were interpreted as specific or non-specific according to the presence of its ncbi accession number in the list of sequences as used for the in silico sensitivity check (table ) . we noticed that the number of specific hits differed from the numbers as scored by the in silico sensitivity check (table ) . however, several reasons for this apparent inconsistency can be considered, see discussion. in summary, using wnv pcr tests as an example, pcrv easily determined the in silico sensitivity and specificity of these pcr tests of different formats in a highly automated manner. all results are included in the validation report generated by pcrv, such as a summarizing table of results, conservation plot and a list of nonspecific hits. the summarizing table clearly demonstrates the differences of the in silico sensitivity and specificity between these pcr tests (table ). in addition, the detailed conservation plot (supplemented data a) and detailed list of nonspecific hits up to mismatches per primer or probe (supplemented data b) support manual check of individual sequences on correctness, background, submission details, and other information. validation of diagnostics by testing all variants of a target pathogen in cultured or field samples, named in vitro and in vivo validation, respectively, is hardly feasible. because of the availability of sequences of pathogens in databases, checking conservation and uniqueness of primer and probe sequences, so-called in silico validation, has become an attractive and reliable alternative to (re-) evaluate specificity and sensitivity of molecular diagnostics. exponential expansion of available sequences, genetic drift of pathogens, and discovery of new pathogens drive the need to frequently validate established pcr tests. this, however, will also become an increasing significant effort. we automated the in silico validation process by integrating freely available software programs into a single tool named pcrv. public databases, such as ncbi as well as other available databases and sequences formatted in single sequence fasta files are compatible with pcrv. pcrv generates a multiple sequence alignment (msa) using a selected reference sequence, which is preferably a full length genome but at least a partially large sequence encompassing the pcr target. software program clustalw . (larkin et al., ) is used to calculate pair-wise alignments of each sequence to the reference sequence, and subsequently a msa is generated using these pair-wise alignments. this strategy exponentially reduces calculation time, in particular for large numbers of sequences. additionally, more than one reference sequence could be used to improve the generation of a msa in case of extreme variability among a group of pathogens. the msa is used to determine the in silico sensitivity, since this is less prone to mismatches in primers or probes (not shown). for example, sequences with numerous mismatches in one of the primers or probes will not be found by an alignment search using these primer or probe sequences as search queries. however, such sequences will be present in the msa, see conservation plots of wnv pcr tests. supplemented data a shows the summarised -without accession numbers -conservation plots of the three wnv pcr tests. pcrv generates a conservation plot listing all hits according to decreasing number of mismatches. hits with the most mismatches needs attention as these could lead to false negative pcr results. we calculated and defined the in silico sensitivity as the percentage of hits with a maximum of one mismatch per primer or probe as these are assumed to be detected with the respective pcr test. the software program ssearch that is available in the fasta sequence analysis package from the university of virginia (pearson, ) uses a calculated expectation value e in combination with a supplied threshold value to determine whether a hit is returned. the expectation value e depends on the number and length of sequences in the database. consequently, the e value of a search hit depends on the location of the found sequence in the database. large sequences are therefore segmented into fragments of maximal nucleotides in length. this reduces the variability in sequence length leading to a more homogenous sensitivity of ssearch across the database and improves the overall sensitivity of ssearch. the sensitivity of the well-known and commonly used blastn alignment search program was compared to that of ssearch (fig. ) . clearly, ssearch returns % of the primers up to six mismatches. in contrast, the percentage of returns with blastn is slightly less than % for three mismatches and rapidly declines by an increasing number of mismatches. we conclude that ssearch is much more accurate, and thus more suitable than blastn to determine the in silico fig. . comparison of the accuracy of an alignment search performed by the blastn and the ssearch software programs. a test database of randomly generated nucleotide sequences was generated containing , sequences of nucleotides in length. sequences contained a primer sequence of nucleotides in length. each primer contained randomly - mismatches. the cut-off expectation value e used in both programs was . the inserted primer with up to mismatches completely returned with blastn, whereas ssearch completely returned the primer with up to mismatches. specificity. we also noticed that blastn tends to find partial/fractional nucleotide alignment hits which is not desirable for primers and probes. in addition, pcrv using ssearch is suitable for use in a laboratory quality control system, since the search process is monitored per mb fasta file for completeness and accuracy/sensitivity by returned hits of flagged internal control sequences (fics). an overview of this monitoring is added to the validation report. examples of incomplete, inaccurate or alignment searches with a low sensitivity are presented (supplemented data c). in case alignment search results are not sufficient, the threshold value can be changed to increase the sensitivity but the calculation time will also increase. here, we showed in silico validation results of wnv pcr tests of different formats as an example. pcrv was also used to validate real time pcr tests at wbvr (fig. ) . ssearch quantifies hits for any combination of primers and probes potentially leading to detectable amplicons, see fig. . this can result in more hits for the in silico specificity check by ssearch than for the in silico sensitivity check by clustalw . . for example, sequences partially overlapping with the pcr target sequence will not be found by the in silico specificity check, since this check only finds complete amplicons. further, ncbi only stores unique nucleotide sequences in its downloadable database export file "nt.gz". identical sequences are combined as one sequence with the sequence name as a concatenation of all individual sequence names separated by the ascii code . pcrv does not recognize merged names as multiple sequences, resulting in less hits by ssearch. detailed analysis of in silico validation results enables a focus on specific test problems, as shown for the pcr test for peste-des-petits ruminants virus (pprv) of wbvr that presumably does not detect pprv strain ghana because of three mismatches in the probe sequence. indeed, the pcr target of this pprv strain was amplified but was not detected by the taqman probe (van rijn et al., a) . we used pcrv to analyse oie-recommended and published pcr tests for other pathogens in order to select the best option for implementation in laboratory diagnostics. upon preparedness on incursions, frequent in silico (re-)validation could also show the need for adaptation of operational pcr tests to emerging epidemics caused by new variants in other parts of the world. pcrv depends on compatible and reliable nucleotide databases. for example, in silico validation by pcrv depends on submission of accurately determined sequences which are coded with the correct taxonomy id number. for example, classical swine fever virus (csfv) sequences that are taxonomy classified as bovine viral diarrhoea virus type (bvdv ii) were consequently interpreted as false positives in the csfv pcr test and as false negatives in the bvdv pcr test. further, in our example of wnv pcr tests, five nonspecific hits appeared to be sequences without taxonomy id. still, these sequences are definitely wnv sequences, although out of nonspecific hits have been synthetically derived (supplemented data b). on the other hand, a more specific taxonomy classification or labelling of sequences in databases could be used for the development of pcr tests specific for subspecies, serotypes or lineages. considering the expected rapid expansion of available sequences, pcrv will be further improved by allowing incremental analyses in which only newly submitted sequences with respect to the previously analysed sequences are processed. this will keep the required analysis time manageable for in silico re-validation of pcr tests. the number of hits for the in silico sensitivity and specificity are not representative for the field situation but represents that of the sequences in the database. in other words, the percentages could be skewed by a small number of sequences in the database, or by a large number of very closely related sequences caused by a huge effort during one epidemic. submitted sequences are sometimes not trimmed for synthetic adaptors like pcr primers causing misleading positive analysis results. synthetic or optimized genes of pathogens can lead to misleading negative pcrv results. synthetic and genetically modified sequences should be labelled as 'nonnatural' in databases to prevent misleading results of in silico validation efforts. finally, negative pcrv results can be created on purpose by development of diva (differentiating infected from vaccinated) vaccine viruses with a deleted or mutated diva target, like ge deletion mutants of bovine herpes virus type and pseudorabies virus (kaashoek et al., ; van oirschot et al., ) , ns deletion mutants of bluetongue virus and african horse sickness virus (feenstra et al., ; van rijn et al., b van rijn et al., , , and liveattenuated lumpy skin disease (lsd) vaccine (agianniotaki et al., ) . viral pathogens belonging to the same taxon showing an extreme variation in their sequence cannot be aggregated in one msa using one reference sequence. further, large scale genomic rearrangements, such as duplication, deletion, insertion, inversion, and translocation, are very common in genomes of bacterial pathogens, and will undoubtedly challenge the calculation of a msa, if this is even possible. currently, we are investigating alignment-free analysis methods to address these challenges. even more, we foresee the development of a next generation in silico tool, partially based on pcrv, to find highly conserved targets for new or confirmatory pcr tests. fig. . overview of the in silico sensitivity and specificity of several real time pcr tests at wbvr as determined by pcrv. the in silico sensitivity of pcr tests is expressed as the percentage of hits with a maximum of one mismatch per primer or probe (squares, line). the in silico specificity is expressed as the percentage of specific hits with mismatches (black) and mismatch per primer of probe (grey). real time pcr tests are indicated: wnv; west-nile virus (eiden et al., ; johnson et al., ) , btv; bluetongue virus (van rijn et al., ; ) , pprv; peste des petits ruminants virus (van rijn et al., a) , ashv_s ; african horse sickness virus segment (van rijn et al., b) , ashv_s ; african horse sickness virus segment (van rijn et al., b) ; in-house developed assays: rvfv; rift valley fever virus, sgpv; sheepand-goat pox virus, ehdv-a; epizootic haemorrhagic disease virus test a, ehdv-b; epizootic haemorrhagic disease virus test b, eav; equine arteritis virus, eblv- ; european bat lyssa virus type , csfv; classical swine fever virus, asfv; african swine fever virus, prv-gb; pseudorabies virus glycoprotein gene gb, prv-ge; pseudorabies virus glycoprotein gene ge. results of pcrv could demonstrate the need to optimize or redesign a pcr test, like for ehdv-a and ahsv_s . note: hits of non-natural sequences were not discarded. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). development and validation of a taqman probe-based real-time pcr method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains biological properties of chimeric west nile viruses assessing sequence comparison methods with reliable structurally identified distant evolutionary relationships nipah virus: a recently emergent deadly paramyxovirus severe acute respiratory syndrome (sars): a review of the history, epidemiology, prevention, and concerns for the future identification of a novel coronavirus in patients with severe acute respiratory syndrome two new real-time quantitative reverse transcription polymerase chain reaction assays with unique target sites for the specific and sensitive detection of lineages and west nile virus strains vp -serotyped live-attenuated bluetongue virus without ns /ns a expression provides serotype-specific protection and enables diva novel orthobunyavirus in cattle genetic characterization of toggenburg orbivirus, a new bluetongue virus, from goats detection of north american west nile virus in animal tissue by a reverse transcription-nested polymerase chain reaction assay a conventionally attenuated glycoprotein e-negative strain of bovine herpesvirus type is an efficacious and safe vaccine clustal w and clustal x version . novel bluetongue virus serotype from kuwait one after the other: a novel bluetongue virus strain related to toggenburg virus detected in the piedmont region searching protein sequence libraries: comparison of the sensitivity and selectivity of the smith-waterman and fasta algorithms query-seeded iterative sequence similarity searching improves selectivity - -fold genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species identification of common molecular subsequences comparative biosequence metrics multiple sequence alignment using clustalw and clustalx genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans marker vaccines, virus protein-specific antibody assays and the control of aujeszky's disease sustained high-throughput polymerase chain reaction diagnostics during the european epidemic of bluetongue virus serotype bluetongue virus with mutated genome segment to differentiate infected from vaccinated animals: a genetic diva approach recombinant newcastle disease viruses with targets for pcr diagnostics for rinderpest and peste des petits ruminants diagnostic diva tests accompanying the disabled infectious single animal (disa) vaccine platform for african horse sickness discovering novel zoonotic viruses monte carlo simulation of the spearman-kaerber tcid novel bluetongue virus in goats the authors are grateful to colleagues of wbvr, in particular to jan boonstra and rené van gennip, for fruitful discussions and suggestions. this research was financially supported by project wot- - - of the dutch ministry of agriculture, nature and food quality (lnv) (wbvr-project number - ). all authors declare no conflict of interest. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jviromet. . . . key: cord- - dq xz authors: do, lien anh ha; van doorn, h. rogier; bryant, juliet e.; nghiem, my ngoc; nguyen van, vinh chau; vo, cong khanh; nguyen, minh dung; tran, tinh hien; farrar, jeremy; de jong, menno d. title: a sensitive real-time pcr for detection and subgrouping of human respiratory syncytial virus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: dq xz improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (rsv) are needed to aid the development and evaluation of novel intervention strategies. a quantitative real-time rt-pcr using specific locked nucleic acid (lna) probes was developed to identify rsv and to distinguish rsv subgroups a and b (rsv lna assay). rsv subgroup diversity and the relationship between viral load and disease severity in confirmed rsv infections were also explored. archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex seeplex™ rv detection kit (seegene) and the novel rsv lna assay. the lna assay demonstrated a significantly higher sensitivity than seeplex, improving overall detection rates from % ( / ) to % ( / ). detection limits of . × ( ) and . × ( ) copies/ml were observed for rsv a and b, respectively. rsv a was detected in / ( %) cases, and / ( %) were positive for rsv b. this novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of rsv. improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (rsv) are needed to aid the development and evaluation of novel intervention strategies. a quantitative real-time rt-pcr using specific locked nucleic acid (lna) probes was developed to identify rsv and to distinguish rsv subgroups a and b (rsv lna assay). rsv subgroup diversity and the relationship between viral load and disease severity in confirmed rsv infections were also explored. archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex seeplex tm rv detection kit (seegene) and the novel rsv lna assay. the lna assay demonstrated a significantly higher sensitivity than seeplex, improving overall detection rates from % ( / ) to % ( / ). detection limits of . × and . × copies/ml were observed for rsv a and b, respectively. rsv a was detected in / ( %) cases, and / ( %) were positive for rsv b. this novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of rsv. human respiratory syncytial virus (rsv) is a pneumovirus and a member of the subfamily of pneumovirinae within the family of paramyxoviridae. based on differences in the antigenicity and nucleotide sequences analysis, rsv strains have been classified in two major antigenic subgroups, denoted rsv a and rsv b (sullender, ) . rsv is a major cause of acute respiratory tract infections in children worldwide (nair et al., ; weber et al., ) , and is responsible for a large proportion of hospital admissions during infancy and early childhood (mejias et al., ) . no vaccine is available, and treatment is limited to supportive care (empey et al., ) . rsv is also a frequent cause of nosocomial infections, and as such may significantly impact the duration of stay in hospital and healthcare costs during childhood (macartney et al., ) . data on epidemiology and molecular virological characteristics of rsv isolates circulating in southeast asia are limited. diagnosis of rsv infection is typically performed on respiratory samples using virus culture, (rapid) antigen detection tests, or molecular assays (henrickson, ) . real-time reverse transcriptase pcr (rt-pcr) provides a rapid and sensitive tool for detection of rsv compared to conventional techniques and enables (semi-)quantitation of viral load (borg et al., ; dewhurst-maridor et al., ; do nascimento et al., ; empey et al., ; falsey et al., falsey et al., , henrickson, ; houben et al., ; hu et al., ; kuypers et al., ; mentel et al., ; perkins et al., ; van de pol et al., van de pol et al., , van elden et al., ) . the locked nucleic acid (lna) technology increases thermal stability of probe-target interaction and improves discrimination of different nucleic acid targets (reynisson et al., ) . the n gene is the most conserved rsv gene across all known genetic clades, and is also expressed most abundantly during viral replication (david and knipe, ; johnson and collins, ) . for these reasons, the n gene was chosen as the target of a novel real-time rt-pcr diagnostic tool, to provide quantitative viral load analysis and discrimination between rsv a and b within a single assay. the quantitative assay was compared to a commercial conventional multiplex pcr method (seeplex tm rv detection kit, seegene, inc., seoul, korea) (kim et al., ; roh et al., ) respiratory samples from a study (do et al., ) on acute respiratory infection in children at the hospital for tropical diseases, ho chi minh city, vietnam. here, the development and validation of this assay is reported, as well as initial observations regarding subgroup diversity within vietnamese rsv strains, and relationships between viral load and disease severity in vietnamese patients. to develop and evaluate this novel rsv diagnostic, respiratory samples, collected and stored as part of a study on acute respiratory infection among children at the hospital for tropical diseases, ho chi minh city, vietnam were used (do et al., ) . specimens were kept at • c for a maximum of h and then aliquoted and stored at − • c until further processing. the samples comprised nasopharyngeal aspirates, throat swabs, and nasal swab collected in viral transport medium, and represented cases admitted to the pediatric intensive care unit (icu) ( / , %) as well as the pediatric respiratory ward ( / , %). one hundred and forty-one ( %) samples were from patients less than years old, ( %) from to years old, ( %) over years old. all samples were analyzed in parallel by the commercial multiplex seeplex tm rv detection kit (seegene, inc., seoul, korea) according to the manufacturer's instructions, to determine the presence of respiratory viruses: human rsv subgroups a and b (rsv a, rsv b); influenza virus a (infv a); influenza virus b (infv b); human coronaviruses ( e, oc ), human metapneumovirus (hmpv), parainfluenza virus , and (piv , , ), human rhinovirus (hrv a) and adenovirus (adv) (kim et al., ; roh et al., ) and by the newly developed rsv lna real-time rt-pcr. rna was extracted from l of specimen and eluted in l on the easy mag . system (biomérieux, marcy l'Étoile, france). an internal rna-virus control (equine arteritis virus [eav] ) was added to each sample prior to extraction at a standard concentration yielding ct values of - as previously described (scheltinga et al., ) . rna was reverse transcribed using superscript iii reverse transcriptase (invitrogen, carlsbad (ca), usa) and random hexamers (roche, mannheim, germany). each l reaction mixture contained l extracted rna, x rt-buffer (invitrogen), . mm of each dntp (roche), ng random hexamer, mm dtt (invitrogen), u of rnase inhibitor and u rt superscript iii. cdna synthesis was performed using an eppendorf master thermocycler gradient system (perkinelmer corporation, foster city (ca), usa) under the following conditions: min at • c, min at • c and min at • c. the assay was designed to detect all known rsv strains and to differentiate rsv subgroups a and b. primers (mth and mth b) were selected using primer express software v . (applied biosystems inc., foster city (ca), usa) and a dataset of n gene sequences available from the ncbi database (table ) to amplify bp for both rsv a and rsv b. rsv subgroups a and b differ by nucleotides within the highly conserved region of the n gene; two lna probes, rsva and rsv b (sigma proligo, singapore), were designed to include three lna residues at these sites (+t, +a and +t for rsv a; +c, +g and +a for rsv b). the choice of reporters and compatible quenchers was based on a bio-rad guideline (biorade, ) . primers and probes were analyzed for false priming sites, hybridizations, and secondary structures at http://lnatools.com/hybridization. plasmids containing the amplicons from rsv a and rsv b clinical strains were constructed for use as positive controls and generation of standard curves for quantitation. briefly, target cdnas amplified via rt-pcr from rsv a and rsv b-positive clinical specimens as identified by seeplex tm rv detection kit, were cloned into pcrii ® -topo ® vector and transformed into the topo- bacterial strain using the topa ta cloning kit (invitrogen). clones were screened by pcr using m primers, and confirmed by sequencing (ceq dtcs quick start kit, ceq capillary sequencer, beckman coulter, inc., fullerton (ca), usa). quantitation of the plasmid preparations (ng/l) was conducted using picogreen dsdna quantitation reagent (invitrogen) in duplicate, and the corresponding copy number of target cdna in the plasmid was calculated (copies/ml). serial -fold dilutions were made for analysis of analytical sensitivity and used as standard curves for viral load quantitation. viral load was expressed as copies per ml of sample, a ct-value of was chosen as a cut-off value for standard positivity. the rsv lna assay was performed on a dna engine peltier thermocycler and chromo real-time pcr system detector (bio-rad, hercules, usa), in a total volume of l containing ul template cdna, . u hotstart taq (invitrogen), × pcr buffer, mm mgcl , . mm of each dntp (roche), , m of each primer (rsv and eav), . m of each probe for rsv, and . m of eav probe (table ) . the pcr employed the following thermal cycler settings: min at • c, followed by cycles of s at • c and min at • c. negative controls and standards were included in every assay run. the number of rsv rna transcripts (copies/ml) of clinical samples was determined based on the standard curve. a ct-value of was chosen as the cut-off value for sample positivity. all clinical samples required a ct value of to for the eav internal control to be considered valid. potential interference between primers/probes for target (rsv) and internal control (eav) was assessed by running serial dilutions of two clinical samples with confirmed high viral loads of rsv a and rsv b (data not shown). the analytical limit of detection was determined using six -fold dilutions of control plasmids from . × to . × copies/ml for rsv a and . × to . × copies/ml for rsv b. the viral load in clinical samples was assessed by running them in parallel with quantitative standards. since the concentration of target cdna in plasmid controls was known, the corresponding concentration of cdna in the clinical sample could be estimated. samples that tested positive by rsv lna but not by seeplex were further confirmed by a seminested rt-pcr targeted at the second hypervariable region of the rsv g gene as described previously (parveen et al., ) . specificity of the assay was assessed by presence of non-rsv viruses within the sample set (as determined by seeplex). specificity was further assessed by attempted amplification of culture supernatants of selected human seasonal influenza isolates [a/sydney/ / (h n ), a/new caledonia/ / (h n ), b/yamanashi/ / ]. statistical comparisons between seeplex rt-pcr and the rsv-lna assay were performed using mcnemar's test. assay variability was quantified by the standard deviation (sd) and coefficient of variation (cv). ct-values (of five -fold dilutions of control the analytical limit of detection was determined using six -fold serial dilutions of control plasmid in sextuplicate. % detection was observed at concentrations ranging from . × to . × copies/ml for rsv a and . × to . × copies/ml for rsv b. the detection rate was % ( / ) for . × copies/ml of rsv a and % ( / ) for . × copies/ml of rsvb. significant negative linear relationships were observed between copies/ml and ct values for the first five -fold dilutions from . × to . × copies/ml for rsv a (adjusted r = . ) and . × to . × copies/ml for rsv b (adjusted r = . ). to assess the analytical intra-and inter-assay reproducibility of the amplification step of the assay, ct values were compared between replicates of five -fold serial dilutions tested in the same batch (table ) or on different days (table ) . coefficient of variation (cvs) for analytical intra-assay variability of rsv a and b ranged from . to . and . to . , respectively. similarly, cvs for analytical inter-assay variability of both rsv a and b ranged from to . specificity of the assay was confirmed by failure to amplify from specimens known to contain other respiratory viruses (as detected by seeplex assay). these included influenza viruses (n = ), rhinovirus (n = ), human coronavirus (n = ), human metapneumovirus (n = ), parainfluenza virus (n = ), and adenovirus (n = ). in addition, no amplification was observed for culture supernatants containing high titer influenza viruses. the additional samples positive by the rsv lna assay were confirmed using an rsv g gene rt-pcr (parveen et al., ) . none of the samples negative by rsv lna assay were positive by seeplex. fluorescence signals of eav internal control were detected for all tested samples at ct values ranging from to . the viral load of rsv detected in samples ranged from . × to copies/ml (median value . × ; interquartile range (iqr) . × to . × ). median (iqr) of viral load in positive samples by both the rsv lna assay and the seeplex kit was . × copies/ml ( . × to . × ). in the samples positive by rsv lna assay alone, median (iqr) of viral load was . × copies/ml ( . × to . × ). there was a significant difference observed in viral load between rsv positive samples diagnosed by both assays and rsv positive samples by only rsv lna assay (mann-whitney u test, p = . ) (fig. ) . among rsv positive samples, were identified as rsv a ( %) and as rsv b ( %). there was % concordance in subgroup identification between the rsv lna assay and the seeplex kit. no association was found between rsv subgroups and disease severity, based on the distribution of cases between pediatric intensive care unit (icu) and other wards. similarly, no significant differences were observed in viral load between rsv subgroups. overall, a total of % ( / ) of patients with severe presentations (pediatric icu admissions) were rsv positive, whereas % ( / ) of patients on other wards were rsv positive (fisher's exact test p = . ). viral loads in nasal pharyngeal aspirates of pediatric icu patients were significantly higher when compared to other cases: median (iqr) of viral load (copies/ml) in the pediatric icu rsv positive samples was . × ( . × to . × ) versus . × ( . × to . × ) in the positive samples from other wards (mann-whitney u test, p = . ). an association . × . . . . × . . . . × . . . . × . . . . × . . . . × . . . . × . . . . × . . . . × . . . . × . . . all values are calculated from ct values of six replicates at each concentration. sd: standard deviation. cv: coefficient of variation. between higher age and lower log-vl was also found (correlation − . ; p = . ) among rsv positive patients, and younger age was associated with more severe disease (correlation . , p = . ). when the dataset was adjusted for age, differences between viral load and severity lost statistical significance (data not shown). no significant differences in rsv viral load were observed between patients with single infections versus co-infections, or between patients requiring oxygen versus patients who did not. detection by seeplex assay revealed an overall co-infection level of % ( / ). these included influenza viruses (n = ), parainfluenza viruses (n = ), coronaviruses (n = ), adenovirus in (n = ), human metapneumovirus (n = ) and rhinovirus (n = ). the rsv lna assay revealed an additional rsv positive samples, none of which were coinfected. the prevalence of coinfection of rsv positive samples was % for rsv a ( / ); % for rsv b ( / ). rsv was the pathogen detected most frequently among hospitalized children with acute respiratory infection in a prospective study conducted between and in pediatric icus and respiratory wards of the hospital for tropical diseases in ho chi minh city, viet nam (do et al., ) . rapid and sensitive detection of rsv is important for installing infection control measures and thus preventing nosocomial spread, which has been recognized as a major risk in pediatric wards (macartney et al., ) . several rt-pcr based methods and real-time rt-pcr have been described for rsv detection (borg et al., ; dewhurst-maridor et al., ; do nascimento et al., ; falsey et al., ; gueudin et al., ; hu et al., ; kuypers et al., kuypers et al., , mentel et al., ; perkins et al., ; van de pol et al., van de pol et al., , van elden et al., ) . an improved pcr diagnostic tool using lna probes targeting the most conserved region of the n gene was developed, to detect and distinguish simultaneously subgroup a and b and to quantify viral rna load. an internal control virus (eav) was added to each specimen in order to monitor efficiency of rna extraction, reverse transcription, and amplification. this assay exhibited an increased diagnostic yield, from % to %, compared to a commercial conventional multiplex pcr assay, the seeplex tm rv detection kit, and produced a diagnostic result within h of sample receipt. previous studies have reported higher sensitivities of pcr methods targeting the n gene, compared to methods targeting other genes (perkins et al., ; rohwedder et al., ) . primers and probes were originally designed based on available n gene sequences from ncbi genbank, including the two reference strains a and b. twenty-four more recent submissions of n gene sequences from cambodia showed perfect matches to these primers and probes. this indicates that these primers and probes theoretically could detect all currently known circulating strains of rsv. the % detection rate for . × copies/ml for rsv a and for . × copies/ml for rsv b are in the same range as previously published methods that used dilution series of plasmid dna and, when re-calculated and assuming a : rt efficiency, as methods that used rna transcripts (ranging from to copies/ml) (borg et al., ; gueudin et al., ; kuypers et al., ) . significant linear correlations of ct values and copies/ml were observed at levels of viral load typically found in clinical specimens. in addition, the assay exhibited good analytical reproducibility as evidenced by intra-and inter-assay cvs ranging from % to %. cvs from other reported diagnostic approaches (based on copies/ml) ranged from % to . % (borg et al., ; gueudin et al., ; kuypers et al., ) . as rsv pathogenesis is multifactorial and varied (collins and graham, ) , the relationships between disease severity, viral load and rsv subgroups were studied. associations between rsv viral load and disease severity have been reported previously (buckingham et al., ; fodha et al., ; houben et al., ) , and several studies have definitively shown a negative correlation between rsv viral load and age (borg et al., ; gueudin et al., ; kuypers et al., ) . the results of the current study confirm a negative correlation between rsv viral load and age, and significant difference in viral load between severe and non-severe patients (defined by admission status to the pediatric icu versus the respiratory ward). however, when the dataset was adjusted for age, differences between viral load and severity lost statistical significance. previous studies have also suggested possible differences in pathogenicity between rsv a and b (campanini et al., ; gerna et al., ; kuypers et al., ; perkins et al., ) . here we report no significant differences in viral load or severity between rsv subgroups. variations in sample collection may also affect observed viral loads, however sample collection for this cohort was conducted by experienced and well-trained hospital personnel using standard operating procedures in conclusion, this novel real-time rsv lna assay offers a rapid, specific, highly reproducible and more sensitive alternative ( %) to a commercial multiplex rt-pcr ( %) for rsv detection, and in addition provides quantitation and subgrouping. this information is useful for epidemiological surveillance and clinical decisionmaking. real-time pcr application guide evaluation of a quantitative real-time pcr for the detection of respiratory syncytial virus in pulmonary diseases nasal 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comparison of the seeplex reverse transcription pcr assay with the r-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses detection of respiratory syncytial virus rna in blood of neonates by polymerase chain reaction diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time rna pcr respiratory syncytial virus genetic and antigenic diversity diagnostic value of real-time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection applicability of a real-time quantitative pcr assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults respiratory syncytial virus infection in tropical and developing countries the authors acknowledge the clinicians in the pediatric intensive care unit and in the pediatric respiratory ward of the hospital for tropical diseases for collecting samples. in addition hoang thi thanh hang and marcel wolbers are acknowledged for statistical advice and tran tan thanh, le van tan and the molecular diagnostic group of the oxford university clinical research unit virology lab for help and technical advice with the work. this study was funded by the wellcome trust of great britain. key: cord- -x qtxaum authors: majchrzykiewicz-koehorst, joanna a.; heikens, esther; trip, hein; hulst, albert g.; de jong, ad l.; viveen, marco c.; sedee, norbert j.a.; van der plas, jan; coenjaerts, frank e.j.; paauw, armand title: rapid and generic identification of influenza a and other respiratory viruses with mass spectrometry date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: x qtxaum the rapid identification of existing and emerging respiratory viruses is crucial in combating outbreaks and epidemics. matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (maldi-tof ms) is a rapid and reliable identification method in bacterial diagnostics, but has not been used in virological diagnostics. mass spectrometry systems have been investigated for the identification of respiratory viruses. however, sample preparation methods were laborious and time-consuming. in this study, a reliable and rapid sample preparation method was developed allowing identification of cultured respiratory viruses. tenfold serial dilutions of ten cultures influenza a strains, mixed samples of influenza a virus with human metapneumovirus or respiratory syncytial virus, and reconstituted clinical samples were treated with the developed sample preparation method. subsequently, peptides were subjected to maldi-tof ms and liquid chromatography tandem mass spectrometry (lc–ms/ms). the influenza a strains were identified to the subtype level within h with maldi-tof ms and h with lc–ms/ms, excluding the culturing time. the sensitivity of lc–ms/ms was higher compared to maldi-tof ms. in addition, lc–ms/ms was able to discriminate between two viruses in mixed samples and was able to identify virus from reconstituted clinical samples. the development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry. respiratory viruses are a major cause of infections, natural outbreaks, and epidemics. approximately million cases of viral community-acquired pneumonia occur every year: million in children and million in adults (ruuskanen et al., ) . a wide range of viruses, including current circulating subtypes of influenza a virus (influenza a(h n )pdm and influenza a h n ), influenza b virus, respiratory syncytial virus (rsv), parainfluenza viruses, adenovirus, and rhinovirus, have been implicated in respiratory tract infections in past decades. in the last ten years, new viruses have emerged as severe acute respiratory syndrome (sars) virus, middle east respiratory syndrome (mers), avian influenza a (h n ) virus, human metapneumovirus (hmpv), coronaviruses nl and hku , and human bocavirus. rapid identification of existing and emerging respiratory viruses is of the utmost importance in combating outbreaks and epidemics, and starting early therapy and prophylaxis. currently, cell culture, serology, and real-time reverse transcription-polymerase chain reaction (rrt-pcr) are used in virological diagnostics. however, there are a few drawbacks with these techniques: (i) serology and rrt-pcr-based assays are target directed and thus potentially miss non-selected or emerging pathogenic viruses (binnicker et al., ; yang et al., ) ; (ii) culturing is time consuming; (iii) serology-based assays are not applicable in the acute phase and/or have low sensitivity. furthermore, multiple tests are needed to detect and subtype mixed infections. matrix-assisted laser desorption/ionization-time of flight mass spectrometry (maldi-tof ms) is a generic technique that can rapidly identify cultured microorganisms (seng et al., ; van veen et al., ) . the analysis of bacteria by mass spectrometry (ms) has made great progress over the last two decades, and it is http://dx.doi.org/ . /j.jviromet. . . - /© the authors. published by elsevier b.v. this is an open access article under the cc by-nc-nd license (http://creativecommons.org/licenses/by-nc-nd/ . /). employed in many hospitals as a rapid and reliable alternative to traditional identification methods. thus far, mass spectrometrybased methods have not been used for virological diagnostics. however, it has been shown that purified influenza virus particles could be identified with either maldi-tof ms or maldi fourier transform ion cyclotron resonance ms (maldi-ft-icr ms). identification was based on the proteolytic digestion of concentrated and purified viral samples and on mass spectrometry analysis of the specific peptide mass profile. the sample preparation methods used in these studies included virus concentration and/or purification with either ultracentrifugation, differential centrifugation, precipitation, filtration, isolation of viral particles or protein(s) with an affinity capture immunoassay or gel electrophoresis (downard et al., ; schwahn et al., a schwahn et al., ,b, a chou et al., ; jang et al., ; nguyen and downard, ; fernandes and downard, ) . these sample preparation procedures were laborious and often time consuming (up to h) and are therefore not applicable for high-throughput virological diagnostics. the aim of this study was to develop a rapid, generic and robust sample preparation method for maldi-tof ms and lc-ms/ms that will enable reliable and fast identification of respiratory viruses. for this purpose, cultured influenza a virus strains and mixed samples of influenza a virus with hmpv or rsv were treated with the developed preparation method. subsequently, obtained peptides were subjected to maldi-tof ms and lc-ms/ms for analyses. the identification of the respiratory viruses was based on peptide sequence differences in abundant viral proteins. to confirm correct identification of peptides by mass spectrometry, the amino acid sequences were compared to the corresponding dna sequences, obtained by sequencing of the viruses. finally, to determine the sensitivity of the in-house developed method the titers of cultured viruses were determined with rrt-pcr. all of the clinical influenza strains, anonymized from routine diagnostics, originated from a collection at the university medical center utrecht (umcu). collection of the samples and analysis of the isolated virus strains were approved by the local medical ethics committee of the umcu. the institutional review board (irb) confirmed (protocol / ) that the viral strains were not regarded as patient-owned material; consequently, the use of these strains was not restricted by dutch law ("law medical scientific research with people", wmo; art. b). nasopharyngeal swabs and tracheal aspirates were collected between and from patients who were hospitalized with respiratory distress symptoms in the wards of the umcu (table s ). the collected samples were cultured immediately in either llc-mk or hep cell lines and were routinely checked for cytopathological effect (cpe) formation. the samples exhibiting specific cpe in % of the cells and testing positive for respiratory viruses by real-time pcr were harvested and stored at − • c. next, eight influenza a h n strains (designated here as bm , bm , and bm through bm ) and two influenza a h n strains (designated here as bm and bm ) were cultured in llc-mk cells in the presence of emem (eagle's minimal essential medium) with trypsin at • c (table s ). in addition, hmpv (designated here as bm ) and rsv type a (designated here as bm ) were cultured in llc-mk cells in the presence of emem with trypsin at • c and in hep cells in the presence of dmem (dulbecco's modified eagle medium) with % fbs (fetal bovine serum) at • c, respectively (table s ) . subsequently, the culture supernatants were collected, and the cell debris was removed by centrifugation ( × g, min). aliquots were frozen at − • c and were used without further purification steps. to determine the sensitivity of the in-house developed sample preparation method the titers of cultured viruses were determined with rrt-pcr by using a standard curve. this standard curve was developed by counting virus particles using electron microscopy and by subsequently performing pcr. viral rna extraction and pcr were performed according to previously described protocols (tan et al., ) . in short, viral genomic rna was isolated using a magnapure lc total nucleic acid kit, according to the manufacturer's guidelines (roche diagnostics, mannheim, germany). murine encephalomyocarditis virus was used as an internal control. reverse transcription of the isolated viral rna was performed using a multiscribe reverse transcriptase kit and random hexamers (applied biosystems, foster city, ca, usa), according to the manufacturer's guidelines. pcr primers and probes were designed on the basis of highly conserved genomic regions of the m gene for influenza a and the n gene for both hmpv and rsv a, and these primers and probes were used for the typing of the viral strains. the primers and probes used are listed in table s . cdna samples were analyzed in a l reaction mixture, containing l of cdna, taqman universal pcr master mix (applied biosystems, abi), primers and fluorogenic probes labeled with the reporter dye -carboxy-fluorescein (fam), and the quencher dye -carboxy-tetramethyl-rhodamine (tamra). amplification and detection were performed with an abi system for min at • c, min at • c, and cycles of sec at • c and min at • c. the samples were assessed for the presence of possible inhibitors of the amplification reaction using the indicated internal control, the signals of which had to range within a clear-cut interval. to confirm the presence and amino acid sequence accuracy of the identified peptides, six of the clinical isolates were sequenced. the rsv strain (bm ) with accession number jq was sequenced previously (tan et al., ) . for sequencing purposes, hmpv (bm ) and influenza (bm , bm , bm , bm , and bm ) pcr fragments were obtained by fractional amplification of magnapure lc genomic rna isolates, using the superscript iii one-step rt-pcr system with platinum taq high fidelity kit (invitrogen) and a fast thermal cycler (abi), according to the manufacturers' protocols. unlike hmpv and rsv, influenza virus contains a segmented genome consisting of segments. all of the segments were completely amplified and purified from an agarose gel prior to fractional amplification, as previously described (zhou et al., ) . the pcr products were applied to a % agarose gel and were purified from the gel with a gene jet gel extraction kit (thermo fisher scientific, landsmeer, the netherlands), according to the manufacturer's protocol. the isolated fragments were used for whole-genome sequencing. the hmpv (bm ) and influenza strains (bm , bm , bm , bm , and bm ) listed in table s were sequenced according to the whole-genome sequence protocol described recently, using the conventional sanger technique (tan et al., ) . fragments ranging between and nucleotides were sequenced with an abi -capillary dna analyzer, using big-dye terminator . (abi). the resulting sequence information was assembled into an hmpv whole-genome sequence through alignment with the corresponding reference sequence (genbank: fj ; http://www.ncbi.nlm.nih.gov/genbank/index.html) using seqman software (dnastar lasergene ); a similar approach was followed for the influenza strains. table s provides an overview of the primers used for influenza, hmpv, and rsv sequencing. the dna and amino acid sequences of the sequenced strains are provided in supplementary data. the starting titers of the viral cultures are shown in table s . the starting titers for the influenza strains and rsv appeared roughly similar (∼ × copies/ml), but the starting titer for hmpv was -fold lower. tenfold serial dilutions for all of the strains (influenza a virus, rsv, and hmpv) were prepared in cymol medium (copan italia, brescia, italy). cymol is a collection, transport, and preservation medium for cells (cytology analysis), viruses, and nucleic acids (pcr-based assays) (luinstra et al., a) . subsequently, l of each tenfold dilution step was treated according to the in-house developed sample pretreatment protocol, as follows. a l aliquot was mixed with l of a . % stock solution of rapigest (waters corporation, milford, ct, usa) and was incubated for min at • c. next, dl-dithiothreitol (dtt, sigma-aldrich) and acetonitrile (lc-ms chromasolv, sigma-aldrich) were added to mm and to % final concentration, respectively. reduction and precipitation of the proteins were performed under controlled microwave radiation in a rapid enzymatic digestion system (reds; hudson surface technology [hst] inc., old tappan, nj, usa) at • c and w for min. the subsequent carbamidomethylation of cysteine residues was executed at • c and w for min in reds, after the addition of iodoacetamide (iaa; sigma-aldrich) to a . mm final concentration. next, the proteinaceous particles were precipitated at , × g for min, and the supernatants were discarded. the pellets were suspended in l of mm ammonium bicarbonate (sigma-aldrich) containing g/ml modified and tpck-treated trypsin from bovine pancreas (sigma-aldrich). digestion was performed for min in reds at • c and w and was terminated by the addition of trifluoroacetic acid (tfa) to % final concentration. after incubation at • c for min, the rapigest precipitates were removed by centrifugation, and the supernatants were filtered through a microcon ym- filter ( mwco, merck millipore ltd.; co. cork, ireland). all of the samples were prepared at least twice. for the analysis of mixtures of two different viral culture samples, each tenfold dilution of influenza a h n (bm ) or h n (bm ) was mixed with the corresponding dilution of the rsv (bm ) or hmpv (bm ) culture samples, respectively, followed by the exact sample preparation procedure described above. all the samples were prepared at least twice. to test whether human proteins present in clinical material interfere with the developed sample preparation method or identification by lc-ms/ms, reconstituted throat swabs were included. a swab that was rubbed around the tonsils of a volunteer was washed by vortexing the swab in cymol medium. subsequently, this so-called clinical sample was spiked with dilutions of cultured influenza a virus (bm , bm ), rsv (bm ), or hmpv (bm ). the same sample preparation and lc-ms/ms analysis procedure as described above were performed on these reconstituted clinical samples. all tested samples were prepared at least twice. a l aliquot of each sample was desalted on a c ziptip (merck millipore ltd.), and the peptides were eluted with l of % acetonitrile containing mg/ml ␣-cyano- -hydroxycinnamic acid (hcca) and . % trifluoroacetic acid (tfa). elution was performed directly onto the maldi target (mtp polished steel, bruker daltonics, gmbh, bremen, germany), and the droplets were air-dried. spectra were recorded in reflector mode, using a maldi-tof ms instrument (autoflex iii, bruker). each spectrum was acquired by averaging laser shots (at % laser power) across a mass range of m/z - followed by noise reduction. the instrument was mass calibrated with an external peptide standard (peptide calibration standard, bruker). the spectra were analyzed with flexanalysis and biotools (bruker). internal calibration was performed using identified peptides derived from viral nucleoprotein or trypsin. from the spectra, peak lists were generated and searched against the ncbi virus database (database collection of viruses: . . ) using mascot, version . . (matrix science, london, uk) with a mass tolerance of ppm (perkins et al., ; koenig et al., ) . the carbamidomethylation of cysteine residues and oxidation of methionine were included in the calculations, and up to two missed cleavages were permitted. the mascot scoring system was used to determine protein expectation values that corresponded to the number of matches that were expected to occur by chance alone. the highest expectation value was considered the best match. all of the assays were performed at least twice. in separate experiments, the peaks that were identified initially as nucleoprotein (np) peptides of influenza a were subsequently confirmed by maldi-tof ms/ms. the ms/ms spectra were recorded in the reflector mode using lift technology (bruker). the digests were analyzed by lc-ms/ms using a nano-advance lc system (bruker) coupled to a q-tof mass spectrometer (maxis impact, bruker). a l aliquot of each digested sample was injected onto a magic c aq uhplc nanotrap column (c , m id × mm, m, Å, bruker) and was washed with loading solvent h o ( . % formic acid) for min at a flow rate of l/min. following valve switching, the peptides were separated on a magic c aq analytical column ( m id × mm, c , m, Å, bruker) at a constant flow of nl/min. the peptide elution gradient was from % a (h o with . % formic acid) to % b (ch cn) in min, followed by an increase to % b for min. optima lc-ms h o ( . % formic acid; fisher scientific) was used for the lc-ms/ms studies. the nanolc system was coupled to the mass spectrometer using a captivespray (bruker) ionization source. the spray voltage was set at . - . kv, and the temperature of the heated capillary was set at • c. the eluting peptides were analyzed using the datadependent ms/ms mode. the ten most abundant ions (charge state + , + , and + ) in an ms spectrum ( - m/z) were selected for data-dependent ms/ms analysis by collision-induced dissociation, using nitrogen as the collision gas. the ms/ms scans were acquired over a mass range of - m/z. peak lists were generated using dataanalysis software, version . (bruker), and the lists were exported as mascot generic (mgf) files. these files were searched against the ncbi database (taxonomy: viruses) with the mascot search algorithm (mascot . . , matrix science). a mass tolerance of ppm and an ms/ms tolerance of mmu were used. up to two missed cleavages for trypsin were allowed; carbamidomethylcysteine was selected for fixed modification and oxidation of methionine for variable modification. only significant protein hits with at least two unique peptides with a score greater than were selected. ten influenza a virus strains, isolated from hospitalized patients and characterized to the subtype level (eight h n and two h n ), were cultured in llc-mk cells, resulting in viral titers ranging from . to . × genome copies/ml (table s ). after the removal of cell debris, tenfold dilution series of the viral cultures were prepared in cymol medium. each dilution was treated according to the in-house developed sample preparation procedure. subsequently, peptides were subjected to maldi-tof ms and were analyzed by mascot software. in two independent experiments, all of the tested influenza a strains (eight h n and two h n ) were correctly identified based on the recognition of peptides derived from nucleoprotein (table and fig. ) . the ten influenza a strains were not only identified to their type, but also correctly identified to their subtype (table ) . the entire procedure, i.e. the dilution series preparation followed by the treatment according to the in-house protocol, data analysis and the obtaining of results, was performed within h for ten samples. the identification limit at which the viral strains were correctly typed and subtyped appeared to be × viral genome copies in the total sample volume deposited onto a maldi plate for analysis (corresponding to a ct value of approximately ; table ). at this viral titer, more than mass peaks could be assigned with high significance to peptides derived from nucleoprotein ( table ). the identity of approximately half of these peptide peaks was confirmed by maldi-tof ms/ms (data not shown). the intensities of the remaining peptide peaks assigned to nucleoprotein were too low for ms/ms analysis. the genomes of five influenza a strains, namely bm , bm , bm , bm , and bm , were sequenced, and the amino acid sequences of all of the identified peptides were found to be encoded in the corresponding genome sequences (supplementary data, genomic and amino acid sequences of sequenced strains). of the other proteins that constitute the viral proteome, only m -derived peptides were identified occasionally (data not shown). the ms spectra also contained peaks from abundant non-viral proteins derived from cell lysates, such as actin and peptides with a s/n of ≥ are listed. these peptides were identified in tested viral strains at titers of × genome copies in the total volume of each sample, deposited onto a maldi plate and analyzed. nd, peptide not detected. ds, detected sequence in the tested strains but in another peptide due to missed cleavage by trypsin. trypsin auto-digestion fragments (shown as un-annotated peaks in fig. ). the same sample preparation procedure was followed as described above, after which the peptides were subjected to lc-ms/ms. virus identification, including the sample preparation and analysis, was achieved in less than h for ten samples. in two independent experiments, all ten influenza strains were correctly identified as either h n or h n influenza a virus, with an identification limit of × genome copies in the total sample volume subjected to lc-ms/ms analysis (corresponding to a ct value of approximately - ; table ). at this viral titer, the influenza viruses were typed and subtyped based on the identification of peptides derived from the nucleoprotein protein, indicated in boldface in table . the nucleoprotein-derived peptides that were identified repeatedly in at least % of the samples containing ≥ × viral genome copies are also shown in table . in addition to the peptides derived from np, the lc-ms/ms analysis identified peptides derived from m , non-structural protein (ns ), ha, and na (table s ). the amino acid sequence coverage of these proteins (for each tenfold dilution of viral culture, an average value of two independent experiments was calculated) is shown in table . similar to the maldi-tof ms results, the identified amino acid sequences of the five sequenced strains (bm , bm , bm , bm , and bm ) were in accordance with their respective genome sequences. the lc-ms/ms spectra also demonstrated proteins derived from cell lysates or culture additives, such as actin, lectin, histone, and enolase (data not shown). moreover, most of the peptides detected with maldi-tof ms were also identified with lc-ms/ms (tables and ). tenfold serial dilutions of strain bm (h n ) were mixed with the corresponding dilution factor of rsv (mixture a), resulting in an approximate : ratio of both strains. similarly, in mixture b, tenfold serial dilutions of strain bm (h n ) were mixed with the corresponding dilution factor of hmpv, although the starting titer of hmpv was times lower than those of influenza and rsv. the mixtures were treated according to the in-house developed sample preparation procedure, subjected to lc-ms/ms, and subsequently analyzed as described for the samples containing a single virus. using lc-ms/ms, both viruses in mixtures a and b were identified simultaneously based on the peptides derived from nucleoprotein proteins. the influenza strains were correctly typed and subtyped from sample dilutions containing approximately ≥ . × genome copies in the total sample volume subjected to lc-ms/ms analysis. for influenza, peptides derived from m , ns , ha, and na proteins were also identified (table ). for the hmpv and rsv strains, in addition to nucleoprotein-derived peptides, those of matrix protein (m), matrix protein - (m - ), phosphoprotein (p), and fusion glycoprotein (f) were also identified (tables , s , and s ). the identification limit at which the rsv strain was identified correctly, was . × genome copy equivalents in the total sample volume subjected to lc-ms/ms analysis (corresponding to a ct value of approximately ; tables and s ). for hmpv, the identification limit was . × genome copy equivalents in the total sample volume subjected to lc-ms/ms analysis (corresponding to a ct value of approximately ; tables and s ). all of the identified peptide sequences of the tested influenza a, rsv, and hmpv strains were confirmed with their respective genome sequence data (supplementary data, genomic and amino acid sequences of sequenced strains). tenfold serial dilutions of influenza a h n (bm ), h n (bm ), rsv (bm ), and hmpv (bm ) were spiked in cymol medium, containing material from a throat swab. these reconstituted clinical samples were treated according to the in-house developed sample preparation method, subjected to lc-ms/ms and analyzed as described above. all the spiked respiratory viruses were identified. in both influenza a and rsv samples, tenfold dilution series of each tested influenza strain was prepared and analyzed by lc-ms/ms. indicated are the total genome copies of the analyzed influenza virus subjected to lc-ms/ms. b amino acid sequence coverage (%) of proteins determined by mascot, as based on identified peptides. percentages are the averages of combined results for the h n or h n strain. proteins were considered significantly identified when the mascot score was ≥ and when a minimum of three peptides each with a score of ≥ was identified. np, nucleoprotein; m , matrix protein; ns , non-structural protein ; ha, hemagglutinin; na, neuraminidase; c identification of type and subtype based on the highest mascot scores of nucleoprotein; thus, the given subtypes should be considered as indicative. identification data were extracted from mascot. nd, peptide sequence not identified. ds, identified sequence in another peptide. the influenza viruses (≥ × genome copies) were typed and subtyped based mainly on the identification of peptides derived from nucleoprotein (shown in bold). influenza a/h n hmpv d a total genome copy number of the tested influenza virus and rsv in a total sample volume subjected to lc-ms/ms analysis. b identified virus. c an average sequence coverage (%) of the identified protein was calculated from two independent experiments. a protein was considered identified when the mascot score was ≥ and when a minimum of three peptides with a score ≥ was identified in the protein. d the starting concentration of hmpv was times lower than that for influenza and rsv. np, nucleoprotein; m , matrix protein; ns , non-structural protein ; ha, hemagglutinin; na, neuraminidase; m - , matrix protein; f, fusion glycoprotein; p, phosphoprotein. identification of viral proteins from reconstituted clinical material spiked with viruses. titer an average sequence coverage (%) of the identified protein was calculated from two independent experiments. a protein was considered identified when the mascot score was ≥ and when a minimum of two peptides with a score ≥ was identified in the protein. np, nucleoprotein; m , matrix protein; ns , non-structural protein ; ha, hemagglutinin; na, neuraminidase; m - , matrix protein; f, fusion glycoprotein; p, phosphoprotein. c positive control; isolation of the virus was without throat swab material. d only in one of the two tested samples peptides were detected. peptides derived from nucleoprotein, matrix protein m , and nonstructural protein ns were identified. in addition, in rsv samples also peptides derived from matrix protein m - and phosphoprotein were identified. in one of the two tested hmpv samples, two peptides derived from phosphoprotein were identified (table ). the identification limit at which influenza a h n was detected, was × genome copies in the total sample volume subjected to lc-ms/ms; for influenza a h n and rsv this was approximately × genome copies; for hmpv × genome copies. the accurate and rapid identification of influenza and other respiratory viruses is important in combating outbreaks and epidemics, detecting newly emerging viruses, and initiating prophylaxis and early therapy. antigen-detection and rrt-pcr tests are used most frequently for the detection of viruses in the acute phase. pcr-based methods can yield results within - h with high sensitivity and specificity (centers for disease control and prevention: guidance for clinicians on the use of rrt-pcr and other molecular assays for diagnosis of influenza virus infection, ), whereas the rapid antigen-detection tests can even yield results within min. however, both methods feature drawbacks. (i) they are target-directed and can potentially miss non-selected or emerging pathogenic viruses that are not covered by the pcr primer sets and antibodies used in the antigen-detection assays. for instance, it was shown that a new type of human papillomavirus (hpv) was not detected by a broad hpv-primer pcr system (johansson et al., ) and newly emerging mutations in the conserved region of m prevented rrt-pcr detection of influenza a h n and h n from clinical specimens (binnicker et al., ; yang et al., ) . (ii) multiple tests (different pcrs or pcr followed by sequencing) are needed for (sub)typing and for detection of mixed infections. (iii) the sensitivity of rapid antigen-detection tests is low. the commonly used rapid influenza diagnostic test (ridt), although fast, lacks accuracy and sensitivity ( - %), resulting in a high percentage of false-negative results (beck et al., ) . theoretically, mass spectrometry-based systems could overcome these drawbacks and they were already investigated for the identification of respiratory viruses. however, until now the used sample preparation methods were too laborious and time-consuming for mass spectrometry to be an identification method of clinical potential. therefore, in this study a rapid, generic and robust sample preparation method for mass spectrometry-based virus identification was developed. subsequently, the sensitivity of the combined procedure of this sample preparation and mass spectrometry analysis was determined. tenfold serial dilutions of ten cultured influenza a strains of two different subtypes, mixed samples of influenza a virus with either hmpv or rsv, and reconstituted clinical samples were treated with the in-house developed sample preparation method without prior purification by laborious procedures, apart from standard removal of cell debris. subsequently, peptides were subjected to maldi-tof ms and lc-ms/ms and identified by use of a web-based mascot search algorithm. to confirm correct identification, the amino acid sequences were compared to the corresponding dna sequences, obtained by sequencing. all of the influenza a strains were successfully identified to the subtype level by both mass spectrometry systems. identification of viruses was achieved within h with maldi-tof ms and within h with lc-ms/ms, excluding the time necessary for culturing the viruses. sensitivity of maldi-tof ms was lower (detection limit of × ; table ) compared to lc-ms/ms (detection limit of × ; table ); the latter corresponding to a ct value of - . earlier studies have already shown that it is possible to identify highly pure and/or concentrated influenza a viruses with maldi-tof ms, maldi-ft-icr ms or lc-ms/ms (schwahn et al., a (schwahn et al., ,b, a chou et al., ; jang et al., ; nguyen and downard, ; fernandes and downard, ; li et al., ) . however, the sample preparation methods used were time-consuming (up to h) and laborious, and are therefore not suitable for rapid applications. in these studies, the same nucleoprotein-derived peptides were found among the identified viral peptides as detected here (schwahn et al., a,b; li et al., ) , which indicates that with the in-house developed sample preparation method, comparable results could be achieved in a much shorter time. furthermore, in the developed sample preparation method, prior purification or extended concentration of cultured viruses, for example by applying ultracentrifugation, is not required for the identification of viral proteins in the presence of other viral or non-viral proteins. the sample preparation time was significantly reduced to approximately . h by the introduction of reds for rapid reduction, alkylation, precipitation, and trypsin digestion. the tenfold serial dilutions of cultured viruses were prepared in cymol, a collection, transport and preservation medium known for its compatibility with multiple diagnostic platforms including cytology and molecular diagnostics (luinstra et al., ) . suspending viruses in cymol significantly (approximately -fold) improved the identification limit compared to the other tested solutions, such as ammonium bicarbonate, nte buffer ( mm nacl, mm tris-hcl [ph . ], mm edta), pbs (phosphate buffered saline), and a universal transport medium (utm-rt; data not shown). besides monocultures of influenza a strains, mixed samples of influenza a virus with either hmpv or rsv were subjected to maldi-tof ms and lc-ms/ms. viruses were identified simultaneously and specifically by lc-ms/ms (table ) , with a detection limit of . × for influenza a (h n and h n ), . × for rsv, and . × genome copies for hmpv. identification was not possible by maldi-tof ms due to its low sensitivity. in addition to monocultures and mixed samples, throat swabs spiked with influenza a, hmpv or rsv were subjected to lc-ms/ms. all the spiked viruses were identified, with detection limits of , , and genome copies for influenza a h n , influenza a h n and rsv, and hmpv, respectively. these results demonstrate that lc-ms/ms is not only able to identify monocultures of viruses, but can also discriminate between two viruses in the same sample and can identify a virus from a sample containing human proteins originating from a throat swab. sensitivity of lc-ms/ms appeared to be lower when mixed samples or reconstituted samples were tested. viral loads in clinical samples vary depending on the type of respiratory material, subtype of the virus, severity and phase of infection, and host factors, such as immunity. in general, viral loads fall within the range of - genome copies/ml (ngaosuwankul et al., ; lee et al., ) . in addition, in a recent pan-european survey (nuttall et al., ) , the average ct values for influenza virus, rsv, and hmpv detected in patients were , , and , respectively (coenjaerts, f.e.j., personal communication). therefore, the ultimate identification limit of a mass spectrometry method should reach genome copies/ml for direct clinical application. the sensitivity of the developed sample preparation method in combination with lc-ms/ms is -fold lower and therefore not good enough for direct application in virological diagnostics. for detection of viruses from clinical samples, viral culture is needed before samples can be prepared and analyzed. because culturing is required, viable virus has to be recovered and successfully grown. for application in virological diagnostics, sensitivity should be increased by developing techniques that enable fast and efficient virus concentration and purification from respiratory samples. furthermore, mass spectrometry systems should be improved to increase sensitivity. in the meanwhile, keeping the drawbacks into mind this in-house developed sample preparation method in combination with lc-ms/ms could be experimentally used for patients with suspicion of a respiratory infection where routine diagnostics are negative. the identification of the influenza a strains, rsv and hmpv was based on detection of peptides derived from different proteins. all these identified peptides were found to be encoded in the corresponding genome sequences. maldi-tof ms and lc-ms/ms both detected nucleoprotein-derived peptides. in addition, lc-ms/msidentified peptides derived from other viral proteins, namely m , ns , ha, and na proteins of influenza viruses and m, m - , p, and f proteins of rsv and hmpv. the identification of peptides from different proteins not only accomplishes identification but also potentially delivers information about virulence, resistance to antiviral medications, and/or transmission efficiency. for instance, an amino acid substitution in nucleoprotein (m l) was shown to enhance human-to-human transmission (zhou et al., ) . interestingly, one of the identified nucleoprotein-derived peptides, i.e., qanngedataglthimiwhsnlndatyqr, contained this substitution. this peptide was found in all of the tested influenza strains ( table ), suggesting that these strains might exhibit increased human-to-human transmission efficiency. in conclusion, mass spectrometry systems have been investigated for the identification of highly pure and/or concentrated influenza a viruses. however, used sample preparation methods were laborious and time-consuming. the developed sample preparation method in combination with lc-ms/ms allowed rapid and reliable identification of cultured respiratory viruses and viruses spiked in throat swabs. to introduce this lc-ms/ms based identification method in virological diagnostics, sensitivity must be improved up to -fold and clinical samples have to be tested. the combination of an improved 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the matrix genes of the human influenza a(h n )pdm and a(h n ) viruses reduce the detection sensitivity of real-time reverse transcription-pcr single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses rapid evolution of h n influenza viruses in chickens in hong kong this work was supported financially by the dutch ministry of defense, grant number v . supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . key: cord- -sj ngpk authors: he, qigai; du, qingyun; lau, suelyn; manopo, ivanus; lu, liqun; chan, shzu-wei; fenner, beau j.; kwang, jimmy title: characterization of monoclonal antibody against sars coronavirus nucleocapsid antigen and development of an antigen capture elisa date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: sj ngpk this report describes the production of several mabs against n protein, a major immunodomain of sars cov nucleocapsid protein [he, q., chong, k.h., chang, h.h., leung, b., ling, a.e., wei, t., chan, s.w., ooi, e.e., kwang, j., . development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. clin. diagn. lab. immunol. ( ) – .]. one representative igg monoclonal antibody (mab), s-a d , was selected and characterized. s-a d reacted specifically react with both recombinant and native nucleocapsid protein of sars cov. the reactivity of s-a d with purified n protein and utilization of the mab as a detector antibody to develop an antigen capture elisa was assessed. as little as . pg of purified n protein and tcid( ) of sars cov could be detected by the antigen capture elisa. specific binding of the mab s-a d to both purified n and sars cov nucleocapsid antigen was effectively inhibited by human sars positive serum and guinea pig anti-n serum. the n protein in n -spike recombinant baculovirus-infected sf- cells could also be identified. n protein was detected in ifa igm-positive serum samples collected from sars confirmed patients, but not in nine samples collected from sars recovery patient. no false positive results were given when samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (ibv), chicken coronavirus, was tested. this monoclonal antibody-based antigen capture elisa is thus a powerful tool for early diagnosis of sars cov infection. severe acute respiratory syndrome coronavirus (sars cov) is the causative agent of a new and emerging disease worldwide marra et al., ) . the disease was widely prevalent in more than countries with reported cases and causing deaths in (who, . thus, the development of diagnostic tests for specific and early detection of sars cov will contribute to the risk management of the disease. at present, sars cov infection is confirmed by the detection of viral rna via pcr or rt-pcr (drosten et al., ; poon et al., ) , however, this is a technically demanding technique and this is susceptible to cross contamination. the determination of infectious virus in samples can be carried out by inoculating cell cultures, such as vero cells, with a patient specimen, though this is relatively time consuming. most serological assays developed so far are based on the detection of specific circulating antibodies he et al., ) . these assays are highly sensitive and specific for detecting antibodies against sars cov. however, a lack of detectable antibodies in sars cov infected patients at early stage or throughout the whole disease course has been reported. these cases oc- - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.jviromet. . . curred in sars patients who were immuno-compromised or who had chronic conditions, e.g., diabetes mellitus or chronic renal insufficiency and may remain afebrile when acutely ill or possess symptoms attributable to underlying diseases, thus delaying sars diagnosis (houng et al., ) . therefore, more effort should be directed towards developing a simple and inexpensive assay for the detection of sars cov proteins. such tests could be used for early detection and follow-up of patients during treatment and thus reducing the workload of laboratory personnel. antibodies against the nucleocapsid protein are longer lived and occur in greater abundance in sars patients than antibodies against other viral components such as the spike, membrane and envelope proteins chen et al., ; huang et al., ; kim et al., ; tan et al., ; timani et al., ; zhu et al., ) . this might be due to the higher expression of nucleocapsid as compared with other viral proteins after sars cov infection (rota et al., ) . these data indicated that nucleocapsid could play a crucial role in antibody response during infection. in our previous work, a major immunodomain of recombinant sars cov nucleocapsid (n ) was identified and used to develop a western blot for the detection of antibodies against sars cov infection, the sensitivity and specificity were . and %, respectively . from these results, we hypothesize that monoclonal antibodies against n would specifically recognize sars cov in immunoassays. in this study, we produced monoclonal antibodies against n protein. the monoclonal antibodies were characterized by sars cov-infected vero cells and nucleocapsid-spike fusion protein-based ifa, western blot, and n proteinbased elisa. the isotype of the promising monoclonal antibody, designated as s-a d , was determined and was further applied to develop a specific and sensitive antigen capture elisa for the detection of sars cov. the sensitivity and specificity of this antigen capture elisa was also assessed. rpmi medium and fetal bovine serum (fbs) were obtained from invitrogen (carlsbad, ca). hypoxanthineaminopeterin-thymidine (hat) supplement used for the propagation of hybridoma was purchased from sigma (st. louis, mo). mice myeloma cell s/p . and spodoptera frugiperda (sf- ) cells were available from our laboratory. human cells (thp- , a ) and vero cells were obtained from the institute of molecular cell biology (imcb), singapore. inactivated sars cov (sin strain) was provided by singapore general hospital. twenty-seven sera that were available from patients presenting symptoms satisfying the world health organization (who) definition of sars and sera were obtained from healthy individuals. sera were provided by tock seng hospital and were heat inactivated at • c for h before use. the recombinant baculovirus bearing the fusion gene encoding n and sc fragment of the spike protein was constructed as described previously (he et al., ) . a commercial sars ifa diagnostic kit (euroimmun, germany), in which inactivated sars cov infected vero cells was used as antigen, was purchased from medizinische labordiagnostika ag, germany. three serum samples from infectious bronchitis virus (ibv)infected chickens and ibv m strain were also tested. the experiments involving the use of the inactivated sars cov or human sars inactivated sera were performed in a bsl laboratory. sars cov n and the n-terminal amino acids of nucleocapsid (n ) protein were expressed and purified as described previously . n was used for animal immunization as well as the antigen in antibodydetection elisa and western blot assay while n acted as a heterologous antigen for optimization of antigen capture elisa. four - -week-old balb/c mice were injected subcutaneously with g of purified n protein emulsified with an equal volume of adjuvant (seppic, france) for three times with a -week interval. mice received a final booster injection with g of antigen in l pbs to the intraperitoneal cavity days prior to hybridoma fusion. mice were sacrificed and their spleen cells harvested. mice s/p . myeloma cells were in log-phase growth prior to fusion with spleen cells (yokoyama, ) . hybridoma culture supernatants were screened using elisa, ifa and western blot as described previously . when the desired clones were identified, they were expanded in cm flasks. one week later, the hybridoma suspension was harvested and cell debris pelleted via centrifugation at g for min, followed by collection of the supernatant and storage at − • c. fifty microliters of hybridoma supernatant was incubated in -well microplates (nunc, denmark) coated with purified nucleocapsid protein, and the bound antibody detected with a : dilution of horseradish peroxidase (hrp) labeled rabbit anti-mouse immunoglobulin (dako, denmark). after extensive washing, the plates were incubated with ophenylenediamine dihydrochloride (opd) (sigma, usa) for min and the reaction was stopped by addition of . l m h so . the absorbances at nm were read using a tecan microplate reader. hybridoma supernatants were diluted : in sample buffer and mixed thoroughly by vortexing, followed by analysis with a commercial kit (euroimmuno, germany), in which sars cov infected vero cells were used as the fluorescence antigen. the two techniques were used to further confirm the reactivity to n protein in accordance with our previous descriptions (he et al., , . the reactivity of mab was evaluated with antigen capture elisa in which purified n was employed as the standard antigen and two-fold serially diluted mab as the detector antibody. hybridoma supernatant that did not produce mab served as a negative control. isotyping was performed using a mouse mab isotyping kit (amersham bioscience, england). guinea pigs were immunized with g of purified n protein. booster injections were administered at week intervals. ten days later the animals were euthanized for serum preparation. samples were evaluated for antibody against the n by n -based western blot. subsequently, igg was extracted from the antiserum using protein a affinity chromatography (sigma, usa) and the concentration determined using a bca measurement kit (sigma, usa). the reactivity of the igg with n protein was assessed by an antigen capture elisa, as described below. the -well flat bottom microtiter plates (nunc, demark) were coated with ng of purified igg in l carbonate buffer ( mm sodium bicarbonate and mm sodium carbonate) per well, incubated at • c for h or at • c overnight. plates were washed three times with pbs-t and blocked with l of blocking solution ( % nonfat milk in pbs-t) and incubated at • c for h. after the plates were rinsed with pbs-t for three times, l of purified n protein in pbst containing % nonfat milk was added. the plate was washed three times with pbs-t, and l of mab was added to each well. the plate was washed again and l of rabbit anti-human immunoglobulin hrp-conjugated antibodies was added at : dilution. after another extensive wash with pbs-t, l of opd was added to each well. plates were incubated at room temperature for min and the absorbance was read at nm. for elisa optimization, monoclonal antibody and purified igg were serially diluted two-fold and were used as detector and capture antibody, respectively, in the new assay. optimization conditions were determined by comparing the homologous (n ) and heterologous (n ) reaction to achieve the highest specificity and the signal-to-noise ratio for this assay. the signal-to noise ratio was calculated by dividing the absorbance of homologous antigen by that of heterologous antigen. purified n ( ng/l) was -fold serially diluted with % nonfat milk in pbs-t as diluent and used as tested antigen in the capture elisa format as described above. the diluent was employed as a blank control. prior to being tested, inactivated sars cov culture ( . pfu/ml) was treated with sample lysis solution ( . % tween- , % triton, and % nonfat milk in pbs) and incubated at • c for h. after centrifugation at g for min, the supernatant was assayed. vero, thp- and a cells were used as negative controls. the detection limits of both standard protein and sars cov were determined according to the cut-off value, which was calculated by the formula (x + s.d.). chicken infectious bronchitis virus (ibv), one of the animal coronaviruses, was propagated in spf embryos. debris was removed from the harvested virus supernatant by centrifugation. the titer of virus was determined to be . eid / . ml. ibv was treated using the same sample lysis buffer and methods as those used for sars cov, and assayed in the same way. twenty five microliters of purified n , at concentration of and ng/l, respectively, were mixed with guinea pig anti-n serum and a serum from a sars convalescent patient. additionally, × and × tcid of inactivated sars cov were incubated with guinea pig anti-n serum. normal guinea pig and human normal sera were included as a negative serum controls. these contents were subsequently incubated at • c for h, followed by incubation at • c for h before sandwich elisa analysis, as described above. the percent inhibition of antibody binding was calculated by the following formula: % inhibition = [ . − (a of n + positive serum)/(a of n + normal serum)] × . the specificity of the blocking was confirmed if the percentage of inhibition was greater than . detection of sars cov protein from human infected cells was mimicked using sf- cells expressing recombinant nucleocapsid, protein according to our previous report (he et al., ) . pellets were resuspended in the sample lysis solution, incubated at • c for h, -fold serially diluted with % nonfat milk in pbs-t before being tested. non-infected sf- cells were used as a negative control. the detection limit of recombinant n protein in the cell lysates was determined according to the cut-off value (x + s.d.). thirty serum samples obtained from healthy individuals were used in the test to determine cut-off value for the new capture elisa. subsequently, serum samples, which were collected from patients infected with sars cov during the sars outbreak in singapore in and were shown to be igm-positive using inactivated sars cov-based ifa, and serum samples available from sars recovery patients and blood donors, respectively, were tested by the antigen capture elisa. fusion of spleen cells from immunized balb/c mice with s/p . myeloma cells produced several hybridoma clones secreting mabs against n proteins (data not shown). positive clones were determined through indirect elisa as having at least a three-fold higher absorbance than that of the background. the positive clones showed great variation in their ability to secret mab. the hybridoma cell lines yielding the highest antibody titer, s-a d , was selected to produce monoclonal antibody for further analyses and experiments. after incubation with mab and fitc-conjugated antibody and subsequent examination by fluorescence microscopy, positive cytoplasmic immunofluorescent stainings of the authentic virus antigen in infected vero cells and recombinant n expressed in sf- cells were shown (figs. a and a), identical to those obtained with guinea pig anti-n monospecific antibody (figs. b and b) and with human sars patient serum (figs. c and c), while fluorescent staining was not observed in the noninfected vero cells and sf- cells ( fig. d and e, fig. d and e). the specific reactivity of the mab s-a d with purified n protein (fig. a ) was identical to that of the human sars positive serum (fig. b) , while no reaction was observed when non-antibody secreting hybridoma was tested (fig. c) . the titrations of mab by elisa, n -based western blot, n -spike fusion protein based ifa and sars cov infected cell-based ifa were : , : , : and : , respectively. finally, the isotypes of the representative mab, s-a d , was determined as igg class; elisa reactivity with purified n is shown in fig. . with the increase in the dilution factors of mab, absorbance gradually decreased, while absorbances of mab at : and : dilution were nearly equivalent to those of the blank controls. thus, the reactivity was dose-dependent. to standardize the sandwich elisa, igg was isolated and purified from pooled guinea pig anti-n sera whose titer was determined to be : by western blot. the concentration of extracted igg was determined to be . g/l. sds-page analysis showed two bands of appropriately . and . kda, representing the heavy and light chain regions (data not shown). as expected, a dose-dependent reactivity of purified igg occurred (fig. ) . the absorbances were far greater than those obtained with the same concentration of nonimmunized guinea pig serum. the absorbances decreased dramatically when the igg concentration was . g/well, and the reactivity of . ng of igg was equal to that of the blank igg control. as a first step in the development of our elisa, mab and purified igg were diluted in coating buffer and was used as capture antibodies to coat the microtiter plates, respectively. however, igg binding to the hydrophobic and hydrophilic microtiter plates was more efficient than the mab. therefore, the elisa plates were coated with purified igg and incubated at • c overnight or at • c for h in the following experiments. when the hrp-conjugated antibody was used at the recommended concentration ( : ) in the test, : dilution of capture antibody ( . g/well) and : dilution of mab were determined as the optimal working conditions based on the signal-to-noise ratio ( . ) ( table ). the antigen capture elisa was standardized using the optimal conditions. the absorbance values of normal vero, thp- and a cells were . , . and . , respectively. therefore, the cut-off value for detection of viruses in cell culture was determined as . (x + s.d.). according to the cut-off threshold ( . ), a − dilution of n protein and a − dilution of sars cov suspension were considered positive (fig. ) . so, it was deduced that as little as . pg of purified n protein, tcid of sars cov could be detected. sf- cells ( × cells/ml) had a % infection rate with recombinant baculovirus at h post-infection based on fluorescence assays using mab against n as the primary antibody. a l aliquot of each dilution of sf- cell lysate supernatant was subjected to elisa. absorbance values of . - . were obtained at dilutions of + − to − . according to the calculated cut-off value ( . + [ × . ] = . ), the test was able to detect a − dilution of infected sf- cells ( × cells/ml) (fig. ) . thus, as few as infected sf- cells could be detected by this elisa. according to the cut-off value based on the detection of samples from healthy individuals, sars cov n protein could be detected in all of the ifa igm-positive sera, but none of the nine samples from sars recovery patients using the new elisa. no false positive results were produced in sera from healthy blood donors. the recognition site of the mab on n protein was blocked by guinea pig anti-n serum and human sars positive serum, leading to the lower absorbance (fig. ) . sars cov could not be detected after incubation with anti-n serum. human and guinea pig normal serum could not inhibit the binding of mab to n or native viral nucleocap- in this test, : and : dilution of igg and mab were used in the standardization of the capture elisa. a this number represents the signal-to-noise ratio of the sample measured at different combinations of the capture and detector antibody in the antigen capture elisa. fig. . inhibition effects of guinea pig anti-n serum and human sars positive serum on the recognition of nucleocapsid antigen by detector antibody. groups and : . g of n ; groups and : . g of n ; groups and : × and × tcid of sars cov. the different concentrations of n protein or sars cov were incubated with guinea pig anti-n serum, human sars positive serum (long bars), guinea pig and human normal serum (short bar), respectively. the contents were subjected to capture elisa. sid antigen (longer bars). the calculated inhibition rates were more than % (shorter bars). in addition, no cross-reaction was observed when ibv was tested. this result demonstrates that the antigen capture elisa is highly specific for detecting the nucleocapsid antigen of sars cov. sars cov is an etiological agent causing severe acute respiratory syndrome, a newly emergent disease. there is a global need to develop a sensitive and specific immunoassay to detect sars cov. in our previous work, a major immunodomain of n protein (n ) was identified and n -based western blot assay was developed to detect antibodies against sars cov. therefore, it is reasonable to assume that a mab against this n protein will improve the test specificity. this paper describes the production of monoclonal antibody against n protein as well as the development of an antigen capture elisa, offering a safe, cost-effective tool for detection of sars coronavirus. in this test, the mab reacted with both n and authentic nucleocapsid in sars cov, indicating the potential of this mab to detect sars cov by immunoassays. moreover, the mab at : dilution was detectable by sf- cell-based ifa whereas mab at : dilution could be detected by sars cov infected vero cells-based ifa. this is partially explained by the fact that the nucleocapsid gene, fused with truncated spike gene, was placed under the transcriptional control of the strong polyhedrin promoter of autographa californica nuclear polyhedrosis virus (acnpv) in the recombinant baculovirus, leading to an abundance of n in infected sf- cells. the recognition of the authentic nucleocapsid antigen indicated that the mab produced in this work can be used to develop immunoassays, for instance, ifa, for direct detection of sars cov from nasopharyngeal aspirates obtained from the patients that are diagnosed as probable or suspected cases under clinical criteria. compared to the mab, igg displayed stronger binding to microtiter plates and was therefore used as capture antibody with the mab as detector antibody in this antigen capture elisa. the detection limit of the test is . pg of standard protein and tcid of sars cov. this sensitivity is consistent with the previous descriptions of sensitivity in other antigen capture elisas. in an attempt to further improve the sensitivity of the assay, mab against the immunogenic sc fragment of spike protein was produced in this experiment and used as a detector antibody to detect spike protein in infected sf- cell lysates and inactivated sars cov. however, this method did not improve sensitivity. in the specificity test, the specific binding of both recombinant and native nucleocapsid by mab was uniformly inhibited by guinea pig monospecific antiserum to n and human sars positive serum, but not by human and guinea pig normal sera. due to a lack of coronavirus infected human samples, ibv was used to assess the specificity of the mab for other coronaviruses. in this experiment, ibv could not be recognized by the mab, indicating the mab's specificity. these observations suggest that the mab-based assay is specific for detection of sars cov infection. moreover, the n protein was consistently detected in our limited igm-positive samples but not in eight igg-positive sera from convalescent patients, indicating the potential of early detection of sars cov infection although it needs clinical trials, consistent with new finding (di et al., ) . theoretically, antigen should be released from infected cells or viruses and appear in the host blood system followed by the presence of relative specific antibody. additionally, due to the abundant expression of n protein compared to other proteins during viral replication in infected tissues, the host immune system is exposed to a larger load of n antigen. therefore, this antigen capture elisa, based on mab to n protein, might provide a more sensitive method for early detection of sars cov infection. antibody detection of sars-cov spike and nucleocapsid protein antigenicity analysis of different regions of the severe acute respiratory syndrome coronavirus nucleocapsid protein monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay reveals high sensitivity of the nucleocapsid protein in acutephage sea of severe acute respiratory syndrome patients identification of a novel coronavirus in patients with severe acute respiratory syndrome evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome novel immunofluorescence assay using recombinant nucleocapsidspike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus evaluation of antibody responses against sars coronaviral nucleocapsid or spike proteins by immunoblotting or elisa development and evaluation of an efficient -noncoding region based sars coronavirus (sars-cov) rt-pcr assayfor detection of sars-cov infections generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus a one step quantitative rt-pcr for detection of sars coronavirus with an internal control for pcr inhibitors profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers cloning, sequencing, expression, and purification of sars-associated coronavirus nucleocapsid protein for serodiagnosis of sars cumulative number of reported probable cases of severe acute respiratory syndrome (sars) production of monoclonal antibodies induction of sars-nucleoprotein-specific immune response by use of dna vaccine we would like to thank dr. ooi eng eong, dr. ling ai ee, dr. chang hiok hee and dr. bernard leung for providing technical support. this project was funded by agri-food and veterinary authority, singapore. key: cord- -nycdhdsd authors: schoenike, barry; franta, amy k.; fleming, john o. title: quantitative sense-specific determination of murine coronavirus rna by reverse transcription polymerase chain reaction date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: nycdhdsd in many applications, it is useful to know the sense and amount of viral rnas present in a sample. in theory, sense-specific measurement of viral rnas may be achieved by reverse transcription polymerase chain reaction (rt-pcr) assays which utilize primers of defined polarity during the rt step. however, in practice, it has been shown that such assays are prone to artifacts, such as non-specific priming, which drastically diminish their reliability. using murine coronavirus mhv- as a model, we describe and validate several modifications of the rt-pcr procedure which eliminate these artifacts. key rt-pcr parameters which were optimized include the design of tagged primers, dnase treatment of in vitro transcribed rna standards, specification of temperature differences between rt and pcr annealing steps, and use of competitive rna templates for quantitative assays. the assays described may be used to determine the sense and abundance of any viral or host rna of interest in complex biological specimens. in the course of many experiments in virology, it is of interest to determine the sense and amount of viral rnas. for example, in the case of viruses with a rna genome, the presence of rnas of antigenomic sense usually indicates active viral transcription. in other studies, in which viral gene expression appears altered, it would be informative to determine whether this change is correlated with corresponding alterations in the level of pos-itive or negative sense viral rna. a striking change in the expected relative or absolute abundance of viral rna of a given sense may provide clues as to the mode of viral replication in vitro or of viral pathogenesis in vivo. in some applications, the sense of viral rnas present in a sample may be determined unambiguously by northern blotting utilizing sense-specific probes. however, in many experiments, particularly with samples obtained in vivo, viral rna may not be of sufficiently high concentration to allow detection by this technique. clearly, rt-pcr assays provide high sensitivity (approximately - logs more than northern blot or nuclease protection assays) (foley et al., ; ausubel et al., ; kö hler et al., ) and are widely used to detect trace amounts of viral rna in many experiments. in theory, it should be straightforward to assay for viral rnas of a given sense by using a primer of defined polarity in the rt step of the procedure. for example, if the rt step is primed by an oligonucleotide which binds negative sense viral rna, cdna should be produced for subsequent pcr amplification if and only if viral templates of negative sense are present in the original specimen; if negative sense viral rna is not present, the reaction should be aborted at the rt step, and theoretically no pcr amplification products will be produced. in fact, several studies have demonstrated that rt-pcr assays based on specific rna template recognition by rt primers of defined polarity will not reliably distinguish between viral rnas of positive or negative sense. for example, in control assays using defined hepatitis c virus (hcv) rna templates, lanford et al. ( ) demonstrated that an rt-pcr product could be obtained with the 'incorrect' sense rt primer or even in the absence of any primer in the rt step. they hypothesized that artifacts arose through a combination of factors, including false priming of the incorrect strand by the rt primer, self-priming of the viral rna template at regions of secondary structure, and random priming by contaminating or irrelevant nucleic acids. similarly, gunji et al. ( ) , also working with hcv, showed that conventional rt-pcr assays were not able to reliably detect viral positive and nega-tive sense rnas in samples which contained cellular rnas. these investigators suggested that fragmented cellular rnas were able to prime viral templates, giving rise to unreliable results. despite the findings above, many published research reports are based on conventional rt-pcr assays, relying on the polarity of primers added to the rt step in putative sense-specific measurements of viral rnas; rarely are control reactions performed to rigorously show that this method is in fact sense-specific. however, upon critical review of the state of available sense-specific rt-pcr assays, mcguinness et al. ( ) indicated '… our results clearly establish that reliable detection of hcv-negative rna strand is not yet achievable by current methods'. recently, lerat et al., ( ) demonstrated that even optimized protocols artifactually detect negative sense hcv rna when these assays are applied to clinical samples. they indicated that artifacts are most likely due to self-priming by abundant viral rnas in infected tissues. collectively, these careful studies, using hcv as a model virus, indicate that technical impediments to unambiguous strand-specific rt-pcr determinations of viral rna may still exist. in the course of studying the persistence of murine coronaviruses in the mouse central nervous system (houtman and fleming, ) it was found necessary to determine the sense and amount of viral rna in the brains of experimental animals. to this end, a rt-pcr procedure was developed and validated which can reliably measure positive and negative sense viral rna present in trace amounts in a complex biological sample. we describe potential pitfalls of sensespecific rt-pcr assays (ssrt-pcr), as well as techniques introduced by other investigators (chaves et al., ; lanford et al., ; lerat et al., ) and our laboratory to circumvent these artifacts. finally, the addition of competitor rna templates to the assay allowed the ssrt-pcr to be performed quantitatively (qssrt-pcr). the method described may easily be adapted to other viral systems, where it is essential to determine the sense and amount of viral rnas in heterogeneous samples which may contain inhibitory or confounding molecules. all experiments were carried out with murine coronavirus mhv- , strain . -v- ; (wang et al., ) , which was propagated and assayed on dbt cells, as previously described (fleming et al., ) . all mice were -week-old male c bl/ j mice (jackson laboratory), seronegative for mhv, which were inoculated with plaqueforming units (pfus) of virus intracerebrally. total cellular rna was extracted from mouse brains, dbt cells, or fl cells which are hela cells expressing the mhv receptor (generously provided by dr thomas gallagher, loyola university, chicago), using rna stat- (teltest) and following the directions of the manufacturer. subsequently, rna was resuspended in depc-treated mm edta ph . and stored at − °c. plasmids were constructed for use in in vitro rna transcription to produce control templates for rt-pcr assays. gene of mhv- , which codes for the nucleocapsid, or n, protein was chosen as a region of interest. because of the redundant, %-nested structure of coronavirus rnas (lai and cavanagh, ) , the sequences of gene , the most % gene, are found in all mhv- rna species. thus, assaying for rnas with the targeted n gene sequence will reflect total mhv- rna abundance. mhv- rna templates were used in rt-pcr reactions to generate two plasmids designated nwt, corresponding to nucleotides - of the mhv- n gene, and n-del, containing a bp deletion and an inserted bgl ii restriction site relative to n-wt. after rt-pcr amplification, rna species transcribed from these plasmids or rt-pcr cdna products made from these template rnas could be distinguished by size or restriction digestions. however, as the n-wt and n-del derived rna templates only differed by approximately % of their lengths and contained identical primer binding sites, the amplification efficiencies of the two templates were expected to be nearly equivalent. plasmid n-wt was constructed by performing pcr reactions using the titan rt-pcr system (boehringer). one microgram of rna from mhv- infected dbt cells was used as the template in rt-pcr reactions primed by . mm of oligonucleotide primers am - and am - (table ). in order to facilitate directional ligation, these primers contain short non-viral sequences encoding a % hind iii or kpn i restriction site respectively. rt was performed at °c for min, followed by treatment at °c for min. the reactants were then subjected to pcr as per the instructions of the titan system protocol, and cycles of amplification, each of which consisted of incubation at °c for min, °c for min, and °c for min were performed. after the last pcr cycle, the reaction was held at °c for min. pcr products were ethanol precipitated, digested with hind iii and kpn i, gel-purified, and ligated into plasmid pgem- z (promega), which was used to transform competent escherichia coli dh a cells. plasmid n-wt dna was prepared with the wizard maxiprep system (promega), following the instructions of the manufacturer. plasmid n-del was prepared by a similar procedure, except that overlap extension techniques horton et al., ; silver et al., ) were used to create the bp deletion and bgl ii site relative to n-wt. briefly, individual pcrs were performed containing . mm of either primer set ( ) am - and am -b or, in the second reaction, ( ) am - and am -c ( table ). the products from reactions ( ) and ( ) were gel purified, mixed, and subjected to a second pcr utilizing . mm each of primers am - and am - . this product was purified subsequently, ligated into pgem- z, and used to produce ndel dna templates as described above. because pgem- z plasmid (promega) contains promoters for sp and t rna polymerases on opposite sides of the sequences inserted into this dna, the system allows in vitro transcription of rnas from either strand of the dna, thus producing rna transcripts which correspond to either sense of the inserted sequences. to produce rna which corresponded to the positive sense of mhv- , either n-wt or n-del plasmids were digested with eco ri, and . mg of the linearized templates were used in an in vitro transcription driven by the ribomax system (promega), following the instructions of the manufacturer and utilizing ml of sp enzyme mix per ml reaction. templates corresponding to negative sense of mhv- were produced using the same protocol, except that plasmids n-wt or n-del were linearized with hind iii, and t rna polymerase was used in the in vitro transcription reactions. after h of incubation, rna templates were obtained from each reaction by three extractions with phenol:chloroform:isoamyl alcohol ( : : , ph . ). subsequently, rna was precipitated by adjusting nh oac to m, fol-lowed by the addition of . volumes of % ethanol. after a wash in % ethanol, the rna pellet was resuspended in ml depc-treated water. in order to inactivate any residual plasmid template, the rna suspension was then subjected to a reaction in a ml volume containing mm tris ph . , mm mncl , . mg/ml bovine serum albumin, mm dtt, u rnasin (promega), and u dnase i (promega). after a -h incubation at °c, the reaction was terminated by three extractions with phenol:choloroform:isoamyl alcohol ( : : , ph . ), followed by ethanol precipitation. the rna pellet was then resuspended in ml mm edta, ph . , and quantitated by absorbency at nm, as well as by comparison to rna standards on formaldehyde gels (promega). rna was then mixed with two volumes of % ethanol, and nacl was added to achieve a final concentration of mm, after which the sample was stored at − °c. a oligonucleotides were used to prime templates corresponding to the n gene of mhv- (genbank database accession number k ). the irrelevant or tag marker sequences correspond to gene ( a protein and coat protein) of the plant virus cowpea chlorotic mottle virus (ccmv) (genbank database accession number m ), control plasmids for which were generously provided by dr paul ahlquist, university of wisconsin, madison. please see text for detailed description of overlap primer use. b all sequences correspond to gene n of mhv- , unless marked with an asterisk, in which case they correspond to gene of ccmv. in most primers, short non-viral sequences were added at the % end to create restriction sites and facilitate cloning. c nucleotides which are italicized are non-viral sequences encoding restriction endonuclease recognition sites to facilitate cloning of rt-pcr products. these sequences are, respectively, hind iii (am - ), kpn i (am - ), ecor i (am - ), bamh i (am - ), and bgl ii (am -b and am -c). the following protocol ( fig. ) was developed after pilot and optimizing experiments. various amounts of rna template were mixed with pmol of tag-am - a primer for positive sense amplification or tag-am - a primer for negative sense amplification respectively (table ) . rt reactions were performed in a final volume of ml containing rna and primers in a buffer with the following final concentrations: mm tris -hcl ph . , mm kcl, mm mgcl , mm dtt, . mm dntps, u rnasin, and u mmlv reverse transcriptase (promega). the reaction was incubated at °c for h. an ampliwax pcr gem wax tab (perkin elmer) was added to each tube, and the tubes were incubated at °c for min to inactivate the reverse transcriptase. the reactions were then cooled on ice. an ml mixture was added on top of the wax tab; this solution consisted of mm kcl, mm tris-hcl ph . , . % triton x- , . mm mgcl , . u taq dna polymerase (promega), and pmol primers am - and imn- for positive sense amplification or primers am - and imn- (table ) for negative sense amplification. the pcr assay was performed in a perkin elmer thermal cycler for cycles, each of which consisted of min at °c, minute at °c, and min at °c. at the termination of the pcr, an additional extension reaction was performed at °c for min. rt-pcr products were visualized after ethidium bromide staining and electrophoresis on % nusieve (fmc) : agarose gel or on % agarose. molecular weights of products were estimated by comparison to puc dna digested separately with sau a i and taq i restriction endonucleases. for quantitative assays, gels were analyzed by computer-assisted quantitative densitometry (nih image, version . , available at http://rsb.info.nih.gov/ nih-image/). quantification of mhv- rna was achieved by competitive rt-pcr reactions in which a known amount of n-del transcript was added as a standard to each rt reaction. rt-pcr products were visualized by ethidium bromide staining and electrophoresis as described above, and the relative intensity of product bands in each reaction was compared by inspection or by densitometry. because n-del has a nucleotide deletion relative to genuine mhv- sequences, reaction products corresponding to this standard template were easily distinguished from full-length product because their migration in electrophoresis was faster than products derived from n-wt or viral rna templates. quantification was achieved either by: ( ) comparison of serial dilutions of n-del standard to unknown sample in the equimolar quantitative rt-pcr (qrt-pcr) method to determine the point of equivalence (gilland et al., ) ; or ( ) comparison of an unknown sample's reactivity versus one concentration of n-del in the standardcurve quantitative qrt-pcr method (tsai and wiltbank, ) . the optimized protocol above was first used to determine the sensitivity, or limits of detection, for the ssrt-pcr assay. serial dilutions of in vitro-transcribed rna n-del templates of either positive or negative sense were amplified with specific primers. the optimized ssrt-pcr could detect approximately . fg of positive sense or negative sense n-del template, either in the pres-ence (fig. ) or absence (data not shown) of mg of total cellular rna derived from uninfected dbt cells. also, we found that the assay for positive sense or negative sense coronavirus rna did not yield a product when fg of the opposite or 'wrong' sense was added to the reaction (fig. ) . however, when fg of the inappropriate rna template was added to the reaction, a non-specific product was detected (data not shown), indicating that the assay has a maximal differential amplification capacity of approximately -fold with regard to detection of a specific rna template relative to an excess of inappropriate or opposite sense rna template. fig. , when compared to fig. , indicates that the inhibition of specific rna detection in the presence of an excess of wrong sense rna is insignificant. finally, in pilot experiments, we have been able to extend the sensitivity of the ssrt-pcr to the attogram (approximately mhv- rna molecules) range by means of nested pcr (zazzi et al., ) methodology using primers am - and am - (data not shown); in this case, it is essential in each experiment to include controls of sense specificity, e.g. a control reaction demonstrating the non-amplification of an appropriate concentration of wrong sense rna. the precision, or reproducibility, of the qssrt-pcr assay was determined by testing aliquots of rna extracted from the brains of an experimental mouse days after mhv- inoculation i.c. an example of this assay is shown in fig. . the assay for positive sense viral rna was conducted in the pg range and the negative sense assay conducted in the fg range on the basis of published studies and pilot experiments showing that positive sense coronavirus rna is in vast excess in most infections. additional bands were sometimes observed during competitive reactions (fig. ) ; these bands likely represent heteroduplexes (schneeberger et al., ) and were not of sufficient magnitude to interfere with quantification. during densitometric analyses, bands corresponding to heteroduplexes were ignored, as these spe-cies would be expected to detract from the intensity of both competitor (mhv- and n-del) rnas equally. three assays were performed by standard curve methodology on separate days ( table ) . as indicated, the coefficients of variation for the positive sense and negative sense assays were . and . %, respectively. similar reproducibility was obtained with rnas obtained from mouse brains during chronic pathogenesis studies and with rnas derived from infected tissue cultures (data not shown). rock ( ) indicates that 'where an accepted standard with a known and agreed upon quantitative result exists, accuracy can be operationally defined as the difference between the measured value of the standard and the value assigned to the standard'. we initially assessed the accuracy of the qssrt-pcr by carefully determining the concentrations of rna template stocks by uv absorbency and comparison to commercial rna standards. reference standards of positive and negative sense n-wt with an assigned value of . fg were then prepared and tested in the qssrt-pcr using equimolar methodology in competitions against n-del. the measured values for n-wt standards were . and . fg for positive and negative sense templates respectively (versus . fg, the expected result in a perfect assay, assuming perfect quantification of input rna stocks against commercial rna standards). unfortunately, it is difficult to produce purified coronavirus genomes in sufficient quantities to serve as biophysically-quantified standards for di- rectly assessing the accuracy of our qssrt-pcr versus authentic viral rna. however, it was possible to isolate and measure viral rna from fl cells after in vitro infection by mhv- and to compare this result with published results based on established methods. using equimolar methodology, qssrt-pcr showed that at maximal infection h after inoculation, fl cells contained . ng or . × molecules of rna competing with n-del template per microgram of total cellular rna and, further, that \ % of the mhv- rna was of positive sense (data not shown). assuming that the yield of total rna extracted from fl cells was approximately . × − mg of rna/cell (rna stat instructions), on a molar basis these results are in close agreement with prior studies based on hybridization kinetics (cheley et al., ) and northern blots (hof-mann et al., ) which indicate that at the peak of coronavirus infection in vitro in the order of molecules of viral rna are present in each infected cell. the consistency among different methods of estimating the level of coronavirus expression suggests that the qssrt-pcr assay yields reasonably accurate results when applied to complex biological samples, such as rna derived from virally-infected cells. in studies of coronavirus pathogenesis during chronic viral infection of the murine central nervous system, it was found essential to determine the sense and amount of viral rna persisting in experimental mice. while rt-pcr in theory should provide the requisite specificity and sensitivity for these determinations, our initial, exploratory experiments showed that conventional rt-pcr assays for viral rnas were not sensespecific, let alone quantitative, findings which were concordant with those of previous investigators (willems et al., ) . in this regard, most rt-pcr protocols are primarily designed to optimize sensitivity rather than discrimination with regard to rna template amplification. in other words, in most applications, low levels of amplification of non-specific products are acceptable and do not detract from the primary purpose of the assay, which is efficient detection of the rna of interest. by contrast, in ssrt-pcr specificity is the over-riding consideration, since the aim of the assay is unambiguous detection of an rna of a given sense, often under circumstances in which the rna is orders of magnitude less abundant than an rna species of the opposite sense (e.g. table ). in this case, assay parameters must be such that non-amplification of incorrect sense rna templates is virtually absolute, since any amplification of the incorrect sense rna, however infrequent, will be geometrically expanded during pcr and may easily lend to invalid results. in order to improve the specificity of ssrt-pcr determinations of viral rna we have adopted and modified several of the procedures described by previous investigators. in our experience, the use of tagged oligonucleotide primers (chaves et al., ; lanford et al., ) was more practical and effective than other proposed specificity-enhancing measures, such as chemical modification of rna templates (gunji et al., ) or the use of thermostable enzymes, such as rtth dna polymerase, in both rt and pcr steps (lanford et al., ) . the key features of our optimized assay are listed in table . we have found that careful attention to each of these parameters is imperati e in order to maintain sense-specificity and consistent quantification in the ssrt-pcr. essentially, during ssrt-pcr there are numerous opportunities for artifacts, and if reliable results are to be obtained, stringent precautions must be taken at every step of the procedure to avoid these artifacts. furthermore, control samples should be run with every assay in order to be certain that non-specific amplification has not occurred. crucial methodological parameters of ssrt-pcr are indicated below. first, a critical consideration in the ssrt-pcr assay developed is the design of oligonucleotide primers for the rt step. these oligonucleotides are hybrid primers in the sense that they contain sequences which are complementary to viral sequences, as well as sequences which are complementary to a non-viral 'tag', or irrelevant, sequence which will be recognized by tag-only primers in the pcr phase of the assay (fig. ) . previously reported rt hybrid primers had approximately equal lengths of anti-viral and tag sequences; as a result, rt reactions were performed at °c, and pcr annealing temperatures were - °c (chaves et al., ; lanford et al., ; lerat et al., ) . by contrast, we have found it essential to use unbalanced or asymmetrical rt hybrid primers which have short ( - mers) anti-viral sequences relative to anti-tag sequences ( - mers) (e.g. primer tag-am - a). pilot experiments demonstrated that symmetrical primers with low pcr annealing temperatures gave rise to non-specific products (data not shown). however, a results are expressed in pg of positive sense or fg of negative sense mhv- rna corresponding to the n-wt amplimer/mg of total brain rna obtained from a mouse days after viral inoculation intracerebrally. three experiments done by standard curve methodology on separate days are shown, and the results are expressed as the mean and standard deviation (s.d.) for the three assays, as well coefficient of variation (cv) for positive and negative qssrt-pcr assays. table key features of the optimized quantitative sense-specific rt-pcr which affect assay performance in the optimized assay, the rt step is performed at °c, close to the melting temperature (tm) of anti-viral sequences of our rt primers, and the pcr annealing step, which depends on longer tag:anti-tag sequence interactions, is set at °c. thus, both the rt and pcr phases of our assay are each conducted under conditions of high stringency, at the threshold at which specific primers are able to anneal correctly to specific templates; in this case, the high stringency in both phases of the assay strongly inhibits non-specific priming. another advantage of asymmetrical primers relates to carryover of hybrid rt primers into the pcr phase of the assay. in reported assays in which the anti-viral and tag sequences of the hybrid primers are both approximately - nucleotides, resulting in rt reaction and pcr annealing temperatures which are close to each other, hybrid rt primers are available and can anneal during the pcr phase of the assay. this availability may result in multiple rounds of cumulative non-specific priming during the pcr, for example, of hybrid primers to aberrant viral rnas, prior mis-primed products, or to plasmidderived dna (in the case of in vitro transcribed rna standards). in the optimized pcr described, primers carried over from the rt step into the pcr steps are unlikely to misprime, since pcr annealing occurs at temperatures which exceed the tm of viral:anti-viral template:primer interactions but are optimal for specific binding of longer tag:anti-tag sequences. also, during the pcr phase of the assay, the primers for this step are at -fold molar excess relative to primers carried over from the rt step. this discrepancy puts primers from the rt step at a further relative disadvantage during the pcr steps and favors specific tag:anti-tag interactions during the pcr. second, we have found that mmlv and °c are the optimum combination of reverse transcriptase and temperature for the rt step of our assay. in competitive assays undertaken at lower temperatures, rt of viral rna was diminished relative to in vitro mhv- transcribed templates (e.g. n-del), suggesting that rna derived from virus may have extensive secondary structure or other features at °c which decrease the proces-sivity of mmlv on this template. under a variety of conditions, the reverse transcriptase amv consistently gave rise to non-specific products, thus limiting its applicability in the sense-specific assay described above. a third methodological consideration relates to the requirement that the specificity of ssrt-pcr assays must be tested with control rna templates of known sense. typically, such rna standards are transcribed in vitro from dna plasmids. paradoxically, these control rna template preparations may introduce artifacts which are not present in actual experimental or clinical samples. for example, traces of plasmid dna, obviously containing strands of both positive and negative sense, virtually always contaminate rnas which are transcribed in vitro by conventional protocols; in this case, strand specificity cannot be demonstrated since control reagents themselves are contaminated by sequences of the 'wrong' sense. in order to overcome this problem, it is essential after in vitro transcription of rna standards to digest the plasmid template with high concentrations of rnase-free dnase in a buffer containing mn (bauer et al., ) (table ) . conventional dnase treatments in mg-containing buffers did not completely degrade plasmid dna (data not shown). in our experience, when non-specific products are observed during ssrt-pcr, trouble shooting usually reveals that this artifact is caused by trace contamination of control rna reagents by residual plasmid dna. this problem may be corrected by either producing new batches of rna standards or by repeated dnase treatment of existing rna standards. also, the conditions of our rt-pcr assay minimize the possibility of amplification of residual, contaminating plasmid dna in two other ways. first, during the rt step at °c plasmid dna templates are likely to remain double-stranded and thus are unavailable for amplification. second, during the pcr step, only primers recognizing tag sequences introduced into prior products will prime taq polymerase (fig. ) ; therefore, carryover plasmid dna, having exclusively viral sequences and lacking tag sequences, will not be amplified. clearly, for assay consistency over time it is necessary to maintain a ribonuclease-free labora-tory environment (blumberg, ; jagus, ) and to avoid rna degradation, for example, during tissue extraction, rna storage, or repeated freeze-thaw cycles of samples (busch et al., ; farkas et al., ; gill et al., ; halfon et al., ) . rna should be isolated immediately, or tissues should be snap-frozen at − °c. the stability of purified rna can be maximized by storing stocks as one-use aliquots or as ethanol precipitants at − °c in dilute nacl and edta. recent evidence suggests that optimum rna storage may be achieved in a solution of mm sodium citrate, ph . (prediger, ) . another essential assay parameter is the institution of a hot start for the pcr, either by means of a heating block (mullis, ) or a wax tab (perkin elmer; chou, et al., ) , in order to avoid non-specific products which result from priming at low temperatures. initially there was considerable, justified scepticism as to the reliability of pcr and related assays with regard to quantitative determination of nucleic acid abundances. however, improved methodology and rigorous control of reactions have permitted unambiguous quantitative pcr and rt-pcr determinations with good correspondence to values obtained with established standard methods, such as northern blot (wang et al., ; yokoi, et al., ) . in fact, the reliability of rt-pcr and similar techniques is such that quantitative assays have been approved for routine clinical use, for example, in determining hiv- mrna levels in patients during antiretroviral chemotherapy (saag, ) . in this regard, once the ssrt-pcr assay was optimized, it was relatively straightforward to extend the method to quantitative determinations by adding competitor rna templates to the reactions. relative to viral sequences, such competitor rnas have identical primer binding sites but contain a small deletion which allows specific products generated from these templates to be distinguished on sizing gels. the power of competitive rt-pcr assays is derived from the fact that the sample and competitor rna templates are amplified in the same reaction tube under identical conditions. assaying both templates in one tube eliminates sources of error which may occur when control and experimental templates are amplified in separate tubes and subsequently compared to each other. competitive qrt-pcr assays may be run in a variety of formats (reviewed in kö hler et al., ) , including equimolar methodology (gilland et al., ) and standard curve methodology (tsai and wiltbank, ) . in our assays of coronavirus rna by qssrt-pcr, we have found that both the equimolar format and standard curve format work well and have similar sensitivity, specificity, precision, accuracy, and practicability (data not shown). we have found it easiest to use the qssrt-pcr in the equimolar mode during initial experiments, when the magnitude of expected coronavirus rna concentration is unknown. by contrast, the standard curve method has been most convenient at later stages of experiments, when the approximate range of coronavirus rna concentrations may be anticipated and many replicate samples in this range must be analyzed. since equimolar and standard curve methods utilize the same reagents and procedures, and differ only in the format in which competitions are conducted, it is easy to switch between these two modes of analysis. in conclusion, after the requirements for production of tagged primers for sense-specificity and competitor rna templates for quantification have been met, the assay we have described may easily be performed in any virology laboratory with rt-pcr capabilities. the sense and quantity of any viral rna may be reliably determined if strict attention is given to controls and to the methodological points listed in table . the assay has been demonstrated to have excellent sensitivity, specificity, and precision, with almost no interference by irrelevant or opposite-sense rnas. it is more difficult to assess the accuracy, defined as freedom from systematic analytical error or correspondence to a true value (rock, ) , of the qssrt-pcr assay we have presented, especially when purified, exactly-quantified standards of genuine viral rna are not available. the experiments presented demonstrate reasonably accurate assay performance with a variety of conditions and standards, including competitions against rna obtained during viral infection in vitro. we suspect that the degree of inaccuracy observed, particularly in the case of negative sense n-wt determinations, is due principally to small errors in measuring the concentrations of our initial rna template stock solutions. in many applications it may be sufficient to determine if a given viral rna species is increasing or decreasing relative to a baseline or standard value during the course of an experiment. if, on the other hand, it is essential to know the concentration of a viral rna in absolute terms, for example, to rigorously determine the average number of viral genomes per infected cell, then additional efforts must be made to precisely measure the concentration of competitor rna standards, followed by careful titration of these standards in competitions against actual viral rnas under realistic experimental conditions. current protocols in molecular biology use of maganese in rt-pcr eliminates pcr artifacts resulting from dnase i digestion creating a ribonuclease-free environment impact of specimen handling and storage on detection of hepatitis c virus rna specific detection of minus strand hepatitis a virus rna by tail-pcr following reverse transcription intracellular murine hepatitis virusspecific rnas contain common sequences prevention of pre-pcr mis-priming and primer dimerization improves low-copy-number amplifications specimen collection and storage for diagnostic molecular pathology investigation pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies quantitation of rna using the polymerase chain reaction ensuring recovery of intact rna from rat pancreas competitive pcr for quantitation of mrna specific detection of positive and negative stranded hepatitis c viral rna using chemical rna modification impact of various handling and storage conditions on quantitative detection of hepatitis c virus rna site directed mutagenesis by overlap extension using the polymerase chain reaction bovine coronavirus mrna replication continues throughout persistent infection in cell culture engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension pathogenesis of mouse hepatitis virus-induced demyelination hybrid selection of mrna and hybrid arrest of translation quantification of mrna by polymerase chain reaction the molecular biology of coronaviruses demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis c virus using strand-specific rt-pcr specific detection of hepatitis c virus minus strand rna in hematopoietic cells false detection of negativestrand hepatitis c virus rna the polymerase chain reaction in an anemic mode: how to avoid cold oligodeoxyribonuclear fusion rna isolation: new ways to handle tissue and store rna laboratory medicine: the selection and interpretation of clinical laboratory studies quantification of hiv viral load: a tool for clinical practice quantitative detection of reverse transcriptase-pcr products by means of a novel and sensitive dna stain site-specific mutagenesis using the polymerase chain reaction quantification of mrna using competitive rt-pcr with standard curve methodology quantitation of mrna by the polymerase chain reaction sequence analysis of the spike protein gene of murine coronavirus variants: study of genetic sites affecting neuropathogenicity non-radioisotopic quantitative rt-pcr to detect changes in mrna levels during early mouse embryo development optimal conditions for detection of human immunodeficiency virus type dna by polymerase chain reaction with nested primers we would like to thank dr thomas gallagher for the gift of fl cells and dr mary lokuta for helpful discussions and suggestions. we also thank meera nathan and joan pooley for excellent technical assistance, and nola mills for excellent editorial assistance. this work was funded by a merit review grant from the veterans administration and by research grant rg -b- from the national multiple sclerosis society. key: cord- -k p fo e authors: nidzworski, dawid; rabalski, lukasz; gromadzka, beata title: detection and differentiation of virulent and avirulent strains of newcastle disease virus by real-time pcr date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k p fo e a rapid diagnostic method based on the melting curve sybr green i real-time pcr analysis was developed to detect and differentiate newcastle disease virus (ndv) strains. degenerated primers based on the cleavage site sequence of the f gene were designed to detect specific sequences characteristic of virulent and avirulent strains of ndv. eighteen strains of ndv from four lineages were identified and grouped into virulent and avirulent strains. peaks on the melting temperature graph with melting temperature values between . and . °c were observed for lentogenic (avirulent) strains. t(m) values higher than . were observed for virulent (mesogenic and velogenic) strains. the detection limit of real-time pcr was × ( ) plasmid copies per reaction or ( ) eid( ) for velogenic strains and ( ) eid( ) for lentogenic strains. the results obtained in this study demonstrate the possible applications for melting curve real-time pcr analysis in laboratory practice for the diagnosis and differentiation of avirulent and virulent strains of newcastle disease virus. belonging to the mononegavirales order, paramyxoviridae family and avulavirus genus, newcastle disease virus (ndv) is an enveloped virus containing linear, non-segmented single-stranded rna (lamb et al., ) . ndv strains are classified into one of three major pathotypes: velogenic, mesogenic and lentogenic (beard and hanson, ) . highly virulent velogenic strains cause acute infections with high mortality, mesogenic (intermediately virulent) strains cause respiratory disease in young chickens and decreased egg production, whereas lentogenic (avirulent) strains cause mild respiratory infection or give no symptoms in poultry (seal et al., ) . studies conducted by rott and klenk ( ) revealed the key role of a small amino acid motif at the cleavage site of the f protein of ndv in the molecular determination of the pathotype. the presence of two pairs of basic amino acids at positions , , and and phenyloalanine at position , at fusion protein cleavage site, makes this site susceptible to cleavage by common subtilisin-like protease and easier spread throughout the host. the sequence (c- r/k-r-q-k/r-r-f -n) is observed in highly virulent strains. in the low virulence strains, there are only two basic abbreviations: aiv, avian influenza virus; apmv, avian paramyxovirus; csfv, classical swine fever virus; eid, egg infective dose; icpi, intercerebral pathogenicity index; ndv, newcastle disease virus; pcr, polymerase chain reaction. * corresponding author. tel.: + ; fax: + . e-mail address: dawid.nidzworski@biotech.ug.gda.pl (d. nidzworski). amino acids at position and , along with leucine at position (c- g/e-k/r-q-g/e-r-l -n). this motif is recognized only by trypsin-like proteases, which are present mainly in the respiratory tract (collins et al., ; nagai et al., ) . because of the role of the cleavage site in pathogenicity, the f gene is the major target for differentiation of newcastle disease virus. the variability in the virulence of ndv causes diagnostic difficulties because only viral infections with an icpi of . or greater are notifiable (oie, ) . therefore, detection of the virus only is not a sufficient for notification and should be followed by methods that evaluate the pathogenicity of isolates. the sensitivity and short time analysis of real-time pcr are its two most important advantages (higuchi et al., ) . several methods for the detection and differentiation of ndv have been previously described. most assays are based on the detection of the fusion gene (pham et al., ) or the matrix and fusion genes (wise et al., ; steyer et al., ) . however, other regions are also used for general detection and differentiation (fuller et al., ; tan et al., ). the first sybr green i assay for the detection of ndv was described by tan et al. ( ) , but this assay could not be used for differentiation of pathotypes of the virus. pham et al. ( ) described a sybr green i real-time pcr melting curve analysis assay for differentiation, although the differences in the tm values between the three genotypes were not very significant and could cause false characterization of the virus. many real-time pcr assays for ndv detection and differentiation using different taqman probes were developed but degenerated primers were not used very often. also, false negative results were reported (cattoli et al., ; wise et al., ) . in some cases, to differentiate between pathotypes of ndv, it was necessary to use two or three pairs of primers and several different taqman probes in one reaction were necessary (wise et al., ; farkas et al., ) . degenerated primers have been proven to be a good tool for the detection of the highly variable newcastle disease virus (farkas et al., ; li et al., ; steyer et al., ) . in this study, a one tube diagnostic method is described for the detection and differentiation of newcastle disease virus using degenerated primers. the ndv strains that were analyzed in this study are listed in table . all of the strains were derived from the collection of the national veterinary research institute (pulawy, poland) and were classified into three major pathotypes based on the intracerebral pathogenicity index (icpi) and comparison with reference strains. avian influenza virus (aiv) subtype h n , coronavirus ( / - ), rotavirus (g / - ), gumboro virus ( / ), and classical swine fever virus (csvf) cellpest strain were from the national veterinary research institute (pulawy, poland). apmv strains were bought from x-ovo limited (dunfermline, united kingdom). reovirus (reo s ) and infectious bronchitis virus (ib k+ / ) were vaccine components of nobilis reo and nobilis ib - (intervet/schering-plough animal health, holland). rna was extracted from allantoic fluid of spf embryonated eggs (lohman, germany), and inoculated with ndv strains in accordance with the oie diagnostic manual (oie, ) using an rneasy mini kit (qiagen, valencia, ca, usa) protocol. additionally, rna from oral and cloacal swabs and homogenized tissues was isolated using the same kit. a set of degenerated primers based on the f gene of ndv was used in this study: ndv-f (position - ) -ctt ggt gay tcy atc cg- ndv r (position - ) -aca cyg ccd ata atg gc- where y = c or t; d = a or g or t. the amplified cdna fragment was base pairs (bp) long and contained the region encoding the f cleavage site. synthesis of the first strand of cdna was carried out according to the procedure described for the transcriptor high fidelity cdna synthesis kit (roche diagnostics, mannheim, germany). the reaction mixture contained: viral rna, random hexamer primers and water that was incubated for min at • c. subsequently, the reaction buffer, rnase inhibitor, dntps, dtt, and reverse transcriptase were added. the final mixture contained: × reaction buffer [ mm tris-hcl, mm kcl, mm mgcl ; ph . ], u of rnase inhibitor [in: mm hepes-koh, mm kcl, mm dtt, % glycerol; ph . ], m of random hexamer primers, table melting-curve analysis of viral strains. lineages by aldous et al. ( ) . apmv - na --- coronavirus mm of each dntps, mm of dtt, and u of reverse transcriptase (in: mm potassium phosphate, mm dtt, . % triton x- , % glycerol, ph . ). it was then incubated for min at • c, followed by min. incubation at • c and chilled on ice. sybr green i real-time pcr was performed using a lightcycler . tm (roche diagnostics, mannheim, germany). l of the final mixture contained l of reaction mixture: × lc dna master sybr green i (roche diagnostics, mannheim, germany), mgcl ( mm), . m of each primer, and l of cdna. a negative control lacking cdna was also prepared. the samples were initially denatured at • c for min, followed by cycles of the following: • c (denaturation) for s, • c (annealing) for s, and • c (extension) for s. to confirm the specificity of the amplified product, a melting curve analysis step was included. the internal temperature of the lightcycler was increased rapidly to • c, then decreased at . • c/s to • c, and then the sample was incubated for s. the fluorescence at nm was measured continuously. the melting peaks were generated using lightcycler software by plotting the first negative derivative of the fluorescence over the temperature versus the temperature (−df/dt). the detection limit was determined using the target copy number and virus titer ( % egg infective dose -eid ) as reference units. (i) a fragment ( bp) of the f gene of the lasota strain was cloned into pgem-t easy vector (promega, madison, wi, usa). plasmid dna was extracted from the bacteria by using a plasmid mini kit (a&a biotechnology, gdansk, poland). the concentration of the plasmid dna was determined spectrophotometrically. the dna copy number was calculated using the formula described by ke et al. ( ) . the plasmid was serially -fold diluted to serve as a standard for determining the sensitivity of the real-time pcr assay. (ii) serial -fold dilutions of the known eid stock solution of lentogenic (lasota) and velogenic (italy/ / ) strains were prepared using sterile dh . rna was extracted and transcribed as described above. cdna was added to the reaction mixture as a template. the sybr green i melting peaks analysis assay was compared with the method described by mia kim et al. ( ) . the sequencing was carried out by genomed ltd. (warsaw, poland) using a roche gs flx/titanium sequencer. multalin software (corpet, ) was used to align the sequences to check for homology with reference ndv sequences. the specificity of the sybr green i real-time pcr assay was investigated using the rna extracted from ndv strains and other avian viruses shown in table . phylogenetic analysis of members of lineages of newcastle disease virus (aldous et al., ) was performed utilizing the neighbor-joining method (saitou and nei, ) in mega software. strains of ndv were derived from genbank or from the polish strains sequenced previously. the representatives of lineages of ndv strains were tested. using the sybr green i real-time pcr melting peak analysis, it was possible to detect and differentiate virulent and avirulent strains of newcastle disease virus. eighteen ndv strains (including ten lentogenic, two mesogenic and six velogenic isolates) were analyzed. melting curve analysis revealed that all strains were divided into two distinct groups (fig. ) . t m values for lentogenic (avirulent) strains were estimated to be between . and . • c, and the t m values for mesogenic and velogenic (virulent) strains were higher than . • c ( table ). the boundary line was calculated as the medium between the highest t m value for avirulent strains and the lowest for virulent strains. the phylogenetic relationship of the ndv strains used in this study is depicted in fig. . the specificity of this real-time pcr assay was investigated by analysing ndv strains and samples of other viruses (table ) . no amplification products were detected when templates originating from these fourteen unrelated viruses were used, while all templates from ndv strains gave distinctive products. the detection limit of this assay was assessed by analysing the serial dilutions of a plasmid carrying s bp fragment of the f gene of ndv and the serial dilutions of the known stock of ndv. in the real-time pcr assay, the detection limits were × copies of dna plasmid per reaction or eid concentration range for the velogenic strain (italy/ / ) and eid for the lentogenic strain (lasota). the results were comparable with the results described by wise et al. ( ) . any results with a ct value higher than were treated as unconvincing and were rejected. the new assay was compared with the method described previously by mia kim et al. ( ) -both gave similar results (table a) in oral and cloacal swabs. the difference in ct values resulted from the use of different methods in the assays (taqman and sybr green i, respectively). the new assay detected ndv in three tissue samples while the one described previously detected ndv only in one sample (table b) . queensland v / (aldous et al., ) . polish isolates are in bold. the phylogenetic tree was generated using the neighbor-joining method (saitou and nei, ) . ulster c (table ) ; queensland v / (m ); roakin (ay ); beaudette c/ (m ); b / (m ); / (table ) ; / (table ) ; / (table ) ; / (table ) ; / (table ) ; / (table ) ; / (table ) ; radom (table ) ; bsdck (ay ); pok/ (y ); bbgck (ay ); bhkpi (ay ); herts/ (m ); texas (m ); mukteswar (ay ); miyadera/ (m ); australia/ (m ); ar (table ) ; ar (table ) ; ar (table ) ; ar (table ) ; ar (table ) ; ar (table ) ; italy (table ) ; gb / (af ); ch / (af ); kuwait (af ); lebanon/ (af ); warwick/ (u ); bsack (ay ); bbeck (ay ); qgb / (af ); fi / (af ); qgb / (af ); cz / (af ); tw / (af ); hfrdk (ay ). a comparison of the sensitivity of sybr green i real-time pcr melting peaks analysis assay (sybr mpa) with the previously published system by mia kim et al. ( ) . (a) three chickens were infected with a lentogenic strain (lasota) with a dose of , eid of the virus. oral (o) and cloacal (c) swabs were collected and days post infection (dpi). (b) one chicken was killed days post infection, and tissues were prepared. rna was extracted as described above from a homogenate of tissues. in this study, a method for the rapid detection and differentiation of newcastle disease virus by sybr green i melting-curve analysis was described. f gene-specific, degenerated primers were used in the assay. the use of degenerated primers made it possible to avoid the previously reported difficulties in the design of primers (aldous and alexander, ) and, additionally, it allowed for the use of only one set of primers for the differentiation of virulent and avirulent strains of newcastle disease virus. this is the first assay based on sybr green i that uses degenerated primers to detect virulent and avirulent strains of ndv in one tube reaction with high resolution. the correlations between virulent (icpi > . ) and non-virulent (icpi < . ) strains and melting temperature values are shown in table . the distinction between lentogenic and mesogenic ( . • c) or lentogenic and velogenic ( . • c) ndv strains was more simple than previously described by pham et al. ( ) , which yielded values of . • c and . • c, respectively. additionally, such a high difference in melting temperature values makes it possible to calculate a safe boundary line ( . • c) for differentiation of ndv strains. this in turn allows for a straightforward method to monitor and classify virulent and avirulent strains of ndv in agreement with european union directive (cec, ) , which calls for the full control of isolates with an intercerebral pathogenicity index (icpi) greater than . . the sensitivity of the method described in this report is similar to the method described by mia kim et al. ( ) and wise et al. ( ) and by pham et al. ( ) . differences in ct values are cause by differences across methods. in real-time pcr using the taqman method (mia kim et al., ) , probes are released and degraded during each cycle and fluorescence increases cumulatively during the reaction. when the sybr green i method is used fluorescence increases proportionally. that explains why higher ct values were obtained while using this method. the advantages of using this sybr green i method are that its operation costs are lower in comparison to either methods using taqman, and it is possible to perform the reaction in a single tube. the previous methods employed two or three pairs of primers (wise et al., ) , and their products for lentogenic and velogenic strains (farkas et al., ) , in some cases, were different in length, which excludes the possibility of using the real-time pcr sybr green i assay. detection and differentiation of newcastle disease virus (avian paramyxovirus type ) a molecular epidemiological study of avian paramyxovirus type (newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene newcastle disease false-negative results of a validated real-time pcr protocol for diagnosis of newcastle disease due to genetic variability of the matrix gene council directive / /eec of july introducing community measures for the control of newcastle disease pathogenicity and phylogenetic evaluation of the variant newcastle disease viruses termed "pigeon pmv- viruses" based on the nucleotide sequence of the fusion protein gene multiple sequence alignment with hierarchical clustering development of an l gene real-time reverse-transcription pcr assay for the detection of avian paramyxovirus type rna in clinical samples real-time pcr-based pathotyping of newcastle disease virus by use of taqman minor groove binder probes simultaneous amplification and detection of specific dna sequences development of a quantitative light cycler real-time rt-pcr for detection of avian reovirus family paramyxoviridae degenerate primers based rt-pcr for rapid detection and differentiation of airborne chicken newcastle disease virus in chicken houses detection of a broad range of class i and ii newcastle disease viruses using a multiplex real time reverse transcription polymerase chain reaction assay proteolytic cleavage of the viral glycoproteins and its significance for the virulence of newcastle disease virus manual of diagnostic tests and vaccines for terrestrial animals: mammals, birds and bees rapid detection and differentiation of newcastle disease virus by realtime pcr with melting-curve analysis molecular basis of infectivity and pathogenicity of newcastle disease virus the neighbor-joining method: a new method for reconstructing phylogenetic tree characterization of newcastle disease virus isolates by reverse transcription pcr coupled to direct nucleotide sequencing and development of sequence database for pathotype prediction and molecular epidemiological analysis a diagnostic method based on mgb probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type detection and differentiation of velogenic and lentogenic newcastle disease viruses using sybr green i real-time pcr with nucleocapsid gene-specific primers detection of newcastle disease virus using a sybr green i real-time polymerase chain reaction development of a real-time reverse transcription pcr for detection of newcastle disease virus rna in clinical samples the authors wish to thank their colleagues from nvri pulawy, poland: krzysztof smietanka and zenon minta for their invaluable suggestions, monika olszewska, elzbieta juszczuk and michal jozwiak for technical support in experiments and tomasz stadejek for providing the csfv strain. key: cord- -ti rpt q authors: zhao, kai; hu, ruili; ni, jianping; liang, jieling; he, xizhong; du, yanan; xu, yan; zhao, binan; zhang, qi; li, chunhua title: establishment of a porcine parvovirus (ppv) lamp visual rapid detection method date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: ti rpt q porcine parvovirus (ppv) is one of the major causes of reproductive pig disease. due to its serious nature, wide spread and consequent great damage to the swine industry, an effective, rapid and convenient method for its detection is needed. a loop-mediated isothermal amplification (lamp) assay was established to detect ppv infection. two pairs of primers were specifically designed to recognize the six different sequences of open reading frame (orf ) gene. the optimized lamp program was as follows: min at °c followed by min at °c.the amplified products were analyzed both by visual inspection after staining with sybr green i dye and by conventional agarose gel electrophoresis. both methods showed the same sensitivity. the limit of detection (lod) for ppv by lamp was copies, which is -fold lower than conventional pcr. our lamp assay did not cross-react with other viruses. we used the established lamp system to test field samples and detected positives. the lamp detection method for ppv represents a visual, sensitive and rapid assay which can detect the virus in the field, offering an attractive alternative for the ppv detection methods currently in use. porcine parvovirus (ppv), a virus belonging to the parvoviridae family, causes maternal reproductive failure of swine known as porcine reproductive system disease which is a serious problem in the pig breeding industry. the characteristics of ppv infection in infected sows (especially primiparous sows) are stillbirth, fetal malformation and mummification, but the infection also can cause neonatal death and piglet disease including diarrhea and dermatitis (yin and liu, ) . all kinds of pigs can be infected by ppv, such as domestic pigs, wild boar, newborn piglets, finishing j o u r n a l p r e -p r o o f pigs and spf pigs. however, the pregnant sow itself and some infected pigs do not have evident clinical symptoms (kennedy et al., ; ellis et al., ) . ppv has caused huge losses to the pig industry. therefore, an effective method is necessary to detect the ppv infection. currently, conventional pcr is used to detect and identify the virus (caprioli et al., ; huang et al., ; jiang et al., ) . but its amplification efficiency is affected by many disturbing inhibitors (wilson, ; abu and rådström, ) . enzyme linked immunosorbent assay (elisa) is also a common way to detect ppv (jenkins, ) . however, infected swine are difficult to diagnose by this method because they are prone to false-positive results during the analytical process (westenbrink et al., ) .though the common pcr, elisa and real-time pcr methods (zheng et al., ; pérez et al., ; ) are also suitable for the qualitative and quantitative analysis of ppv, it requires highly skilled laboratory technicians. therefore, an alternative quick, accurate and simple method is still needed to detect ppv. some years ago, a novel nucleic acids amplification technique was introduced which was called loop-mediated isothermal amplification (lamp) (notomi et al., ) . the method is simple and extremely specific to the target sequence, since the four primers can identify the six target sequences and amplify it (mori et al., ; zhang et al., ) . compared with other detection methods, the lamp method has many advantages, in particular specificity, sensitivity and rapidity. the lamp products have a typical ladder-like pattern and can be detected by adding sybr green i dye (zhang et al., ; iwamoto et al., ) . the lamp amplification solution can be visually turned to green in the presence of a dye sybr green i, while the lamp solution remains orange in the absence of amplification (iwamoto et al., ) . the lamp method has become a useful assay for the fast detection of food borne pathogenic microorganisms and infectious diseases (he et al., ) . other examples are the detection of heat-labile i and heat-stable i enterotoxin genes of enterotoxigenic escherichia coli by lamp (yano et al., ) . in this study, a detection method based on the lamp technology is described which is suitable for the clinical detection of porcine parvovirus. the diagnostic kit was j o u r n a l p r e -p r o o f developed, tested and applied. the present study offers the necessary technological basis for the prevention and control of porcine parvovirus infection. the viral strains used for the lamp assays were obtained from the institute of animal husbandry and veterinary science, shanghai academy of agricultural sciences. porcine parvoviruse (ppv), classical swine fever virus (csfv), porcine circovirus type (pcv ), porcine pseudorabies virus (prv) and porcine reproductive and respiratory syndrome virus (prrsv) were included. the geographical origin, and year of isolation of these viruses were summarized in table . pig sera were gathered from a slaughterhouse in shanghai (china) and used as clinical samples for the detection of ppv by lamp. dna was extracted from ppv, pcv and prv by the blood viral dna/rna kit (biomiga inc, san diego, ca). the dna from ppv obtained in the previous step was used as template to optimize the test reaction temperature. rna from prrsv and csfv was extracted following the same method as the dna. the process from rna to cdna was achieved by reverse transcription (takara corp., japan). these cdna templates were used for the next specific experiment. two pairs of primers were designed by primer explorer based on the ppv vp gene (capsid protein ) gene of ppv genome (https://www.ncbi.nlm.nih.gov/ nuccore kf . ). they were called fip, bip, f and b and the information of them were shown in table . the f and b primers were used in the pcr reaction and the target sequence was bp. pcr assays were performed in μl reaction volumes containing . μl ×buffer (takara), . mm dntps, . μm each of f and b , . u taq dna polymerase j o u r n a l p r e -p r o o f (takara biotechnology co., ltd, dalian, china) and μl template dna. the program consisted of an initial denaturation at °c for min, followed by cycles at °c for s, °c for s, and °c for s, and a final extension at °c for min in an applied biosystem thermal cycler (applied biosystem., us). the pcr products were sequenced and analyzed. meanwhile, the bp pcr products were purified using the qiaquick pcr purification kit (qiagen, germany),following the manufacturer's instructions. then the fragments were cloned into peasy®-t cloning vector and transformed into trans -t competent cell using peasy®-t cloning kit (transgen biotech co., ltd., beijing, china). the positive plasmid were obtained according to blue/white selection and identified by colony pcr and sequencing. the resulting positive plasmid containing vp gene fragment of ppv was extracted for further experiments. lamp reactions were performed in volumes of μl, which contained . μl ×buffer (-mg + )(takara), . mm mg + , . mm dntps, . μm each of fip and bip, . μm each of f and b , m betaine (sigma), and . u bst dna polymerase (vazyme biotech co.,ltd). after adding μl template dna, the mixture was incubated for min at °c and cooled on ice for min, after which the bst polymerase was added. the lamp reaction was performed in a conventional heating block. to optimize the reaction temperature, lamp was carried at ℃, . ℃, . ℃, . ℃, . ℃, . ℃, . ℃, . ℃, . ℃, . ℃ . ℃ and ℃ for min and terminated at ℃ for min, respectively. the reaction system was the same as method . mentioned above, and the template was plasmid containing ppv vp gene fragment with the concentration copies per μl. two reactions with and without inner set of primers were carried out at . ℃ as control. j o u r n a l p r e -p r o o f to verify the specificity of ppv detection by lamp, the dna (ppv, pcv , prv) and cdna samples (prrsv, csfv) were amplified as sample templates by the lamp reaction at . ℃ for min and terminated at ℃ for min, respectively. as a positive control we used the ppv vp plasmid; as a no template control (ntc) water was used. all reactions were repeated in duplicates. ten-fold serial dilutions of the ppv plasmid were made to obtain a gradient of to copy per μl. these plasmids of different concentration were prepared to define the limit of detection (lod) of ppv dna by lamp and pcr assays. the amplification products were checked on agarose gel electrophoresis. in addition, the lamp products were also checked by adding a dye sybr green i. lamp and pcr amplified products were detected by % (w/v) agarose gel electrophoresis and observed by staining with goldview (sbs genetech co., ltd., shanghai). the lamp products were also directly observed depending on their color by mixing each sample with μl : -diluted sybr green i (thermo fisher scientific, usa). the positive sample will turn green while the negative sample will still remain orange. whole blood samples were collected as random from "duroc ×landrace ×yorkshire" pigs with the weight of - kg and - days old. these blood samples were placed at ℃ for hours, then the sera were separated by centrifugation at r/min for minutes. serum samples were tested using the lamp and pcr method for the presence of ppv to determine if their source were infected with ppv. we also compared the lamp results of straightly using the serum samples with the dna extraction by the blood viral dna/rna kit. samples of serum were used as template in lamp reactions, meanwhile the genomic dna of these serum samples were extracted as template to perform this experiment. all reactions were carried out at . ℃ for min and terminated at ℃ for min, respectively. the optimum temperature for the lamp reaction was determined to be ± °c ( fig. ). at this temperature, the typical ladder-like pattern was the brightest and clearest, corresponding to the highest amount of product. the comparison between reactions with and without inner set primers confirmed the feasibility of this lamp protocol. in this assay, only the genomic dna samples of ppv strains and ppv plasmid (positive control) used as template gave the typical ladder-like bands and a green color, while the dna/cdna samples obtained for other virus species as well as the ntc had no bands and showed an orange color (fig. a and b) . the results indicated the primers could only amplify ppv nucleic acid. consistently, the length of the amplified product was bp as predicted. its sequence was confirmed to be % identical to the corresponding sequence in the ppv vp gene. the lower lod of ppv by lamp was found to be copies based on the results of the agarose gel electrophoresis analysis (fig. a) and visual observation (fig. b ). in contrast, the lod for the pcr was copies (fig. ) . these results indicate that the lamp method is about times more sensitive than the conventional pcr assay and this sensitivity is in line with the daily testing requirements. a total of serum samples were tested using the lamp and pcr methods to determine whether their sources were infected by ppv. among them, clinical samples were found to be positive by lamp and samples were positive by pcr (table ). in summary, serum samples were detected positive and samples were negative by both lamp and pcr. the coincidence rate of these two methods was . % ( / ) for clinical samples detection. the comparison between serum template and genomic dna template showed that dna extraction is not necessary and can be omitted. using the serum samples straightly as template doesn't affect the lamp results ( figure ). in this study, we developed a method for the visual and rapid detection of ppv using an optimized lamp technique. lamp has a number of advantages when compared to pcr, particularly its high sensitivity, easy manipulation in addition to its visual and time saving detection. we investigated the optimized ppv lamp method and observed its high specificity for ppv, showing no amplification products for any of the other viruses tested. importantly, two pairs of primers were used to identify the target gene by lamp (nagamine et al., ) , while only one pair of primers was used in conventional pcr. our lamp method has a high specificity because it targets the conserved region of ppv orf gene in the design of the two pairs of primers. the lamp reaction can be carried out under isothermal conditions in a relatively short time, without specific equipment like a pcr thermo cycler. the lamp reaction took only min total time and did not need either intricate pretreatment or an expensive apparatus. the reaction was more quickly than the enzyme linked immunosorbent assay and the real-time pcr. hence, the lamp method we developed has the advantages of easy manipulation and easy popularization. the lamp detection limit for ppv based on visual observation by addition of j o u r n a l p r e -p r o o f sybr green i and by gel electrophoresis analysis was copies. the sensitivity of detection by lamp was times higher than by conventional pcr. this indicated that the visual observation method could be used to analyze the lamp product and could reliably replace the conventional agarose gel electrophoresis . the sensitivity of the lamp method is in line with reports about other virus species, such as swine transmissible gastroenteritis coronavirus, h avian influenza virus and yellow head virus imai et al., ; mekata et al., ) . in the analysis of clinical samples, dna extraction and purification steps were not needed. serum samples can be used straightly as templates for the lamp reaction. the sensitivity of the lamp method was less affected by the composition of the clinical samples than observed with pcr. this feature not only can decrease the time and cost of the lamp reaction, but also can simplify many troublesome programs. for the evaluation of clinical samples, we tested randomly sera by lamp and pcr, and verified this feature. compared to conventional pcr, the detection rate of ppv by elisa was . % (jekins, ) . furthermore, elisa is known to easily cause false-positive results (westenbrink et al., ) . moreover, the elisa method is troublesome and time-consuming in contrast of the lamp method. although the lod by real-time pcr is times lower than by conventional pcr, the detection rate of real-time pcr for ppv is only . % ~ % (zheng et al., ; ). lamp method can make it applicable to laboratories, small-scale hospitals, private clinics and pig industry. for a reliable lamp test, some precautions should be adopted to prevent the occurrence of false positive results. for example, separate work areas and aerosol-resistant pipette tips should be used. meanwhile, the used pipette tips and reaction vessels should also be collected in airtight containers. moreover, it is advisable to divide reagents into aliquots in order to avoid contaminations. notably, negative control samples should be firstly finished as soon as possible when total reaction system is finished to aliquot. a lamp method for ppv has been developed successfully by others (chen et al., j o u r n a l p r e -p r o o f ; liu et al., ; qu et al., ) . chen et al chose to amplify the vp gene of ppv by using a set of four primers at ℃ for min. in this study, we selected four different primers to amplify the vp gene and used the sybr green i dye for the detection of ppv. although both lamp methods were established using specific primers based on the highly conserved ppv ns protein gene (qu et al., ) , the primers we designed are in different region of ns from them. we had aligned many ppv genome sequences and chose the most conserved region for primer design. furthermore, we combined with the dye sybr green i and realized the visual detection for ppv lamp instead of by the conventional gel electrophoresis analysis or fluorescent detection . in addition, dna extraction was omitted in order to save time. in the present study, porcine serum as sample can be used straightly in the lamp assay without dna extraction, as the lamp reaction is well tolerant against biological substances. therefore, in the lamp assay the dna extraction step can be ignored (kaneko et al., ) . thus, the lamp method can be used to detect ppv in the field without the need of a pcr thermocycle instrument, electrophoresis apparatus or turbidimeter. hence, our lamp protocol provides an attractive new method for the detection of ppv. the results of this study illustrate that lamp detection offers a convenient visual approach to detect ppv rapidly, sensitively, specifically and simply. above all, the lamp method was improved from the point of high reaction efficiency and accurateness. for ppv detection, it supplements and extends the former approach; the method was developed into a diagnostic kit that is well received and applied in the field. the authors declare no conflict of interest. all relevant data are within the paper and its supporting information. and ℃, respectively. the other reactions with and without inner set of primers were carried out at . ℃ as control. plasmids containing ppv vp gene fragment with the concentration copies per μl were as template. all reactions were incubated at above temperature for min and terminated at ℃ for min. the amplification products were detected by % (w/v) agarose gel electrophoresis staining with goldview. all reactions were carried out at . ℃ for min and terminated at ℃ for min. a. results on % (w/v) agarose gel electrophoresis staining with goldview. b. visual results by adding sybr green i. m: dl dna marker; n: no template control; pcr was carried out at to copies of plasmids of ppv as template, respectively. the amplification products were detected by % (w/v) agarose gel electrophoresis staining with goldview. 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rapid and sensitive detection of lily symptomless virus by reverse transcription loop-mediated isothermal amplification multiplex pcr for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses development of h -rt-lamp (loop-mediated isothermal amplification) system for rapid diagnosis of h avian influenza virus infection loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, m. avium, and m. j o u r n a l p r e -p r o o f intracellulare in sputum samples an enzyme-linked immunosorbent assay for detection of porcine parvovirus in fetal tissues simultaneous detection of porcine circovirus type , classical swine fever virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus in pigs by multiplex polymerase chain reaction tolerance of loop-mediated isothermal amplification to a culture medium and biological substances reproduction of lesions of post weaning multisystem wasting syndrome by infection of conventional pigs with porcine circovirus type alone or in combination with porcine parvovirus development of a loop-mediated isothermal amplification assay for the detection of porcine hokovirus establishment of porcine parvovirus detection by loop-mediated isothermal amplification assay detection of yellow head virus in shrimp by loop-mediated isothermal amplification (lamp) detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation accelerated reaction by loop-mediated isothermal amplification using loop primers loop-mediated isothermal amplification of dna isolation and identification of porcine parvovirus s- strain (in chinese) isolation and identification of porcine parvovirus s- strain (in chinese) a multiple sybr green i-based real-time pcr system forthe simultaneous dete ction of porcinecircovirus type , porcine parvovirus, pseudorabies virus and to rque teno sus virus and inpigs rapid and sensitive diagnosis of porcine parvovirus by loop-mediated isothermal amplification (lamp) method some multiplication properties of the lapinized chinese strain (c strain) of classical swine fever virus in primary bovine testicular cells (in chinese) an enzyme-linked immunosorbent assay for detection of antibodies to porcine parvovirus inhibition and facilitation of nucleic acid amplification rapid and sensitive detection of heat-labile i and heat-stable i enterotoxin genes of enterotoxigenic escherichia coli by loop-mediated isothermal amplification the pseudorabies vaccination research. i: pseudorabies attenuated vaccine research (in chinese) p r e -p r o o f rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines isolation and identification of nj strain of porcine parvovirus(in chinese) simultaneous detection of porcine parvovirus and porcine circovirus type by duplex real-time pcr and amplicon melting curve analysis using sybr green we are grateful to professor peter j.m. rottier for editing the manuscript. key: cord- -jzdlvo p authors: bennett, susan; gunson, rory n.; maclean, alasdair; miller, rhona; carman, william f. title: the validation of a real-time rt-pcr assay which detects influenza a and types simultaneously for influenza a h n ( ) and oseltamivir-resistant (h y) influenza a h n ( ) date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: jzdlvo p influenza a h n ( ) was declared by the world health organisation (who) as the first influenza pandemic of the st century. rapid detection of influenza a and differentiation of influenza a h n ( ) and seasonal influenza a is beneficial. in addition the rapid detection of antiviral resistant strains of influenza a h n ( ) would be useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. it was the aim of this study to develop a real-time rt-pcr that can detect all influenza a viruses and type simultaneously for influenza a h n ( ) and oseltamivir resistant (h y) influenza a h n ( ). this multiplex assay will allow laboratories to screen respiratory samples for all types of influenza a, influenza a h n ( ) virus and oseltamivir resistant (h y) influenza a h n ( ) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. since most virology laboratories already offer a molecular service for influenza a this assay could easily be implemented into most areas at little cost therefore increasing local access to resistance testing. the world health organisation (who) received the first report of an oseltamivir-resistant influenza a h n ( ) virus in july (who, a) . sequence analysis revealed that the resistance was associated with a histidine to tyrosine substitution in the viral neuraminidase (n ) at position . since this discovery a total of oseltamivir-resistant influenza a h n ( ) (h y mutant) cases have been reported to the global influenza surveillance network (gisn) (who as of . . ) (who, ) . of these cases, % have been in immunocompromised patients who have received prolonged oseltamivir treatment, % have been associated with oseltamivir treatment (who, ) . several cases have also been associated with chemoprophylaxis (mmwr cdc, a) and there have also been cases found in individuals who had not received treatment (chen et al., ) . despite the increasing number of cases there has been two reports of person to person transmission of resistant virus (gulland, ; who, a) . there has been one report of a community cluster of oseltamivir resistant cases (le et al., ) however there has been no evidence of ongoing circulation of the virus in the community. furthermore, the resis-tance strain has not been shown to cause altered or more severe infection. being able to detect rapidly the emergence of resistant virus is important as it allows clinicians to change rapidly patient treatment to a more effective drug (all cases bar one remain sensitive to zanamivir (who, ) ). such information also informs infection control procedures and public health surveillance. numerous methodologies are available for the detection of antiviral drug resistance in influenza viruses. phenotyping assays are the gold standard for oseltamivir resistance testing (zambon and hayden, ) . however, this method is time consuming, expensive and is only available at specialist centres. newer methods such as pyrosequencing are rapid and sensitive in detecting h y but few laboratories are equipped with these machines (cdc, b) . a group in rotterdam developed recently a real-time pcr based method for the detection of h y (personal communication with m. schutten). this method was shown to be sensitive, rapid and, unlike the aforementioned methods, uses a chemistry available to most diagnostic laboratories. the incorporation of this h y resistant typing assay into an established duplex assay that detects influenza a while typing simultaneously all that are h n ( ) positive is described. the new assay will allow rapid diagnosis, typing and resistance testing of respiratory samples in real time. the universal influenza a and influenza a h n ( ) n assays were developed in-house and are published previously . the universal influenza a assay is a widely used assay that targets the matrix region of the virus. participation in various external quality assessment (eqa) schemes has shown this assay to detect influenza a viruses from humans and animals with high sensitivity . the n assay is specific for influenza a h n ( ). the h y resistance assay was developed by a group in rotterdam and was supplied as personal communication (by m. schutten) . this allelic discrimination assay was designed to distinguish between the cytosine (oseltamivir sensitive) and the thymidine (oseltamivir resistant) mutation at the nucleotide position in the n gene of influenza a h n ( ) that gives rise to the antiviral resistance. the probe of the h y resistance assay was modified by the developers of the assay by adding two nucleotides (tt) at the end at a later date and published (van der vries et al., ) . it should be noted that we did not use the adapted probe. the n probe from this paper was also not used in this study as there was a well validated n probe in use in the laboratory . the primer probe concentrations for each assay were optimised individually using in-house protocols (gunson et al., (gunson et al., , . the h y resistance assay used the primers at an optimised concentration of m and the probe at . m in a l reaction volume. the universal influenza a h n ( ) duplex used the primers at an optimised concentration of m and the probes at . m in a l reaction volume . the triplex used the primers at an optimised concentration of m, universal influenza a and h n ( ) probe at . m, and the h y probe at . m in a l reaction volume (all primers and probes are shown in table ) . for all assays, one-step rtrt-pcr was performed on l of rna extract with the platinum one-step qrt-pcr kit (invitrogen) on an abi prism sds real-time platform (applied biosystems). the following thermal profile was used: a single cycle of reverse transcription for min at • c, min at • c for reverse transcriptase inactivation and dna polymerase activation followed by amplification cycles of s at • c and s at • c each (annealing-extension step). thresholds were set manually for analysis. the specificity of the triplex assay was assessed using who panels ( , ). both panels contained seasonal influenza a h n , h n , and avian h n viruses. the panel also contained influenza a h n ( ). a pool containing the following commonly encountered respiratory pathogens was also tested: influenza b, adenovirus, coronavirus ( e, oc , nl , hku ), parainfluenza - , rhinovirus, respiratory syncytial virus, human metapneumovirus, and mycoplasma pneumoniae (all the components present in the pool were present at ct values of between and ). the endpoint detection limit of the universal influenza a and h n ( ) components of the triplex was directly compared to the published duplex using a dilution series of an influenza a h n ( ) clinical sample and clinical samples containing seasonal h n and h n viruses. each dilution was tested in duplicate. these were carried out to ensure that the addition of the h y assay to the universal influenza a h n ( ) duplex did not result in a reduction in endpoint detection limit of the universal influenza a and h n ( ) components. the endpoint detection limit of the h y component of the triplex was compared directly to the h y single assay using a dilution series of influenza a h n ( ) h y positive clinical sample. each dilution was tested in duplicate. this was carried out to ensure that the addition of the h y assay to the universal influenza a h n ( ) duplex did not result in a reduction in the endpoint detection limit of the h y component. the new triplex assay was also directly compared to the universal influenza a h n ( ) to assess inter-assay variability (reproducibility/long-term precision) positive extraction run controls for flua, n and h y were monitored over pcr runs. to assess intra-assay variability (repeatability/short-term precision) a positive extraction run control for flua, n and h y were tested in wells on the same pcr run. finally the ability of the triplex assay to detect minor populations of the oseltamivir resistant influenza a virus in samples containing a mixture of resistant and sensitive viruses was assessed. to do this similar concentrations of h y and wild-type influenza a h n ( ) rna controls were mixed and then diluted the h y positive rna further in wild-type (wt) rna. diluting resistant virus in wild-type virus represents decreasing populations of resistant rna in a mixed strain sample, allowing to predict the approximate level which minor populations of resistant rna could be detected. all clinical samples were extracted on the qiagen mdx using the qiaamp viral rna kit (qiagen, crawley, uk) according to manufacturer's instructions. the triplex was evaluated using who influenza a quality control panels. the universal influenza a component of the triplex assay detected successfully all the samples that contained influenza a subtypes. the h n ( ) (table ). the h y component of the assay did not detect any of the samples as positive. a pool of other respiratory pathogens was tested using the triplex and no cross-reaction was observed. the endpoint detection limit of the universal influenza a and the h n / component of the triplex assay in comparison to the duplex assay was assessed using serial dilutions of clinical samples containing influenza a h n ( ) and seasonal h n and h n viruses (table ) . when testing the dilution series of the influenza a h n ( ) sample, the influenza a component of the duplex detected the − dilution in out of occasions whereas the influenza a component of the triplex assay detected down to the − dilution in out of occasions. the h n ( ) components in the duplex and triplex both detected down to the − dilution. the h y component of the triplex did not detect any samples in the dilution series as positive (table ) . when testing the dilution series of the seasonal h n and h n clinical samples, the influenza a component of the duplex assay detected the − dilution of the h n dilution series whereas the influenza a component of the triplex detected down to the − dilution in out of occasions. the influenza a component of the duplex and triplex detected the − dilution of the h n dilution series. the h n ( ) and h y components of the triplex did not detect any of the samples in the dilution series as positive ( table ) . the final endpoint detection experiment compared the h y component of the triplex assay to the single h y assay by testing serial -fold dilutions of an influenza a h n ( ) clinical sample known to contain the h y mutation. the h y single assay detected down to − whereas h y component of the triplex detected down to − . it should be noted that all components of the triplex had similar endpoints in detecting resistant rna (influenza a and h n ( ) − , h y − out of ) ( table ). the triplex was then compared to the duplex assay on clinical samples taken from patients with a clinical diagnosis of influenza a h n ( ). in total, were positive by both assays and there were discrepancies. of theses discrepancies were positive by the duplex but negative by the triplex ( influenza a positive only and h n positive only), were positive by the triplex but negative by the duplex ( influenza a positive only and h n positive only). all of these samples that were discrepant had high threshold cycle (ct) values (ranging from . to . ) and were at the endpoint of detection limit of the assays (see tables and ) . the inter-assay variability was assessed by monitoring positive run controls over pcr runs and intra-assay variability by testing a positive control in wells on one pcr run ( table ). the results suggest that there is little inter-assay variability as the ct of each component was similar over different runs with low standard deviation (sd) and coefficient of variation (cov) values. the results also suggest that the intra-assay variability of the assay is also good for each component as little variation was observed when the positive control was repeatedly tested. it should be noted that for h n the control used was weaker than that of the other components of the assay and so was nearer the endpoint detection limit (see tables and ) of this test which is likely to be the cause of the slightly higher sd and cov values. overall these results suggest that the triplex assay is precise and robust. finally the ability of the triplex assay to detect minor populations of h y was assessed by testing dilutions of h y rna diluted in wt rna. the results suggest that there is reliable detection of the h y to a dilution of : (table ). this represents a minor population of . %. in the present study, a multiplex assay which detects influenza a and simultaneously types those that are influenza a h n ( ) and highlights whether the virus contains the h y mutation was developed. adding the h y discrimination assay to the published duplex assay was shown to have no effect on the performance of any of the individual components. this was shown using several dilution series, a number of eqa panels and on prospective clinical samples taken from patients with clinically diagnosed h n ( ). multiplexing the methods had no detrimental effect on the ct values of each component and slightly improved the performance of each component in the triplex. the multiplex was also precise and robust as shown by assessment of inter-assay and intra-assay variability of the individual components of the assay. multiplexing these different test components offers several advantages during future outbreaks of this virus. as mentioned in the previous publication multiplexing the universal assay and the h n ( ) typing assay will allow laboratories to screen respiratory samples for influenza a h n ( ) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. the incorporation of the h y resistance assay is achieved at little extra cost (gunson et al., ) . since most virology laboratories already offer a molecular service for influenza a this assay could be easily implemented into most areas therefore increasing local access to resistance testing. access to a local service offers several clinical advantages over the current system which relies upon samples being referred to specialist centres. for example this assay allows the detection of resistant virus while the patient is being monitored for the presence of influenza a. consequently clinicians can change to an effective treatment at a much earlier stage than currently achievable (who, b). as mentioned above, a number of the reports of h y resistant virus were found in patients with no history of receiving oseltamivir. as a result this assay would also prove useful in the surveillance of oseltamivir resistant influenza a h n ( ) strains in the community (lackenby et al., ) . during the - seasonal influenza a (h n ) season the oseltamivir-resistant strain became the dominant strain circulating in the community, therefore it is possible that this could happen again with the pandemic strain in the next influenza season (harvala et al., ) . it is important to note that this assay only detects the h y mutation in influenza a h n ( ) virus and will not detect other mutations associated with resistance such as e v and n s. consequently if resistance is still suspected in an influenza a positive patient that has tested h y negative then other oseltamivir resistant mutations should be considered and samples should be sent for specialist testing (collins et al., ) . oseltamivir-resistant pandemic influenza a (h n ) virus structural basis for oseltamivir resistance of influenza viruses pyrosequencing assay to detect h y mutation in the neuraminidase of the novel a (h n ) viruses oseltamivir-resistant pandemic influenza a (h n ) virus infection in summer campers receiving prophylaxis-north carolina first cases of spread of oseltamivir resistant swine flu between patients are reported in wales optimisation of pcr reactions using primer chessboarding practical experience of high throughput real time pcr in the routine diagnostic virology setting using multiplex realtime pcr in order to streamline a routine diagnostic service development of a multiplex real-time rt-pcr that allows universal detection of influenza a viruses and simultaneous typing of influenza a/h n / virus the emergence of oseltamivir-resistant pandemic influenza a(h n ) virus amongst hospitalised immunocrompromised patients in scotland emergence of resistance to oseltamivir among influenza a (h n ) viruses in europe a community cluster of oseltamivir-resistant cases of h n influenza evaluation of a rapid molecular algorithm for detection of pandemic influenza a (h n ) virus and screening for a key oseltamivir resistance (h y) substitution in neuraminidase weekly epidemiological record th october oseltamivir resistance in immunocompromised hospital patients pandemic (h n ) briefing note pandemic (h n ) -update position statement: global neuraminidase inhibitor susceptibility network key: cord- -t y wg authors: decaro, nicola; pratelli, annamaria; campolo, marco; elia, gabriella; martella, vito; tempesta, maria; buonavoglia, canio title: quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: t y wg a taqman(®) fluorogenic reverse transcriptase-polymerase chain reaction (rt-pcr) assay was developed for the detection and quantitation of canine coronavirus (ccov) rna in the faeces of naturally or experimentally infected dogs. the ccov fluorogenic rt-pcr assay, which targeted the orf (m gene), was more sensitive than a conventional rt-pcr assay targeting the same gene, showing a detection limit of copies of ccov standard rna, and was linear from to ( ) copies, allowing quantitation of samples with a wide range of ccov rna loads. a total of faecal samples of diarrhoeic dogs were tested simultaneously by conventional and fluorogenic rt-pcr: were negative by both techniques, whereas tested positive by conventional rt-pcr and by the established ccov fluorogenic assay. one sample, which was positive by conventional rt-pcr, gave no signal in the fluorogenic assay. in addition, by the fluorogenic assay ccov shedding in the faecal samples of an experimentally infected dog was monitored for days. the high sensitivity, simplicity and reproducibility of the ccov fluorogenic rt-pcr assay, combined with its wide dynamic range and high throughput, make this method especially suitable for efficacy trials on ccov vaccines. canine coronavirus (ccov), a member of the family coronaviridae, is an enveloped, single-stranded, positive-sense rna virus, responsible for mild to severe enteritis in pups. ccov belongs to the group i coronaviruses, which also include the transmissible gastroenteritis virus of swine (tgev), the porcine epidemic diarrhoea virus (pedv), the porcine respiratory coronavirus (prcov), the feline coronaviruses (fcovs) and the human coronavirus e (hcov e). about two-thirds of the ccov genomic rna is occupied by two large, partially overlapping open reading frames (orfs), orf a and orf b, which encode two polyproteins leading to the viral replicase formation. the ' one third of the genome consists of other orfs encoding the struc-tural proteins and the other non-structural ones. the structural proteins comprise the s, e, m and n proteins encoded by orf , orf , orf and orf , respectively (enjuanes et al., ) . in young pups, sometimes in combination with other pathogens, ccov infection may cause severe diarrhoea, vomiting, dehydration, loss of appetite, and, occasionally, death. ccov shedding in faeces occurs for - days post-infection (keenan et al., ) . nevertheless long term viral shedding has been detected by pcr in ccov infected pups (pratelli et al., b (pratelli et al., , b . traditionally, diagnosis of ccov infection was made using virus isolation or electron microscopy, but these methods have been demonstrated to be poorly sensitive or specific. recently, the establishment of reverse transcriptase-polymerase chain reaction (rt-pcr) assays has led to an increase of both sensitivity and specificity, so that pcr has become the "gold standard" technique for the ccov diagnosis (pratelli et al., (pratelli et al., , . several rt-pcr based methods have been developed for detecting ccov rna in the faeces of dogs, but none of these were designed to be quantitative (bandai et al., ; naylor et al., ; pratelli et al., pratelli et al., , c . moreover, conventional rt-pcr assays are time consuming and contain a certain risk of carryover contamination due to the post-pcr manipulations and a second amplification step in nested pcr systems, especially when a high sample throughput is required. finally, those methods are limited in sensitivity and allow only relatively few samples to be processed at one time. conversely, it is well-known that real-time taq-man rt-pcr enables a reproducible, sensitive and specific quantitation of viral rna (budevi and weinstock, ; gut et al., ; martell et al., ; callahan et al., ; balasuriya et al., ; reid et al., ; smith et al., ; spackman et al., ) . the present study describes a real-time fluorogenic rt-pcr assay for ccov. the method is based on the taqman ® technology, which uses a dual-labeled fluorogenic probe combined with the → exonuclease activity of taq polymerase, resulting in an increase of the reporter dye's fluorescence released in the course of the pcr amplification (holland et al., ; heid et al., ) . the analytical performance of the ccov fluorogenic rt-pcr was evaluated in comparison to that of a conventional qualitative rt-pcr assay. the fluorogenic assay was then applied to detect and quantify viral load in ccov naturally infected dogs and to trace the course of ccov infection in the faeces of a dog experimentally infected with a field ccov strain. a total of faecal samples, collected from diarrhoeic pups in different geographical areas of italy, were processed in order to detect ccov and quantify viral rna amounts in the faeces. in addition, one dog, months of age, which had been tested negative for ccov antigen in the faeces by rt-pcr (pratelli et al., ) and for ccov antibodies by elisa (pratelli et al., a) , received ml ( ml intranasally and ml orally) of a cell culture medium containing tcid / l of a ccov field strain, as previously described (pratelli et al., b (pratelli et al., , . faecal samples of the infected dog were collected daily for days and subjected to both ccov conventional rt-pcr and real-time testing. total rna was extracted from each faecal sample with qiaamp ® rneasy mini kit (qiagen gmbh, hilden, germany) in accordance with the manufacturer's protocol. the starting material consisted of mg of faeces for each sample. template rnas were eluted in l of rnase-free water and stored at − • c until their use. to obtain a standard for the fluorogenic rt-pcr, the orf (m-protein gene, bp) of ccov strain / (buonavoglia et al., ) was cloned into pcr ® . -topo vector (topo ta cloning ® , invitrogen, milan, italy) and transcribed with ribomax tm large scale rna production system-t (promega italia, milan, italy) from the t promoter, according to the manufacturer's guidelines. after a dnase treatment to remove all dna, the transcripts were purified using a commercial column (qiaamp ® rneasy mini kit, qiagen gmbh) and quantified by spectrofotometrical analysis. ten-fold dilutions of the rna transcripts were carried out in te (tris-hcl, edta, ph . ) buffer containing g carrier rna (trna from escherichia coli, sigma-aldrich srl, milan, italy) per ml. aliquots of each dilution were frozen at − • c and used only once. the orf nucleotide sequences of several ccov strains (pratelli et al., a) were aligned using the bioedit software package (http://www.mbio.ncsu.edu/bioedit/bioedit. html). primers and taqman probe were designed using beacon designer software, version . (premier biosoft international, palo alto, ca, usa) to amplify a conserved -bp fragment within the aligned orf sequences. primers and probe were synthesized by mwg biotech ag (ebersberg, germany). the taqman probe was labeled with -carboxyfluorescein (fam) at the end and with -carboxytetramethylrhodamine (tamra) at the end. the position and sequence of the primers and probe used for taqman rt-pcr amplification are reported in table . triplicates of the standard dilutions and rna templates were subjected simultaneously to reverse transcription (rt) with geneamp ® rna pcr (applied biosystems, applera italia, monza, italy). one microliter of each triplicate of standard dilutions or template rna was reverse transcribed in a reaction volume of l containing pcr buffer × (kcl mm, tris-hcl mm, ph , ), mgcl mm, mm of each deoxynucleotide (datp, dctp, dgtp, dttp), rnase inhibitor u, mulv reverse transcriptase . u, random hexamers . u. synthesis of c-dna was carried out at • c for min, followed by a denaturation step at • c for min. the -l pcr mixture for one reaction contained l of iq tm supermix (bio-rad laboratories srl, milan, italy), nm of each primer (ccov-for and ccov-rev), nm of probe ccov-pb and l of c-dna. the thermal cycle protocol used was the following: activation of itaq dna polymerase at • c for min and cycles consisting of denaturation at • c for s and primer annealing-extension at • c for min. fluorogenic pcr was performed in an i-cycler iq tm real-time detection system (bio-rad laboratories srl) and the data were analysed with the appropriate sequence detector software (version . ). the accumulation of the pcr products was detected by monitoring the increase in fluorescence of the reporter dye. signals were regarded as positive if the fluorescence intensity exceeded times the standard deviation of the baseline fluorescence (threshold cycle [c t ]). conventional rt-pcr, amplifying a bp fragment of the orf of ccov, was carried out as previously described (pratelli et al., ) . briefly, pcr amplification was carried out geneamp ® rna pcr (applied biosystems, applera italia) and the following thermal conditions: reverse transcription at • c for min, inactivation of mulv reverse transcriptase at • c for min, cycles of • c for min, • c for min, • c for min, with a final extension at • c for min. the pcr products were detected by electrophoresis through a . % agarose gel and visualization under uv light after bromide ethidium staining. the position and sequence of the primers used for conventional amplification are reported in table . to compare the analytical sensitivity, -fold dilutions of the standard rna, ranging from to copies/l, were tested by both fluorogenic and conventional rt-pcr. in addition, -fold dilutions in dulbecco's minimal essential medium (d-mem) of the ccov vaccinal strain / - c (pratelli et al., ) , starting from . tcid / l, were processed. each standard or virus dilution was quantified three times separately. as shown in table , the detec-tion limit of the taqman rt-pcr was - log higher than that of conventional rt-pcr, ranging around copies/l and − . tcid / l for standard rna and ccov strain, respectively, with a detection rate of % for each positive dilution. serial -fold dilutions of standard rna (from to copies/l) were used to generate a standard curve to quantify ccov rna in faecal samples from naturally or experimentally infected dogs. the standard curve was created automatically by the i-cycler iq optical system software, version . (bio-rad laboratories srl), by plotting the c t values against each standard dilution of known concentration. the standard curve that was generated spans eight orders of magnitude and shows linearity over the entire quantitation range (slope = − . ), providing an accurate measurement over a very large variety of starting target amounts. the coefficient of linear regression (r ) was equal to . and the pcr efficiency ranged around % (fig. ) . the reproducibility of the method was established with the c t values obtained for the same standard dilution in different assays and within an assay, in order to calculate the interassay and intra-assay coefficient of variation (cv). the cv was obtained by dividing the standard deviation of the standard dilution by its mean and multiplying that result for . to estimate the interassay reproducibility, copies of the standard rna were submitted in triplicate to consecutive runs. the intra-assay reproducibility was determined by pipetting the same standard copy number ( molecules) times on the same -well reaction plate. the cv between runs and within-run was . and . %, respectively. twenty-nine of the faecal samples examined were negative for ccov by both conventional and fluorogenic rt-pcr; in / ccov negative samples, canine parvovirus type was detected by a specific haemagglutination assay (data not shown). as shown in fig. , by conventional rt-pcr samples were found to be positive and negative for ccov. conversely, samples tested positive and negative by real-time rt-pcr. totally, faecal samples were in agreement by both tests ( positive and negative samples). twenty-two samples, ccov negative by conventional amplification, resulted positive by the fluorogenic rt-pcr assay. one sample tested positive by conventional amplification and negative by real-time analysis. four samples which gave a positive signal in the fluorogenic rt-pcr assay could not be quantified, since their viral titre was below the sensitivity limit of the assay ( copies). regarding the ccov rna loads assessed by real-time analysis, it was demonstrated that the faeces of the naturally infected dogs contained a wide range of ccov rna amounts, from to . × /l of template, with a median titre of about /l of template. the results of the conventional amplification and real-time analysis carried out on the faecal samples of the ccov experimentally infected dog are summarized in fig. . the dog tested positive for ccov by conventional rt-pcr for days, from day to post-infection (dpi). in contrast, by quantitative fluorogenic rt-pcr, ccov shedding was demonstrated during the entire observation period ( days), reaching a peak at dpi ( . × rna copies/l of template). the infected dog showed mild diarrhoea from dpi to (pratelli et al., b (pratelli et al., , , concomitantly with the faecal shedding of the highest ccov titres. the development is described of a quantitative, simple, rapid and reproducible method for the detection and quantitation of ccov rna in faecal samples of dogs infected experimentally or naturally with ccov. the minimum copy number which could be detected by the ccov fluorogenic assay was approximately copies of standard rna. on the other hand, this assay was able to quantify correctly samples with more than copies/l of template, since the linearity of the generated standard curve persisted up to the highest titre of in vitro-transcribed rna analysed. in contrast, the detection limit of conventional rt-pcr was about molecules. this considerably wide range of linearity allows the use of this system to analyse samples with a wide range of ccov loads. in comparison with conventional rt-pcr, the fluorogenic assay presents many advantages. in addition to its greater sensitivity, this technique is rapid, allowing several samples to be processed in few hours, with a large increase in throughput. the assay is a closed system in which the tube is never opened post-amplification, and this eliminates the possibility of cross-contamination of new samples with products amplified previously. a limited carryover may occur due to the separation between rt and fluorogenic pcr, but we preferred a two-step assay, since one-tube methods are less cheap and sensitive than a two-step rt-pcr procedure (nakamura et al., ) . in order to reduce the risk of contamination, we have separated strictly the different working steps and carried out pipetting in different laminar flow hoods. however, the main advantage of the fluorogenic dye system consists of quantifying ccov rna amounts in faecal samples with a high degree of reproducibility and precision (cv between runs = . %, cv within-run = . %). quantitative gel-based pcr assays have been established for measuring several cellular and viral rnas, but they required time-consuming and potentially contaminating post-amplification steps and showed a lower precision (wang et al., ; kinoshita et al., ; nagano and kelly, ; hua et al., ; martell et al., ) . nevertheless, quantitation of ccov in the faeces is essential to trace the course of natural as well as experimental infection in dogs. among the faecal samples collected from the ccov experimentally infected dog, four were only found to be positive by the fluorogenic assay, probably due to their low viral load combined with the higher sensitivity of the taqman assay. conversely, one of the faecal samples of dogs infected naturally were only found to be positive by conventional rt-pcr. a possible explanation for this ambiguous result may be that the m gene, which is the target for both the conventional and fluorogenic assay, presents a certain degree of variability, so that mismatches in the binding site of primers and probe may reduce and, eventually, prevent an efficient amplification (pratelli et al., a (pratelli et al., , b . finally, ccov quantitation by real-time analysis could be a useful and complementary method to evaluate the efficacy of vaccines in challenged dogs. usually, dogs experimentally inoculated with field ccov strains do not develop considerable clinical signs, impairing any comparison between vaccinated and unvaccinated dogs. thus, the efficacy of ccov vaccines may be evaluated, as described previously (pratelli et al., b (pratelli et al., , , by monitoring viral shedding in the faeces of vaccinated dogs after ccov challenge, using virus isolation and conventional rt-pcr. theoretically, ccov amounts in the faeces could be assessed by virus titration on cell cultures. however, the low sensitivity of ccov isolation and, mainly, the appearance of ccov specific antibodies in the faeces after the challenge may affect this virological assay (personal observation). conversely, the use of the ccov fluorogenic rt-pcr assay could give a quantitation of viral loads, potentially showing differences in ccov shedding. detection of equine arteritis virus by real-time taqman ® reverse transcription-pcr assay canine coronavirus infections in japan: virological and epidemiological aspects fluorogenic rt-pcr assay (taqman) for detection and classification of bovine viral diarrhea virus l'infezione da coronavirus del cane: indagine sulla presenza del virus in italia development and evaluation of serotype-and group-specific fluorogenic reverse transcriptase pcr (taqman) assay for dengue virus coronaviridae one-tube fluorogenic reverse transcription-polymerase chain reaction for the quantitation of feline coronaviruses real time quantitative pcr detection of specific polymerase chain reaction product by utilizing the - exonuclease activity of thermus aquaticus dna polymerase quantitation of sheep-associated malignant catarrhal fever viral dna by competitive polymerase chain reaction intestinal infection of neonatal dogs with canine coronavirus - : studies by virologic, histologic, histochemical and immunofluorescent techniques quantification of gene expression over a wide range by the polymerase chain reaction high-throughput real-time reverse transcription-pcr quantitation of hepatitis c virus rna tissue distribution and regulation of rat prolactin receptor gene expression quantitative analysis by polymerase chain reaction amplification and detection of a single molecule of human immunodeficiency virus rna identification of canine coronavirus strains from faeces by s gene nested pcr and molecular characterization of a new australian isolate development of a nested pcr assay for the detection of canine coronavirus diagnosis of canine coronavirus infection using nested-pcr variation of the sequence in the gene encoding for transmembrane protein m of canine coronavirus (ccv) severe enteric disease in an animal shelter associated with dual infections by canine adenovirus type and canine coronavirus prevalence of canine coronavirus (ccov) antibodies in dogs in bari, italy, by an enzyme-linked immunosorbent assay m gene evolution of canine coronavirus in naturally infected dogs pcr assay for the detection and the identification of atypical canine coronavirus in dogs identification of coronaviruses in dogs that segregate separately from the canine coronavirus genotype efficacy of an inactivated canine coronavirus vaccine in pups safety and efficacy of a modified-live canine coronavirus vaccine in dogs detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay detection of australian bat lyssavirus using a fluorogenic probe development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h and h hemagglutinin subtypes quantitation of mrna by the polymerase chain reaction key: cord- -asjvf authors: lee, yu-ching; leu, sy-jye c.; hu, chaur-jong; shih, neng-yao; huang, i-jen; wu, hsueh-hsia; hsieh, wen-shyang; chiang, bor-luen; chiu, wen-ta; yang, yi-yuan title: chicken single-chain variable fragments against the sars-cov spike protein date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: asjvf the major concern for severe acute respiratory syndrome (sars), caused by the sars-associated coronavirus (sars-cov), is the lack of diagnostic and therapeutic agents. using a phage display technology in a chicken system, high-affinity monoclonal antibody fragments against the sars-cov spike protein were characterized. ten truncated spike protein gene fragments were expressed in escherichia coli cells. following the immunization of chickens with these recombinant spike proteins, two single-chain variable fragment (scfv) antibody libraries were established with short or long linkers to contain × ( ) and × ( ) transformants, respectively. after four rounds of panning selection, the scfv antibodies of randomly chosen clones were demonstrated by coomassie blue staining, and verified by western blot analysis. in a comparison of nucleotide sequences with the chicken germline gene, we found that all clones varied in the complementarity-determining regions, that two scfv antibodies reacted significantly with sars-cov-infected vero cells, and that those two specific scfv antibodies recognized the same region of the spike protein spanning amino acid residues – . in conclusion, the results suggest that the chicken scfv phage display system can be a potential model for mass production of high-affinity antibodies against the sars-cov spike protein. the severe acute respiratory syndrome (sars) is a newly emerging disease caused by a sars-associated coronavirus (sars-cov) ksiazek et al., ; peiris abbreviations: sars-cov, severe acute respiratory syndrome-associated coronavirus; s, spike; scfv, single-chain variable fragment; e. coli, escherichia coli; rt-pcr, reverse-transcription polymerase chain reaction; cdr, complementarity-determining region; fr, framework region; v h , heavychain variable region; v l , light-chain variable region * corresponding author. tel.: + x ; fax: + . e-mail address: yangyuan@tmu.edu.tw (y.-y. yang) . et al., ) . the virion consists of the following four major structural proteins: spike (s), membrane (m), envelope (e), and nucleocapsid (n) (marra et al., ; rota et al., ) . the s protein has two functional domains (s and s ) based on the predicted localization of their amino acid residues: - and - , respectively (he et al., ) . a region located between amino acids and on the s domain serves as a receptor-binding site (dimitrov, ; li et al., ; wang et al., ) . the c-terminal s domain has been shown to mediate membrane fusion during sars-cov infection (tripet et al., ) . further, immunization of mice with recombinant s protein can protect them from sars-cov infection (bisht et al., ; yang et al., ) . the results suggested that the s pro- tein is a good candidate for developing vaccines and antiviral drugs, and that generating monoclonal antibodies to recognize specifically the s protein would be valuable. although monoclonal antibodies with high specificities have been favored for both research and clinical applications in recent years, using the traditional hybridoma approach to generate human monoclonal antibodies for therapeutic purposes is still difficult because it is a tedious and expensive process (groves and morris, ; lillehoj and malik, ) . in contrast, the phage display system is a safe and effective procedure because the process involves the in vitro cloning of antibody repertoires, and subsequent isolation of monoclonal antibodies from combinatorial antibody libraries (barbas et al., ; winter et al., ) . of many recombinant antibody forms, the single-chain variable fragment (scfv) is a small protein entity retaining the variable regions of both heavy and light chains of an entire antibody molecule (bird et al., ; huston et al., ) which can be efficiently generated in a phage display system (chi et al., ; pavoni et al., ; wang et al., ) . antibody production in the chicken is efficient (abouzid et al., ; leclaire et al., ) . it has been reported that constructing chicken antibody libraries using the phage display technology can generate high-affinity scfvs for diagnostic applications (fehrsen et al., ; finlay et al., ; park et al., ) . performing reverse-transcription polymerase chain reaction (rt-pcr) to amplify the entire v region repertoire using one set of primers is simple and convenient because all avian immunoglobulin genes are derived from single light-and heavy-chain variable (v l and v h ) germline sequences (andris-widhopf et al., ; mccormack et al., ; yamanaka et al., ) . using the phage display technology, monospecific scfv and fab antibodies neutralizing the sars-cov infection have been generated from non-immunized individuals and convalescent sars patients (kang et al., ; sui et al., ) . the current study aimed to show that monoclonal igy scfv antibodies which bind specifically to the s protein and sars-cov-infected vero cells can be isolated from chickens immunized with escherichia coli-derived s proteins. ten sets of primers were synthesized to amplify s gene fragments using the sars-cov rna genome as a template (genbank accession no. nc ). the entire procedure was performed using a one-step rt-pcr kit as described by the manufacturer (qiagen, valencia, ca, usa). the amplified s gene products of - bp in length were individually digested with bamhi and xbai restriction enzymes and ligated into the pet- expression vector (novagen, darmstadt, germany). the resultant plasmids were transformed into the e. coli bl- (de ) strain for protein expression. clones were grown in ml lb medium containing ampicillin ( g/ml) at • c overnight. the bacterial culture was then diluted fold in the same lb medium and further grown until the od reached . . for his-fused s protein expression, iso-propyl-␤-d-thiogalactopyranoside (iptg) was added to a final concentration of . mm in the culture for induction. the cell pellet was resuspended in ml of binding buffer ( mm nacl, mm tris-cl, and m urea; ph . ) and lysed by three cycles of freezing (− • c) and thawing ( • c). after centrifugation, the resulting cellular lysate was loaded onto a ni + -charged resin column for protein purification according to the manufacturer (amersham biosciences, uppsala, sweden). female white leghorn (gallus domesticus) chickens were immunized with mixed s protein fragments ( g/each) in an equal volume of freund's complete adjuvant by intramuscular injection. three additional immunizations were carried out in intervals of days. after each immunization, blood was obtained and titrated by an enzyme-linked immunosorbent assay (elisa) to determine the presence of an antigen-specific immune response. the spleens of these chickens were harvested for rna isolation. phage libraries displaying scfv antibodies were constructed according to published protocols with minor modifications (andris-widhopf et al., ; barbas et al., ) . after pcr amplification with chicken-specific primers, gene products of heavy-chain (v h ) and light-chain variable (v l ) regions were subjected to a second round of pcr with a short or long linker to form full-length scfvs, which were cloned further into the pcomb h vector. to increase the cloning efficiency, g of pcomb h and . g of the full-length scfv product were applied in the ligation reaction. the recombinant dnas were transformed into the e. coli xl- blue strain by electroporation, recombinant phage production was initiated by the addition of the helper phage, vcs-m , and were precipitated with % polyethylglycol and % nacl (w/v) and finally resuspended in phosphate-buffered saline (pbs) containing % bovine serum albumin (bsa) and stored at • c. microtiter plates precoated with mixed s protein fragments ( . g/well) at • c overnight were blocked with % bsa for h at • c. then, plaque-forming units (pfu) of recombinant phages from the library preparation was added to each well, and the plates were incubated at • c for h. unbound phages were removed, and the wells were washed vigorously with tbst (tbs with . % tween ) buffer. bound phages were eluted with . m hcl/glycine (ph . )/ . % bsa and neutralized with m tris-base buffer. eluted phages were used to infect the e. coli xl- strain in the log phase for amplification, and they were recovered with % polyethylglycol and % nacl for the next round of selection. the panning procedure was repeated in the wells four times. phagemid dna from the final enriched phage was prepared and digested with nhei and spei to remove the phage protein iii gene. the digested dna with compatible cohesive ends was self-ligated, and the resultant phagemid was electroporated into e. coli. individual clones were grown in the presence of . mm iptg for protein induction. cells were pelleted and lysed by three cycles of freezing and thawing. after the final thawing, the supernatants were harvested by centrifugation for subsequent assays. the scfv-expressing lysates were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (sds-page). the proteins were transferred onto nitrocellulose membranes (amersham biosciences, little chalfont, uk), which were then blocked with % skim milk in tbst for h. polyclonal goat anti-chicken igy antibodies (bethyl laboratories, montgomery, tx, usa) were added and incubated for an additional hour. the membranes were washed with tbst three times for min each. the bound antibodies were detected by adding horseradish peroxidase (hrp)-conjugated donkey anti-goat immunoglobulin (ig) antibodies (sigma, st. louis, mo, usa). after three washings, the membranes were developed with diaminobenzidine (dab) substrate until the desired intensity was achieved. to examine the binding ability of the scfv-expressing lysate against the s protein, microtiter plates precoated with sars-cov-infected vero cell lysates (euroimmun, lueberk, germany) were used for the elisa. after rinsing with pbst (pbs with . % tween ), test lysates were distributed to wells in duplicate and incubated at • c for h. the bound scfvs were detected with enzyme-labeled antibodies as described above. the nucleotide sequences of heavy-and light-chain genes from chosen clones were determined by an auto-sequencer machine (abi prism ; perkin-elmer, national health research institute). the sequencing primer, ompseq ( -aagacagctatcgcgattgcagtg- ) as described by andris-widhopf et al. ( ) , was used for the v l and v h gene analyses. biochip slides (euroimmun) were used to test the binding reactivities of antibodies in sera or scfv-expressing lysates to native s protein in sars-cov-infected vero cells. slides were incubated with either sera or lysates at room temperature (rt) for h. after washing with tbst, the bound antibodies or scfvs were incubated with mouse anti-human (sigma) or goat antichicken antibodies (bethyl laboratories) at rt for h. after washings, slides were incubated for h at rt with corresponding fluorescein isothiocyanate (fitc)-labeled antibodies (sigma). finally, slides were coated with mounting oil and examined with an immunofluorescent microscope. epitopes were mapped with specific scfvs by detecting their reactivities with truncated s protein fragments by western blot analysis. ten purified his-fused s fragments were transferred onto a nitrocellulose membrane after electrophoresis. following blocking with % skim milk, membranes were incubated with scfv-expressing lysates for h at rt. then, the bound scfv antibodies were detected as described above. the binding affinities of specific scfvs were determined by competitive inhibition assays. briefly, l of each scfvexpressing lysate were first incubated with equal volumes of a series of two-fold-diluted soluble s fragments ( . - g/ml) at rt for h. the reactivities of the mixtures with each fragment individually precoated on a microtiter plate ( . g/well) were measured as described above. the dissociation constant (k d ) of the scfv binders was calculated according to the klotz method (friguet et al., ) . ten truncated fragments of the sars-cov s gene were amplified by pcr, cloned into the pet- vector, and expressed as his-fused recombinant proteins. table lists the locations and predicted gene lengths of these fragments. after transformation and induction, these proteins were expressed successfully in e. coli bl- cells and purified using ni + -charged resin. fig. a shows that these s protein fragments, s -s , had the predicted molecular weights of about - kda after being analyzed by sds-page. to construct the scfv libraries, two female white leghorn chickens were chosen for immunization. after the final immunization step, the chickens were killed, and the rna was extracted from their spleen cells. one set of specific primers were designed to amplify the v l and v h regions of the scfv fragment genes. after a second and consecutive pcr step, the v l and v h genes were joined with a short (ggssrss) or long linker (ggssrssssggggsgggg) to form a full-length scfv gene conformation (data not shown). two libraries, ssc (with the short linker) and lsc (with the long linker), containing × and × transformants, respectively, were constructed in this study. fig . a shows that scfv antibodies were well amplified and expressed. as predicted, the molecular weights of these expressed scfv antibodies with short or long linkers were and kda, respectively. their identities were verified further using specific anti-chicken igy antibodies in the western blot analysis (fig. b) . the binding abilities of clones against the s protein from each library were analyzed using elisa. fig. a shows five clones obtained from the ssc library to be strongly positive (with ods of . - . ) for binding activity, while others showed weak or no reactivity. fig. b illustrates four clones selected from the lsc library to have higher activity (with ods of . - . ). the nucleotide sequences of the v l and v h regions of clones (seven each from the ssc and lsc libraries) with different binding abilities were analyzed. the cdrs of the v l region of five of the clones showed more than % variation. from among those clones, ssc showed the lowest and ssc the highest mutation rates, of % and %, respectively. on the other hand, the v h region analysis showed that ten of the clones showed more than % cdr variations. among those clones, ssc showed the lowest and ssc the highest mutation rates, of % and %, respectively (fig. ) . the binding activities against the s protein expressed by sars-cov-infected cells of clones with od values greater fig. . binding activity of randomly selected clones analyzed by elisa. cellular lysates containing single-chain variable fragment (scfv) antibodies from various ssc (a) and lsc (b) library clones were examined for their binding to sars-cov-infected cell lysates using a commercially available kit. negative control (n) is a cellular lysate lacking scfv expression. bound scfv was detected using anti-chicken light-chain antibodies and was measured at nm. the elisa data were represented as mean of the duplicated wells ± s.e.m. using sigmaplot statistical analysis software. (table ). as represented in fig. , the immunocytochemical staining results showed that clones ssc (c), ssc (d), and lsc (e) had better or similar reactivities compared with that of convalescent serum (a), while clone lsc (f) exhibited no significant binding signal. two negative controls including normal human serum (b) and bacterial cell lysate without scfv expression (g) exhibited no reactivity at all. panels h-n were used to demonstrate the total cell numbers, morphology, and distribution under light microscopy. clones ssc and lsc were used to identify possible antigenic sites on the s protein with western blot analysis. the purity of the s protein fragments (s to s ) was examined and are shown in fig. a. fig. b and c shows that ssc and lsc scfv antibodies recognized mainly the s protein, suggesting that a potential antigenic epitope is located in the region of amino acid residues - of the intact s protein. fig. illustrates the results of the competitive inhibition assays of two representative scfv antibodies against the s fragment. eighy-three percent and % inhibitory effects were found on the binding reactivity of ssc and lsc antibodies, respectively, against the s fragment in the presence of a concentration of g/ml of the free s fragment. the k d values calculated using the klotz plot for scfvs ssc and lsc were . × − and . × − m, respectively. to face a possible sars outbreak in the future, it is necessary to have better diagnostic and therapeutic agents. to overcome the problems which occur frequently during traditional antibody production, such as obtaining low quantities of monoclonal antibodies (mabs) and the occasional loss of their efficiency due to persistent culturing, a phage display system was used to develop avian anti-sars-cov antibodies. the highly conserved and regions of fr and fr facilitate the use of a set of oligonucleotide primers to amplify the v region repertoire for library fig. . competitive inhibition assay of two representative single-chain variable fragment (scfv) antibodies against the s fragment. the amount of bound ssc and ssc scfv in the presence of free s inhibitor was measured and expressed as a percentage of the binding of scfv in the absence of an inhibitor. b and b are the amounts of bound scfv in the presence and absence of the inhibitor, respectively. construction (davies et al., ; mccormack and thompson, ) . two scfv antibody libraries were established which contained × and × clones showing that construction of the chicken scfv antibody library was easier compared to the construction of antibody libraries for humans or other mammals. thus, the clinical and scientific aspects of chicken scfv generation have received recently much attention (fehrsen et al., ; finlay et al., ; park et al., ) . as shown in fig. a and b, the expression and presence of scfv antibodies were examined by coomassie blue staining and western blot analysis. using elisa, it was observed that more than half of the selected clones in both the ssc and lsc libraries could react significantly with sars-cov-infected cell lysates (fig. ) . interestingly, the ssc and lsc scfv antibodies showed strong reactivity in the elisa, but weak binding signals in the subsequent immunocytochemical staining analyses. this discrepant result may have been due to the conformational modification of the s protein in the different preparation processes. some chosen enriched binders against target antigens immobilized on elisa wells have been found to react poorly against the same antigens located on the surface of cells (parren et al., ) . despite this potential problem in our study, three clones (ssc , ssc , and lsc ) were found to bind equally or more efficiently to the s protein compared with the patients' sera by immunocytochemical staining (fig. ) . as shown in fig. , sequence comparison of the clones (seven each from the ssc and lsc libraries) with the chicken germline revealed that all of the cdrs were dissimilar. somatic hypermutations to increase antibody affinity have been found to occur more frequently in cdrs than frs of the rearranged v gene (gearhart and bogenhagen, ) . in fact, the ig genes of the selected clones from hyperimmunized chickens had more mutations in the cdrs as a result of the affinity selection of b cells. therefore, this conclusion is in agreement with that obtained by other researches (finlay et al., (finlay et al., , sapats et al., ) and suggests that high mutation rates in these variable genes indicate an antigen-driven response in the chicken induced by the s proteins. the result of the present study showed that three clones (ssc , ssc , and lsc ) had significant binding signals to intact sars-cov-infected vero cells (fig. ) . to map the potential antigenic epitopes with western blotting, it was found that clones ssc and lsc recognized the purified s fragment ( fig. b and c) , but that clone ssc showed no reactivity (data not shown). conformational folding of the s protein present on the surface of sars-cov-infected cells differs from that of the truncated s proteins immobilized on nitrocellulose membranes, thus leading to differential recognition of clone ssc 's scfv antibody. the western blotting results in the study indicated further an antigenic epitope that spanned amino acids - . previous studies have shown an antigenic domain that spanned amino acid residues - can produce antibodies against sars-cov infection wang et al., ) . based on these results, it is concluded that the ssc and lsc scfv antibodies have specific binding abilities against the s protein and may be able to neutralize the infectivity of sars-cov in susceptible host cells. in conclusion, the identified epitope is a potential candidate for vaccine development, and these specific monoclonal scfv antibodies can help in developing valuable reagents for both scientific and clinical applications. genetic vaccination for production of dna-designed antibodies specific to hepadnavirus envelope proteins methods for the generation of chicken monoclonal antibody fragments by phage display assembly of combinatorial antibody libraries on phage surfaces: the gene iii site phage display: a laboratory manual single-chain antigen-binding proteins severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice isolation and characterization of rabbit single chain antibodies to human regi alpha protein epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated chinese isolate of severe acute respiratory syndromeassociated coronavirus (sars-cov) selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes the secret life of ace as a receptor for the sars virus identification of a novel coronavirus in patients with severe acute respiratory syndrome serogroup-reactive and type-specific detection of bluetongue virus antibodies using chicken scfvs in inhibition elisas exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins generation of highaffinity chicken single-chain fv antibody fragments for measurement of the pseudonitzschia pungens toxin domoic acid measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay clusters of point mutations are found exclusively around rearranged antibody variable genes veterinary sources of nonrodent monoclonal antibodies: interspecific and intraspecific hybridomas identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain fv analogue produced in escherichia coli human neutralizing fab molecules against severe acute respiratory syndrome coronavirus generated by phage display a novel coronavirus associated with severe acute respiratory syndrome protection against bacterial superantigen staphylococcal enterotoxin b by passive vaccination angiotensin-converting enzyme is a functional receptor for the sars coronavirus the new antibody technologies the genome sequence of the sars-associated coronavirus chicken igl variable region gene conversions display pseudogene donor preference and - polarity immunoglobulin gene diversification by gene conversion development and characterization of a recombinant chicken single-chain fv antibody detecting eimeria acervulina sporozoite antigen in vitro antigen challenge of human antibody libraries for vaccine evaluation: the human immunodeficiency virus type envelope selection, affinity maturation, and characterization of a human scfv antibody against cea protein coronavirus as a possible cause of severe acute respiratory syndrome chicken recombinant antibodies specific for very virulent infectious bursal disease virus potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association structural characterization of the sars-coronavirus spike s fusion protein core expression cloning of functional receptor used by sars coronavirus identification of two neutralizing regions on the severe acute respiratory syndrome coronavirus spike glycoprotein produced from the mammalian expression system construction of single chain variable fragment (scfv) and biscfv-alkaline phosphatase fusion protein for detection of bacillus anthracis making antibodies by phage display technology chicken monoclonal antibody isolated by a phage display system a dna vaccine induces sars coronavirus neutralization and protective immunity in mice this study was supported by a grant (nsc - -b- - -y) from the national science council (nsc) of taiwan. prof. winston w. shen made editing comments on a previous version of this manuscript. key: cord- - zwrqjj authors: geller, chloé; fontanay, stéphane; finance, chantal; duval, raphaël e. title: a new sephadex™-based method for removing microbicidal and cytotoxic residues when testing antiseptics against viruses: experiments with a human coronavirus as a model date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zwrqjj the relative lack of efficient methods for evaluating antiseptic antiviral activity, together with weaknesses in the existing european standard (i.e. nf en +a ), underlines the need to seek a new method which could allow a more precise evaluation of the antiseptic antiviral activity of chemical agents. this protocol is based on an original gel-based filtration method, using “in-house” g- and g- sephadex™ columns. this method allows the neutralization of both the activity and the cytotoxicity of a large range of molecules, according to their molecular size, in only min. the viral model used was the human coronavirus (hcov) e chosen for (i) its increasing medical interest, (ii) its potential resistance and (iii) its representing enveloped viruses mentioned in the european standard. first, the protocol was validated and it was demonstrated that it was fully operational for evaluating antiviral antiseptic potentiality and useful to screen potentially antiseptic molecules. second, chlorhexidine (chx) and hexamidine (hxm) were assessed for their potential anti-hcov e antiseptic activities. it was demonstrated clearly that (i) hxm had no activity on the hcov e and (ii) chx showed a moderate anti-hcov e activity but insufficient to be antiseptic. antiviral antisepsis and disinfection are crucial for preventing the environmental spread of viral infections. indeed, very few efficient and specific treatments are available to control most of these infections. furthermore, emerging viruses and associated diseases as well as nosocomial viral infections have become an important issue in medical fields. the coronaviridae family, an enveloped rna virus family, is a representative example of this issue. since the s, only two human pathogen members of this family have been identified: the human coronavirus, strain e (hcov e) and the human coronavirus, strain oc (hcov oc ). known to be responsible for a large proportion of common colds (hamre and procknow, ; holmes, ; larson et al., ) , they have been implicated in more serious diseases, along with the newly identified hcov: the nl strain (van der hoek et al., ) and the hku strain (woo et al., ) . indeed, these viruses have been recognized as causing lower respiratory tract infections, van elden et al., ) . they have also been involved in nosocomial viral infections, especially in young children and neonates (gagneur et al., a,b; moes et al., ; vabret et al., ) , in the elderly (falsey et al., ; nicholson et al., ) and in immunocompromised patients (pene et al., ) . furthermore, the outbreak of severe acute respiratory syndrome (sars) in - , responsible for the first worldwide epidemic of the st century, was due to a newly discovered coronavirus, the sars associated coronavirus (sars-cov) (drosten et al., ; ksiazek et al., ; peiris et al., ) . this outbreak led to a new awareness of the medical importance of the coronaviridae family. another interesting feature of coronaviruses is their potential environmental resistance, despite the accepted fragility of enveloped viruses. indeed, a hcov e survival of % after h at • c and % relative humidity has been observed, as well as a hcov e survival of % at • c and a relative humidity ≥ %, which could account for airborne transmission and for winter epidemics of hcov e, respectively (ijaz et al., ) . another study showed % and % of virus residual infectivity for the hcov e and the hcov oc , respectively, after days in suspension in phosphate buffered saline (pbs) at • c and % of residual hcov e infectivity after h of drying on different supports found in hospital settings, i.e. aluminium, sterile sponges or latex surgical gloves (sizun et al., ) . more recently, numerous studies have underlined an important sars-cov survival under various conditions: in serum, in : diluted sputum, in faeces for at least h and in urine for at least h. sars-cov also retained infectivity at • c, at room temperature and at • c for at least h (duan et al., ; rabenau et al., ) . furthermore, some studies have shown the transfer of viruses from contaminated hosts (hands, nasal mucosa) to surfaces and from environmental surfaces to hands and therefore a potential contamination of susceptible hosts (sattar et al., ; winther et al., ) . another study has also shown a longer survival of a herpesvirus on human skin than on an inanimate carrier (i.e. a stainless steel disk) (graham et al., ) . thus, considering the potential resistance of hcov for some hours in the environment, their pathogenesis and the absence of specific treatment, an efficient skin antisepsis, to stop eventual dissemination by the hands of healthcare workers, is a meaningful way to fight virus transmission and associated diseases. a fundamental point is to evaluate the efficiency of antiseptics-disinfectants (ats-d) on viruses properly. the principle of antiseptic antiviral activity evaluation is (i) to collect viruses and the product under test for an appropriately defined and precise contact time, (ii) to neutralize product activity, i.e. stopping product activity and removing its potential cytotoxicity, and (iii) to estimate the loss of viral infectivity due to the product activity. these tests require appropriate controls especially to check the absence of interference due to the test itself on viral infectivity, the efficiency of neutralization, the removal of cytotoxicity, and also, to establish reproducible and well defined conditions, e.g. contact time, temperature, interfering substances ( fig. ) . to date, only one european standard (nf en +a ) on virucidal ats-d activity testing in human medicine was published in january (afnor, ) . it specifies the test method and minimum requirements to establish virucidal activity according to the potential use of the products tested, e.g. disinfection of surfaces and instruments, hygienic hand wash or thermo-chemical disinfection. virus strains, temperatures, contact times, interfering substances are specified for each potential use. according to this standard, a product is considered to have an ats-d antiviral activity if it induces a loss of infectivity of at least log in viral titers during an accurate contact time. some parameters need to be extended to improve antiviral ats-d activity testing. for instance, one proposed method in the european standard to neutralize product activity is a : dilution of product/virus mix in iced medium. however, considering the very large range of ats-d nature (single molecules or a mixture of different ones), this method is not always reliable and furthermore, it may lengthen contact time. the european standard suggests other methods, based on gel-filtration techniques, using products as sephadex tm lh- , microspin tm columns s hr or minicon ® concentrators, but they lead to problems of contact times, separation capacity and cost (supplementary data, fig. ). with these aims in mind, a protocol was developed that allows neutralization, in only min, of the activity and cytotoxicity of a large range of molecules, according to their molecular size. this procedure also involves a gel-filtration-based method but uses other types of sephadex tm gels: sephadex tm g- and g- . their retention capacities are [ - ] g mol − and [ - ] g mol − , respectively, which is consistent with the molecular mass of most ats-d (supplementary data, fig. b ). to validate this protocol and establish its potential limits, antiseptic antiviral activities of two ats-d, the chlorhexidine (chx) and the hexamidine (hxm), were tested on hcov e. minimum essential medium (mem; . , fetal calf serum (fcs; ), trypsine-edta ( - ), earle's balanced salt solution (ebss; ), trypan blue (tb; - ) and mem without glutamine and without phenol red ( - ) were purchased at invitrogen (cergy pontoise, france). for culturing cells, cm cell culture flasks ( ) human embryonic pulmonary epithelial cells, l- cells (atcc ccl- ), kindly provided by dr h.f. hildebrand (faculty of medicine, lille, france), were grown in mem supplemented with % of fcs, at • c in a humidified atmosphere containing % co . they were split - times a week. cells, grown in cm cell culture flasks were washed times with phosphate buffered saline (pbs) and . ml of trypsine-edta was added. flasks were incubated for a few minutes at • c with % co and . ml of mem % fcs were added to achieve cell dissociation. stock cells were prepared with the cellular suspension obtained after trypsinization and mixed with mem containing % (v/v) fcs and % (v/v) dimethylsulfoxyde. they were then aliquoted and conserved at − • c in liquid nitrogen. human coronavirus, strain e (atcc vr ), kindly provided by dr. s.a. sattar (faculty of medicine, ottawa, canada) was cultivated on l- cells, in mem supplemented with % fcs, at • c in a humidified atmosphere containing % co . • c has been shown to be the optimal growth temperature for the hcov e (bradburne, ) . stock viruses were prepared by infection of cm cell culture flasks with a confluent monolayer of l- cells, not much older than days. when confluence was reached, medium was removed and cells were washed times with pbs. cells were then inoculated with ml of stock virus suspension diluted at : in ebss and incubated for adsorption virus time (i.e. h), at • c with % co . culture flasks were then filled out with ml of mem % fcs and re-incubated at • c with % co for h, before the cytopathic effect (cpe, i.e. cell lysis), was completely achieved. three cycles of freezing/thawing of min each were performed in order to liberate virions. media were centrifuged at × g for min to eliminate cellular fragments and, supernatants containing viruses were aliquoted in cryotubes and conserved at − • c. l- cells were seeded at × cells/well in -well flatbottom culture in mem % fcs. cell counts were performed with the tb exclusion method. cells were incubated at • c in % co , for h to obtain a confluent monolayer. after removing medium, l of fresh mem % fcs were added to each well. cells were then infected at a multiplicity of . , and -fold serial dilutions were carried out. infected cells were incubated at • c in % co for days to obtain the optimal cpe. at the end of the incu-bation period, cells were fixed and stained with may-grünwald and giemsa. fifty microliters/well of may-grünwald solution were allowed to fix and stain cells for min and after a washing step with tap water, l/well of giemsa solution were added for min to achieve the final staining before a last washing step with tap water. infected wells were counted under a phase-contrast inverted microscope (olympus, rungis, france) and viral titers or % cell culture infective dose (ccid ), expressed as viral infectious particles ml − , were estimated according to the reed and muench method (reed and muench, ) . all molecules tested were prepared, extemporaneously, as aqueous solutions and filter-sterilized on . m filters. chlorhexidine (chx) is a bisbiguanide, used widely as antiseptic and disinfectant. it has been used at concentrations from . % (m/v), i.e. . × − mol l − , in aqueous solutions for skin damage disinfection to . % (m/v), i.e. . × − mol l − , in alcoholic solutions for pre-operative skin disinfection. a chx salt, the chx digluconate, was used because of its greater water solubility and its commercial use under this form. its molecular weight is . g mol − . the aqueous solution is colourless. hexamidine (hxm) is also used widely for skin disinfection and external use. it belongs to the aromatic diamidine group. hxm is insoluble in water; therefore a hxm diisethionate salt solution, of which the water solubility limit is g l − (fleurette, ) , is also used in commercial formulations as in this study. in commercial formulations, hxm is used at . % (m/v), i.e. . × − mol l − , or at . % (m/v), i.e. . × − mol l − . its molecular weight is . g mol − . the aqueous solution is colourless. mtt viability assays and nr cytotoxicity assays were carried out to evaluate the cellular impact of molecules tested on l- cells. in both assays, l- cells were seeded into -well culture plates in mem with % fcs. they were incubated at • c with % co for h prior to the addition of the appropriate dilutions of the product to be tested and then re-incubated for h, h and h. for mtt tests, a modified mosmann's protocol was used (mosmann, ) . after medium removal, l/well of pbs and l of mtt solution at mg ml − in pbs were added. after h of incubation at • c in % co , l of sodium dodecyl sulfate were added to dissolve formazan dark blue crystals produced by reduction of mtt by succinate mitochondrial deshydrogenase. plates were re-incubated for h and measurements were performed with a scanning multiwell spectrophotometer (elisa reader, multiscan ex, thermo fisher scientific, saint herblain, france) using a test wavelength of nm and a reference wavelength of nm. cell viability was validated, which is proportional to the produced quantity of formazan crystals and expressed as % inhibitory concentration (ic , mol l − ). nr assays were performed according to a modified borenfreund's protocol (borenfreund and puerner, ) . an aqueous stock solution at mg ml − of nr, subjected to a centrifugation at × g for min to eliminate eventual aggregates, was prepared. for the assay, a "ready to use" solution of nr at g ml − in mem without glutamine and without phenol red was prepared extemporaneously to avoid fine precipitates which could form when nr is mixed with medium. after medium removal, cells were rinsed with l of pbs/well prior to the addition of l/well of "use" nr solution. plates were re-incubated for h at • c in % co to allow the uptake of the dye by living cells. the dye-medium was then taken off and cells were washed with l/well of "a" solution ["a" solution: % (v/v) formaldehyde at . % (v/v), % (m/v) cacl in distilled water]. to release nr accumulated in lysosomes of living cells, l/well of "b" solution were added ["b" solution: % (v/v) acetic acid, % (v/v) absolute ethanol in distilled water]. the quantity of nr released was estimated by spectrophotometric measurement at nm in a scanning multiwell spectrophotometer. it was proportional to the cytotoxicity of molecules tested and expressed as % cytotoxic concentration (cc , mol l − ). these tests were also performed with solutions of chx and hxm at various concentrations and after their filtration on the "in-house" sephadex tm columns to evaluate their retention and the elimination of potential cytotoxicity. sephadex tm media are a range of cross-linked dextran gels of variable porosity according to the degree of cross-linking. this property, based on the principle of exclusion-diffusion, allows the separation of different molecules according to their molecular size. they are manufactured in a bead form and need to be suspended in a buffer. two different kinds of sephadex tm were used in this protocol, the sephadex tm g- and the sephadex tm g- , which have resolution capacities of - g mol − and - g mol − , respectively. according to the manufacturer's instructions, sephadex tm g- and g- should be suspended for minimum h at room temperature, in at least times their volume in pbs (i.e. about g l − ), to equilibrate the ph at and to allow the gel to swell. suspensions were sterilized by autoclaving and excess of pbs was eliminated to obtain a suspension with a volume ratio of sephadex tm /pbs of / . columns were made up with a ml syringe, stuffed with carded cotton, associated with a drilled eppendorf type . ml microtube; each part was sterilized by autoclaving. the body of the syringe was filled with ml of sterile sephadex tm gel, prepared as described above. the system with the syringe and the eppendorf tube was placed into a ml conic bottom centrifuge tube to conserve sterility and centrifuged at × g for min. the definitive "in-house" sephadex tm column was then ready (supplementary data, fig. ). ninety-six-well flat bottom culture plates were seeded at × cells/well in mem % fcs and incubated at • c in humidified atmosphere with % co for h to obtain a confluent monolayer. after days of incubation, the appropriate dilution of the product to be tested was prepared in distilled water and filter-sterilized on a . m filter. viral suspensions of hcov e were allowed to thaw; l were mixed with . ml of the diluted product and allowed to stay in contact for the desired contact time. to separate viruses and the product, l of this mixture were placed into the "inhouse" sephadex tm column, s before the end of the contact time, to leave sufficient time to centrifuge. the system was placed in a ml centrifuge tube to maintain sterility and centrifuged at × g for min. this centrifugation retained the product according to its molecular size and the kind of sephadex tm used and to recover viruses in the filtrates. viral titers were then evaluated with the end point dilution method (section . ). the potential loss of hcov infectivity was estimated by the difference between viral titer of positive control, which was also subjected to filtration on the "in-house" sephadex tm columns but without the presence of the product; and viral titer obtained after contact with the product tested for desired contact time and neutralization by filtration on the "in-house" sephadex tm columns. three types of controls were performed in each experiment to validate the results: (i) non-retention of viruses after filtration on sephadex tm columns, (ii) neutralization of the potential antiviral activity of the product tested, and (iii) elimination of its cytotoxicity. as the european standard (nf en +a ) states, the difference in viral titer before and after treatment, (i.e. in this case, gel filtration), without product tested, should not excess . log . to assess the non-retention of viruses, e.g. hcov e, viral suspensions were mixed with sterile distilled water for the contact time defined for the experiment, filtered on the "in-house" sephadex tm columns and titrated as describe above (section . ). neutralization assays were performed by filtration of l of the solution to be tested on the "in-house" sephadex tm columns and l of the filtrates were mixed with l of viral suspension. this mixture was then inoculated to the l- cells monolayers. the viral titer obtained should be equivalent to viral titer without filtration (i.e. a log difference ≤ . ), since the product should be retained in the column. this experiment also verified that filtrates did not have any influence on virus infectivity. cytotoxicity controls were also carried out for each experiment, as a complement to mtt and nr assays undertaken previously. these controls were performed by filtration of l of the product tested and then filtrates were inoculated on l- cells. after days of incubation, cells monolayers were observed under an inverted microscope and any sign of cytotoxicity should be visible. retention of the molecules tested by the "in-house" sephadex tm columns was also confirmed by uv-visible spectrophotometry. the range of dilutions was assayed to establish specific spectrophotometric parameters: maximum absorption wavelength max , molar absorption coefficient ε and detection limits, for each molecule. dilutions were prepared in distilled water to mimic the test conditions. spectra were performed on a uv-visible spectrophotometer (uvmc , safas, monaco) within a - nm window. calibration curves (a max = f(c)) were established to determine the molar absorption coefficient (ε) for each molecule. different dilutions of products tested were prepared in distilled water and filtered through the "in-house" sephadex tm columns. their absorbances were then measured at max to determine the residual concentration. to validate this process, statistical analysis of the different steps was undertaken using statview ® version . . for all tests, the maximum acceptable risk was %. to assess homogeneity of columns, i.e. volumes of gel after the first centrifugation and volumes of filtrate after the second centrifugation, kolmogorov-smirnov tests were used to assure distribution normality of each independent tested batch, and then bartlett's tests were used to compare variances. in case of variances in homogeneity, an anova test was used to compare means of the different batches; in case of non-homogeneity of variances, a non-parametric test of kruskall-wallis was used to compare averages. finally, correlation studies, using a fisher transformation, were realised to estimate whether there was an association gel volumes and either filtrates volumes or residual concentrations after filtration on sephadex tm . for uv-spectrophotometry, regression analysis (a = f(c)) was carried out to establish specific spectrophotometric parameters of each molecule tested. as specified in section . . , two centrifugation steps were needed: (i) to prepare optimized columns of . ml of gel, whether for sephadex tm g- or sephadex tm g- and (ii) to filter the drugvirus mixture to achieve the neutralization step with retention of molecules tested, non-retention of viruses and homogeneous quantity of filtrates. different speeds and times of centrifugation were then tested and best parameters were determined: min at × g for the first centrifugation and min at × g for the second centrifugation. the homogeneity of (i) gel volumes obtained after the first centrifugation and (ii) filtrate volumes obtained after the second centrifugation; was first established on independent batches of table uv-visible spectrophotometric parameters of molecules tested and retention rates. molecules tested were suspended in sterile distilled water (ph ). calibration curves were established for each molecule on a uv-visible spectrophotometer within a - nm wavelength window. the concentrations tested ranged from − mol l − to − mol l − except for trypan blue (tb) and neutral red (nr) for which concentrations ranged from − mol l − to . × − mol l − and from × − mol l − to × − mol l − , respectively. regression analyses were then performed to determine specific spectrophotometric parameters: (i) linearity limits (mol l − ), (ii) minimum detection limits (mol l − ); within these limits, (iii) the specific maximal absorption wavelength ( max , nm) and (iv) the specific molar absorption coefficient (ε, l mol − cm − ). retention rates (see formula in section . . ) of each molecule by the specified type of sephadex tm media are indicated, i.e. tb and chlorhexidine (chx) retention rates by sephadex tm g- ; nr and hexamidine (hxm) retention rates by sephadex tm g- . ci corresponds to the initial concentration or concentration before filtration. absorption wavelength used to estimate the retention rate is specified each time it was necessary. specific spectrophometric parameters for each molecule tested retention rate columns for each sephadex tm gel, by statistical analysis using statview ® v. . (section . ). retention rates of dye molecules were measured: the tb ( . g mol − ) for the sephadex tm g- and the nr ( . g mol − ) for the sephadex tm g- . to determine this rate, the spectrophotometric parameters of tb and nr (table ) were established by measuring absorbances of concentrations ranging from − mol l − to . × − mol l − for tb and from × − mol l − to × − mol l − for nr, followed by a regression analysis (section . ). the residual concentrations of filtrates were estimated by measuring absorbances in uv-visible and calculating the retention rate according to the formula: retention rate = [(ci − cf)/cf] × , where ci was the initial concentration or concentration before filtration and cf was the final concentration or the residual concentration after filtration. retention rates obtained for tb after filtration on sephadex tm g- were ( . ± . )% and ( . ± . )% after filtration of tb solutions at . × − mol l − and . × − mol l − (which was the commercial concentration), respectively. for nr solutions, either at − mol l − or − mol l − , after filtration on sephadex tm g- , concentrations were under the detection threshold. thus, the retention rates obtained were > . % after filtration of nr solutions at − mol l − and − mol l − . it was found that, within a range of [ - ]l of gel volumes after the first centrifugation, there was no significant variation in filtrate volumes and in retention rates. in further experiments, all gel volumes were included within these limits. basically, the "in-house" sephadex tm columns should retain the product but not viruses. according to the european standard nf en +a (afnor, ) , the difference between viral titer before and after treatment, i.e. here, after filtration, should not exceed . log . to ensure this non-retention of viruses, independent experiments for each sephadex tm type and for each contact time tested, were performed. the hcov e stock suspension was diluted : in sterile distilled water and filtered on the "inhouse" sephadex tm columns. the viral titers were compared with and without filtration (table ) . for sephadex tm g- , log differences between viral titers were . ± . , . ± . and . ± . for contact times of min, min and min, respectively (n = for each contact time). for sephadex tm g- , log differences between viral titers were . ± . , . ± . , . ± . , . ± . and . ± . for contact times of min, min, min, min and min, respectively (n = for each contact time). consequently, sephadex tm columns of each sephadex tm type did not retain hcov e and did not induce a loss of infectivity. elimination of cytotoxicity was a crucial control before inoculation of filtrates onto the cells. it allowed the assessment of antiviral table elimination of potential cytotoxicity by filtration on the "in-house" sephadex tm columns. % inhibitory concentration (ic ) and % cytotoxic concentration (cc ) of chlorhexidine (chx) and hexamidine (hxm), without filtration, on l- cells were evaluated with methyl thiazol tetrazolium (mtt) assays and neutral red (nr) assays, respectively (in bold). ic and cc of (i) chx solutions at − mol l − and − mol l − , and (ii) of hxm solutions at − mol l − and − mol l − , after filtration on the "in-house" sephadex tm g- and sephadex tm g- columns, respectively, were then evaluated. results, expressed in mol l − , represent the average of independent experiments. ci h h h chx ic without filtration ( . ± . ) × − ( . ± . ) × − ( . ± . ) × − − mol l − > − > − > . × − − mol l − > − > − > − cc without filtration ( . ± . ) × − ( . ± . ) × − ( . ± . ) × − without filtration ( . ± . ) × − ( . ± . ) × − ( . ± . ) × − − mol l − ( . ± . ) × − ( . ± . ) × − ( . ± . ) × − − mol l − > − > − > − cc without filtration ( . ± . ) × − ( . ± . ) × − ( . ± . ) × − − mol l − ( . ± . ) × − ( . ± . ) × − ( . ± . ) × − − mol l − > − > − > . × − activity, eliminating an eventual cellular impact of the product on l- cells. mtt and nr assays were carried out to determine ic and cc , respectively. the ic and cc of chx and hxm solutions (without filtration) were first determined on l- cells (section . ). then, ic and cc of chx and hxm solutions, at different concentrations and after filtration on the "in-house" sephadex tm columns, were determined to make sure of the elimination of the eventual cytotoxicity by the filtration on the "in-house" sephadex tm columns. each experiment was repeated times independently to find the ic and cc corresponding to the average results of those experiments. as shown in table , ic of chx on l- cells were ( . ± . ) × − mol l − , ( . ± . ) × − mol l − and ( . ± . ) × − mol l − at h, h and h, respectively, and cc were ( . ± . ) × − mol l − , ( . ± . ) × − mol l − and ( . ± . ) × − mol l − at h, h and h, respectively. after filtration of chx solutions at − mol l − and − mol l − on sephadex tm g- columns, cc and ic were > − mol l − , according to the detection limit of the method, except at h, where ic was > . × − and cc > . × − mol l − after filtration of − mol l − chx solution. the same experiments were carried out with hxm. ic of hxm without filtration were ( . ± . ) × − mol l − , ( . ± . ) × − mol l − and ( . ± . ) × − mol l − at h, h and h, respectively, and cc were ( . ± . ) × − mol l − , ( . ± . ) × − mol l − and ( . ± . ) × − mol l − at h, h and h, respectively. after filtration of hxm solution at − mol l − on the sephadex tm g- columns, ic and cc were > − mol l − , except at h where cc was > . × − mol l − . table non-retention of hcov e by the "in-house" sephadex tm columns. results, expressed as log of % cell culture infective dose (ccid ), represent the average of viral titers of at least independent experiments. these assays allowed to evaluate the non-retention of the human coronavirus e (hcov e), for each sephadex tm type and each contact time tested. viral suspensions were diluted at : in sterile distilled water for the specified contact time before filtration on the "in-house" sephadex tm columns and inoculation to the l- cells. nd: not determined. sephadex tm g- sephadex tm g- without filtration after filtration difference without filtration after filtration difference . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) nd nd nd . ± . (n = ) . ± . (n = ) . ± . (n = ) nd nd nd . ± . (n = ) . ± . (n = ) ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) . ± . (n = ) hxm solution at − mol l − was also assayed. ic and cc were shown to be very close to those without filtration (table ) and thus retention was shown to be incomplete for this concentration. consequently, the "in-house" sephadex tm columns clearly demonstrated that they efficiently eliminate cytotoxicity of solutions at concentrations ≤ − mol l − . as for dye molecules (tb and nr), specific spectrophotometric parameters and retention rates of chx and hxm were determined using uv-visible spectrophotometry (section . ). testing different dilutions of chx, i.e. from − mol l − to − mol l − , the specific spectrophotometric parameters of chx were established (table ) . two maximum absorption wavelengths ( max ) at nm and nm were observed, with molecular absorption coefficients of . × l cm − mol − and . × l cm − mol − , respectively. these coefficients were obtained by means of a regression analysis realized with statview ® v. . (n = measurements, p < . ). the relation a = f(c) was linear between [ − to . × − ] mol l − for max = nm (correlation coefficient = . ) and between [ − to − ] mol l − for max = nm (correlation coefficient = . ). the detection limit was − mol l − for both of the max . these results were consistent with data from the literature (bonazzi et al., ; european pharmacopeia, a; ha and cheung, ; havlikova et al., ; hebert et al., ) , where uv detection, especially after high performance liquid chromatography, was done either at nm or between nm and nm. the retention rates of chx solutions were then evaluated at different concentrations by the "in-house" sephadex tm g- columns (table ) . three independent experiments were realized in duplicate (n = ). a retention rate > % was observed for chx solutions at − mol l − and − mol l − . chx solution at − mol l − was not completely retained and retention rates were of ( . ± . )% at nm and ( . ± . )% at nm. in the same way, hxm was assayed from − mol l − to − mol l − ( table ). the max was observed at nm associated to a molar absorption coefficient of . × l cm − mol − , with a linear relationship between [ − to − ] mol l − (n = , p < . , correlation coefficient = . ). the detection limit of hxm with uv-visible spectrophotometry is − mol l − . these data were consistent with the european pharmacopeia, which recommends hxm detection at nm (european pharmacopeia, b) . hexamidine retention rates by the "in-house" sephadex tm g- columns were evaluated. they were > % for hxm at − mol l − and − mol l − . as for other molecules, retention limits were reached for hxm at − mol l − and retention rate could not be determined accurately because residual concentrations were above the linearity limits of detection, i.e. − mol l − . thus, the retention rate was < %. verifying neutralization control was essential to assess the stop of potential antiviral activity and the respect of the defined contact time. furthermore, this test ensured that there was no interference of the filtrates with virus infectivity (fig. ) . as recommended by the european standard nf en +a , the difference between virus control and neutralization control should not excess . log (section . . ). neutralization controls were done for each independent experiment (each product concentration and each contact time). neutralization assays were performed for chx at − mol l − and − mol l − . the mean of log differences obtained (n = independent assays for each concentration) were . ± . and fig. . evaluation of antiseptic antiviral activity of chx and hxm on the hcov e. this figure shows the log reduction in viral titers obtained after the action of chlorhexidine (chx) and hexamidine (hxm) at different concentrations and at specified contact times: (a) represents the log reduction in human coronavirus e (hcov e) titers obtained after action of chx at − mol l − ( ) and − mol l − ( ) and (b) represents the log reduction in hcov e titers obtained after action of hxm at − mol l − ( ). the bold line in both graphs represents the threshold of log reduction to reach to pretend to an antiviral antiseptic activity. . ± . , respectively. the same assays were carried out with hxm at − mol l − and the mean of log difference obtained (n = independent assays) was . ± . . thus, potential antiviral antiseptic activity was correctly neutralized for each molecule and for each concentration tested. furthermore, no interference with virus infectivity was observed. assays were performed as described in section . . . each assay corresponded to the evaluation of potential ats anti-hcov activity of one concentration of each molecule tested and one contact time. each assay was repeated independent times and included necessary controls, i.e. positive virus control, non-retention of hcov, efficiency of neutralization and elimination of cytotoxicity, to validate the test (fig. ) . chx was tested at concentrations, − mol l − and − mol l − and each concentration was tested during different contact times, i.e. min, min, min and min ( fig. a) . the log reduction in viral titers induced by the action of chx at − mol l − was of . ± . , . ± . , . ± . and . ± . for min, min, min and min, respectively. at the concentration of − mol l − , chx induced a reduction of . ± . , . ± . , . ± . and . ± . for contact times of min, min, min and min, respectively. according to the european standard nf en +a , the log reduction is lower than log , so, even if chx had a certain activity on the hcov e, it had no ats anti-hcov e properties in these conditions. hxm showed an even worse activity on the hcov e. at − mol l − , it induced only a reduction of . ± . log and . ± . log for min and min of contact time, respectively, so very far from the threshold recommended by the european standard (fig. b) . given these results, concentration − mol l − and contact times of min and min were not tested. to fight viral infections, ats-d seem crucial considering: (i) the relative lack of efficient treatments and vaccines available, and (ii) the risk of outbreak of new viral pathogens. thus, it is essential to evaluate their antiviral activity properly and reliably. the major items proposed in the european standard nf en +a were followed, such as (i) contact times, (ii) the maximum of . log differences between control viral titers (section . . ) to validate the assays and (iii) the log reduction to assess an antiseptic antiviral activity of drugs tested. however, some parameters of this standard needed to be intensified, especially the neutralization method and the choice of virus strain. indeed, the neutralization step is fundamental: (i) to ensure a precise contact time and (ii) to remove any potential cytotoxicity before inoculation to the cells, which could involve confusion in product activity estimation. in the european standard nf en +a , two global techniques are proposed: dilution in iced medium and gel filtration techniques, but they involve different problems such as lengthening of contact times, efficiency and also cost. in the dilution method, the neutralization step requires min, inconsistent with recommended contact times for the different applications mentioned in the european standard. also, not all contact times are suited to the potential use of the product, e.g. min is the mandatory contact time for testing disinfectants for surfaces and instruments and facultative times are min, min and min, but in practice, a surface would rarely be disinfected during min. furthermore, dilution has not always been shown to be efficient. for instance, the ic and cc of chx on l- cells are higher than × − mol l − at h, h and h. so even after a : dilution of a − mol l − solution of chx (the european standard recommends a : dilution or : if not efficient), the solution remains cytotoxic. nonetheless, the european standard also proposes gel filtration techniques to neutralize product activity, using sephadex tm lh- , an ultrafiltration system (minicon ® millipore) or pre-packed columns microspin tm s hr. the major problem of these methods is the lengthening of contact time. indeed, they need at least min to achieve the neutralization step, sometimes min or more, depending on the techniques. furthermore, separation capacities are not well adapted to the molecular mass of most ats-d. another drawback of minicon ® concentrators is that they are non-sterile devices and do not bear sterilization, so they cannot be used to filter viruses. the american standard (astm e - ) recommends also a gel filtration method, using sephadex tm lh- or sephacryl tm superfine s- , to neutralize virucidal activity (astm, ) . the virucidal efficacy criterion for the astm standard is a log reduction > . the astm procedure is close to the one developed here. however, the centrifugation time: (i) to eliminate the void and (ii) to separate the product tested and viruses is min instead of min with the "in-house" sephadex tm columns. the first gel proposed, sephadex tm lh- , has well adapted retention capacities to most ats-d molecular mass (supplementary data, fig. b) . unfortunately, it is not sold anymore. the other gel proposed, sephacryl tm superfine s- , is commercialized in a suspension form in dis-tilled water containing % ethanol as conservative. the presence of ethanol could affect the tests, emphasizing the effect of the ats-d tested so leading to an overestimation of the product activity. the cost was also higher than the "in-house" sephadex tm g- or g- columns and its retention capacity was not well adapted (supplementary data, fig. ) . the choice of virus strain could also be discussed and has recently been questioned by ijaz and rubino ( ) . the european standard nf en +a proposed viruses: a poliovirus (type , lsc- ab), an adenovirus (type ) and a bovine parvovirus (haden strain). poliovirus was chosen for its potential high resistance to chemicals but taking into account the worldwide program of poliomyelitis eradication, the legitimacy of this choice is questionable. in the same way, the choice of b parvovirus and adenovirus is arguable. although b parvovirus is responsible for serious but rare materno-foetal infections and also infections in immunocompromised patients and/or patients with a haemolytic anaemic disorder, it has not been shown to be a frequent nosocomial pathogen. its interest, however, lies in its high resistance to chemicals and to a wide range of ph and temperatures and its potential blood transmission route (morinet et al., ) . however, the strain used in the european standard is a bovine parvovirus, so there is a question of representativeness. in contrast, human serovar adenovirus is more interesting notably for its responsibility in mild upper respiratory tract infections, ophthalmologic infections and also gastro-enteritis, particularly in new-borns and young children. it can cause rare but serious (mortality up to %) infections in immunodepressed patients, especially in bone-marrow transplanted patients (carret and le faou, ) . it has also probably been chosen for its high resistance in environmental conditions. all these viruses are naked viruses and not always representative of majors risks encountered in community or in hospital settings. when establishing this protocol, the first challenge was to elaborate a unique protocol for both sephadex tm gels, in order to make assays easier to perform. the best parameters for the needed centrifugation steps were determined for conserving the sterility during the entire assay. these parameters were first validated through statistical analysis, without drugs tested, to ensure the repeatability of columns yield and to fix the acceptability limits of the protocol. thus, this protocol was made to perform neutralization in only min, which was better than all the other methods. therefore, a different virus to those proposed in the european standard was chosen: the hcov e, not only for its medical interest but also for its presence in the annexe b of the european standard nf en -a as potential contaminant for surfaces, instruments and hands. furthermore, it belongs to enveloped viruses, which are absent from the standard and which may behave differently to naked viruses under the action of ats-d. it was also chosen for its relative resistance in different conditions taking into account that enveloped viruses are most often considered fragile. during the assays to show the non-retention of the hcov e by the "in-house" sephadex tm columns, whether g- or g- , the importance of ph was highlighted as an important cause of variability in results, i.e. in viral titers. these difficulties were experienced, mainly with sephadex tm g- that needed excess of solvent (i.e. pbs) when swelling to reach neutrality, and also with sterile distilled water, owing to the sterilization process, which often involved acidification. this could be due to a high sensitivity of the hcov e to the ph. rapid loss of infectivity and an aggregation of virions have been shown at ph and • c, whereas the virus was quite stable at ph and • c (sturman et al., ) . so ph should be verified and adjusted to the neutrality to allow a standardization of the protocol. to test this protocol, anti-hcov e activity of chx and hxm were evaluated because of their wide use and their molecular size, which allowed testing both types of sephadex tm , g- and g- . first, they were shown to be retained through the "in-house" sephadex tm columns and their potential cytotoxicity was removed. furthermore, the neutralization technique did not interfere with virus infectivity. at this stage, some limits of the gel filtration method in the concentration of the product tested appeared. above concentrations of − mol l − , molecules were not retained completely and there was a phenomenon of column overloading. however, this did not seem serious, since products are used rarely at this concentration or above, notably because of their potential toxicity. at this stage, the anti-coronavirus activity of chx and hxm could be assessed. thus, the results demonstrated clearly that hxm had no ats-d activity on the hcov e, but to our knowledge, little data concerning a possible ats-d antiviral activity of hxm is available in the literature. in addition the results showed that despite activity on the hcov e, chx could not pretend to have ats-d activity on the hcov e in testing conditions and according to the european standard. however, according to the american standard, of which the virucidal efficacy criterion is a log reduction, chx had an ats-d antiviral activity at − mol l − and min, but this activity is not really representative of field use conditions. in most studies, chx has been shown: (i) to be inefficient against naked viruses (bernstein et al., ; kawana et al., ; narang and codd, ; wood and payne, ) and (ii) to be efficient at concentrations > − mol l − on enveloped viruses but its activity depends on viruses tested and testing conditions (bernstein et al., ; kawana et al., ; platt and bucknall, ; tyler and ayliffe, ; wood and payne, ) . for instance, chx reduced herpes simplex virus viral titers of > log in suspension tests for under min (kawana et al., ; wood and payne, ) and only of log on a dried sample of hsv in min (tyler and ayliffe, ) . it is also important to note that most antiseptics and other chemicals are used as a mix of different molecules and the diluent, often ethanol, has its own virucidal activity. for instance, different formulations containing chlorhexidine digluconate were assayed for ats antiviral activity. it did not respond to the american virucidal criterion when tested at . % (m/v), i.e. . × − mol l − with cetrimide at . % (m/v). however, it induced a log reduction > in hcov viral titers when tested at . % (m/v), i.e. . × − mol l − , with cetrimide at . % (m/v) and ethanol at % (sattar et al., ) . this data showed that testing conditions must be taken into account to compare the antiseptic antiviral activity, i.e. suspension or carrier tests, presence or not of interfering substances and the chosen criterion to assess a virucidal activity. the possibility of misestimating product activity due to different testing conditions makes it essential to determine the best parameters so as to be as close as possible to the real conditions of application. the validation of this protocol was the first step in the improvement of antiviral ats-d activity evaluation and there is still a long way to go to get the perfect test. as expected, the goal of this method is to eliminate or at least reduce to the lowest possible level, the concentration of products tested in order to evaluate, with precise contact time, their antiviral potentiality, without toxicity for host cell and without modifying virus infectivity. even if this protocol had been validated by means of an in vitro suspension test, it could be extrapolated to in vitro carrier tests, in vivo or ex vivo protocols. such protocols can use support as plastic, steel disk (sattar et al., ) , fingertips (sattar and ansari, ) or disks of human skin removed during plastic surgery (graham et al., ) . carrier tests are recommended in different standards to simulate at best field situation, where viruses are dried and often embedded in organic material limiting the access to ats-d and thus their potential activity. ex vivo protocols are of importance to test antiviral ats activity of either pathogenic viruses or toxic compounds that, for healthy and ethic reasons, could not be assayed with in vivo protocols, using for instance, fingertips of volunteers. furthermore, they are also useful to evaluate pathogen survival on human skin. carrier tests need a dilution step to recover the dried virus suspension from the support after action of a chemical. this dilution step plays the role of neutralizer and ensures the contact time. it could be achieved by addition of culture medium or any chemical, which could neutralize specifically product activity (e.g. use of sodium thiosulfate for neutralizing povidone-iodine). however, the diluent itself could be toxic for the cells. thus, this method could be applied in such a case to eliminate the chemical neutralizer and inoculate the viral suspension safely. this potential is also found in the astm standard e - . this study describes a global method of chemical neutralization when ensuring the non-retention of viruses and the maintenance of their infectivity, in min. therefore, this method could be used to test chemicals for different applications from surfaces to water disinfectants. it could be used in the human medical field, as well as in veterinary or industrial (e.g. food industry) applications and also to screen the antiseptic antiviral potency of new drugs. chemical disinfectants and antiseptics-virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine-test method and requirements 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a novel coronavirus, coronavirus hku , from patients with pneumonia the action of three antiseptics/disinfectants against enveloped and non-enveloped viruses we sincerely thank dr. s.a. sattar key: cord- - gej co authors: marcekova, zuzana; psikal, ivan; kosinova, eva; benada, oldrich; sebo, peter; bumba, ladislav title: heterologous expression of full-length capsid protein of porcine circovirus in escherichia coli and its potential use for detection of antibodies date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: gej co a capsid protein of porcine circovirus (pcv ) serves as a diagnostic antigen for the detection of pcv -associated disease known as a postweaning multisystemic wasting syndrome (pmws). in this report, a bacterial expression system was developed for the expression and purification of the full-length pcv capsid (cap) protein from a codon-optimized cap gene. replacement of rare arginine codons located at the ′ end of the cap reading frame with codons optimal for e. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. the cap protein was purified to greater than % homogeneity by using a single cation-exchange chromatography at a yield of mg per litre of bacterial culture. despite the failure of the e. coli-expressed cap protein to self-assemble into virus-like particles (vlps), the immunization of mice with recombinant cap yielded antibodies with the same specificity as those raised against native pcv virions. in addition, the antigenic properties of the purified cap protein were employed in a subunit-based indirect elisa to monitor the levels of pcv specific antibodies in piglets originating from a herd which was experiencing pcv infection. these results pave the way for a straightforward large-scale production of the recombinant pcv capsid protein and its use as a diagnostic antigen or a pcv subunit vaccine. porcine circoviruses (pcv) are small non-enveloped, singlestranded circular dna viruses of the circoviridae family . two distinct types of pcv have been described: the non-pathogenic pcv type (pcv ) (tischer et al., ) and the pathogenic pcv type (pcv ) nayar et al., ) , which is associated with a newly emerged disease called postweaning multisystemic wasting syndrome (pmws) (clark, ) . four-to twelve-weeks old piglets which are affected with pmws display clinical symptoms of wasting, respiratory distress, anaemia, diarrhoea, jaundice and enlarged lymph nodes (for review, see chae, ) . pmws is considered to be an important porcine disease worldwide which is reported to have a serious economic impact on the global pig farming industry. the . kb pcv genome contains three functional open reading frames (orfs) . orf encodes several forms of non-structural replicase proteins (mankertz and hillenbrand, ; mankertz et al., ) , orf encodes the capsid protein (nawagitgul et al., ) , and orf encodes a -amino acid protein which appears to be involved in virus-induced apoptosis of infected cells (liu et al., ) . the capsid protein is a unique structural protein of the viral coat (nawagitgul et al., ) that is formed by protein subunits in an icosahedral t = capsid structure (crowther et al., ) . the n-terminus of the capsid protein is rich in basic amino acid residues and displays a nuclear localization signal (nls) which is important for capsid assembly (liu et al., a) . the capsid protein is highly immunogenic and reacts strongly with serum from pcv -infected pigs (mahe et al., ; fenaux et al., ) , and thus, it is the preferred antigen in a variety of serological tests (nawagitgul et al., ; blanchard et al., ; liu et al., ; shang et al., ) . the expression of recombinant capsid protein was achieved successfully in the baculovirus system (nawagitgul et al., ; mahe et al., ; blanchard et al., ; fan et al., ) , where the expressed capsid protein assembled spontaneously into virus-like particles (vlps) (nawagitgul et al., ; liu et al., ) . vlps represent non-infectious structures which completely lack the dna or rna genome of the virus. therefore, vaccines based on vlps trigger an immune response to the host immune cells by mimicking native virus morphology (ludwig and wagner, ) . however, the production of vlps for vaccination in eukaryotic systems is costly for veterinary applications. on the other hand, heterologous protein expression in e. coli offers an alternative to the production of large amounts of protein. the expression of full-length capsid protein in a standard bacterial expression system, such as e. coli bl (de ), has not been reported. only certain regions of the cap protein (wu et al., ) or a fusion protein with maltose-binding protein (liu et al., b) or truncated variant of cap lacking the nls (zhou et al., ; trundova and celer, ) have been expressed in e. coli. in this study, a genetic approach which takes advantage of a codon-optimized synthetic oligonucleotide and a synthetic codonoptimized gene is described to obtain the high-level production of the full-length pcv capsid protein in e. coli bl (de ) cells. the purified capsid protein is used as antigen to develop an indirect elisa for monitoring the levels of pcv specific antibodies in piglets originating from a herd experiencing pcv infection. the czech field-strain isolate of porcine circovirus type (l- , brno, czech republic) was used in this study. the virus stock was prepared from the supernatant of organ homogenate from a pig which fulfilled the diagnostic criteria for pmws (sorden, ) . samples of enlarged lymph nodes were pooled and homogenised in a fivefold volume of phosphate-buffered saline (pbs, ph . ). two volumes of chloroform were added to volumes of the homogenate and the mixture was shaken at • c for min and centrifuged at × g for min. the supernatant was subjected to a cushion of cscl density gradient ( . g/ml) and centrifuged at , × g for h in a beckman sw ti rotor (beckman coul-ter, fullerton, usa). the purified pcv virions were resuspended in pbs and subsequently used to infect the circovirus-free pk cells which were maintained in a d-mem medium (paa laboratories, pasching, austria) supplemented with % heat-inactivated fetal calf serum (gibco, invitrogen, carlsbad, usa) at • c with % co . the viral dna was purified from pk -infected cells using a dnazol genomic dna isolation reagent (molecular research center, cincinnati, usa) according to manufacturer's instructions. the genomic dna of pcv isolate was sequenced by abi prism xl analyzer (applied biosystems, foster city, usa) using bigdye terminator . cycle sequencing kit (applied biosystems, foster city, usa). the nucleotide sequence encoding the cap protein was . % identical with that of orf of pcv strain fd (genbank accession no. ay ) (de boisséson et al., ) . a bp sequence encoding the cap protein was amplified using polymerase chain reaction (pcr) with the following primers: the upstream primer -ccccatggcgatgacgtatccaaggaggc- containing the ncoi site and the downstream primer -tgtctcgagagggttggggggtc- containing the xhoi site. the purified pcr product was cut with ncoi and xhoi (new england biolabs, ipswich, usa), cloned into the pet b expression vector (novagene, merck kgaa, darmstadt, germany), and the vector was designated as pet b-cap-his (fig. b) . to generate a truncated version of the cap protein ( cap-his) lacking the first n-terminal amino acid residues, the pet b-cap-his was used as a template for pcr with the following primers: the upstream primer -agaccatgggcagcc-atcttggccagatc- containing ncoi site and downstream primer -tgtctcgagagggttggggggtc- containing the xhoi site. the bp pcr product was cut with ncoi and xhoi, cloned into the pet b vector, and designated as pet b-cap-his (fig. b) . for the construction of a vector carrying the codon-optimized nucleotide sequence of the first amino acid residues of the cap protein, the pet b-cap-his expression vector was cut with ncoi and msci. the resulting fragment was ligated with a dsdna adaptor (sense oligonucleotide -catgacctatccgcgccgccgttacc-gccgccgccgccaccgcccgcgcagccatctggg- and anti-sense oligonucleotide -cccagatggctgcgcgggcggtggcggcggcg-gcggtaacggcggcgcggataggt- ) creating the ncoi and msci overhangs at and end of the adaptor molecule, respectively. this construct was designated as pet b-o-cap-his (fig. b) . codon-optimized gene encoding the pcv capsid protein was obtained from genscript (piscataway, usa) and the nucleotide sequence was deposited in genbank (accession no. eu ). the plasmid carrying the nucleotide sequence of the cap protein was cut with ncoi and xhoi and cloned into pet b expression vector. to enhance the expression of the synthetic cap gene in e. coli, a pet b expression vector was modified by replacement of the nucleotide sequence between the ndei and saci sites with a dna adaptor molecule annealed from a pair of synthetic oligonucleotides (sense oligonucleotide -tatgcaccaccaccacc-accacgccatgggagct- and anti-sense oligonucleotide -cccatggcgtggtggtggtggtggtgca- ) which encoded the n-terminal histidine tag of the synthetic cap gene. this construct was cut with ncoi and xhoi and ligated with the synthetic cap gene which had been cut with the same restriction enzymes to obtain the expression vector carrying s-cap with the flanking n-and cterminal histidine tags (pet b-s-cap) (fig. b) . all the constructs were confirmed by dna sequence analysis with abi prism xl analyzer (applied biosystems, foster city, usa) using a big dye terminator cycle sequencing kit. recombinant proteins were expressed in e. coli bl (de ) (novagene, merck kgaa, darmstadt, germany). cells containing the expression plasmid were grown at • c in luria-bertani (lb) medium supplemented with g/ml kanamycin to the optical density of . at nm and then isopropyl ␤-dthiogalactopyranoside (iptg) (alexis corporation, lausen, switzerland) was added to a final concentration of mm. after h of growth, cells were harvested by centrifugation ( × g for min) and resuspended in mm tris-hcl (ph . ) containing mm edta. the expression in e. coli bl -codonplus(de )-ripl strain (stratagene, agilent technologies, santa clara, usa) was done under the same conditions, except for antibiotics concentrations: kanamycin ( g/ml), chloramphenicol ( g/ml) and spectinomycin ( g/ml). this strain contains extra copies of the argu, iley, prol, and leuw genes for rare trnas, which can rescue protein production restricted by either agg/aga or ccc codons. the expression of desired proteins was documented by sds-polyacrylamide gel electrophoresis (sds-page) and western blot analysis. bacterial pellet was washed and resuspended in sonication buffer ( mm mes, ph . , mm nacl) containing mm phenylmethylsulfonyl fluoride. the cells were disrupted by sonication ( w, misonix sonicator , misonix, farmingdale, usa) on ice and the homogenate was centrifuged at , × g at • c for min. solid urea was added to a final concentration of m, and mixed gently for min. after centrifugation at , × g for min at • c, the supernatant was loaded to a unosphere-s cation-exchange column (bio-rad, hercules, usa) which was connected to an akta prime chromatography system (ge healthcare, chalfont st. giles, united kingdom). after washing with column volumes of sonication buffer, the cap protein was eluted with a continuous gradient of nacl ( - m) in a buffer containing mm mes, ph . and m urea. pooled fractions of purified cap protein were concentrated by using centricons (amicon mw cutoff, millipore, billerica, usa), and stored at − • c for further use. total protein concentration was determined by the bradford assay (bio-rad, hercules, usa) using bovine serum albumin as a standard. proteins were separated by sds-page using % polyacrylamide gels and either stained by coomassie brilliant blue r- or transferred to a nitrocellulose membrane (pall corporation, new york, usa) in a transfer buffer ( mm tris-hcl, ph . , mm glycine, . % sds, % methanol) using a te xp semi-dry transfer unit (hoefer, holliston, usa) at v/cm for h. the membranes were blocked with % non-fat milk in pbs-t (phosphate-buffered saline containing . % tween- ), incubated with anti-his monoclonal antibody ( : ) (sigma, st. louis, usa) followed with a peroxidase-conjugated anti-mouse antibody. the signal was developed using an enhanced chemiluminiscence system (ge healthcare, chalfont st. giles, united kingdom). the experimental work with mice was conducted according to the certificate of animal welfare authority commission of the ministry of agriculture of the czech republic no. mze / in accordance with current czech legislation on animal welfare. a total of balb/c mice ( weeks of age) were used in the experiment. a group of mice was bled before the immunization to obtain pre-immune serum. a group of mice received intramuscular injection with recombinant s-cap ( g protein per mouse) mixed with a complete freund's adjuvant (sigma, st. louis, usa). three weeks later, the mice were boosted intramuscularly with the same dose of recombinant cap protein mixed with incomplete freund's adjuvant (sigma, st. louis, usa). the mice were euthanised and bled days after the last injection prior to the sera were screened for the presence of the cap-specific antibodies by using an immunoperoxidase monolayer assay (ipma). for ipma, confluent monolayers of pk cells were mixed with a reference pcv isolate (stoon- , after passages) and incubated at • c for h prior to fixation with % paraformaldehyde. the fixed cells were then incubated with mice sera ( : dilution in pbs-t buffer) at • c for h, followed by incubation with a peroxidase-conjugated goat anti-mouse secondary antibody ( : dilution in pbs-t buffer) and the peroxidase activity was detected with -chloro- -naphthol (bio-rad, hercules, usa) as a substrate. e. coli cells were washed twice in mm phosphate buffer (ph . ) and fixed in a solution containing % glutaraldehyde in mm phosphate buffer (ph . ) for h. after three washes, the cells were post-fixed in buffered % oso ( mm phosphate buffer, ph . ) for h, washed three times in mm phosphate buffer (ph . ), dehydrated in ethanol series, and then transferred into absolute acetone and embedded in vestopal w resin (sigma, hercules, usa). ultrathin sections were cut with glass knife using a lkb ultratome (lkb, bromma, sweden) and mounted on formvar-coated copper grids. the grids were stained in saturated aqueous uranyl acetate followed by lead citrate. samples were examined under philips cm electron microscope (fei, eidhoven, the netherlands) at kv. images were recorded by using a megaviewii slow scan camera controlled by analysis . software (olympus soft imaging solutions, münster, germany). pcv antibodies were determined with an indirect enzymelinked immunosorbent assay (elisa) using the purified s-cap and cap-his proteins as coating antigens (cap-elisa). the -well elisa plates (nunc, thermo fisher scientific, waltham, usa) were coated with l of mm sodium carbonate buffer (ph . ) containing g/ml of the proteins and incubated overnight at • c. the plates were washed with phosphate-buffered saline (pbs) containing . % tween- (pbs-t) and blocked with a blocking buffer (pbs containing % bovine serum albumin) at • c for h. after washing, l of each serum sample (diluted : ) was added into each well and tested in quadruplicate: two wells for a negative control antigen glutathione s-transferase (sigma) and two parallel wells for the antigens. the plates were washed with pbs-t and l of blocking buffer containing a peroxidase-conjugated goat anti-swine antibody (bethyl laboratories, montgomery, usa) was added into each well and incubated for h. finally, the plates were washed with pbs-t three times and the colour reaction was developed with , , , -tetramethylbenzidine (tmb) as a substrate. the optical density at nm (od nm ) was read using a microplate reader. the final od nm value was calculated by subtracting the mean value of od nm of wells containing a negative antigen from that of the parallel wells containing the capsid proteins. the cut-off value was determined by using specific pathogen free (spf) pig's serum samples diluted at : which were negative for anti-pcv antibodies as determined by ipma. the mean od nm value was . with a standard deviation (s.d.) of . and the final cut-off value was calculated by adding the mean od nm value + s.d. to the value of . . pig serum with a high titre of pcv antibodies : , as determined by ipma, was used as a positive control (kindly provided by annette mankertz from robert-koch institut, berlin, germany). the spf pig sera were negative for pcv and used as negative controls in each plate. a limited serological and genomic load survey of specific pcv antibodies and equivalent pcv genome copy numbers, respectively, was carried out on pig sera collected at , , , and weeks of age. selected piglets were selected from a pig herd experiencing pcv infection, marked with tags and monitored during their nursery and fattening period. at the regular intervals, a minimum of ml of blood was collected from each piglet and the sera were stored at − • c for further use. total genomic dna was extracted from a l volume of each serum sample using a nucleospin blood isolation kit (macherey-nagel, düren, germany) according to manufacturer's instructions. quantification of pcv dna levels was performed using a realtime pcr by roche light cycler (roche, basel, switzerland) in a taqman format as previously described (brunborg et al., ) . in short, the taqman probe was labelled with -fam and -bhq fluorophores (generi biotech, hradec kralove, czech republic) and the primers were designed to amplify a bp dna segment within the nucleotide sequence of the pcv cap gene. the absolute quantification of pcv dna was carried out using calibration curves generated by means of external standard dna obtained by cloning the pcv cap gene into a pcr . vector (invitrogen, carlsbad, usa). standard curves for pcv dna quantification were gener- ated using tenfold dilution of the linearised plasmids in the range of log . statistical analyses were carried out using the one-way analysis of variance (anova) test running in spss software (base . , spss, chicago, usa). values of p < . were considered significant. the open reading frame encoding cap protein was amplified using pcr from genomic dna of pcv (l isolate) and the bp long dna fragment was cloned into pet b expression vector (fig. b) . the expression was documented in crude cell lysate by sds-page and western blotting. as demonstrated in fig. , no expression of cap-his was detected in coomasie-stained gels and by western blot using anti-his antibody. frequently, an insufficient production of heterologous proteins in e. coli is due to the presence of codons that are used rarely in bacterial proteosynthesis (zahn, ) . forced high-level expression of genes with rare codons may lead to the depletion of the endogenous pools of the corresponding trnas, resulting in the abortion of the translation process and the degradation of the mrna (mcnulty et al., ) . examination of the nucleotide sequence of the cap gene revealed a cluster of rare codons within the first codons of the gene (fig. c) . the presence of such rare codon tandems near the end of a coding sequence has been reported to cause ribosomal frameshifts and codon skipping with a strong inhibitory effect on protein translation (gurvich et al., ) . in order to eliminate the cluster of rare codons from the end of the cap gene, the expression vector encoding a truncated variant of cap protein ( cap-his), which was lacking the first amino acid residues, was constructed (fig. b) . expression of the truncated gene in iptg-induced e. coli bl (de ) cells resulted in the production of a protein with an apparent molecular weight of kda ( fig. a) which corresponded to the cap-his protein. this suggested that the removal of the first codons from the end of the cap gene allowed the production of cap protein in e. coli. in previous report, the expression of the truncated variant of cap protein lacking the nuclear localization signal (nls) has been reported (zhou et al., ; trundova and celer, ) . the nls consists of amino acid residues at the n-terminus of cap and the nls-defective constructs have been expressed also as fusion proteins with glutathione s-transferase (zhou et al., ) or maltose-binding protein (liu et al., b) . based on these results, the authors concluded that the expression of the capsid protein in e. coli was inhibited by the nucleotide sequence encoding the nls domain. however, the expression of the truncated cap protein, which lacks the first amino acids, was achieved which suggested that the production of the full-length capsid protein in e. coli was hindered only by the occurrence of rare arginine codons near the end of the cap gene. the heterologous expression of the entire cap gene was examined also in e. coli bl -codonplus(de )-ripl cells. this strain contains extra copies of the argu, iley, prol, and leuw genes for rare trnas, which rescue protein production restricted by either agg/aga or ccc codons. the expression of the cap gene in iptginduced e. coli bl -codonplus(de )-ripl yielded the production of a protein with an apparent molecular weight of kda which corresponded to the full-length cap protein ( fig. a) . this indicated that the expression of rare aminoacyl-trnas allowed the production of the full-length cap protein. however, the use of e. coli bl -codonplus(de )-ripl for a high-yield production of recombinant proteins is inconvenient as the expression system depends on using a combination of three different antibiotics: one for the maintenance of the expression vector, and the other for the maintenance of plasmids carrying trna genes. moreover, due to the presence of high amounts of antibiotics the growth rate of bacterial cells is reduced and expensive. a promising approach to maximise heterologous production of proteins in e. coli is codon optimization strategy which makes codon usage in the gene of interest to match the available trna pool within the cells (jana and deb, ; peti and page, ; burgess-brown et al., ) . to facilitate the cap gene expression in e. coli bl (de ), the sequence of the first codons was optimized as described in fig. c . ten codons, rare in e. coli, were replaced by the most frequent ones, including six arginine, two proline, and single leucine and threonine codon substitutions. the codon-optimized sequence was introduced into pet b-cap-his using ncoi and msci restriction sites to obtain pet b-o-cap-his (fig. b) . the expression of the o-cap-his gene in e. coli bl (de ) cells yielded the production of a protein with a molecular weight of kda (fig. a) . the corresponding band was recognised by the anti-his antibody which confirmed the presence of the o-cap-his protein (fig. b) . these data indicated that the replacement of the first codons of the cap gene with a codon-optimized sequence enabled the production of cap protein in a conventional prokaryotic expression system, such as e. coli bl (de ). different bioinformatics tools are available for codon optimization which considers many factors, such as rna secondary structure, gc content, repetitive and rare codons (grote et al., ) . these approaches were used to design a synthetic cap gene with nucleotide sequence optimized for prokaryotic expression in e. coli (genbank accession no. eu ). the synthetic gene with introduced ncoi and xhoi restriction sites was cloned into pet b vector carrying the c-terminal polyhistidine tag and the expression of this construct was analyzed by sds-page. however, no protein product was detected in coomasie-stained gel (data not shown). to force the expression of the capsid protein from the synthetic gene, additional codons encoding the polyhistidine tag were added at the end of the gene. the expression of this construct (pet b-s-cap) in e. coli bl (de ) yielded the s-cap protein with a molecular mass of kda, which was a slightly higher (+ kda) than the o-cap-his protein due to the presence of the additional polyhistidine tag at the n-terminus of the protein (fig. a) . the levels of the s-cap protein expression were comparable to those of the o-cap-his protein which indicated that codon optimization of the entire sequence of the cap gene did not enhance significantly the heterologous expression of the capsid protein in e. coli. the analysis of protein distribution in e. coli cells revealed that all the cap proteins were recovered in the soluble (cytoplasmic) fraction of the iptg-induced cell lysate. however, initial attempts to purify native cap proteins gave unsatisfactory results. using standard chromatographic procedures, no significant binding of cap proteins was detected while loading the material on either cationexchange or nickel immobilised affinity columns in native buffer conditions (data not shown). to enable purification of cap proteins, denaturating conditions in the presence of m urea were utilised. the cytosolic extract of the cell lysates was supplemented with solid urea to a final concentration of m and the mixture was loaded onto a unosphere-s cation-exchange column at ph . . a typical elution profile of protein fractions from the unosphere-s column is shown in fig. a . fractions - contained large amounts of unrelated bacterial proteins, while the later fractions, eluting after min, were enriched with cap proteins (fig. b) . the identity of the cap proteins was confirmed also by western blotting with anti-his antibody (fig. c) . however, some apparent differences between the s-cap protein and the remaining capsid proteins were observed during purification procedures. while the s-cap protein was recovered in a high purity in the distinct peak eluting at min (fig. b, fraction ) , both the o-cap-his and cap-his proteins were eluted from the column in a shorter time (about - min). moreover, these fractions were enriched partially with a protein with a molecular weight of about kda and some low molecular weight proteins below kda (data not shown). the original aim of using the polyhistidine tag was to isolate the cap proteins on a metal affinity chromatography column. however, the presence of two polyhistidine tags within the s-cap protein resulted in the increase of the basicity of the protein, which enabled simple purification of the protein by a single step cation-exchange chromatography. the next purification steps did not increase the purity of proteins (data not shown) and these steps were omitted in the purification protocol. the purity of the s-cap protein was greater than % after the cation-exchange chromatography and approximately mg of purified protein was obtained from l of bacterial cell culture. these data suggested that the yield of the s-cap protein is much higher than the yield of the o-cap-his and cap-his proteins in terms of purity, time and costs for large-scale production of the recombinant pcv capsid protein. to examine the immunogenicity of the s-cap protein, mice were immunized with the purified protein and the collected sera were analyzed for the presence of cap-specific antibodies. western blot analyses revealed that a single -kda band in the bacterial lysate enriched in the s-cap protein was recognised by the cap-positive sera, but not by the pre-immune sera (data not shown). moreover, a typical dense nuclear staining of pcv -infected pk cells was seen using the cap-positive sera compared to the pre-immune sera by in situ immunohistochemistry (data not shown). these results documented the immunogenicity of the s-cap protein isolated under denaturating conditions. the majority of vlps have been produced using insect or mammalian cell expression systems, but some vlps have been found to assemble also in e. coli cells (noad and roy, ) . in order to detect whether the pcv capsid protein is able to self-assemble into vlps in e. coli, the ultrastructure of the bacterial cells expressing the o-cap protein, which lacks any of the additional tag (fig. ) , was examined by using electron microscopy (em) of ultrathin sections. as shown in fig. , no vlps were observed in either non-induced cells or e. coli cells expressing the o-cap protein. in contrast, in vivo vlps assembly was detected unambiguously following the expression of a fusion protein containing the capsid and nucleocapsid protein obtained from the gag polyprotein of mason-pfizer monkey virus (ulbrich et al., ) (fig. a and b ). in addition, the sucrose density gradient fractionation of cytosolic content of e. coli cells expressing o-cap protein did not reveal any presence of o-cap protein in the form of vlps. the absence of vlps was corroborated also by em of negatively stained preparations (data not shown). these results demonstrated that the pcv capsid protein was unable to self-assemble into vlps in the cytosol of e. coli cells. in contrast, self-assembly of the pcv capsid protein into vlps has been observed repeatedly in baculovirus expression systems (nawagitgul et al., ; kim et al., ; liu et al., ) . the vlps were of similar morphology as the intact pcv virions and appeared to be empty capsids with a less ordered structure (nawagitgul et al., ) . however, the question why the pcv capsid protein does form vlps in insect cells and not in e. coli remains to be addressed. sequence analysis of the pcv capsid protein revealed amino acid residues with consensus patterns for potential posttranslational modifications (n-glycosylation, phosphorylation) (liu et al., b) , which are known to occur in eukaryotic but not in prokaryotic expression systems. another explanation could be a specific need for chaperones, scaffolding proteins, or ssdna, which might play important roles in a proper pcv assembly (ludwig and wagner, ) . a plausible hypothesis is that the n-terminal portion of the pcv capsid protein is enriched with basic amino acids that could bind preferentially to the bacterial genomic dna, and therefore, prevent a regular arrangement of the capsid subunits into vlps. this hypothesis would be supported by the observation that the e. coli cells expressing cap protein produced large amounts of outer membrane vesicles (fig. d ) compared to the non-induced cells or the cells expressing the capsid protein of mason-pfizer monkey virus. indeed, the release of outer membrane vesicles has been attributed to a novel envelope stress response (mcbroom and kuehn, ) , which might result from the binding of the cap protein to genomic dna. specificity and sensitivity of both the full-length capsid protein (s-cap) and its truncated variant ( cap-his) to pig pcv -positive sera was determined using an indirect elisa format (cap-elisa). using a chequer-board titration of pcv -positive serum (ipma titre of ), optimal antigen concentrations and serum dilutions were selected to be g/ml and : , respectively. when the positive/negative cut-off value was set to . (see section ), all the pcv -positive sera tested were % specific (data not shown). cap-elisa showed a low background, and significant differences between pcv -negative and pcv -positive pig sera were detected (fig. ) . cap-elisa did not exhibit any cross-reactivity with pig sera obtained from spf pigs which were infected experimentally with coronavirus causing a transmissible gastroenteritis (tge) or a porcine epidemic diarrhoea (ped), respectively. moreover, no significant differences in specificity and sensitivity of cap-elisa were observed between the s-cap and cap-his proteins when used as coating antigens, indicating that the n-terminus of the capsid protein is not specifically recognised by antibodies in the pcv positive sera (wu et al., ) . this showed that the recombinant full-length cap protein could be used as coating antigen to develop the indirect cap-elisa for specific and sensitive detection of pcv antibodies. pcv antibodies are detected currently by indirect immunofluorescence , ipma , competitive elisa (walker et al., ) and by indirect elisa based on either pcv viral particles (nawagitgul et al., ) or recombinant pcv capsid protein expressed in baculovirus (nawagitgul et al., ; blanchard et al., ; liu et al., ) . however, while both the indirect immunofluorescence and ipma assays are highly demanding and not suitable for large-scale survey of pcv infection, the cap-his proteins ( g/ml) was analyzed by a colorimetric reaction at the optical density at nm (od nm). od nm of . represents a cut-off value for seropositivity of the samples. tge-specific and ped-specific pig sera were obtained from spf pigs which were experimentally infected with the coronavirus causing transmissible gastroenteritis (tge) and porcine epidemic diarrhoea (ped), respectively. data represent the means ± s.d. for the three independent experiments. current elisa formats are less specific and display antigenic crossreactivity to non-pathogenic pcv (magar et al., ) . recently, an indirect elisa based on the recombinant nls-truncated pcv capsid protein expressed in e. coli has been established by shang et al. ( ) . although a direct comparison in the sensitivity and specificity of the full-length (this study) and the nls-truncated (shang et al., ) proteins as antigens would be interesting, it is very likely that both elisa formats are comparable in the detection of pcv antibodies. considering these data, the cap-elisa based on the recombinant full-length cap protein as coating antigen would provide a simple and reliable tool for standard serodiagnosis of pcv infection. to further analyze the capacity of cap-elisa to detect the pcv specific antibodies, a limited serological and genomic load survey of pcv infection was monitored in piglets during the first weeks of age. the piglet's antibody response along with total amounts of pcv dna per ml of serum was determined by cap-elisa and quantitative pcr, respectively. as shown in fig. a , the levels of pcv -specific antibodies showed an initial drop within the first weeks of age, from then on the levels increased gradually until th week. in contrast, the dna copy numbers of pcv increased gradually during the st weeks of age to reach the maximum level in the th week, after which they decreased slightly until the th week (fig. b) . these results are in good agreement with the time course dynamics of pcv infection in newborn piglets (mckeown et al., ) . a dramatic decrease of the pcv -specific antibodies in the th week of age corresponds to the period in which the passive intake of maternal antibodies from breast milk during the weaning period (weeks - ) is discontinued. this results in the onset of pcv infection after the th week of age prior to the active production of the intrinsic pcv -specific antibodies (weeks - ). taken the final values were expressed as a signal-to-positive (s/p) ratio. s/p value was determined according to the following formula: (od nm of sample − od nm of negative control)/(od nm of positive control − od nm of negative control). s/p value of . represents the cut-off signal for seropositivity of the samples. (b) time course analysis of the pcv dna copy numbers per ml of serum as detected by the quantitative pcr. total genomic dna was extracted from l of each serum sample and quantified by the pcr amplification of bp dna segment within the nucleotide sequence of the pcv cap gene using a taqman format. results of statistical analysis using one-way analysis of variance (anova) between serum samples obtained at given time intervals from piglets are represented by the box plots. the median value for each dataset is indicated by the black center line. the vertical height of each box indicates the - % data range. the upper and lower bars denote the largest and smallest data values. the marker ( ) denotes the extreme value of the individual serum sample that is > . times the inter-quartile range from the upper and lower quartile. the marker (᭹) denotes the extreme value of the individual serum sample that is > times the inter-quartile range from the upper quartile. data represent the means ± s.d. for the three independent experiments. together, these data confirmed that cap-elisa was developed with a high specificity and sensitivity for detection of pcv antibodies in pig sera and could be used for large-scale surveys of pcv infection at low cost. in summary, a bacterial expression system has been developed for the production of the full-length recombinant capsid protein of porcine circovirus type . here, the codon optimization strategy was used to obtain high yields of the recombinant capsid protein in an inexpensive cultivation system. purification protocol based on the single step cation-exchange chromatography provided a cost effective procedure to obtain substantial quantities of the cap protein in a high purity. although the recombinant capsid protein expressed in e. coli did not self-assemble into vlps, the antigenic properties of the cap protein resembled that of intact pcv virions. in addition, the recombinant full-length cap protein was used as antigen to develop the indirect cap-elisa for specific and sensitive detection of pcv infection. porcine circoviruses: a review isolation of porcine circovirus-like viruses from pigs with a wasting disease in the united states of america and europe an orf protein-based elisa for porcine circovirus type antibodies in post-weaning multisystemic wasting syndrome quantitation of porcine circovirus type isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a taqman-based realtime pcr codon optimization can improve expression of human genes in escherichia coli: a multi-gene study postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology post-weaning wasting syndrome comparison of the structures of three circoviruses: chicken anemia virus, porcine circovirus type , and beak and feather disease virus molecular characterization of porcine circovirus type isolates from post-weaning multisystemic wasting syndrome-affected and non-affected pigs isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome construction and immunogenicity of recombinant pseudotype baculovirus expressing the capsid protein of porcine circovirus type in mice immunogenicity and pathogenicity of chimeric infectious dna clones of pathogenic porcine circovirus type (pcv ) and nonpathogenic pcv in weanling pigs jcat: a novel tool to adapt codon usage of a target gene to its potential expression host expression levels influence ribosomal frameshifting at the tandem rare arginine codons agg-agg and aga-aga in escherichia coli strategies for efficient production of heterologous proteins in escherichia coli characterization of the recombinant proteins of porcine circovirus type field isolate expressed in the baculovirus system nuclear localization of the orf protein encoded by porcine circovirus type bacterial expression of an immunologically reactive pcv orf fusion protein development of an elisa based on the baculovirus-expressed capsid protein of porcine circovirus type as antigen characterization of a previously unidentified viral protein in porcine circovirus type -infected cells and its role in virus-induced apoptosis efficient production of type porcine circovirus-like particles by a recombinant baculovirus virus-like particles-universal molecular toolboxes retrospective serological survey of antibodies to porcine circovirus type and type differential recognition of orf protein from type and type porcine circoviruses and identification of immunorelevant epitopes replication of porcine circovirus type requires two proteins encoded by the viral rep gene identification of a protein essential for replication of porcine circovirus release of outer membrane vesicles by gramnegative bacteria is a novel envelope stress response effects of porcine circovirus type (pcv ) maternal antibodies on experimental infection of piglets with pcv mistranslational errors associated with the rare arginine codon cgg in escherichia coli characterization of novel circovirus dnas associated with wasting syndromes in pigs detection and characterization of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs modified indirect porcine circovirus (pcv) type -based and recombinant capsid protein (orf )-based enzyme-linked immunosorbent assays for detection of antibodies to pcv open reading frame of porcine circovirus type encodes a major capsid protein virus-like particles as immunogens strategies to maximize heterologous protein expression in escherichia coli with minimal cost development and validation of a recombinant capsid protein-based elisa for detection of antibody to porcine circovirus type update on porcine circovirus and post-weaning multisystemic wasting syndrome (pmws). j. swine health prod a very small porcine virus with circular single-stranded dna expression of porcine circovirus orf gene requires codon optimized e. coli cells distinct roles for nucleic acid in in vitro assembly of purified mason-pfizer monkey virus canc proteins overexpression of an mrna dependent on rare codons inhibits protein synthesis and cell growth in vitro expression, monoclonal antibody and bioactivity for capsid protein of porcine circovirus type ii without nuclear localization signal expression of the porcine circovirus type capsid protein subunits and application to an indirect elisa development and application of a competitive enzyme-linked immunosorbent assay for the detection of serum antibodies to porcine circovirus type the excellent technical help of sona charvatova, hana kubinova, zuzana vecerkova and petra klodnerova (proteix, s.r.o.) is acknowledged. we also wish to thank pavel ulbrich (institute of chemical technology, prague, czech republic) for providing mason-pfizer monkey virus canc expression vectors. this work was supported by grants gacr / /p (l.b.), npvii b (p.s.) of the ministry of education, youth and sports of the czech republic, npv b (i.p.) and (i.p.) of the ministry of agriculture of the czech republic, and institutional research concept av z and av z . key: cord- -ww t k authors: sakthivel, senthilkumar k.; whitaker, brett; lu, xiaoyan; oliveira, danielle b.l.; stockman, lauren j.; kamili, shifaq; oberste, m. steven; erdman, dean d. title: comparison of fast-track diagnostics respiratory pathogens multiplex real-time rt-pcr assay with in-house singleplex assays for comprehensive detection of human respiratory viruses() date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: ww t k fast-track diagnostics respiratory pathogens (ftdrp) multiplex real-time rt-pcr assay was compared with in-house singleplex real-time rt-pcr assays for detection of common respiratory viruses. the ftdrp assay correctly identified diverse respiratory virus strains, of ( %) external quality assessment samples spiked with cultured virus and of ( %) archived respiratory specimens that tested positive for respiratory viruses by in-house assays. of prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, ( . %) and ( %) were positive by ftdrp and in-house assays for one or more viruses, respectively, with combined test results showing good concordance (k = . , % ci = . – . ). individual ftdrp assays for adenovirus, respiratory syncytial virus and rhinovirus showed the lowest comparative sensitivities with in-house assays, with most discrepancies occurring with specimens containing low virus loads and failed to detect some rhinovirus strains, even when abundant. the ftdrp enterovirus and human bocavirus assays appeared to be more sensitive than the in-house assays with some specimens. with the exceptions noted above, most ftdrp assays performed comparably with in-house assays for most viruses while offering enhanced throughput and easy integration by laboratories using conventional real-time pcr instrumentation. respiratory viruses are among the most important causes of human morbidity and mortality worldwide (nair et al., ; pavia, ) . clinically indistinguishable, respiratory virus infections require accurate laboratory diagnosis to guide treatment effectively and prevention decisions. polymerase chain reaction (pcr) and other molecular assays are now routinely used for diagnosis ଝ the contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the us centers for disease control and prevention (cdc) or department of health and human services (dhhs). names of specific vendors, manufacturers, or products are included for public health and informational purposes; inclusion does not imply endorsement of the vendors, manufacturers, or products by the cdc or dhhs. of respiratory virus infections (beck and henrickson, ; kehl and kumar, ), but the large and increasing number of viruses makes laboratory testing with individual (singleplex) virus assays challenging. conversely, multiplex pcr assays that combine multiple individual assays in a single reaction facilitate more rapid, high throughput and cost-effective testing and are generally preferred in the clinical setting (elnifro et al., ; jansen et al., ) . numerous laboratory-developed and commercial multiplex pcr assays using different amplification platforms have been described for respiratory viruses and have been generally shown to be superior to traditional diagnostic methods, such as virus culture and antigen detection for sensitive and specific detection of respiratory viruses (arens et al., ; bibby et al., ; brittain-long et al., ; caliendo, ; gadsby et al., ; kim et al., ; lamson et al., ; mahony et al., ; raymond et al., ). the us food and drug administration (fda) has cleared recently two commercial assays, the xtag ® rvp fast (luminex molecular diagnostics, austin, tx) and filmarray ® respiratory panels (idaho technology inc., salt lake city, utah) (rand et al., ) , for invitro diagnostic use for multiplex detection of respiratory viruses. however, many of these assays are costly, require specialized laboratory equipment and use highly multiplexed reactions that may be deficient in individual assay performance and can be difficult to modify without extensive assay reoptimization (gunson et al., ) . the ftd respiratory pathogens (ftdrp) multiplex assay kit (fast-track diagnostics, luxembourg) uses standard commercial one-step reverse transcription (rt)-pcr hydrolysis probe chemistry and common real-time pcr instrumentation. the ftdrp assay consists of discrete primer/probe mixes that together cover common human respiratory viruses. this study reports the results of a comparison of the ftdrp multiplex assay with a panel of validated in-house singleplex real-time rt-pcr assays developed at the centers for disease control and prevention (cdc). virus isolates and archived clinical specimens were obtained from cdc collections acquired during routine surveillance and outbreak investigations. these included laboratory reference virus strains and field isolates and geographically (u.s., central and south america and africa) and compositionally diverse specimens [nasopharyngeal and oropharyngeal swabs ( ), nasal washes and aspirates ( ), sputum ( ), lung autopsy tissue ( ) and unidentified ( )] collected from children and adults with acute respiratory illnesses (aris) acquired between and and previously testing positive for respiratory viruses by the in-house singleplex assays. all residual samples and extracts were stored at − • c. whenever possible, archived specimens were selected to achieve a proportional representation of viral loads. forty-six mock human specimens spiked with moderate-to-low concentrations of virus were available from the quality control for molecular diagnostics (qcmd, glasgow, scotland) external quality assessment (eqa) programs for rhinovirus/coronavirus, adenovirus, parainfluenza viruses, human metapneumovirus/respiratory syncytial virus, and influenza a & b viruses (wallace, ) . pooled nasal wash specimens from consenting healthy new military recruits was kindly provided by dr. lisa lott, eagle applied sciences, l.l.c., san antonio, tx. finally, a subset of nasopharyngeal aspirates (npas) from an etiologic study of children < years of age hospitalized with ari at a tertiary hospital in são paulo, brazil, between march and september , were selected from the seasonal peaks of respiratory virus circulation for each of the study years based on local surveillance data. the npas were collected directly into liquid nitrogen, aliquoted and transferred to − • c and retained until retrieved for this study. this study was approved by institutional review boards at the university of são paulo and santa casa de misericórdia de são paulo hospital, brazil, and cdc. total nucleic acid (tna) extracts were prepared from samples using the nuclisens ® easymag ® (biomérieux). because of their multiple study origins and testing histories, residual archived specimen extraction volumes varied from to l and tna elution volumes from to l. rnase-free water was added to a few archived extracts (≤ -fold dilution) to obtain sufficient volume for comparison testing. for prospectively tested nasal aspirate specimens, l of each sample was extracted and the tna recovered in l of elution buffer which was then split into aliquots and frozen at − • c until testing. all extracts were subjected to identical freeze-thaw cycles for comparison testing. all extracts were confirmed positive for human rnase p gene by real-time rt-pcr before inclusion in the study. the ftdrp multiplex real-time rt-pcr assay (ver. , cat. no. ftd - / ) consists of separate primer/probe mixes covering human respiratory viruses and brome mosaic virus (bmv), an rna plant virus that serves as an internal extraction control when spiked into the sample (virus provided); mix # : influenza a virus (inf a), influenza b virus (inf b), bmv; mix # : coronavirus (cov) nl , e and oc and enterovirus/parechovirus (ev/pev); mix # : parainfluenza virus (piv) , and ; mix # : piv , human metapneumovirus (hmpv) and human bocavirus (hbov); mix # : rhinovirus (rv), respiratory syncytial virus (rsv) and adenovirus (adv). individual assays within each pool are distinguished by use of different probe fluorophores, with the exception of the ev and pev assays, where both probes are rox-labeled and therefore cannot be distinguished. each kit also contains a positive plasmid control pool and detailed instructions on test performance. the ftdrp assay was performed following the manufacturer's recommendations. briefly, l of × rt-pcr buffer was combined with . l of each primer/probe pool and . l of × enzyme mix (agpath-id tm one-step rt-pcr kit, applied biosystems), and l of each mixture was then added to wells of a pcr plate ( sample reactions plus one positive and one negative virus control). ten l of sample tna extract or controls were then added to the respective wells of each primer/probe pool. the following cycling conditions were performed on a fast dx real-time pcr instrument (applied biosystems): min at • c, min at • c and cycles of s at • c and s at • c. threshold cycle (ct) values were determined by manually adjusting the fluorescence baseline to fall within the exponential phase of the amplification curves and above any background signal. a positive test result was considered a well-defined curve that crossed the threshold cycle within cycles. positive and negative virus plasmid controls provided in the kit were included in all runs to monitor assay performance. the bmv internal control was spiked into clinical specimens to monitor sample extraction and reverse transcription. previously extracted tna samples were evaluated for rnase p only. in-house singleplex real-time rt-pcr assays for rsv, hmpv, piv - , rv, adv, hbov and covs ( e, oc , nl , hku , sars-cov) as previously described (dare et al., ; emery et al., ; fry et al., ; heim et al., ; kodani et al., ; lu et al., lu et al., , morgan et al., ) were performed on a mx p qpcr system (agilent technologies) using agpath-id tm one-step rt-pcr reagents (applied biosystems) with the following cycling conditions: • c for min, • c for min and cycles of • c for s and • c for min. primer/probe sequences are available from d.e. on request. the in-house ev and pev assays as modified from previous reports (kilpatrick et al., ; nix et al., ) were performed on a mx p qpcr system using the superscript iii platinum ® one-step quantitative rt-pcr system reagents (invitrogen) with the following cycling conditions: • c for min, • c for min and cycles of • c for s, • c (ev) or • c (pev) for s and • c for s. universal inf a and inf b assays were performed on a fast dx real-time pcr instrument with sds software ver. . (applied biosystems) using the superscript iii platinum ® one-step quantitative rt-pcr system with the following cycling conditions: • c for min, • c for min and cycles of • c for s and • c for s (stephen lindstrom, cdc, personal communication). following standard operating procedures, all in-house assays were performed in l final reaction volumes containing l of sample tna extract. a positive test result was considered a welldefined curve that crossed the threshold cycle within cycles. positive and negative virus rna transcript or whole virus extract controls were included in all runs to monitor assay performance. percent sensitivity and specificity of the ftdrp assay for prospectively collected specimens were calculated using the inhouse assays as the reference standard. agreement between assays was measured using the kappa statistic (cohen, ) where indicates no agreement and indicates perfect agreement. the ftdrp assay was first evaluated with undiluted tna from cultures of respiratory virus strains corresponding to most assays in the multiplex to assess assay specificity and virus strain inclusivity (table ) . although no ftdrp assay for sars-cov was available, this virus was tested to assess the specificity of the other ftdrp cov assays. hbov and cov nl and hku isolates were not available for testing. positive results were obtained with both assays for all viruses with no cross-reactions detected. ftdrp and in-house assay results were within ct values for ( %) of the viruses tested. notably, the ftdrp rv assay gave a substantially higher ct value ( . ct) with one rv isolate (rv-b ). serial dilutions of rv-b tna showed the ftdrp assay to be > -fold less sensitive than the corresponding in-house assay with this virus strain (data not shown). the specificity of the ftdrp assay was further evaluated with pooled nasal wash samples from consenting normal healthy adults to represent diverse microbial flora in the human respiratory tract. positive results were obtained with the in-house assays for rv (ct . ), cov e (ct . ) and adv (ct . ) which were confirmed by alternate rt-pcr assays and sequencing. the ftdrp assay was positive for rv (ct . ) and cov e (ct . ), but did not detect the adv on initial or repeat testing. all other in-house and ftdrp assays were negative. forty-one mock respiratory samples spiked with low to moderate levels of different viruses and negative control samples selected from qcmd eqa programs for hrv/cov, adv, piv, rsv/hmpv and inf a/b, were tested to assess assay performance against the reference qcmd assays (table ) . overall, expected results were obtained with ( %) and ( %) of positive eqa program samples with the in-house and ftdrp assays, respectively. all program negative control samples were negative by both assays. one sample (adv - ), with low concentration adv-f , was initially negative by the in-house assay, but positive on repeat testing. the ftdrp assay gave expected results with all piv ( ), cov ( ) inf table comparison of ftdrp and in-house assays with archived respiratory specimens previously positive for respiratory viruses. in-house assay results classified as strong (ct < ), moderate (ct ≥ to ≤ ) or weak (ct > ) positive. c ftdrp ev/pev assay does not distinguish between ev and pev. eight specimens separately tested positive for ev ( ct < ; ct ≥ to ≤ ) and pev ( ct < ; ct ≥ to ≤ ) by in-house assays. comparison of ftdrp and in-house assays with archived respiratory specimens with sequence confirmed rhinovirus (rv) or enterovirus (ev and adv-b (adv - ) were consistently negative and rv-b (rv.cv - ) showed substantially higher ct values ( . ct) by the ftdrp rv assay. two hundred sixty-five diverse respiratory specimens that previously tested positive for respiratory viruses by in-house assays were selected for comparison with the ftdrp assay. of these, were positive for at least one of the assays available in the ftdrp multiplex; two specimens positive for cov hku for which there was no corresponding ftdrp assay were also tested to assess the specificity of the other ftdrp cov assays (table ) . because of limited available sample volume, only ftdrp multiplex mixes containing the virus-specific assay were performed and virus codetections by the other assays in each multiplex mix were not included in the analysis. all specimens were confirmed positive by in-house singleplex assays on retesting. the ftdrp assay identified all specimens that were positive for hbov ( ), cov nl ( ), inf a ( ), inf b ( ), hmpv ( ), piv ( ) and ev/pev ( ev and pev); > % for piv ( / ) and piv ( / ); % for piv ( / ); % for rv ( / ); % for rsv ( / ); and % for adv ( / ). overall, the ftdrp assay identified correctly % of the archived specimens positive for respiratory viruses by the in-house assays and % of specimens with lower ct values (< ). two specimens positive for cov hku by in-house singleplex assay were negative by the ftdrp cov e, oc and nl assays. the ftdrp adv, rsv and rv assays gave the lowest relative sensitivities with the archived specimens at %, % and %, respectively. with the exception of rv, most discrepancies occurred with samples containing low levels of viral target. for example, most ftdrp adv false-negatives occurred with moderate to high ct value specimens (mean ct . ; range . - . ), but this did not appear to be associated with any particular adv type. a wide range of sequence-confirmed adv types were represented among the archived specimens, including species b (types , and ), c (types , , and untyped) and f (types and ), suggesting that the ftdrp adv assay is inclusive for all recognized human adv types. in contrast, ftdrp rv assay failed to detect rv positive samples with low ct values by the corresponding in-house assay. to further assess the ftdrp rv and ev/pev assays for virus type/strain inclusivity and group exclusivity, archived samples with high rv ( ) or ev ( ) loads and typed by partial vp and/or vp / rt-pcr and sequencing (protocols available from x.l. on request) were retested (table ). the ftdrp rv assay gave negative results with samples and was ≥ ct values higher than the inhouse assay with others, all species b or c rvs. the ftdrp ev/pev assay was also positive with sequence-confirmed rv positive specimens of which was also positive by the in-house ev assay; a second sample also gave an exponential fluorescence amplification curve with the in-house ev assay, but with a > ct value and was therefore classified as ev-negative based on test cutoff criteria. although ev was not detected in of these samples by vp / rt-pcr, and pev was not detected by the in-house assay, the presence of these viruses could not be ruled out definitively. nevertheless, the most probable explanation for these results is that the ftdrp and in-house ev assays cross-react with some rv strains. five sequence-confirmed ev-positive samples were positive by the ftdrp ev/pev assay with no evidence of cross-reactions with the rv and pev assays. three hundred-eight nasopharyngeal aspirates selected from a study of infants and young children hospitalized with acute respiratory infection were tested prospectively by both in-house and ftdrp assays. of these, ( . %) were positive for one or more of the viruses by either the in-house singleplex or ftdrp multiplex assays, with ( . %) positive by the in-house assay and ( %) positive by ftdrp assay alone (table ) . overall, the inhouse and ftdrp assays showed good concordance (k = . , % ci = . - . ) ( table ). as seen with the archived specimens, however, the ftdrp adv, rsv and rv assays gave consistently lower detection rates than the corresponding in-house assays, at . %, . % and . %, respectively, and missed some specimens with high virus loads. coincidently, these three assays are combined in the same reaction mix (mix # ) and had the highest co-detection rate for these viruses by in-house assays at . %; followed by mix # (piv , hbov, hmpv) at . %; mix # (cov e, cov oc , cov nl , ev/pev) at . %; mix # (piv , piv , piv ) at . %; and mix # (inf a, inf b) at . %. simultaneous presence of multiple targets in the same specimen may have led to competitive inhibition of amplification of less abundant targets and may explain some loss of assay sensitivity. the ftdrp hbov assay appeared to be more sensitive than the corresponding in-house assay (lu et al., ) with specimens containing low levels of hbov. to further investigate this finding, limited sequencing studies were performed using a newly developed semi-nested pcr assay specific for the hbov ns gene that amplifies all recognized hbov types (protocol available from x.l. upon request). of the specimens positive for hbov by both inhouse and ftdrp assays, of (mean in-house ct . ; range . - . ) were successfully sequenced (all hbov type ). in contrast, only of in-house assay positive (mean ct . ; range ct . - . )/ftdrp negative and none of the ftdrp positive (mean ct . ; range ct . - . )/in-house negative specimens could be confirmed by ns pcr and sequencing. failure to resolve these discrepancies may be due to (i) a higher sensitivity of the ftdrp assay with specimens containing low levels of hbov dna, possibly attributable to the larger volume of tna extract used in the ftdrp assay ( l vs. l), (ii) failure of both assays to detect some variant hbov strains and/or (iii) non-specific amplification or amplicon contamination in these samples. the ftdrp ev/pev assay also appeared to be more sensitive and specific than the corresponding in-house ev assay with some specimens. of specimens positive by the ftdrp ev/pev assay (mean ct . ; range . - . ), and negative by in-house ev and pev assays, had recoverable vp and/or vp / sequences representing different evs (echovirus , , ; enterovirus ; poliovirus ; coxsackievirus a , b , b ); , that were also positive by in-house and ftdrp rv assays (ct < ), had sequence-confirmed species a rv of which also had type-indeterminate ev sequences present; gave a fluorescence amplification curve with the in-house pev assay, but with a ct value > and therefore was classified as pev negative; and could not be sequenced. of samples positive by the in-house ev assay and negative by the ftdrp ev/pev assay, all were strongly positive for rv (ct < ) by both in-house and ftdrp rv assays and were confirmed positive for rv species a or c by vp and/or vp / sequences. all specimens positive by the in-house table comparison of ftdrp and in-house assays with prospectively tested respiratory specimens. in-house + ftdrp + in-house diagnosis of ari in both clinical care and public health settings has greatly advanced in recent years with the increased availability of rapid, sensitive and specific molecular tests for the simultaneous detection of multiple respiratory pathogens. some commercial assays in particular that have received fda (k) clearance have made substantial inroads into the diagnostic laboratory (rand et al., ) . however, these assays are often costly, require dedicated laboratory equipment, use highly multiplexed reactions where individual assay performance may be compromised, and can be difficult to modify quickly in response to the emergence of new medically important virus strains, as occurred during the h n influenza pandemic. the commercial multiplex ftdrp real-time rt-pcr assay addresses some of these limitations by offering a complete kit with moderate throughput for detection of respiratory viruses that could be easily integrated into the workflow of laboratories using conventional real-time pcr platforms. the ftdrp assay setup and runtime requires approximately . h for samples and controls (assay reagents are aliquoted in sample test units), excluding sample extraction, and with a kit list price of $ . /sample (pcr enzyme kit costs not included). by combining assays into multiplex reaction mixes, individual mixes could more easily modified if needed without impacting the other mixes and could allow for more efficient targeted testing based on epidemiologic findings. in this study, the ftdrp multiplex assay was compared with inhouse singleplex assays corresponding to each of the test viruses. overall, the ftdrp and in-house assays performed comparably for most viruses tested, particularly when the virus was abundant in the sample (low ct values). with exceptions noted below, most discordant results were seen with samples containing lower concentrations of virus (high ct values), suggesting that differences in assay sensitivity near their detection limits was responsible for these discrepancies rather than failure of primer/probe hybridization due to critical target sequence mismatches. ftdrp assays for rsv, rv and adv in particular showed lower relative sensitivities than the corresponding in-house assays with some clinical specimens. the ftdrp rv assay showed clear evidence of dropouts with some rv strains (see further discussion below), and some prospectively tested specimens were negative for rsv and adv, even when the viruses were abundant. it is notable that these three ftdrp assays are combined in the same reaction mix and these three viruses showed the highest co-detection rates by singleplex in-house in these specimens. it is possible that competing amplification reactions in some specimens containing multiple virus targets may have reduced the sensitivity of some assays for low abundant targets. this may have had a more noticeable impact on detection of adv, where a disproportionate number of adv positive specimens had lower virus loads. this would be expected in a population comprised of infants and young children where persistent low level adv shedding is common. development of real-time rt-pcr assays that can detect all rv and ev strains and distinguish between both groups is challenging due to the extensive sequence diversity within each group and sequence similarity between some ev and rv strains. these data confirmed previous experience with the in-house ev and rv assays: both assays cross-react with some rv and ev strains, particularly if present in high copy number (lu et al., ; oberste et al., ) . although this complicated efforts to evaluate the ftdrp ev/pev and rv assays, several conclusions can be drawn from these findings. the ftdrp ev/pev assay appeared to be more sensitive and specific than the in-house assay for detection of some ev strains, including the recently emergent ev (cdc, ), although cross-reactions with some rv strains identified in the archived sample collection could not be ruled out. of particular concern, the ftdrp rv assay was insensitive with some sequence confirmed rv species b and c strains. this finding suggests that critical primer/probe mismatches with these viruses substantially diminished target amplification and/or probe hybridization. more extensive testing of culture purified rv and ev strains will be necessary to assess the full extent of this deficiency. limited discrepancy testing was conducted and focused primarily on samples with large differences in ct values between the in-house and ftdrp assays. to resolve all discrepant results for all assays would require extensive confirmatory testing with additional molecular tests possessing equal or greater sensitivity than those evaluated here, which was beyond the scope of this study. limited specimen volume prevented additional testing in some cases. the apparent false-positive results by either assay were most likely true positives occurring as the result of the conditions noted above. following completion of this work, fast-track diagnostics introduced a new version of the ftdrp assay (ftd respiratory pathogens ) that expands testing to include new respiratory pathogens and modifies of some of the existing assays to enhance performance (miriam steimer, fast-track diagnostics, personal communication) . given these changes, and the promising potential of the ftdrp multiplex assay for diagnosis of respiratory virus 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taqman real-time pcr and semi-nested vp pcr for detection of enteroviruses in clinical specimens viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp comparison of automated microarray detection with real-time pcr assays for detection of respiratory viruses in specimens obtained from children linkage between the journal and quality control molecular diagnostics (qcmd) funding for this project was awarded by the high priority pandemic and seasonal influenza scientific proposal request initiative. key: cord- -zarq s authors: pulford, david; meyer, hermann; brightwell, gale; damon, inger; kline, richard; ulaeto, david title: amplification refractory mutation system pcr assays for the detection of variola and orthopoxvirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zarq s pcr assays that can identify the presence of variola virus (varv) sequences in an unknown dna sample were developed using principles established for the amplification refractory mutation system (arms). the assay’s specificity utilised unique single nucleotide polymorphisms (snp) identified among orthopoxvirus (opv) orthologs of the vaccinia virus copenhagen strain a l and a r genes. when a variola virus specific primer was used with a consensus primer in an arms assay with different orthopoxvirus genomes, a pcr product was only amplified from variola virus dna. incorporating a second consensus primer into the assay produced a multiplex pcr that provided orthopoxvirus generic and variola-specific products with variola virus dna. we tested two single nucleotide polymorphisms with a panel of variola virus strains, collected over years from countries across the world, and have shown that they provide reliable markers for variola virus identification. the variola virus specific primers did not produce amplicons with either assay format when tested with other orthopoxvirus dna samples. our analysis shows that these two polymorphisms were conserved in variola virus genomes and provide a reliable signature of orthopoxvirus species identification. the world health organisation (who) announced the eradication of variola virus (varv), the causative agent of smallpox in and subsequently recommended that global vaccination should cease (fenner et al., ) . reference stocks of variola virus are currently maintained in who licensed repositories at the centers for disease control & prevention (cdc), atlanta and vector laboratories, novosibirsk. there is no information on possible unlicensed stocks that may be held elsewhere in the world, however the deliberate releases of a virulent anthrax strain in the usa in has highlighted the hazards that unlicensed stocks may represent. today the majority of children and adults are not vaccinated against smallpox, and the consequences of a re-emergence of variola virus, by whatever means, would be far reaching without effective public health interventions (gani and leach, ; meltzer et al., ) . the clinical presentation of ordinary, haemorrhagic and flat forms of smallpox have historically been confused with monkeypox, chickenpox, meningococcal and other diseases that produce generalized skin lesions (henderson et al., ; breman and henderson, ) . laboratory diagnosis of smallpox can take several days if virus culturing is performed requiring biosafety level iv laboratories. a simple, reliable and sensitive diagnostic assay would offer improvement for effective medical and public health countermeasures in the event of a release. the first genetic techniques employed for specific identification of variola virus utilised restriction fragment length polymorphisms (rflp) of viral genomic dna (mackett and archard, ; esposito and knight, ; dumbell et al., ) . this approach has been used extensively to differentiate between other orthopoxvirus (opv) species including vaccinia, cowpox, camelpox, monkeypox, mousepox or ectromelia viruses and for other members of the genus. rflp of genomic dna requires lengthy virus culture to generate suitable quantities of high quality dna. today, pcr based methods offer considerable improvement in sensitivity and specificity for diagnosis and the subsequent sequencing of a large number of orthopoxvirus genomes (goebel et al., ; smith et al., ; massung et al., ; shchelkunov, ; antoine et al., ; shchelkunov et al., shchelkunov et al., , shchelkunov et al., , gubser and smith, ; afonso et al., ) , has significantly increased the potential for developing new genetic based assays. rflp analysis of pcr products has been widely adopted for orthopoxvirus species identification. rflp of amplified a-type inclusion body protein gene sequences have been employed for differentiation of orthopoxvirus species (meyer et al., neubauer et al., neubauer et al., , . a collection of assays have been developed around genetic studies of the haemagglutinin (ha) protein, another orthopoxvirus non-essential gene (ropp et al., ) . rflp examination of the cytokine response modifier b gene has also demonstrated that polymorphisms can be exploited by restriction endonuclease digestion (loparev et al., ). an extensive rflp survey of strains has also been performed on a set of kb pair amplicons that represent the entire virus genome (leduc et al., ) . recently, lightcycler pcr of the ha gene was employed to amplify products from members of the genus, and could differentiate from other orthopoxvirus species on the basis of melt curve analysis (espy et al., ) . however, only a very limited number of strains were investigated in this study. rapid assays based on fluorogenic taqman pcr of the ha gene have demonstrated the utility of single nucleotide polymorphisms (snp) for orthopoxvirus identification (ibrahim et al., (ibrahim et al., , sofi ibrahim et al., ) . rapid fluorogenic assays offer benefits for rapid diagnosis but require expensive equipment and reagents. a variola-specific pcr assay was described that could differentiate between variola major and alastrim minor isolates based on the size of the amplified product . however, a recent evaluation of this pcr revealed that a subset of cowpox viruses also produced amplicons of the same size described for variola minor strains . consequently, more sequence information on variola, vaccinia, cowpox, camelpox and monkeypox virus isolates is required to demonstrate the existence of species-specific sequences. studies of orthopoxvirus species to date have shown that unique genes are very rare, (shchelkunov et al., (shchelkunov et al., , (shchelkunov et al., , gubser and smith, ) but species-specific snps are frequently observed in most genes. we have sequenced several genes from a large collection of orthopoxviruses to identify virus specific polymorphisms and have observed that snps are usually the only reliable genetic elements for virus identification (pulford et al., ) . frequently no restricticion endonucleases exist that can exploit these unique markers by rflp. to test this hypothesis, we constructed pcr assays based upon the amplification refractory mutation system (arms) (newton et al., ) which is a sensitive technique for interrogating single base pair differences between dna templates. arms has been used to detect a number of different variants of the hepatitis b virus (gramegna et al., ; liang et al., ) as well as identify genetic profiles of virulent, attenuated or vaccine strains of transmissible gastroenteritis virus and porcine respiratory coronavirus (lai et al., ) . using a modification of arms we have developed novel gel based single-tube assays which not only detect old world orthopoxviruses, but are also specific for variola virus. these multiplex assays employ three primers; two consensus primers generate an amplicon diagnostic of an old world orthopoxvirus and the third primer simultaneously binds to a variola-specific polymorphism and initiates extension of a shorter pcr product to detect the presence of variola virus. examination of amplified pcr products by agarose gel electrophoresis allows the distinction of one (orthopoxvirus present) or two (variola virus detected) pcr products. we show here the examination of these assays with dna samples from an extensive panel including variola and other orthopoxvirus isolates. all variola virus dna was prepared from strains kept at the who repository at the cdc, atlanta, usa. the panel included dna from isolates obtained from five continents from between and (table ) . variola virus isolates were grown on bsc cells whereas other orthopoxviruses (table ) were grown on ma cells (pulford et al., ) . archived clinical tissue samples were recovered from liquid nitrogen. all chemicals were purchased from sigma aldrich unless otherwise stated. assays were initially developed using orthopoxvirus dna prepared from partially purified virus. ma cells infected with orthopoxviruses were dounce homogenised, centrifuged for min at ×g and the supernatant was layered onto ml of % (w/v) sucrose and centrifuged at , × g. the pellet was resuspended in l mm tris ph . , mm edta containing . g proteinase k (boehringer mannhiem), mm nacl, % sds and % -mercaptoethanol and incubated at • c for min. the sample was then phenol:chloroform extracted and the aqueous phase collected, l m nacl was added and the genomic dna was precipitated by addition of . vol. % ethanol and centrifugation for min. the dna pellets were washed with % ethanol, air dried, resuspended in l distilled water and stored at + • c. all variola virus dna samples prepared by the cdc were obtained from . ml of infected cell suspension. samples were heat inactivated at • c overnight and the samples were checked for sterility in tissue culture before processing with the aquapure genomic lysis kit (biorad) according to manufacturer's instructions. dna prepared from clinical specimens was performed using the aquapure genomic lysis kit for tissues according to manufacturer's instructions. final dna pellets were resuspended in up to l of dna hydration buffer and incubated overnight at room temperature before storage at • c. the variola virus specific primers were designed after compiling sequences for orthologs of the vaccinia virus copenhagen a l and a r genes (pulford et al., ) . primers were designed to interrogate a specific polymorphism and specificity was increased by incorporating an adjacent mis-match base (fig. c -aagattattgttgcctcctttgac- antisense a l s -tgtttctggaggaggcĀ gḠ g- antisense a r c -tcttatcacagtgaccgtagttgc- sense a r c -gtaatgaacggatttgacttgctac- antisense a r s -tttgttcattacaatcattatttattaggc - antisense control templates (ct) a l ct -tgtttctggaggaggcĀ aḠ tttaaattcggact- a r ct -tttgttcattacaatcattatttattagcc cgcgtgcttccag- design at primer -end primer types include orthopoxvirus consensus (c) and variola virus specific (s) oligonucleotides. variola virus specific polymorphisms are shown underlined and mismatch bases introduced into each primer sequence are shown in lower case. et al., ) . a third orthopoxvirus primer upstream of the variola-specific primer was included to generate a multiplex assay (fig. ) . the orthopoxvirus generic product in both multiplex assays is larger than the variola virus specific amplicon. high quality hplc purified primer sets were used for multiplex and arms assays (cruachem). primers were designed with oligo . software to have matching melting temperatures with minimum secondary structure and primer-dimer character. ten micromoles working stocks of each primer were prepared and were added in varying ratios for optimal multiplex assay performance. positive control templates (ct) were generated from vaccinia virus ihd-j dna by pcr using synthetic oligonucleotide primers that contained variola virus specific polymorphisms (table , underlined). the ct primers were combined with the consensus primer in a pcr and the products were purified using a qiaquick column and diluted / , before they were used as control samples in pcr (see fig. ). all pcr reagents were purchased from roche. master mixes of reagents were prepared and l volumes were dispensed with l of sample dna for pcr. each a l pcr assay included nm of generic primers and nm of variola-specific primer, whereas a r pcr assays used nm of generic primer and nm of variola-specific primer. all pcr assays used nmol of dntps (roche), mm mgcl and . u/l taq dna polymerase (roche). variola virus pcr assays contained between . and . ng of infected cell culture dna per reaction. camelpox virus cp , ( ) cowpox virus brighton, ( ) cowpox virus ep , ( ) cowpox virus norway, ( ) ectromelia virus mp , ( ) monkeypox virus z , ( ) racoonpox virus vr , ( ) variola virus synthetic control template, ( ) water and (m) bp ladder (roche). ten microliters of pcr product was loaded on a % agarose gel. all pcr assays were performed with an mj research ptc . assays were optimised for specificity using the mj v thermal gradient block and with a panel of orthopoxvirus dna or synthetic control templates. multiplex and arms assays with variola virus genomic dna were performed at the cdc, atlanta using thermal cycling programs with predicted temperature algorithms. the optimised protocol for both assays was • c min; ( • c s, • c s, • c s) × cycles; ( • c s, • c s, • c s + s/cycle) × cycles; • c min; + • c. to evaluate the specificity of the multiplex pcr we tested the a l and a r multiplex assays with a small panel of orthopoxvirus dna samples (fig. ) . synthetic control templates were used to confirm the functioning of the variola virus specific primer in each multiplex assay (lane ). the shorter variola-specific amplicon was absent from all the orthopoxvirus genome samples (lanes - ) but was produced by the control template (lane ) confirm- ing assay specificity. the consensus primers for the a l and a r genes produced pcr amplicons for all the old world (lanes - ), but not for the new world (lane ) orthopoxviruses. a more extensive survey was then performed to evaluate the reliability of variola virus specific amplification using a larger panel of orthopoxvirus dna samples. these cross-reactivity assays are summarised in table . multiplex and arms assay formats were employed with camelpox, cowpox, ectromelia, monkeypox and vaccinia virus isolates. in all assays performed no variola virus product was generated in the a l or a r arms or multiplex assay formats. however, the a l consensus primers proved unreliable at amplifying a generic orthopoxvirus product with some cowpox virus strains (table ) . to establish the specificity of the variola-specific primer we then performed some arms assays using a small panel of orthopoxvirus genomes including variola genomic dna (fig. ) . the combination of the a l and primers produced a bp product with variola dna (lane ), but no product with any other orthopoxviruses in this experiment (lanes - ). a similar result was obtained with the a r and primers which produced a bp variola-specific product (lane ), but no product with the other orthopoxviruses (lanes - ). the arms is an imperfect method for identifying snps because the absence of an amplified product reveals lit- tle information about the assay performance. therefore, we tested all variola dna samples with the multiplex assay format to report the presence of orthopoxvirus dna and to confirm if the hypothesised variola-specific snps were present. as anticipated, all the variola virus genomes, produced two pcr amplicons, the smaller band represented a variola-specific product and the larger band was an orthopoxvirus generic amplicon (fig. ) . no bands were amplified from mock-infected bsc cell dna in any assay (lane ). a second panel of more variola isolates was also tested with the a l multiplex assays (fig. , a l multiplex b). two pcr products were always amplified from all variola strains tested demonstrating that the polymorphisms exploited by these multiplex assays were conserved. to evaluate the performance of the multiplex for diagnosis we performed assays using dna extracted from lesion or scab samples obtained from smallpox and chickenpox skin biopsies. these samples offered a significant test for the variola virus specific multiplex pcr because the dna was of unknown quality being prepared from archived patient scabs collected during the eradication campaign. seven separate variola virus isolates (fig. , lanes - ) and one chickenpox (lane ) sample prepared from scabs were compared using the a l multiplex assay only. all seven biopsy samples produced two amplicons. no products were amplified from varicella zoster dna (lane ) confirming the suitability of this assay for diagnosis from clinical specimens. this paper describes an easy method for performing fast, reproducible and specific pcr assays with the minimum of equipment or reagents. to achieve this goal we compared orthopoxvirus ortholog sequences of the vaccinia virus copenhagen a l and the a r genes using a panel of african and eurasian viruses. these two genes both code for virus membrane proteins, and although they are present in all orthopoxviruses so far sequenced, they display remarkable sequence heterogeneity (pulford et al., ) . we exploited this diversity to design some new variola virus specific pcr assays. the principles established for the arms pcr were exploited to design an oligonucleotide capable of specifically priming and extending from variola virus snp's. the a l- primer utilised two variola unique polymorphisms in close proximity and introduced a mismatch base a → g to generate further instability at the primer -end (table ) . this primer produced a characteristic bp amplicon with variola virus dna (fig. , lane ) in the arms assay. priming events from orthopoxvirus genomes other than variola virus were inhibited strongly by this cluster of three non-matched bases at the -end of the a l oligonucleotide primer making it highly specific. the a r- primer contained a single variola polymorphism and an adjacent base mismatch c → g to create further instability at the -end. this primer worked specifically with variola dna in an arms assay when combined with the a r- primer, and also worked specifically in a multiplex with all variola viruses tested. the consensus primer pair a r- and a r- faithfully produced a larger ∼ bp amplicon with all orthopoxviruses tested. the quantity of shorter variola virus specific amplicons should normally exceed longer pcr products because they are synthesised more efficiently. however, the incorporation of a mis-match base into the variola-specific primers reduces their binding energy and subsequently the efficiency of product amplification (fig. ) . the a l multiplex also revealed minor ∼ bp bands on gels with samples containing variola virus dna only (fig. ) . the size of this extra band corresponds to the sum of the consensus and specific products together, and may represent a heteroduplex. the production of potential minor heteroduplex complexes was also observed for other multiplex assays we performed (not shown) which may be the result of the repetitive sequence elements identified in the a l orthologs (pulford et al., ) . orthopoxvirus genes frequently contain repetitive sequence elements (massung et al., ) . consequently, the structural characteristics of this sequence might explain the low abundance of variola virus specific products in the a l- multiplex assays. knight et al. ( ) described a variola virus specific pcr assay that could differentiate between variola alastrim minor and major isolates based on the size of the amplified product. a recent evaluation of this published diagnostic method with a large panel of other orthopoxviruses revealed that a subset of cowpox viruses also produce amplicons corresponding exactly with the size described for variola minor strains . we analysed the performance of the arms and multiplex a l and a r assays with the same cowpox virus subset (table , cowpox viruses ep- , opv / , opv / , opv / , opv / and opv / ) and observed that no variola-specific amplicons were produced from these or any other orthopoxvirus dna samples. it is clear that the production of an ever-expanding orthopoxvirus sequence database (http://www.poxvirus.org/ index.html) has given researchers the opportunity to produce many new assays (espy et al., ) , but the real value of any diagnostic assay can only be provided by the quantity and diversity of the samples tested. the arms and multiplex assays described herein were performed and validated with a very large panel of variola virus and orthopoxvirus strains to confirm that the snps probed by these assays are preserved and unique to variola virus genomes. the clinical presentation of a generalised exanthum is not uncommon. smallpox was often confused with a range of common skin conditions, and frequently chickenpox and monkeypox have been difficult to differentiate from smallpox at early times in clinical presentation (fenner et al., ; breman and henderson, ) . the assays described here, were able to differentiate between dna from smallpox, monkeypox and chickenpox and have demonstrated their reliability and specificity with a large collection of orthopoxvirus samples, including clinical specimens. the application of these multiplex and arms pcr assays offer a simple and inexpensive alternative to the sequencing of amplicons generated from a l or a r orthologs for orthopoxvirus identification. the genome of camelpox virus the complete genomic sequence of the modified vaccinia ankara strain: comparison with other orthopoxviruses diagnosis and management of smallpox a variant of variola virus, characterized by changes in polypeptide and endonuclease profiles orthopoxvirus dna: a comparison of restriction profiles and maps detection of smallpox virus dna by lightcycler pcr smallpox and its eradication, who development, multiplexing, and application of arms tests for common mutations in the cftr gene transmission potential of smallpox in contemporary populations the complete dna sequence of vaccinia virus detection of the hepatitis b virus major pre-core mutation by the amplification refractory mutation system technique the sequence of camelpox virus shows it is most closely related to variola virus, the cause of smallpox smallpox as a biological weapon: medical and public health management the potential of nucelase pcr for detecting a single-base polymorphism in orthopoxvirus real-time microchip pcr for detecting single-base differences in viral and human dna polymerase chain reaction identification of smallpox virus the use of arms pcr and rflp analysis in identifying genetic profiles of virulent, attenuated or vaccine strains of tgev and prcv smallpox research activities: u.s. interagency collaboration presence of hepatitis b and c viral genomes in us blood donors as detected by polymerase chain reaction amplification detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmb gene region conservation and variation in orthopoxvirus genome structure analysis of the complete genome of smallpox variola major virus strain bangladesh- terminal region sequence variations in variola virus dna modeling potential responses to smallpox as a bioterrorist weapon amplification of 'variola virus-specific' sequences in german cowpox virus isolates sequence alterations within and downstream of the a-type inclusion protein genes allow differentiation of orthopoxvirus species by polymerase chain reaction gene for a-type inclusion body protein is useful for a polymerase chain reaction assay to differentiate orthopoxviruses specific detection of mousepox virus by polymerase chain reaction specific detection of monkeypox virus by polymerase chain reaction analysis of any point mutation in dna. the amplification refractory mutation system (arms) orthologs of the vaccinia a l and a r virion membrane protein genes display diversity in species of the genus orthopoxvirus pcr strategy for identification and differentiation of small pox and other orthopoxviruses functional organization of variola major and vaccinia virus genomes the genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact orfs for immunomodulatory and host range proteins alastrim smallpox variola minor virus genome dna sequences analysis of the monkeypox virus genome nucleotide sequence of kbp of vaccinia virus strain wr from near the right inverted terminal repeat real-time pcr assay to detect smallpox virus the authors wish to thank gudrun zoeller of the institute of microbiology, munich, for her technical assistance. the authors would also like to acknowledge the help of dr. yu li at the cdc for his advice. key: cord- - c ymc authors: huang, yu-liang; pang, victor fei; pan, chu-hsiang; chen, tsu-han; jong, ming-hwa; huang, tien-shine; jeng, chian-ren title: development of a reverse transcription multiplex real-time pcr for the detection and genotyping of classical swine fever virus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: c ymc a reverse transcription multiplex real-time pcr (rt-mrt-pcr) was developed for rapid detection and genotyping of classical swine fever virus (csfv). the universal primers and specific taqman probes for each of the three genotypes, genotypes , , and , were designed within the ′-utr of the csfv. non-csfv swine virus and clinical samples from specific pathogen-free (spf) pigs were both demonstrated to be csfv-negative by rt-mrt-pcr. the diagnostic sensitivity of rt-mrt-pcr was determined to be viral copy/μl for each genotype of standard plasmid. for the analytical sensitivity experiment, samples of csfv genotype strains and samples from csfv outbreak farms were all detected as csfv-positive by rt-mrt-pcr, and the genotype results were consistent with the results of sequencing from a previous study. the intra-assay and inter-assay variations of rt-mrt-pcr were below % in all experiments. the sensitivity of rt-mrt-pcr was the same as the reverse transcription nested pcr (rt-npcr) and higher than reverse transcription pcr (rt-pcr) and viral isolation from clinical samples. this assay was used further to evaluate the duration of viremia of wild-type csfv in vaccinated exposed pigs. the results indicated that pigs vaccinated with the e subunit vaccine had longer viremia than pigs given the c-strain vaccine, which is compatible with the findings of previous studies. thus, the new rt-mrt-pcr is a rapid, reproducible, sensitive, and specific genotyping tool for csfv detection. classical swine fever (csf) is a highly contagious and multisystemic hemorrhagic disease that results in economic losses in the swine industry worldwide and is a notifiable disease to the office international des epizooties, according to the terrestrial animal health code (oie, ) . the course of disease can be acute, subacute, chronic, or late onset, but csf can also remain unnoticed in infected pigs (van oirschot, ) . classical swine fever virus (csfv), which is an enveloped, positive-sense, single-stranded rna virus, is classified in the genus pestivirus within the family flaviviridae, which also includes bovine viral diarrhea virus (bvdv) and border disease virus (bdv). the genus pestivirus, including csfv, bvdv, and bdv, can be differentiated by the sequences of their -utr fragments (hofmann et al., ) . at the genetic level, csfvs can be divided into genotypes , , and , based on the partial sequences of the e and ns b genes. each genotype can be classified further into three or four sub-genotypes, referred to as . , . , and . ; . , . , and . ; and . , . , . , and . , respectively (paton et al., ) . detailed genetic information about csfv field isolates can be used to form an important database for molecular epidemiology of csfv, which allows for a greater understanding of the origin of csfv as well as for improvements in eradication programs against csf (vilcek and nettleton, ) . currently, csf is still spreading gradually and evolving worldwide. in europe, all csfv strains were genotype prior to the s, but the various sub-genotype strains . , . , and . were isolated from different european countries during the s and s (paton et al., ; stegeman et al., ; biagetti et al., ) . csf is still endemic currently in various european countries. in latin america, only strains of genotype have been reported thus far, and sub-genotypes . , . , and . are circulating currently within that region (paton et al., ; pereda et al., ; sabogal et al., ) . in asia, csf epidemics are also fairly ubiquitous and strains of genotypes , , and have been isolated in different asian countries (paton et al., ; blacksell et al., ) . in taiwan, all csfv strains were found to be subgenotype . prior to . however, genotype strains of csfv, identified as sub-genotype . or . , have been isolated since and have replaced gradually the sub-genotype . strain (pan et al., ) . currently, genotyping of isolated csfv is based on the amplification of the e and ns b sequences by reverse transcription pcr (rt-pcr), which requires nucleotide sequencing for further confirmation and takes approximately - days for completion. from the perspective of csf control, a more rapid and accurate method for viral detection and genotyping is essential for the emergency responses to the disease outbreak. reverse transcription multiplex real-time pcr (rt-mrt-pcr) has been successfully demonstrated for simultaneous detection of many pathogens, including bvdv (baxi et al., ) , dengue virus (lai et al., ) , influenza virus (payungpom et al., ) , yellow fever virus, japanese encephalitis virus, west nile virus, and st. louis encephalitis virus (chao et al., ) . the aim of this study was to develop rt-mrt-pcr for the detection and genotyping of csfv. the porcine kidney cell line (pk- ) was maintained in minimal eagle medium (mem) with % heat inactivated fetal calf serum, units/ml penicillin g, g/ml streptomycin, and . g/ml amphotericin b. the cells were used to isolate csfv. classical swine fever virus, bovine viral diarrhea virus types and , japanese b encephalitis virus, swine influenza virus, porcine reproductive and respiratory syndrome virus, porcine respiratory coronavirus, transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine astrovirus, porcine teschovirus type , porcine enterovirus types and , foot-and-mouth disease virus type o, reovirus, pseudorabies virus, and porcine circovirus types and were used in this study. the classical swine fever virus variants used included strains of c-strain, non-c-strain strains (ald, gp − , a , and cap strain) of genotype , the q - strain of genotype , and the - strain of genotype . field samples were collected from three different sources: ( ) samples, including buffy coats and tissue extracts, were collected from a specific pathogen-free (spf) farm which were free of hog cholera virus, pseudorabies virus, actinobacillus, and toxoplasma gondii, ( ) tissue extracts, including samples of genotype and samples of genotype , were collected from farms that had a csfv outbreak during the period - , and ( ) tissue extracts from different vaccinated farms that had been submitted to the animal heath research institute for routine detection of csfv by local animal disease control centers. trizol ® reagent (invitrogen, carlsbad, ca, usa) was used to extract total rna from plasma, buffy coat, tissue extracts, and pk- cells inoculated with tissue extracts. to do this, l of each sample was first mixed with ml of the trizol ® reagent and incubated for min at room temperature. then . ml of chloroform was added and the samples were incubated for min at room temperature. all samples were centrifuged at , × g for min at • c. the supernatants were transferred to fresh tubes and . ml of isopropyl alcohol was added to each. samples were then incubated at room temperature for min. centrifugation was performed again at , × g for min at • c. the supernatants were removed and the rna pellet from each sample was washed with % ethanol. finally, each rna pellet was air dried and resuspended with l of depc-treated water. the primers and probes used for the study were designed based on the csfv sequences published by the national center for biotechnology information, and sequence alignment was performed using dnastar version (dnastar, madison, wi, usa). universal csfv primers, two pairs (cp , cp , cp f, and cp r), and specific taqman probes for genotypes , , and (g , g , and g ) were designed based on the -utr of csfv (table ). the -ends of g , g , and g were labeled with -hexachloro-fluorescein-ce phosphoramidite (hex), cyanine (cy ), and -carboxy-flourescein (fam), respectively. the -ends of g , g , and g were all labeled with , -bis-( -butyloctyloxy)p-quaterphenyl (bbq). the primers cp and cp were used in both rt-pcr and rt-mrt-pcr combined with taqman probes g , g , and g . the primers, cp f and cp r, were used in reverse transcription nested pcr (rt-npcr). the extracted rna from each sample was transcribed reversely and amplified in a one-tube reaction using rt-pcr. the rt-pcr reaction was carried out in a final volume of l containing l of extracted rna, × dna polymerase buffer, units of recombinant rnase inhibitor (promega, madison, wi, usa), units of amv reverse transcriptase (promega, madison, wi, usa), unit of dna polymerase (jmr, uk), . m of deoxyntp mixture, and . m of each primer (cp and cp ). the reaction was carried out in a thermal cycler (applied biosystems, foster city, ca, usa). the reaction condition for rt-pcr included samples incubation at • c for min followed by • c for min. this incubation was then followed by cycles of denaturation ( • c for s), annealing ( • c for s), and extension ( • c for s). a final extension at • c for min was performed before storing the sample at • c. for the subsequent nested pcr, l of rt-pcr product was used as the template. the reaction condition was similar to that of the rt-pcr described previously, and the primer pairs cp f and cp r were used. . . two step rt-mrt-pcr . . . cdna synthesis the extracted rna from each sample was reverse transcribed into cdna. the reverse transcription was carried out in a volume of l containing l of extracted rna, × amv reaction buffer (promega, madison, wi, usa), units of recombinant rnase inhibitor (promega, madison, wi, usa), units of amv reverse transcriptase (promega, madison, wi, usa), . m of deoxyntp mixture, and . m of cp primer. the reaction was carried out in the thermal cycler (applied biosystems, foster city, ca, usa). the reaction conditions involved incubation at • c for min followed by • c for min. finally, the reaction was held at • c and the products were used as templates for multiplex real-time pcr. the multiplex real-time pcr was carried out in the iq tm multicolor real-time pcr detection system (bio-rad, hercules, ca, usa) in a reaction volume of l, containing l of cdna, × powermix (bio-rad, hercules, ca, usa), . mm mgcl , . m g , g , and g each of the taqman probe, and . m each of primer cp and cp . the reaction condition involved incubation at • c for min followed by cycles of denaturation ( • c for s), and annealing/extension ( • c for min). all samples were simultaneously detected for hex, cy , and fam fluorescence by the monitoring system. the threshold level was fixed at , , and rfu in hex, cy , and fam fluorescence, respectively. samples were recognized as csfv-negative when the threshold cycle rose above . the rt-pcr products of c-strain (genotype ), q - (genotype ), and - a (genotype ) were cloned using the pgem-t easy vector system (promega, madison, wi, usa) and were propagated in e. coli, jm , according to the manufacturer's instructions. plasmids were purified by the qiaamp plasmid maxi kit (qiagen, valencia, ca, usa) and were quantified by measuring od with spectrophotometer du ® (beckman, palo alto, ca, usa). the viral copy of the extracted plasmids was calculated using the formula: x (g/l) × × plasmid length (bp) × = y viral copy/l each plasmid was optimized to × viral copy/l. following optimization of concentration, the standard plasmid was equally mixed with plasmids of genotypes , , and . the standard plasmid was then used to develop standard curves and evaluate the diagnostic sensitivity of the rt-mrt-pcr. the non-csfv swine viruses were used to determine the analytical specificity of the primers and the taqman probes used in the experiment. to evaluate the diagnostic specificity, samples from the spf farm were used. a total of samples, including strains of genotype , strains of genotype , and strains of genotype , were examined by rt-mrt-pcr for the presence of csfv and their genotypes. for genotype , samples comprising c-strain viruses and ald, gp − , cap, a strains. the samples of genotypes and were collected from farms with csfv outbreaks. the genotypes of all samples used in this experiment had also been identified by sequencing and described in a previous study (pan et al., ) . -fold serial dilutions of the standard plasmid from to viral copy/l of each genotype were tested to determine the detection limits of rt-mrt-pcr. the serially diluted plasmid was also used to establish a standard curve for genotypes , , and by plotting the threshold cycle and the viral copy logarithm. the standard plasmid, including , , or viral copy/l in each genotype, was used to evaluate the inter-assay and intra-assay reproducibility of rt-mrt-pcr. each concentration was detected in triplicate. the threshold cycle of each concentration was obtained and calculated. c-strain (genotype ), q - (genotype ), and - (genotype ) strains were -fold serially diluted and their viral copies quantified by rt-mrt-pcr. linear regression was used to calculate the linear correlations (r ) between tcid and the viral copy logarithm. -fold serially diluted samples of c-strain, q - , and - strains were determined to be csfv-positive by viral isolation, rt-pcr, rt-npcr, and rt-mrt-pcr. a total of clinical samples were used to detect csfv by viral isolation, rt-pcr, rt-npcr, and rt-mrt-pcr. these samples included samples collected from spf pigs, samples from csfv outbreak farms, and samples from local animal disease control centers. the agreement among tests was based on kappa statistics (thrusfield, ) . the agreement was classified by kappa statistic values into five groups: almost perfect ( . or higher), substantial ( . - . ), moderate ( . - . ), fair ( . - . ), slight ( . - . ), and poor ( ). in order to use the rt-mrt-pcr to study the duration of viremia of wild-type csfv in vaccinated exposed pigs, plasma samples were collected from pigs as described below. biocontainment facilities at the animal health research institute, council of agriculture, taiwan, were used to house spf pigs. these pigs were randomly divided into c-strain, e subunit, and control groups with , , and pigs, respectively. the pigs in the c-strain group were vaccinated once at weeks of age with one dose of attenuated lapinized c-strain vaccine for csfv produced by the division of biologics at animal heath research institute. the pigs in the e subunit group were vaccinated twice with one dose of e subunit vaccine (bayer, munich, germany), at and weeks of age. the control pigs were not vaccinated. at weeks of age, all pigs were placed in the same room with a csfv-infected pig that had been infected with × tcid of the ald strain of csfv at days prior to contact with the other pigs. plasma samples were collected from all pigs at , , , , , , , , and days post-contact (dpc). the standard plasmids were detected simultaneously using the hex, cy , and fam fluorescence by rt-mrt-pcr, and the fluorescence levels were all higher than the threshold level. the level of the hex, cy , and fam fluorescence of the non-csfv swine viruses and the samples from spf farm were all below the threshold level (fig. ) . a total of samples were detected as csfv-positive by rt-mrt-pcr. the genotyping results of the rt-mrt-pcr were all consistent with the results of the genotypes verified by sequencing of the samples (table ) . the detection limit of rt-mrt-pcr was determined to be viral copy/l for each genotype of the standard plasmid. the threshold cycles for standard plasmid, including copy number/l in each genotype, were . , . , and . in the genotypes , , and samples, respectively (fig. ) . the standard curves of rt-mrt-pcr analytical sensitivity of rt-mrt-pcr. number were established from to viral copy/l. the linear correlations (r ) between the threshold cycle and the viral copy logarithm were . , . , and . for genotypes , , and , respectively (fig. ) . the coefficient of variation (cv) of genotype for the threshold cycle values ranged between . % and . % for the intra-assay and . % and . % for inter-assay (table ). the cvs for both intra-assay and inter-assay of genotype ranged between . % and . % and between . % and . %, respectively (table ). the cvs for intraassay and inter-assay of genotype ranged between . % and . % and between . % and . %, respectively (table ) . to investigate the correlation between tcid and the viral copy of rt-mrt-pcr, csfv was diluted serially -fold and a each concentration of standard plasmid contained plasmids of genotypes , , and and simultaneously detected three genotypes by rt-mrt-pcr. each standard plasmid was detected in triplicate by rt-mrt-pcr in the intra-assay and inter-assay. was assayed by rt-mrt-pcr. the r s of c-strain (genotype ), q - (genotype ), and - (genotype ) strains between tcid and the viral copy were . , . , . , respectively (fig. ) . to compare the sensitivity of the different methods, the c-strain, q - , and - strains of csfv were diluted serially -fold and were examined by viral isolation, rt-pcr, rt-npcr, and rt-mrt-pcr. the sensitivities of viral isolation, rt-npcr, and rt-mrt-pcr were all found to range between and . tcid /ml, which was a -fold greater sensitivity than rt-pcr (table ) . agreement of viral isolation, rt-pcr, rt-npcr, and rt-mrt-pcr, of total clinical samples were detected as csfv-positive, and of these samples were csfv-negative by viral isolation. all csfv-positive samples detected by viral isolation were also shown to be csfv-positive by rt-mrt-pcr, rt-pcr, and rt-npcr. among csfv strains were -fold serially diluted and csfv was detected from viral isolation, rt-pcr, rt-npcr, and rt-mrt-pcr. the tcid of csfv strains was calculated from result of viral isolation. the agreement among viral isolation, rt-pcr, rt-npcr, and rt-mrt-pcr a . a a total of clinical samples was detected for the presence of csfv and the results included and used to evaluate agreement among viral isolation, rt-pcr, rt-npcr, and rt-mrt-pcr. agreement among tests was based on kappa statistics (thrusfield, ) . detection of wild-type csfv in plasma of vaccinated exposed pigs by rt-mrt-pcr a . the pigs in the c-strain group were vaccinated once at weeks of age with one dose of attenuated lapinized c-strain vaccine of csfv. the pigs in the e subunit group were vaccinated twice with one dose of e subunit vaccine, at and weeks of age. the control pigs were not vaccinated. at weeks, all pigs were placed in the same room with the wild-type csfv-infected pig. plasma samples were collected from all pigs at , , , , , , , , and days post-contact (dpc). b the unit of concentration of csfv in plasma determined by rt-mrt-pcr was log viral copy/ml. c the pig was death at dpc. the csfv-negative samples detected by viral isolation, , , and were shown to be csfv-positive by rt-mrt-pcr, rt-pcr, and rt-npcr, respectively. the kappa values were . for viral isolation and rt-mrt-pcr, . for viral isolation and rt-pcr, and . for viral isolation and rt-npcr (table ). the sensitivity values of the three assays were all % or equal to viral isolation. regarding the viral isolation with the three assays, the specificity values were . %, . % and . % in rt-mrt-pcr, rt-pcr, and rt-npcr, respectively. the plasma samples of vaccinated exposed pigs were collected and assayed by rt-mrt-pcr (table ). in the c-strain group, wildtype csfv was detected from the plasma of vaccinated exposed pigs only at the time point of days post-contact (dpc). two of the four pigs in this group revealed . and . log viral copy number/ml in the plasma samples. in the e subunit group, wild-type csfv was detected between and days post-contact (dpc). all four pigs in this group showed serum viral loads ranging from . to . log copy number/ml. in the non-vaccinated control group, csfv could be detected from to dpc, and the serum viral loads ranged from . to log viral copy number/ml. the rt-mrt-pcr developed in the present study is a rapid and highly specific technique for the detection and the genotyping of csfv. rt-mrt-pcr shows no inter-genotypic cross-reactivity among different csfv strains or with other swine viral pathogens ( fig. and table ). in addition, this method is more rapid for the identification of genotypes than other csfv-detecting assays, such as rt-pcr, rt-npcr, or real-time pcr, which require nucleic acid sequencing of the amplified products for further genotype identification (greiser-wilke et al., ) . in addition, the rt-mrt-pcr can distinguish simultaneously different genotypes of csfv and can be used further for epidemiological studies of csfv infection, which cannot be achieved by viral isolation, rt-pcr, and rt-npcr. rt-mrt-pcr has the ability to identify the genotypes of clinical samples much more quickly than a sequencing assay. this method was used for a retrospective study of the epidemiology of csfv that occurred in taiwan from to , and all results were consistent with the sequencing analysis (table ) . however, there is difficulty in utilizing rt-mrt-pcr in areas that are endemic for wild-type csfv genotype virus and are using currently the attenuated csfv genotype vaccine, such as c-strain, to control csfv. in this case, the g probe is not able to differentiate between the wild-type and attenuated csfv genotype vaccine virus using the rt-mrt-pcr products, except when further electrophoresis of the rt-mrt-pcr products is applied (pan et al., ) . the difference in product size is due to the -end sequences of the attenuated csfv genotype vaccine strains, which contain a t-rich insertion that the wild-type csfv lacks (pan et al., ) . the sensitivity of the csfv detection by rt-mrt-pcr could be equivalent to that of detection using real-time pcr. it has been widely considered that real-time pcr is one of the most sensitive methods for csfv detection, more sensitive than the methods of viral isolation, antigen elisa, rt-pcr, and rt-npcr (hoffmann et al., ; haegeman et al., ; depner et al., ) . it was shown that the sensitivity of rt-mrt-pcr is comparable to those of rt-npcr and viral isolation, and higher than rt-pcr for the samples of csfv diluted serially (table ). however, it was also found that among the csfv-negative samples of clinical samples tested by viral isolation, - samples were detected as csfv-positive by rt-pcr, rt-npcr, and rt-mrt-pcr (table ). the false-positive samples were repeated three times by rt-pcr, rt-npcr, and rt-mrt-pcr and the sequenced pcr products were all highly homologous to the attenuated lapinized c-strain (data not shown). the results indicated that rt-mrt-pcr, rt-pcr, and rt-npcr have higher sensitivity than viral isolation for clinical samples. the discrepancy in the sensitivity of those methods between samples of csfv diluted serially and clinical samples could be due to the different detection targets of each method. the detection targets for viral isolation are infectious csfv, whereas those for rt-pcr, rt-npcr, and rt-mrt-pcr are all nucleic acids isolated from csfv. the detection of infectious csfv by viral isolation is affected by several factors, including the viral stage, anti-csfv antibody, storage stage of clinical samples, and stage and type of cell lines used, resulting in lower positive results than expected. however, it must be noted that molecular assays cannot replace completely viral isolation. this is due to the fact that the presence of viral genetic materials detected by molecular assays does not represent viral infectivity. for example, the viral genetic material may be isolated from inactive viruses trapped in, for example, the phagocytic cells or immune complexes, in addition to infectious viruses. on the other hand, the sensitivity of the rt-npcr was equivalent to the rt-mrt-pcr; however, the rt-npcr has lower reproducibility than that of the rt-mrt-pcr (data not shown) and requires a longer processing time than rt-mrt-pcr. thus, the rt-mrt-pcr is a superior method for csfv detection as compared to virus isolation, rt-pcr, and rt-npcr. when rt-mrt-pcr was applied for evaluation of viremia caused by the wild-type csfv in vaccinated exposed pigs, a different viremic pattern was demonstrated in preliminary results. these data are in agreement with the shorter duration of viremia in the viral challenged-pigs with c-strain group vaccination, as compared to that with the e subunit group in this rt-mrt-pcr study and previous studies. previous studies have demonstrated that vaccination with the c-strain vaccine could induce complete protection against subsequent csfv challenge (ferrari, ; uttenthal et al., ; van oirschot, ; suradhat et al., ) , in which neither viremia nor viral shedding was observed in vaccinated pigs that had been challenged week after vaccination. however, the pigs vaccinated with c-strain in this study showed a further transient viremia in the plasma at dpc (table ) . it was also found that viremia in e subunit vaccinated pigs was similar to c-strain results in that vaccinated exposed pigs in our experiment had longer durations of viremia than those in other studies. a period of wild-type csfv viremia in pigs treated with the e subunit vaccine was detected by rt-mrt-pcr (table ). this period was longer than the one reported in the study by uttenthal et al. ( ) in which either complete protection or only transient viremia at days post-infection was detected. the differences in the viremic periods of wild-type csfv in this study and previous studies might be due to the different challenge methods used or the different sensitivities of the methods of detection used. the efficacy of the csfv vaccines is correlated with the amount of viral particles contained in each dose of the vaccine. it has been shown that pigs vaccinated with a dose of less than tcid or / dose of c-strain viral particles do not develop complete protection and often have viremia and viral shedding after challenge with virulent csfv (jong et al., (jong et al., , . in taiwan, there are two types of commercial c-strain vaccines, which are prepared either from cell cultures or from visceral organs of rabbits. the viral titer of the cell culture-derived c-strain vaccine can be evaluated and titrated during its preparation; however, such a procedure cannot be performed in the preparation of the c-strain vaccine from the visceral organs of rabbit. according to official regulations, the viral load per dose of cell culture-derived c-strain vaccine must contain at least . of % of the fluorescence assay infected-dose (faid ), but no official regulations have been established for the rabbit visceral organ-derived vaccine. the . faid is equivalent roughly to . viral copy, as determined by the rt-mrt-pcr used in this study (data not shown). the lack of any evaluation systems and official regulations on rabbit visceral organ-derived c-strain vaccine may have certain negative impacts on the quality of this kind of vaccine. this may result in further adverse effects on the csfv prevention program in the field. the rt-mrt-pcr was also used for evaluation of the viral load on all available commercial c-strain vaccines in taiwan, including two cell culture-derived vaccines and six rabbit visceral organ-derived vaccines. these results indicate that none of the commercial c-strain vaccines have met the minimum requirement of the official regulation. the established rt-mrt-pcr could be utilized to evaluate not only the quality of the rabbit visceral organ-derived c-strain vaccine, but it could also be used as an alternative for monitoring the quality of cell culturederived c-strain vaccine. however, since real-time pcr does not differentiate between infective and non-infective viral particles, then if the viral copy number in the vaccines tested, determined by rt-mrt-pcr, meets the official regulation, the assay measuring of viral infectivity, such as viral isolation and titration, should be a supplemental analysis to confirm the potency of the tested vaccines. real-time pcr may be an alternative to viral isolation for the study of the pathogenesis of csfv infection, which includes tissue distribution, viral load, and the routes of viral shedding (kamolsiriprichaiporn et al., ; ophuis et al., ; koenig et al., ) , as this method was able to quantify the copy numbers of csfv in the samples examined. the rt-mrt-pcr is capable of simultaneously detecting and quantifying three genotypes of csfv. thus, this method should be an effective tool for any of the studies on chronological changes in viral prevalence, such as the gradual replacement of the sub-group . strains of csfv with the subgroups . and . strains in taiwan after . in summary, the rt-mrt-pcr is a rapid, reproducible, specific, and sensitive assay for the detection, quantitation, and genotype identification of csfv. additionally, rt-mrt-pcr can also be used in csfv studies in various areas, including epidemiology, pathogenesis, and vaccine quality evaluation. rt-mrt-pcr appears to be more functional than all of the existing assays and may be suitable for routine laboratory diagnosis, both for the detection and the genotyping of csfv. in the future, the two-step rt-mrt-pcr could be improved into a one-step approach in which reactions are performed generally in smaller reaction volumes and a positive control can be included to preclude false negative results. a one-step multiplex real-time rt-pcr for detection and typing of bovine viral diarrhea viruses molecular epidemiology of classical swine fever in italy genetic typing of classical swine fever viruses from lao pdr by analysis of the non-coding region development of multiplex real-time reverse transcriptase pcr assays for detecting eight medically important flaviviruses in mosquitoes evaluation of real-time rt-pcr assay for the routine intra vitam diagnosis of classical swine fever a tissue culture vaccine with lapinized chinese (lc) strain of hog cholera virus (hcv) genetic typing of classical swine fever viruses-a review characterisation of the discrepancy between pcr and virus isolation in relation to classical swine fever virus detection rapid characterization of new pestivirus strains by direct sequencing of pcr-amplified cdna from the noncoding region validation of a real-time rt-pcr assay for sensitive and specific detection of classical swine fever immunoprotection induced by tissue culture-adapted lpc strain of hog cholera virus development of tissue culture hog cholera vaccine a comparison of the pathogenicity of two strains of hog cholera virus. . virological studies detection of classical swine fever vaccine virus in blood and tissue samples of pigs vaccinated either with a conventional c-strain vaccine or a modified live marker vaccine 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 detection and quantitative pathogenesis study of classical swine fever virus using a real time rt-pcr assay world organisation for animal health. classical swine fever phylogenetic analysis of classical swine fever virus in taiwan rapid detection and differentiation of wild-type and three attenuated lapinized vaccine strains of classical swine fever virus by reverse transcription polymerase chain reaction genetic typing of classical swine fever virus single step multiplex real-time rt-pcr for h n influenza a virus detection phylogenetic analysis of classical swine fever virus (csfv) field isolates from outbreaks in south and central america phylogenetic analysis of recent isolates of classical swine fever virus from colombia the - epidemic of classical swine fever in the netherlands factors critical for successful vaccination against classical swine fever in endemic areas diagnostic testing classical swine fever (csf) marker vaccine. trial i. challenge studies in weaner pigs vaccinology of classical swine fever: from lab to field pestiviruses in wild animals this research was supported in part by grant as- . . -hi-h from the council of agriculture. we especially thank drs. sue-min huang (animal health research institute) for her technical support on the iq tm multicolor real-time pcr detection system, yeou-liang lin (animal health research institute) for his assistance in the animal experiment, and fan lee (animal health research institute) for his valuable suggestions regarding experimental design. key: cord- -wrrqb authors: pratelli, a; tempesta, m; greco, g; martella, v; buonavoglia, c title: development of a nested pcr assay for the detection of canine coronavirus date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: wrrqb a diagnostic test for canine coronavirus (ccv) infection based on a nested polymerase chain reaction (n-pcr) assay was developed and tested using the following coronavirus strains: ccv (usda strain), ccv ( / , field strain), feline infectious peritonitis virus (fipv, field strain), trasmissible gastroenteritis virus (tgev, purdue strain), bovine coronavirus (bcv, wbl- strain), infectious bronchitis virus (ibv, m- strain) and fecal samples of dogs with ccv enteritis. a -bp segment of the gene encoding for transmembrane protein m of ccv is the target sequence of the primer. the test described in the present study was able to amplify both ccv and tgev strains and also gave positive results on fecal samples from ccv infected dogs. n-pcr has a sensitivity as high as isolation on cell cultures, and can therefore be used for the diagnosis of ccv infection in dogs. canine coronavirus (ccv) is a single-stranded rna virus belonging to the coronaviridae family and is responsible for mild to severe gastroenteritis in dogs (binn et al., ; appel, ) . in-fected dogs shed ccv in feces for - days (keenan et al., ) but shedding can be prolonged in some pups. the virus content of feces is very high at the time clinical signs first appear (appel, ) . electron microscopic (em) examination of negatively stained fecal suspensions or viral isolation in cell cultures are the most commonly used methods for the diagnosis of ccv infection in dogs. both of those methods are time-consuming, however, and em is not available in many laborato-ries and may produce artifacts. because it is sometimes difficult to differentiate ccv infection from infection caused by the more virulent canine parvovirus, it is important to be able to obtain a rapid etiological diagnosis in kennels experiencing an outbreak of acute gastroenteritis. recently a nested polymerase chain reaction (n-pcr) assay was developed for detection of feline infectious peritonitis virus (fipv), a coronavirus closely related to ccv, in clinical specimens (gamble et al., ) . the present report describes the development of a n-pcr for the diagnosis of canine coronavirus infection. the n-pcr procedure was chosen because it is more sensitive and specific than tests which employ a single target sequence (porter-jordan et al., ) . the target sequence for amplification was a segment of the gene encoding for the transmembrane protein m of ccv. the above sequence, comprising bp, straddles nucleotides and , as described by herrewegh et al. ( ) . the following primers were prepared:ccv : %-tcc aga tat gta atg ttc gg- % sense primer ( - nucleotides);ccv : %-tct gtt gag taa tca cca gct- % antisense primer ( - nucleotides);ccv : %-ggt gtc act cta aca ttg ctt- % internal primer ( - nucleotides). the cdna was synthesized in a ml total reaction volume containing . ml of rna, pcr buffer × (kcl mm, tris -hcl mm, ph . ), mm mgcl , . mm of each dntp, u/ml rnasi, u/ml rt, . mg/ml ccv primer. the cdna was synthesized at °c for min with the final stage at °c for min. pcr was undertaken in ml volumes in a mixture containing pcr buffer × , mm mgcl , mm of each dntp, u amplitaq gold dna polymerase, and . mg/ml of ccv primer. the amplification reaction was carried out in a dna thermal cycler (perkin elmer cetus, usa) for cycles with denaturation at °c for min., annealing at °c for min. and polymerization at °c for min. all reactions were preceded by an activation phase of amplitaq gold at °c for min. a -ml aliquot of the amplified product was then visualized by agarose gel electrophoresis ( % agarose, v for min) and subsequent uv transillumination after ethidium bromide staining. for the n-pcr, a -ml aliquot of the : dilution of the first amplicon was subjected to a second round of amplification using the ccv and ccv primers and the same cycling procedures. the pcr and n-pcr trials were carried out on the following ccv strains: 'usda strain' and strain / , that was isolated from a dog with enteritis (buonavoglia et al., ) . four additional coronaviruses were also examined: fipv, isolated from a cat with infectious peritonitis (buonavoglia et al., ) ; porcine transmissible gastroenteritis virus (tgev), purdue strain; bovine coronavirus (bcv), strain wbl- ; infectious bronchitis virus (ibv), strain m- . the pcr and n-pcr were carried out on fecal samples from six dogs with enteritis. em examinations were previously carried out on only fecal samples from two dogs and typical coronavirus particles were demonstrated. fecal samples from two normal dogs were included as controls. genomic rna was extracted from the cryolysate of cell cultures infected with the examined coronavirus strains, using the rneasy total rna kit (qiagen gmbh germany). similar preparations were made from the : dilution of the fecal samples. serial -fold dilutions of ccv strain / were inoculated into monolayer cultures of a- cells and those dilutions were used for both pcr and n-pcr. the virus titre was calculated days after infection, assaying for median tissue culture infectious dose (tcid ) by typical viral cytopathic effects. as shown (fig. ) , ccv and ccv primers specific for the target sequence of m gene generated, as expected, a bp amplified product with the following viral strains: ccv usda, ccv / , fipv and tgev, but not with bcv and ibv. ccv and ccv primers used for n-pcr generated a bp amplicon in the same coronavirus strains amplified by the first pcr, with the exception of fipv (fig. ) . the ccv infectious titer in a- cells was . tcid , whereas the detection limit by pcr was the − dilution (fig. ) . using the same virus dilutions, the genomic rna was detected by n-pcr up to the − dilution (fig. ) . the pcr carried out on fecal samples gave positive results for three of the six diarroheic specimens. two of three samples pcr-positive, previously resulted positive in em examinations. the fecal samples from normal dogs consistently gave negative results. the n-pcr gave positive results for four of the six diarroheic fecal samples, whereas the samples from normal dogs gave negative results. . sensitivity of pcr on log dilutions of ccv: amplified products of bp using ccv and ccv primers. lane , marker (gene ruler tm bp dna ladder plus, fermentas); lanes - , undiluted, − , − , − , − , − , − , − . fig. . sensitivity of n-pcr on log dilutions of ccv: amplified products of bp using ccv and ccv primers. lane , marker (gene ruler tm bp dna ladder plus, fermentas); lanes - , undiluted, − , − , − , − , − , − , − . ccv, fipv and tgev by pcr; ccv and tgev by n-pcr. however, this limit does not restrict the use of either method in routine laboratory diagnostic tests. our principal interest was to develop both pcr and n-pcr as methods to rapidly differentiate ccv infections from other canine enteric pathogens which cause similar clinical illness. the diagnosis of ccv enteritis would be more rapid with the pcr assay than with virus isolation in cell cultures, and would obviate the need for em, which may provide spurious results. moreover, the pcr test allows the detection of denatured ccv in fecal samples. pcr carried out with ccv and ccv primers specific for the target sequence - of m gene revealed high sensitivity; tests performed on corresponding viral dilutions, which also were inoculated into cell cultures, gave positive results to the − dilution (approximately - tcid of virus). when viral titrations in cell culture were compared with pcr and n-pcr results, the n-pcr was found to be the most sensitive, giving positive reactions up to the − virus dilution. the higher sensitivity of n-pcr was also evident in assays carried out on dog fecal samples. in regard to the specificity of the n-pcr, the primers ccv and ccv were able to amplify the m gene segment of ccv, fipv and tgev genomes, but not the m gene segment of ibv and bcv. this is expected because ccv, fipv and tgev are closely related (horsburgh et al., ) . n-pcr, with the primers ccv and ccv , amplified both ccv and tgev genomes, but not that of fipv, thus revealing higher specificity than the conventional pcr. the pcr and n-pcr assays described in the present study revealed a relatively low specificity with respect to the amplification of the gene segments of the different coronaviruses studied, i.e. canine coronavirus recovery and characterization of a coronavirus from military dogs with diarrhea l'infezione da coronavirus del cane: indagine sulla presenza del virus in italia. notiziario farmaceutico veterinario isolamento e caratterizzazione di uno stipite di virus della peritonite infettiva felina development of a nested pcr assay for detection of feline infectious peritonitis virus in clinical specimens feline coronavirus type ii strains - and - originate from a double recombination between feline coronavirus type i and canine coronavirus analysis of a . kb sequence from the %-end of canine coronavirus genomic rna intestinal infection of neonatal dogs with canine coronavirus - : studies by virologic, histochemical and immunofluorescent techiques nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented dna the authors thank mr donato narcisi for his excellent technical assistance. key: cord- - n cvgs authors: dhar, arun k.; roux, michelle m.; klimpel, kurt r. title: quantitative assay for measuring the taura syndrome virus and yellow head virus load in shrimp by real-time rt-pcr using sybr green chemistry date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: n cvgs taura syndrome virus (tsv) and yellow head virus (yhv) are the two rna viruses infecting penaeid shrimp (penaeus sp.) that have caused major economic losses to shrimp aquaculture. a rapid and highly sensitive detection and quantification method for tsv and yhv was developed using the geneamp(®) sequence detection system and sybr green chemistry. the reverse transcriptase polymerase chain reaction (rt-pcr) mixture contained a fluorescent dye, sybr green, which exhibits fluorescence enhancement upon binding to double strand cdna. the enhancement of fluorescence was found to be proportional to the initial concentration of the template cdna. a linear relationship was observed between input plasmid dna and cycle threshold (c(t)) values for ( ) down to a single copy of both viruses. to control for the variation in sample processing and in reverse transcription reaction among samples, shrimp β-actin and elongation factor- α (ef- α) genes were amplified in parallel with the viral cdna. the sensitivity and the efficiency of amplification of ef- α was greater than β-actin when compared to tsv and yhv amplification efficiency suggesting that ef- α is a better internal control for the rt-pcr detection of tsv and yhv. in addition, sample to sample variation in ef- α c(t) value was lower than the variation in β-actin c(t) value of the corresponding samples. the specificity of tsv, yhv, ef- α and β-actin amplifications was confirmed by analyzing the dissociation curves of the target amplicon. the c(t) values of tsv and yhv samples were normalized against ef- α c(t) values for determining the absolute copy number from the standard curve of the corresponding virus. the method described here is highly robust and is amenable to high throughput assays making it a useful tool for diagnostic, epidemiological and genetic studies in shrimp aquaculture. taura syndrome virus (tsv) and yellow head virus (yhv) are the two most important rna viruses of penaeid shrimp (penaeus sp.) (lightner et al., ) . in the western hemisphere, tsv has caused serious economic losses, whereas, yhv is viewed as one of the most significant viral pathogens in the eastern hemisphere (brock, ; flegel, ) . the cumulative losses due to tsv in the americas from to were estimated to be us$ . - . billion (lightner et al., ) . yhv has affected significantly shrimp farming in south east asian countries including thailand, china, malaysia, indonesia and india (lightner et al., ) . taura syndrome disease, caused by the tsv, was first described in samples collected from shrimp farms located near the mouth of the taura river in ecuador in (jimenez, ; hasson et al., ) . tsv virions are non-enveloped, icosahedral, - nm in diameter and contain a single stranded positive sense rna genome of kb capable of encoding three major ( , and kda) and one minor ( kda) capsid proteins bonami et al., ) . we have cloned and sequenced the %-end of tsv genome (robles-sikisaka et al., ) . sequence analysis showed that, unlike mammalian picornaviruses, tsv capsid protein genes are located at the %-end of the genome and the tsv genome organization is similar to insect picornaviruses (robles-sikisaka et al., ) . yhv was first reported in with the occurrence of mass mortalities in farm reared black tiger shrimp (p. monodon) in thailand (chantanachookin et al., ) . yhv virions have an enveloped bacilliform shape of - × - nm in size (wongteerasupaya et al., ) . the viral genome contains a single stranded, positive sense rna and encodes four major structural proteins of , , and kda. the partial nucleotide sequence (open reading frame b) revealed that the genome organization of yhv is very similar to the gill-associated virus (gav), reported from australia. it has been proposed that yhv and gav should belong to a new taxon (proposed name oka irus) in the order nidovirales that also included coronaviruses, toroviruses and arteriviruses (walker et al., ) . the current diagnostic methods for tsv and yhv include bioassay using indicator hosts, monitoring clinical signs, histopathology, dot blot, in situ hybridization using virus specific gene probe, immunohistochemistry and by the polymerase chain reaction (pcr) (lightner and redman, ) . although conventional pcr is most sensitive among these methods, it is unable to detect a single copy of the viral genome in the infected tissue. this is critical for the development of a specific pathogen free shrimp-breeding program and for monitoring movement of live and frozen shrimp between countries. to address these issues, we have developed a rapid and highly sensitive real-time quantitative pcr method using the ge-neamp ® sequence detection system coupled with sybr green chemistry. sybr green dye has a high affinity for double-stranded dna (ds-dna) and exhibits enhancement of fluorescence upon binding to the dsdna. in the geneamp ® sequence detection system, the fluorescence of the sybr green dye is monitored at the end of the each cycle and the increase in fluorescence above background is dependent on the initial template concentration (pe biosystem geneamp ® user manual, ). the method does not need any post pcr analyses and the specificity of the product is monitored by analyzing the melting curve (ririe et al., ) . the objectives of the present study were ( ) to determine the sensitivity and specificity of sybr green rt-pcr using the geneamp sequence detection system in detecting tsv and yhv; and ( ) to determine the tsv and yhv load in laboratory challenged shrimp. of arizona, arizona) was prepared by homogenizing pcr confirmed tsv and yhv infected tail tissue in % saline ( : w/v) and centrifuging the homogenate in a tabletop centrifuge (beckman microfuge lite model) at rpm for min. the supernatants were diluted to : before injecting the animals. healthy juvenile shrimp were injected with a virus inoculum ( ml : copies) using a -gauge needle and ml tuberculin syringe between the last - tail segments on the ventral surface. control group animals were injected with a tail muscle homogenate from pcr-confirmed virus negative healthy animals. healthy tissue homogenate was prepared same as described above. animals were kept indoors within environmentally controlled tanks, reared on a commercially available feed formulation (madmac-ms dry pellet, bio-marine, inc. hawthorne, ca) and routinely monitored during the course of the study. virus challenged moribund animals were killed at - days post-injection (p.i.) for tsv susceptible p. annamei and yhv susceptible p. stylirostris. for tsv resistant super shrimp p. stylirostris, animals were sacrificed - days p.i. the sampling time was based on the observation that in super shrimp p. stylirostris tsv titer attains a high level after - days p.i., as determined by real-time reverse transcriptase polymerase chain reaction (rt-pcr) (k.r. klimpel, unpublished) . tail muscle tissue ( mg) from virus challenged as well as control animals were taken for the extraction of rna using tri reagent™ (molecular research center, inc. ohio). the rna pellets were dissolved in dnase, rnase free distilled water and the yield of total rna was measured by using a spectrophotometer (shimadzu uv- ). the rna quality was assessed by running the samples in a % formaldehyde agarose gel following standard protocol (sambrook et al., ) . total rna was treated with dnase i using the messageclean ® kit of genhunter corp. (nashville, tn) before synthesizing cdna for sybr green rt-pcr. a list of primers used for the rt-pcr amplification of tsv, yhv, b-actin, and elongation factor- a (ef- a) is given in table . for tsv, yhv and b-actin rt-pcr, cdna was synthesized using omniscript™ cdna synthesis protocol (qiagen, ca) and mg total rna in a ml reaction volume. the rt-pcr mixture contained, ml cdna reaction mixture, × pcr buffer (sigma, st. louis, mo), mm dntp, . mm of each forward and reverse primer and . u of red taq dna polymerase (sigma) in a ml reaction volume. the temperature profile for the pcr amplification was °c min followed by cycles of °c min, °c min, °c min with extension at °c min. the pcr amplified products were run in a % agarose gel at v for h and stained with ethidium bromide to visualize the products on a uv transilluminator. the ef- a gene was previously isolated from a white spot syndrome virus (wssv) challenged p. stylirostris shrimp by using the mrna differential display technique (dhar et al., b) . tsv, yhv and b-actin cdnas were cloned into a topo cloning vector (invitrogen, ca) and the ef- a cdna was cloned into a pcr-trap vector (genhunter corp., inc.). the recombinant plasmid dna was sequenced in an automated dna sequencer (model abi a, pe applied biosystems). the sequence analyses were carried out using the ncbi blast search program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) to confirm identity between the cloned and the published sequences based on which the primers were designed (table ). the primers used for sybr green rt-pcr are listed in table . the primers were designed based on the sequence of the cloned segment of tsv, yhv, b-actin and ef- a genes and using the primer express software version . (pe ap-plied biosystem). the primers were checked by running a virtual pcr and the amplifications were analyzed for expected product, mispairing and primer dimer formation using a computer program (amplify v . b, dr william engles, university of wisconsin, department of genetics). the best primer set was taken for amplification. the sybr green rt-pcr amplifications were undertaken in a geneamp thermocycler coupled with a geneamp ® sequence detection system (pe applied biosystems). the cdna synthesis was carried out in a ml reaction volume containing mg dnase i treated total rna, × rt-pcr buffer, mm dntps (pe applied biosystems), . mm oligo dt, u of rnase inhibitor (pe applied biosystems) and u of mutiscribe™ reverse transcriptase (pe applied biosystems). the cdna reaction mixture was diluted : using dnase, rnase free molecular biology grade water and ml was taken for each amplification reaction. the amplifications were carried out in a well plate in a ml reaction volume containing . ml of × sybr ® green master mix (pe biosystems), . mm each of forward and reverse primers and ml of the : diluted cdna. the thermal profile for sybr rt-pcr was °c min, °c min followed by cycles of °c s and °c min. in each well plate, a dilution series of the plasmid standard for the respective virus was run along with the unknown samples for the corresponding virus and the ef- a control. each sam-ple had - replicates and all reactions were repeated at least times independently to ensure the reproducibility of the results. for comparing the efficiency of amplification of ef- a and b-actin genes with tsv and yhv, cdna was synthesized in a ml reaction volume as described above. a serial dilution was then made using sheared salmon sperm dna ( ng/ml) as a diluent. sybr green rt-pcr was performed in a well plate using ml of each of the cdna dilutions for tsv and yhv detection along with ef- a and b-actin controls following the reaction parameters as described above. the plasmid dnas containing bp tsv insert and bp yhv insert were separately linearized by hindiii (promega, wi) digestions. an aliquot of the digested plasmids were run in a % agarose gel to confirm the digestion before purifying the remaining digestion reactions by qiaquick gel purification kit (qiagen, ca). dna was quantified using a spectrophotometer (shimadzu uv- ) and dilutions were made using sheared salmon sperm dna ( ng/ml) as a diluent. after a sybr green pcr run, data acquisition and subsequent data analyses were done using the sequence detection system (sds version . ). in the sequence detection system, the fluorescence of sybr green against the internal passive reference dye, rox (dr n ) is measured at the end of each cycle. a sample is considered positive when dr n exceeds the threshold value. the threshold value is set at the midpoint of dr n vs. cycle number plot. for all the amplifications described in this paper, the threshold value of dr n was taken as . . the threshold cycle (c t ) is defined as the cycle at which a statistically significant increase in r n is first detected. target cdna copy number and c t values are related inversely. a sample containing higher copies of the target cdna will cross the threshold at an earlier cycle compared to a sample with lower copies of the same target. the copy number of tsv and yhv samples were determined by normalizing the c t values of the samples with respect to ef- a and then extrapolating the normalized c t values to the standard curve of the corresponding virus. for further statistical analyses, the c t values were exported into a microsoft excel worksheet. regression analyses of the c t values of the cdna dilution series were used to determine the amplification efficiency for tsv and yhv compared to the corresponding ef- a and b-actin controls. the analytical sensitivity of sybr green pcr was determined by using a serial dilution of tsv and yhv plasmid dna as template for amplification. dilution series of plasmid standard contained . - . × copies for tsv and . - . × copies for yhv. a linear relationship between the input plasmid dna and the c t values with regression coefficient (r ) greater than . were obtained for both the viruses. the mean c t values of replicate assays ranged from . . (for . × copies) to . . (for . copies) for tsv and . . (for . × copies) to . . (for . copies) for yhv, respectively (fig. , table ). the coefficient of variation was less than . % for both tsv and yhv samples (table ) . to compare the amplification efficiency of tsv and yhv with the internal control genes, ef- a and b-actin, a serial dilution of the cdna derived from tsv and yhv infected samples were made. if the amplification efficiency of tsv and yhv with the corresponding internal controls, ef- a and b-actin, is very similar then the difference in slope (ds) of curves for the virus and the corresponding internal controls will approach to . the ds value of tsv and ef- a was − . and tsv and b-actin was − . ( fig. a) . the ds value of yhv and ef- a (+ . ) was closer than the ds value of yhv and b-actin (+ . ) (fig. b) . to determine the sample to sample variation in the ef- a and b-actin c t values, sybr green rt-pcr was run for tsv, ef- a and b-actin or yhv, ef- a and b-actin in parallel in the same well plate. for the tsv samples, the c t values for ef- a ranged from . to . and the c t values of b-actin ranged from . to . (table ). for the yhv samples, the c t values for both ef- a and b-actin genes were quite variable although the variability for b-actin c t values ( . - . ) were slightly higher than ef- a c t values ( . - . ) for the corresponding samples (table ). since the sybr green rt-pcr does not involve any post-pcr analysis, amplification of specific vs. non-specific products was confirmed by analyzing the dissociation curve of the target amplicons. a dissociation curve with a single peak at temperature expected for that amplicon indicated specific amplification. the amplification profiles and the dissociation curves for tsv and yhv along with their corresponding internal controls (ef- a and b-actin) are shown in fig. and fig. . when amplification was undertaken with cdna from tsv infected shrimp, a significant increase in sybr green fluorescence was recorded with a c t value of . (fig. a) . amplification using cdna from healthy shrimp, did not provide any significant increase in fluorescence indicating absence of tsv specific target (fig. a) . the dissociation curves showed a single peak at melting temperature (t m = . °c) expected for the tsv amplicon only in the tsv infected, but not in the healthy sample (fig. b) . however, both healthy and the tsv infected sample provided successful amplification of ef- a and b-actin genes ( fig. c and e) with a single peak at expected melting temperature (t m = . °c for ef- a and t m = . °c for b-actin) ( fig. d and f) . for yhv sample, only the cdna from infected but not healthy animals provided the am- fig. . the standard curve for tsv (a) and yhv (b) obtained by sybr green pcr using plasmid dna as template. the number of copies of tsv plasmid dna added to each reaction mixture (corresponding to the numbers on the linear curve in panel a) were as follows: ( ) . × , ( ) . × , ( ) . × , ( ) . × , ( ) . × , ( ) . × , ( ) . , ( ) . , ( ) . and ( ) . . for yhv sample, the plasmid copy numbers (corresponding to the numbers on the linear curve in panel b) were as follows: ( ) . × , ( ) . × , ( ) . × , ( ) . × , ( ) . × , ( ) . × , ( ) . , ( ) . , ( ) . and ( ) . . plification of virus-specific product (fig. a ). the dissociation curves indicated that the amplicon had melting temperature (t m = . °c) as expected for the yhv specific product (fig. b) . however, both healthy and infected samples provided successful amplification of ef- a and bactin genes ( fig. c and e) with each dissociation curve showing a single peak at the expected melting temperature for ef- a and b-actin ( fig. d and f). to assess the reproducibility of sybr green assays, amplifications were carried out independently on different days. in a well plate, each sample had - replicates. the coefficient of variation for the c t values of tsv, yhv, ef- a and b-actin genes were less than . % indicating that the assay was highly reproducible (table ). the tsv and yhv viral load in the laboratorychallenged shrimp was determined by normalizing the c t values of the virus with ef- a c t values and then extrapolating the normalized c t values of the samples to the standard curves of the corresponding virus. the tsv load in p. annamei (kona stock) varied from to copies/mg of total rna and the tsv load in super shrimp p. stylirostris was - copies/mg of total rna. the yhv load in the super shrimp p. stylirostris varied from . × to . × copies/mg of total rna. this indicated that super shrimp p. stylirostris has greater resistance to tsv compared to p. annamei (kona stock) but it is highly susceptible to yhv. the shrimp aquaculture industry has expanded rapidly over the last three decades. this has coincided with the emergence of new viral pathogens that were unknown previously to shrimp farming. in addition, there have been considerable movements of live and frozen shrimp from one country to another increasing the risk of spread of diseases into naive populations (lightner et al., ) . for example, until the geographic distribution of tsv was restricted to the americas. from late to early , tsv epizootics were recorded in taiwan that was attributed to the introduction of tsv contaminated postlarvae and spawners from ecuador and elsewhere in the latin america to taiwan (tu et al., ) . to prevent the spread of viral epizootics and to monitor the movement of live and frozen shrimp among countries and continents, there is a growing and urgent need to develop rapid and highly sensitive detection methods. the real-time rt-pcr described here is highly sensitive. it is capable of detecting up to a single copy equivalent of the tsv or the yhv genome (fig. ) . in sybr green rt-pcr, it takes cycles (c t = ) to detect a single copy of a viral genome (perkin elmer user manual, geneamp ® sequence detection system, user manual, ) . a linear relationship between the input plasmid dna and the c t values was observed from down to a single copy of both tsv and yhv. detection of viruses over such a large dynamic range is useful for measuring the viral load in animals with different levels of infection. thus sybr green rt-pcr provides a continuous scale for measuring the viral load. in addition, since sybr green rt-pcr is capable of detecting a single copy of viral genome, it will be useful to detect sub-clinical infections. due to exquisite sensitivity of sybr green pcr, it is highly susceptible to pcr carry over or other contamination. therefore, laboratory hygiene practices should be followed very strictly to pre-vent any potential contamination that may give false positive result. in addition, any negative result as well as samples with c t values close to should be tested at least twice for confirmation. the sybr green rt-pcr was not only highly sensitive but also very specific for detecting tsv, yhv and the internal control genes, ef- a and b-actin. the specificity of sybr green rt-pcr was determined by monitoring the amplification profile and the dissociation curve of the target amplicons. in sybr green rt-pcr, a sample is considered positive when the amplification plot crosses the threshold value. for example, in fig. a the amplification plot of tsv infected sample exceeds the threshold value at cycle number . whereas the amplification plot of the healthy sample did not exceed the threshold line. to ensure that the amplification plot obtained for tsv infected sample was indeed due to the amplification of tsv specific product, the dissociation curve of the product was analyzed (fig. b ). since the dissociation curve of a product depends on its gc content, length and sequence composition; amplification of a specific versus non-specific product could be differentiated by examining the dissociation curve. the tsv amplicon provided a dissociation curve with a single peak at . °c which is expected for the tsv specific amplicon. to determine the quality and any variation in the amount of input rna as well as the efficiency of the reverse transcriptase reaction in both healthy and tsv infected samples, ef- a and b-actin genes were amplified in parallel to the target virus. both healthy and tsv infected samples provided successful amplification of ef- a and b-actin genes with the dissociation curve showing a single peak at the expected temperature (t m for ef- a was . °c and t m for b-actin was . °c, fig. c -f). similar observations were recorded for yhv amplicon and the ef- a and b-actin controls for the corresponding samples ( fig. a-f) . in addition to sensitivity and specificity, sybr green rt-pcr is very rapid and robust in nature. it takes about h to run a well plate from the time the plate is put into the instrument. after amplification, the data analysis takes a few minutes. in a well plate, samples can be run at a time with two replicates for each virus sample, internal control, positive control and negative control. thus, sybr green rt-pcr can be used for high throughput assays for yhv and tsv detection. in recent years, real-time rt-pcr based on taqman chemistry, has been used for the detection of rna viruses infecting plants (roberts et al., ) , animals (moody et al., ; komurian-pradel et al., ; oleksiewicz et al., ) and to quantitate cellular transcripts in yeast and mammals (kang et al., ; leutenegger et al., ; schmittgen et al., ) . schmittgen et al. ( ) compared the endpoint rt-pcr to taqman and sybr green real-time rt-pcr to evaluate the time course of mrna fig. . the amplification plots and the dissociation curves of yhv, ef- a and b-actin genes. the melting temperature (t m ) of each amplicon is shown alongside its dissociation curve. formation and decay of human chimeric b globin gene. both real-time rt-pcr methods produced a -to -log dynamic range of amplification compared to -log dynamic range for endpoint rt-pcr. they reported that although both real-time rt-pcr methods provided comparable dynamic range and sensitivity, sybr green detection was more precise and produced a more linear decay plot than the taqman detection system. in contrast to sybr green detection, multiple fluorogenic probes can be used in a taqman assay to detect more than one target in a reaction. however, taqman assay is more costly than sybr green assay. recently, we isolated ef- a by mrna differential technique while comparing the rna fingerprints of healthy and wssv, a double stranded dna containing virus, infected shrimp (dhar et al., b) . the ef- a was expressed constitutively in both healthy and wssv infected shrimp. we compared the amplification efficiency and sensitivity of ef- a and b-actin to that of tsv and yhv to determine which of these two genes could serve as a better internal reference. ideally, the amplification efficiency as well as the sensitivity of an internal control should be comparable to the rna under study and the internal control should be expressed at an equivalent level irrespective of tissue used, stages of development and the experimental treatments (bustin, ) . the sensitivity (y intercepts) and amplification efficiency (slope) of ef- a was greater than b-actin when compared to both tsv and yhv amplification. for example, the sensitivity (y intercept . ) and amplification efficiency (slope − . ) of tsv was more similar to ef- a (y intercepts . , slope − . ) than b-actin (y intercepts . and slope − . ) ( fig. a) . similarly, the slope and the intercepts of yhv curve were more similar to the ef- a than b-actin (fig. b ). there is considerable evidence that the bactin transcription varies widely in response to experimental treatment in human breast epithelial cells, porcine tissues and canine myocardium (reviewed in bustin, ) . in the current study, both ef- a and b-actin showed variation in their level of expression in tsv and yhv in-fected samples. however, based on the sensitivity and the amplification efficiency, as well as the level of variation, ef- a appeared to be a better internal reference for sybr green rt-pcr detection of tsv and yhv. one of the obstacles in the development of virus resistant lines in shrimp is the lack of method(s) for quantification of viruses. lack of established crustacean cell lines further emphasizes the need to develop methods for virus quantitation. recently, we have developed a real-time pcr assay based on sybr green chemistry for the detection and quantification of two penaeid dna viruses, infectious hypodermal and haematopoietic necrosis virus (ihhnv) and wssv (dhar et al., a) . we used the sybr green pcr method, along with random amplified polymorphic dna (rapd) technique, to identify genetic markers in p. stylirostris shrimp populations that differ in their ihhnv load (hizer et al., ) . thus, sybr green rt-pcr, along with other molecular techniques, will be useful for developing tsv and yhv resistant lines in shrimp. in summary, the sybr green rt-pcr method described above is a major development in the detection and quantification of tsv and yhv in shrimp. the method is very rapid, highly sensitive and is applicable to routine high throughput assay making it a suitable tool for diagnostic, epidemiological and genetic studies in shrimp aquaculture. taura syndrome of marine penaeid shrimp: characterization of the viral agent special topic review: taura syndrome, a disease important to shrimp farms in the swimming through troubled water. proceedings of the special session on shrimp farming absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays histology and ultrastructure reveal a new granulosis-like virus in penaeus monodon affected by yellow-head disease detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative pcr and sybr green chemistry use of mrna differential display to identify the differentially expressed genes from white spot virus infected shrimp (penaeus stylirostris), plant and animal genome ix special topic review: major viral diseases of the black tiger prawn (penaeus monodon) in thailand taura syndrome in penaeus annamei: demonstration of a viral etiology rapd markers as predictors of infectious hypodermal and hematopoietic necrosis virus (ihhnv) resistance in shrimp (penaeus stylirostris) sindrome de taura (resumen) transcript quantitation in total yeast rna using kinetic pcr quantitative real-time pcr for the measurement of feline cytokine mrna risk of spread of penaeid shrimp viruses in the americas by the international movement of live and frozen shrimp shrimp diseases and current diagnostic methods measuring infectious bursal disease virus rna in blood by multiplex real-time quantitative rt-pcr reverse transcription polymerase chain reaction (rt-pcr) used for the detection of taura syndrome virus (tsv) in experimentally infected shrimp development of a novel real-time rt-pcr assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues product differentiation by analysis of dna melting curves during the polymerase chain reaction real-time rt-pcr fluorescent detection of tomato spotted wilt virus nucleotide sequence of %-end of the genome of taura syndrome virus of shrimp suggests that it is related to insect picornaviruses molecular cloning: a laboratory manual, second ed. cold spring harbor laboratory quantitative reverse transcription-polymerase chain reaction to study mrna decay: comparison of endpoint and real-time methods a yellow head virus gene probe: application to in situ hybridization and determination of its nucleotide sequence taura syndrome in pacific white shrimp penaeus annamei cultured in taiwan yellow head complex viruses: transmission cycles and topographical distribution in the asia-pacific region yellow-head virus of penaeus monodon is an rna virus the authors would like to thank dr k.w. hasson for his comments on the manuscript and tony dettori and dorain thompson for their help in maintaining the animals during the virus challenge experiments. the research was partly funded through a grant from the us department of commerce, sbir grant -dkna- - to krk. dna sequencing was performed by the molecular pathology shared resource, university of ca, san diego, cancer center, which is funded in part by nci cancer center support grant c p ca - . key: cord- - g tuyrc authors: liang, xiao; chigerwe, munashe; hietala, sharon k.; crossley, beate m. title: evaluation of fast technology analysis (fta) cards as an improved method for specimen collection and shipment targeting viruses associated with bovine respiratory disease complex date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: g tuyrc in order to improve the analytic quality of respiratory specimens collected from cattle for nucleic acid-based diagnosis, a study was undertaken to verify realtime pcr efficiency of specimens collected and stabilized on fta cards™, filter paper which is treated chemically. nucleic acids collected using fta cards without the need for a cold-chain or special liquid media handling provided realtime pcr results consistent ( . % agreement, kappa . [ % ci = . – . ]) with the same specimens collected using traditional viral transport media and shipped on ice using the u.s. department of transportation mandated liquid handling requirements. nucleic acid stabilization on fta cards was evaluated over a temperature range (− °c to + °c) for up to days to mimic environmental conditions for diagnostic sample handling between collection and processing in a routine veterinary laboratory. no significant difference (p ≥ . ) was observed in realtime pcr cycle threshold values over the temperature range and time storage conditions for bovine viral diarrhea virus, bovine respiratory syncytial virus, bovine coronavirus, and bovine herpesvirus i. the four viruses evaluated in the study are associated with bovine respiratory disease complex where improvements in ease and reliability of specimen collection and shipping would enhance the diagnostic quality of specimens collected in the field, and ultimately improve diagnostic efficiency. in order to improve the analytic quality of respiratory specimens collected from cattle for nucleic acidbased diagnosis, a study was undertaken to verify realtime pcr efficiency of specimens collected and stabilized on fta cards tm , filter paper which is treated chemically. nucleic acids collected using fta cards without the need for a cold-chain or special liquid media handling provided realtime pcr results consistent ( . % agreement, kappa . [ % ci = . - . ]) with the same specimens collected using traditional viral transport media and shipped on ice using the u.s. department of transportation mandated liquid handling requirements. nucleic acid stabilization on fta cards was evaluated over a temperature range (− • c to + • c) for up to days to mimic environmental conditions for diagnostic sample handling between collection and processing in a routine veterinary laboratory. no significant difference (p ≥ . ) was observed in realtime pcr cycle threshold values over the temperature range and time storage conditions for bovine viral diarrhea virus, bovine respiratory syncytial virus, bovine coronavirus, and bovine herpesvirus i. the four viruses evaluated in the study are associated with bovine respiratory disease complex where improvements in ease and reliability of specimen collection and shipping would enhance the diagnostic quality of specimens collected in the field, and ultimately improve diagnostic efficiency. © elsevier b.v. all rights reserved. testing of specimens collected from live animals for pathogen detection has become increasingly important for the control and prevention of production-limiting diseases. although the detection of a specific virus does not correlate perfectly with presence of clinical disease, the identification of specific viruses which animals are exposed to can help determine production management decisions. nucleic acid detection assays, predominately realtime pcr, have become a routine and highly valued component of laboratory diagnosis, providing increased timeliness and reliability for detection of a wide range of animal pathogens. the accuracy and diagnostic sensitivity of nucleic acid-based methodologies are however impacted critically by the quality and integrity of the diagnostic specimens. factors contributing to low-quality, non-representative diagnostic specimens often include microbial overgrowth and enzyme-induced degradation of nucleic acids (blacksell et al., ; wang et al., ; malentacchi et al., ; pazzagli et al., ) which occur between the time of sample collection in the field and processing in the laboratory. this is of particular concern for veterinary diagnostics where animals, particularly wildlife and range animals such as sheep and cattle, are not located near a veterinary clinic, laboratory, or appropriate courier system. a variety of stabilizing agents and fixatives are available for the preservation of dna and rna, but for various reasons, including cost and complexity of use, they have not found wide application for collection and transport of veterinary diagnostic specimens. a commercial technology used initially to preserve dna for long-term storage, has since been applied to stabilization of both rna and dna for use in forensic and diagnostic applications (salvador and de ungria, ; picard-meyer et al., ; tack et al., ) . the commercial product, fta cards tm , is comprised of filter paper treated chemically, that when the biological specimen is applied to the paper, serves to lyse cells, denature proteins, and stabilize the nucleic acids. the nucleic acids in the specimen captured by and dried onto the fta card are protected from nucleases (liang et al., ) , oxidation, uv damage, and microbial overgrowth. importantly, the stabilized specimen can be transported between the collection site and the laboratory without the need for a cold chain, liquid media handling concerns, time-sensitive shipping arrangements, or associated risk of disseminating an infectious agent. the fta card technology has been applied successfully to a range of tissues to capture and stabilize nucleic acids for subsequent testing, including human dna (dobbs et al., ) , wildlife dna (smith and burgoyne, ) , pathogen nucleic acids from plant specimens (ndunguru et al., ) , and a range of human and animal pathogen nucleic acids, including from parasites, bacteria, and viruses. (moscoso et al., ; purvis et al., ; picard-meyer et al., ; kraus et al., ) . the purpose of the current work was to verify the use of the fta technology for collection and transport of diagnostic specimens used in the detection and identification of key viral pathogens associated with, or predisposing cattle to, bovine respiratory disease (brd) complex (fulton, ) including bovine viral diarrhea virus (family flaviviridae, genus pestivirus, bvdv), bovine respiratory syncytial virus (family paramyxoviridae, subfamily penumovirinae, genus pneumovirus, brsv), bovine coronavirus (family coronaviridae, genus coronavirus, bcov), and bovine herpesvirus (family herpesviridae, subfamily alphaherpesvirinae, bhv- ). bovine respiratory disease was targeted specifically for this study as a disease syndrome impacting the beef cattle industry (fulton, ) and where early and reliable diagnosis is considered to be compromised due to the difficulty of collecting and submitting samples to the laboratory in a sufficiently suitable manner to fully insure the integrity and quality of the specimens. the fta card was considered an easy and reliable field-sampling and shipping option not requiring liquid handling, shelf-life sensitive laboratory media that must be pre-ordered or stored on-site by veterinary practitioners and producers, and having no requirements for cold storage. these characteristics are lacking for liquid media traditionally used, and thus the fta card approach offers an improvement in the practicality and feasibility of sampling from at-risk animals in the field, and in turn providing enhanced nucleic acid-based diagnostic and disease surveillance support for producers and practitioners in rural or remote environments. the objective of the study was to verify that nucleic acids from specimens collected onto fta cards would be of equal or improved quality for pcr-based diagnosis (horwood and mahony, ; thonur et al., ) as compared to specimens collected and transported using traditional liquid media. the study design included bench validation to demonstrate the successful recovery of target viral nucleic acids, both dna and rna, from the selected pathogens following stabilization on fta cards. the efficiency of nucleic acids recovered from fta cards and used in realtime pcr testing for the representative brd-associated viruses was compared to nucleic acids recovered from the traditional method of specimen collection and transport using viral transport media. the study assessed the stability of nucleic acids stored on fta cards at a temperature range representing the extremes of environmental heat ( • to • c) and cold specimen handling conditions (− • to − • c), and a timeframe from specimen collection to laboratory processing consistent with the expected extremes of diagnostic sample shipping ( - days). for field validation, a combination of archival virus positive diagnostic specimens (n = ) and specimens collected by cooperating large animal veterinarians who were provided with the fta cards and minimal sampling instruction were utilized (n = ). the samples included respiratory tract specimens obtained from animals in beef herds presenting with clinical respiratory disease and consistent with current brd herd surveillance and veterinary diagnostic practices. archival samples included diagnostic swab fluids previously tested as realtime pcr positive for brsv (n = ), bhv- (n = ), bcov (n = ), and bvdv (n = ). thirty virus negative archival samples were additionally tested for each of the four viruses. samples were removed from a − • c freezer archive, thawed, and aliquots were spotted onto fta cards and allowed to air-dry at room temperature. additionally, eighty-one deep nasal swabs were collected in duplicate in the field using the traditional method of a dacron or cotton-tipped swab, transferred into tubes of viral transport media, packaged with wet ice packs and absorbent packing materials, then shipped via overnight road delivery. the second swab collected at the same time from the same animal was transferred onto fta cards, allowed to air dry, and shipped in a sealed envelope (fig. ) . reference strains of the brd-associated viruses used in the study included bovine viral diarrhea virus tgac - - kindly provided by dr. j. ridpath, national animal disease center (nadc) in ames, iowa; bovine respiratory syncytial virus strain nvsl bvd ; bovine coronavirus strain nvsl bdv ; and bovine herpesvirus- stain vr- (lot number ) obtained from the american tissue culture collection. stock virus was serially diluted from : to : − in viral transport media, and stored at − • c prior to use. four drops of each virus dilution were spotted onto individual indicator fta cards, each drop containing approximately l of fluid. the fta cards were air dried at room temperature for approximately min. each specimen collected onto fta cards and into viral transport media was extracted and tested in triplicate by the routine realtime pcr methodology used by the california animal health and food safety laboratory system for diagnostic sample testing. an internal control, xeno tm , was added prior to processing in order to evaluate the extraction efficiency from the fta cards. the fta cards were processed by punching mm disks from the inoculated area of , placing the punches in individual wells of a well microtiter plate, and adding l of rapid extraction solution to release the nucleic acids from the fta paper. after min of incubation at room temperature, the plate was shaken for min at rpm. fifty microliters of fluid was transferred into a new well and the plate with the disks was discarded. from this step on, the extraction procedure was identical for the fta card and viral transport media specimens. the magmax- tm viral isolation kit was used to prepare samples for realtime pcr testing following the recommendations of the manufacturer. real-time pcr was performed using previously published primer sets for bvdv (mahlum et al., ) , bhv- (brower et al., ) and brsv (boxus et al., ) ; the primer and probe information for bcov was kindly provided by dr k. kurth (wvdl, madison, wi). all pcr reactions utilized the path-id tm multiplex one-step rt-pcr kit and were performed under the same conditions on an applied biosystems fast real-time pcr system. the pcr thermocycler setting for the path-id tm multiplex one-step rt-pcr kit was as follows: stage at • c for min; stage at • c for min; and stage at • c for s; followed by stage at • c for min; stages - were repeated for cycles, whereas the commercial bvdv detection kit was performed per recommendation of the manufacturer. to evaluate operator variability, side-by-side testing was performed and compared between two laboratory technicians. no difference in nucleic acid recovery was detected between technicians. fig. . nasal swabs were obtained from calves. the sampling method was nasal swabs placed in viral transport media or onto fta cards. each sample was tested for the presence of viruses using independent real-time pcr assays, resulting in pcr tests per sample collection method. the realtime pcr ct values for the xeno tm internal control provided a standard deviation of less than . cycle threshold (ct) values, indicating consistent recovery of the viral nucleic acids from the fta cards. there was % agreement of pcr results for the fta card specimens and the specimens in viral transport media across all dilutions for the individual viruses in the bench validation phase of the study. all virus dilution and nucleic acid recovery series showed linearity for the respective assays over at least dilutions with r values above . . a t-test assessing variations among the different storage conditions revealed no statistically significant difference (p ≥ . ) in ct values for the range of temperature and time storage conditions for any of the viruses tested. realtime pcr efficiencies calculated within the temperature studies were above % for each of the viruses, and linearity measurements were consistently above r values of . . inter-assay variability was highest in the bvdv assay with a standard deviation of . cts among all dilutions evaluated and lowest in the brsv assay with a calculated standard deviation of . cts. agreement for the archival samples when comparing fta-stabilized to the original swab fluid specimens was . %, kappa . ( % ci = . - . ), related to a single bhv- discrepant specimen that was detected as positive from viral transport media and negative from the fta card. (tables and ) . a noteworthy observation was higher ct values indicating less viral nucleic acid detected for the archived samples on fta cards for brsv (average difference . ct), bhv- (average difference . ct), and bcov (average difference . ct). the lower detection sensitivity for fta card samples generated from archived case material is presumed to be due to the dilution of the original swab material in viral transport media prior to archiving, as opposed to collecting directly onto fta cards at the time of sampling. in a recent field study (foster et al., unpublished) % agreement was shown for fta card samples collected from nasal cavities of known bvdv pi and direct contact animals (n = ) when compared to edta blood from the same animals. for the field validation component of the current study where samples were collected directly onto fta cards prior to laboratory submission (fig. ) , agreement was . % ( / ) based on dichotomous results (positive versus negative) comparing the specimens on fta cards to those in viral transport media; . % ( / ) of specimens were pcr positive only using the fta card, and . % ( / ) were positive only using the traditional viral transport media method of collection and shipping. kappa comparison (bland and altman, ; von kummer et al., ) yielded a . ( % ci = . - . ) agreement (good) between the fta cards and the viral transport media method of sample stabilization and transport for the field sample portion of the study (table ) . bovine coronavirus was the most prevalent virus detected by realtime pcr during this phase of the study, with % animals testing table kappa values ( % ci) and percentage agreement for specimens collected into viral transport media compared to specimens collected onto fta cards. positive ( / ). in of the positive animals, bcov was detected using both sample handling approaches, in / cases only the fta card specimen yielded a positive result, and in / only the specimen in viral transport media tested pcr positive for bcov. the overall agreement between the fta card specimens and vtm specimens in viral transport media for bcov was . % ( / ). for the remaining viruses, there was an insufficient incidence of the virus among the field specimens collected directly onto fta cards to statistically compare the fta card to the viral transport media approach to sample handling. a single specimen collected onto a fta card yielded a low concentration of bvdv (near the assay's defined limit of detection), leading to a predicted agreement of . % ( / animals) for bvdv. bovine herpesvirus- was detected in a low concentration in two specimens in viral transport media, but not in the paired fta specimens yielding a predicted agreement for bhv- of . % ( / ). bovine respiratory syncytial virus was detected using both the specimens in viral transport media and those on fta cards for % agreement; however the realtime pcr positive test result was for only one animal (tables and ) . the study provides bench data and initial field data to support the use of fta card technology as a simple and efficient alternative method for diagnostic sample collection and shipment. the overall agreement between fta cards and viral transport media for sample handling was . ( % ci = . - . ) (tables and ). the primary drawback for the use of fta card technology is that the sample collected cannot be used for virus isolation attempts, and is suitable only for nucleic acid-based diagnostic testing. the fta card approach demonstrates equivalent and potentially superior performance in delivering nucleic acids for nucleic acid-based testing, while overcoming critical sample quality drawbacks frequently associated with inconsistent cold chains, producer/practitioner onsite storage or rapid accessibility to quality laboratory transport media and supplies, and shipping concerns related to liquid biological materials. in the course of the field study, an unexpected observation was that practitioners and producers selected animals for sampling that were showing relatively advanced clinical signs of respiratory disease, apparently assuming that clinical signs equated with better diagnostic recovery of the infectious agent. in the case of brd, as with many other disease syndromes, the inciting agent is often not detectable by the time the clinical signs are severe or even observable. this sampling observation clearly indicates that in addition to ease and convenience of the sampling tool, an equally important criterion for quality specimens must include appropriate educational efforts to ensure that specimen collection occurs in the optimum timeframe and from the animals most likely to be shedding the specific infectious agent(s) of interest. the effect of sample degradation and rna stabilization on classical swine fever virus rt-pcr and elisa methods statistical methods for assessing agreement between two methods of clinical measurement real time rt-pcr for the detection and quantitation of bovine respiratory syncytial virus encephalitis in aborted bovine fetuses associated with bovine herpesvirus infection use of fta gene guard filter paper for the storage and transportation of tumor cells for molecular testing bovine respiratory disease research multiplex real-time rt-pcr detection of three viruses associated with the bovine respiratory disease complex avian influenza surveillance with fta cards: field methods, biosafety, and transportation issues solved rnase l: its biological roles and regulation detection of bovine viral diarrhea virus by taqman reverse transcription polymerase chain reaction spidia-dna: an external quality assessment for the pre-analytical phase of blood samples used for dna-based analyses inactivation, storage, and pcr detection of mycoplasma on fta filter paper application of fta technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues spidia-rna: first external quality assessment for the preanalytical phase of blood samples used for rna based analyses use of filter paper (fta) technology for sampling, recovery and molecular characterisation of rabies viruses evaluation of fta paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus dna stability of forensic str markers in ftaextracted buccal dna of betel-quid chewers automated forensic dna purification optimized for fta card punches and identifiler str-based pcr analysis one-step multiplex real time rt-pcr for the detection of bovine respiratory syncytial virus, bovine herpesvirus and bovine parainfluenza virus interobserver agreement in assessing early ct signs of middle cerebral artery infarction stability and infectivity of novel pandemic influenza a (h n ) virus in blood-derived matrices under different storage conditions the authors declared no potential conflict of interest with respect to the research, authorship, or publication of this article. financial support for the execution of this study was provided by the rustici rangeland foundation, . key: cord- - notzm s authors: elia, gabriella; cavalli, alessandra; desario, costantina; lorusso, eleonora; lucente, maria stella; decaro, nicola; martella, vito; buonavoglia, canio title: detection of infectious canine parvovirus type by mrna real-time rt-pcr date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: notzm s a taqman real-time rt-pcr assay was developed for detection of rna transcripts produced by replicating cpv- . a pair of primers and a taqman probe targeting the spliced ns mrna were designed. a synthetic dna fragment was constructed to mimic the spliced ns mrna by pcr-based gene assembly and was used for generation of standard rnas. the detection limit of the assay was × ( ) rna copies and standard curve displayed a linear range from × ( ) to × ( ) copies and a good reproducibility. the assay was then applied to determine the mrna loads in the tissues of dogs naturally infected by cpv- . mrna was detected in a variety of tissues, including the central nervous system. canine parvovirus type (cpv- ) emerged as causative agent of severe hemorrhagic gastroenteritis in dogs in (kelly, ; appel et al., ) and spread rapidly all over the world. since the original type underwent extensive antigenic changes and was fully replaced by the antigenic variants cpv- a and cpv- b (mochizuki et al., a; de ybanez et al., ; greenwood et al., ; truyen et al., truyen et al., , sagazio et al., ; buonavoglia et al., ; pereira et al., ) . the variants - a and - b differ from the original type cpv- by a few amino acid changes in the vp protein, that account for an extended host range in vivo ed in vitro and for increased affinity to canine transferrin receptors (parker et al., ; hueffer et al., ) . a new antigenic variant, type c, has been reported in dogs in europe and southern asia nakamura et al., ; decaro et al., c) . cpv- is a member of the parvoviridae family and is closely related to feline panleukopenia virus (fpv) and mink enteritis virus (mev) (parrish et al., ) . parvoviruses possess a nonenveloped capsid surrounding a single-stranded dna genome * corresponding author. tel.: + ; fax: + . e-mail address: g.elia@veterinaria.uniba.it (g. elia) . of about kb in length. the genome has two open reading frames (orf). orf encodes the two capsid proteins, vp and vp while orf encodes two nonstructural (ns) proteins, ns and ns , that are translated by alternative splicing of the transcribed rna (mrna). as a result, ns contains amino-terminal amino acids in common with ns joined to amino acids from an alternative open reading frame at the -end of the orf (wang et al., ) . several assays are available for detection of cpv- in the faeces of infected dogs. hemagglutination or virus isolation may fail to detect small amounts of virus or the virus neutralized by secretory antibodies. cpv shedding in the faeces reaches high loads in the first days after infection, with a rapid decrease at - days post-infection (decaro et al., a; elia et al., ) . also, in the late stage of infection the antibodies in the gut lumen may sequestrate most virus particles (decaro et al., c; desario et al., ) . furthermore, as cpv- -induced cytopathic effect in cell cultures is often not evident, staining by immunofluorescence is required, with additional laboratory work. more recently, molecular methods such as pcr (mochizuki et al., b; hirasawa et al., ; truyen et al., ; schunck et al., ; senda et al., ; uwatoko et al., ; tempesta et al., ; pereira et al., ; buonavoglia et al., ) and real-time pcr (decaro et al., c) proved to be rapid, sensitive and specific assays for detection of cpv in clinical samples. however, detection of parvovirus dna by pcr does not provide any information on virus infectivity. parvovirus dna appears to be very stable and low levels of dna can persist in serum and a range of tissues for months following acute infection (soderlund-venermo et al., ) . accordingly, detection of viral dna does not equate necessarily with active viral infection and diagnostic assay targeting parvovirus b rna transcripts have been developed (bostic et al., ) . in this study a taqman rt-pcr method was developed for detection of rna transcripts produced by replicating cpv- . the load and distribution of cpv- mrna in samples from infected dogs were estimated in comparison with the load of virus dna, as evaluated by real-time pcr. a synthetic dna fragment was constructed to mimic the spliced parvovirus mrna encoding for the ns protein. a bp fragment within the orf sequence was chosen, that spans the rna splicing region (wang et al., ) . to generate synthetic dna, overlapping primers were designed and used in repeated pcrs for the assembly and the amplification steps. the assembly was carried out in a reaction mix of l consisting of one unit of kod dna polymerase (novagen, madison, wi, usa), l of × buffer supplied by the manufacturer, m dntp and m of each primer (table , a + b + c). the thermal conditions were as follows: s at • c, min at • c and min at • c repeated for cycles with a final extention at • c for min. because single-stranded ends of complementary oligonucleotide dna fragments are filled in by pcr, cycling with dna polymerase results in increasingly larger dna fragments until the full-length sequence is obtained. in the amplification step an aliquot of annealed oligonucleotide mixture was used as template for a second pcr reaction containing two outside primers, s /as (table ) in order to amplify the full-length sequence. amplification was performed as follows: cycles at • c for s, • c for min, • c for min with a final extension at • c for min. the pcr product was cloned into a linearized plasmid vector with overhanging -deoxythymidine (t) residues (topo ta cloning kit, invitrogen, milan, italy). rna transcripts were generated in vitro with ribomaxtm large scale rna production system-t (promega italia, milan, italy) from the t promoter, according to the manufacturer's guidelines. residual dna was eliminated by dnase treatment and the transcript was purified with rneasy columns (qiagen s.p.a., milan, italy) and quantified by spectrophotometrical analysis. rna copy numbers were then calculated and -fold serial dilutions were prepared in te (tris-hcl, edta, ph . ) buffer containing g carrier rna (trna from escherichia coli, sigma-aldrich srl, milan, italy) per millilitre. aliquots of each dilution were frozen at − • c and used only once. primers and taqman probe were designed using beacon design software version . (premier biosoft international, palo alto, ca, usa) to amplify a bp fragment of the spliced mrna. primers and probe were synthesized by mwg biotech ag (ebersberg, germany). in order to avoid the amplification of genomic dna, the taqman probe was designed such that the probe binding region encompassed the rna splicing site (fig. ) . the probe was labelled with -carboxyfluorescein (fam) at the -end and with -carboxytetramethylrhodamine (tamra) at the -end. the position and sequence of the table sequence of primers and taqman probe used in the study antisense fam-tggcgagactatcaacttccgat-tamra a primers used for assembly and amplification steps of standard template. b primers used in real-time rt-pcr. primers and probe used for taqman rt-pcr amplification are reported in table . duplicate of the standard dilutions and rna templates were subjected simultaneously to reverse transcription (rt). one microliter of each duplicate of standard dilutions or template rna was reverse transcribed in a reaction volume of l containing pcr buffer × (kcl mm, tris-hcl mm, ph . ), mgcl mm, mm of each deoxynucleotide (datp, dctp, dgtp and dttp), rnase. inhibitor u, mulv reverse transcriptase . u, random hexamers . u. synthesis of c-dna was carried out at • c for min, followed by a denaturation step at • c for min. for the real-time assay, l of c-dna was added to l of reaction master mix. the master mix consisted of l of iqtm supermix (bio-rad laboratories srl, milan, italy), nm of each primer (r for and r rev), nm of probe r pb. fluorogenic pcr was carried out in a i-cycler instrument (iqtm real-time detection, bio-rad laboratories srl) with the following steps: activation of itaq dna polymerase at • c for min and cycles consisting of denaturation at • c for s, primer annealing extension at • c for min. standard rna ( copies/ml of faecal suspension) used in a real-time rt-pcr assay for avian influenza virus was employed as an internal control (ic) in order to confirm the successful extraction of rna, conversion to c-dna and the taqman pcr reaction, as previously demonstrated (elia et al., ) . the analytic specificity of spliced cpv- mrna detection by real-time rt-pcr was assessed by testing rna and dna preparations of other canine pathogens, including canine coronavirus types i and ii (ccovi, ccovii) (decaro et al., d) , canine distemper virus (cdv) (elia et al., ) , canine adenovirus (cadv) (decaro et al., a) , reoviruses (decaro et al., b) and rotaviruses . within-and between-run precision of the quantitative realtime rt-pcr assay was assessed by multiple measurements of samples containing different amounts of spliced mrna ( . × , . × , . × and . × ). the samples were evaluated in five replicates (within-run precision) and in four separate experiments (between-run precision). for all measurements, mean value, standard deviation and coefficients of variations (cvs) were calculated for the threshold cycle (c t ) values. tissue samples including brain, cerebellum, spleen, lungs, liver, kidneys, bladder and mesenteric lymphnodes from nine cpv- -positive dogs were collected at necroscopy. infection by cpv- in the dogs had been diagnosed by real-time pcr (decaro et al., c) and viral characterization (cpv- , - a, - b and - c) was achieved by minor groove binder (mgb) probe assays (decaro et al., b) . twenty-five milligrams of each tissue sample were homogenized and used for rna extraction (qiaampviral rna mini kit, qiagen s.p.a., milan, italy), according to manufacturer's instructions. to asses the kinetic of mrna production during cpv infection, the cpv- b strain / (buonavoglia et al., , was inoculated onto canine a- cell line maintained in dulbecco minimal essential medium with % fcs. the titre of the viral stock was . tcid . each well of well plates was seeded with ml of cell suspension containing about × cells. the monolayers were infected shortly after trypsinization with l of a viral dilution containing . × dna copies/ l of template (m.o.i. of . ). madin darby bovine kidney (mdbk) cells, that are nonpermissive to cpv- infection, were inoculated in parallel to compare cpv- replication in permissive and non-permissive cultural systems. supernatants and criolysates were taken separately at , , and h post-infection (p.i.) from the plates and used to evaluate the load of cpv- mrna and dna. viral rna was isolated using a commercial rna isolation kit (rneasy total rna kit, qiagen s.p.a., milan, italy) according to manufacturer's instructions. the rna was eluted in l of nuclease free water and stored at − • c. viral dna was extracted using qiaamp viral kit, following the standard protocol (qiagen s.p.a., milan, italy). rna extracted from mock-infected a- cells and three tissue samples (brain, spleen, liver) from an uninfected dog were included as negative controls. to compare the amount of viral dna in the tissue samples and in the infected cell cultures, a real-time pcr assay was used as described previously (decaro et al., c) . the specificity of the system for cpv- mrna was assessed by running in the assay samples known to be positive for other infectious agents. no cross-reactivity with ccovi and ii, cdv, cadv, reoviruses and rotaviruses was observed in this assay. similarly, no detectable fluorescence signal was obtained in the negative controls (mock-infected cells and virus-negative tissue samples), confirming the assay was highly specific for the detection of cpv- spliced mrna. the linear range of quantitation of the real-time rt-pcr assay for cpv- mrna was determined by using -fold serial dilutions of the standard rna ranging from × to × copies. standard rna was first reverse transcribed and c-dna used to determine the detectability and the linearity of the assay (fig. ) . threshold cycle (c t ) values were measured in duplicate and were plotted against the known copy number of the standard sample. the generated standard curve covered a linear range of eight orders of magnitude and showed linearity over the entire quantitation range (slope = − . ). the coefficient of linear regression (r ) was equal to . . to assess the within-and between-run precision, samples containing different amounts of cpv- mrna were assayed in replicate on the same plate or in multiple experiments (fig. ) . the intra-assay cvs were in the range of . % (samples containing . × mrna copies/l of template) to . % (samples containing . × mrna copies/l of template), whereas the inter-assay cvs were comprised between . % (samples containing . × mrna copies/l of template) and . % (samples containing . × mrna copies/l of template). table shows the loads of mrna at different hours p.i. in the cells inoculated with cpv- b. intracellular cpv- fig. . coefficient of variation intra-assay and inter-assay over the dynamic range of the real-time rt-pcr for cpv- mrna. mrna was detected as early as h p.i., with significant increase from . × to . × copies/l of template after h p.i. a parallel increase in the number of viral dna copies was detected both in the criolysates and in the supernatants. as expected, in the a- cells a few copies of mrna were detected in the supernatants collected at h p.i. and the mrna load did not increase significantly at and h p.i. in the non-permissive mdbk cells, cpv- b infection did not result in virus replication, as viral rna and dna were not detected at all (data not shown). the newly developed real-time rt-pcr assay was applied to determine the viral mrna loads in different tissues of cpv- naturally infected dogs. the results were therefore compared with the real-time pcr assay specific for viral dna (table ) . spliced mrna was demonstrated in a variety of tissues, showing a wide bodily distribution of cpv- , as previously demonstrated (decaro et al., ) . the mrna load in the tissues markedly varied. in the tissues with the highest virus dna loads (up to . × dna copies/ l of template) such as spleen, liver, lungs and mesenteric lymphnodes, the mrna loads ranged from undetectable levels to . × mrna copies/l of template. the highest viral mrna load was found in the central nervous system (cns), including brain and cerebellum, with the titres ranging from . × to . × mrna copies/l of template. in these tissues a correlation was found between the viral dna and mrna loads, as the mrna was detected in most dna-positive tissue samples. in this study, a real-time rt-pcr assay was designed to provide a complementary molecular tool for the study of cpv- pathogenesis. thus far, a number of nucleic acid amplification techniques for the detection of cpv- have been described. however, all such techniques are targeted to cpv- dna and a specific molecular method for detection of infectious cpv- particles was not available. parvovirus dna appears to be stable and low levels of dna of human parvovirus b have been detected in serum and tissues for months after acute infection (soderlund-venermo et al., ) . high titres of cpv- dna can been detected for several weeks in blood in dogs even after the virus has disappeared from the intestinal content (decaro et al., unpublished data) . also, during parvovirus replication in vitro, defective genomes may be generated and the real infectious virus titres may be overrated (holland, ; tempesta et al., ) . the synthesis of parvovirus mrnas is considered a marker of virus infection in cells (bostic et al., ) and therefore accurate estimate of the viral rna transcripts may be exploited as a good proxy for evaluation of virus replication. to achieve selective amplification of cpv- mrna, a realtime rt-pcr system was designed to target the spliced rna transcript specific for the ns protein. the real-time rt-pcr assay proved to be highly specific, as no cross-amplification of other canine viruses was observed. the assay was highly reproducible and linear over a range of eight orders of magnitude, from to copies, allowing a precise calculation of cpv- mrna loads. the reproducibility of the assay was high with relatively small intra-and inter-assay variability. the taqman assay was used to investigate the viral mrna loads in tissue samples from naturally infected dogs. cpv- mrna was demonstrated in a variety of tissues, showing a wide distribution of the infectious virus in the organism. however, viral mrna loads were not strictly correlated with the counterpart dna titres, since in some tissues there were high viral dna loads, but low rna titres or no mrna at all. the reasons for these inconsistencies may be threefold: (i) some tissues are not permissive to cpv- replication and the detection of viral dna could be a consequence of the viraemia; (ii) the quantity of mrna may depend on the stage of cell infection, decreasing as the infection course proceeds; (iii) viral rna in some tissues may undergo quick degradation by the tissue rnases and therefore improper conservation after repeated cycles of freezing and thawing or even delayed storage of samples may result in the loss of virus mrna (peirson and butler, ) . an intriguing finding of our study was the detection of cpv- mrna in cns of infected dogs. involvement of the cns tissues during parvovirus infection has been described in cats and virus replication in the nervous tissues may result in the onset of neurological signs (csiza et al., ; wilcox et al., ; url et al., ) . by converse, in dogs cpv antigen has never been detected in neurons, despite the presence of neurodegeneration (agungpriyono et al., ; url and schmidt, ) . in a recent study on cpv- distribution in tissues (decaro et al., ) , high titres of viral genome were detected in the cns with median titres above dna copies/ l of template. in agreement with these results, we could observe the presence of cpv- mrna in the brain and cerebellum tissues. this finding does not necessarily support the conclusion that neurons are permissive to cpv- replication as the presence of a full set of viral mrnas may not be followed by the production of a full set of viral 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nonstructural protein- and the replication of canine parvovirus recovery of viral agents from the central nervous system of cats key: cord- -qbskh uu authors: de arriba, m.l; carvajal, a; pozo, j; rubio, p title: lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: qbskh uu lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (pedv) strain cv- and challenged days later with the virulent isolate of the same virus. a lymphoproliferative assay was developed in which mononuclear cells isolated from lymphoid tissues at different postinoculation and postchallenge days underwent a secondary in vitro stimulation with semipurified antigen obtained from pedv-infected cell cultures. vigorous lymphocyte proliferative responses were detected in the pigs inoculated with the virulent pedv at postinoculation days – , especially in the mesenteric lymph nodes and the blood; however, in the spleen this response was lower and less regular. the pigs inoculated with the attenuated virus showed a less intense response, the higher lymphocyte proliferation also corresponded to the mononuclear cells from mesenteric lymph nodes. lymphocyte proliferation responses showed high correlations with protection against homologous challenge with virulent pedv, and this correlation was higher in the gut associated lymphoid tissues (mesenteric lymph nodes). the cell proliferation response detected in blood mirrored that detected in the mesenteric lymph nodes, and showed also good correlation with protection. the results confirm that t-cell-helper function, assessed by lymphocyte proliferation responses, contributes to establishing a protective immune response against pedv infections. porcine epidemic diarrhoea is an enteric disease in pigs caused by a member of the coronaviridae family, porcine epidemic diarrhoea virus (pedv) (cavanagh et al., ; murphy et al., ) . because of the high prevalence and the severity of clinical symptoms, porcine epidemic diarrhoea represents the most important viral infection of swine intestinal tract in spain and in many other european and asean countries (carvajal et al., b; pensaert, ; sueyoshi et al., ; van reeth and pensaert, ) , although not in the usa where the pedv is not present but a high prevalence of transmissible gastroenteritis virus infections is still present (saif, ; saif and wesley, ) . the disease is characterized by acute watery diarrhoea, depression and anorexia. although morbidity is high and most of the animals in the herd can be affected within a week, mortality is usually low in adult animals ( %), which can recover in - days. however, when piglets less than - weeks are infected, mortality is usually % and can reach % in severe outbreaks, as usually happens in transmissible gastroenteritis. besides the mortality, pedv infection causes important economic loses due to the diminishing of the productive indexes (pensaert, ) . as in many other viral infections of food animals, the lack of effective treatment makes immunity the main key for prevention and control of the disease. however, the development of candidate vaccines needs previous knowledge of immunological aspects related to the infection. due to the enteric nature of the disease and the special configuration of the mucosal immune system, protection depends mostly on the local immune response (corthesy and kraehenbuhl, ; kagnoff, ; saif et al., ; van cott et al., ) , and is the reason why these studies cannot be limited to blood but require organs containing gut associated lymphoid tissues, in which local immune response can be measured. in spite of the fact that the importance of humoral immune response in gastroenteric viral infections of porcine is well recognized (corthesy and kraehenbuhl, ; saif et al., ; tô et al., ; van cott et al., ; yuan et al., ) , little is known about cell-mediated immunity, particularly in pedv infections. however, it has been proposed that cellular immune response may play an important role in the protection and recovery from infection, besides the fact that production of antibodies is regulated by cytokines produced by activated t lymphocytes and other mononuclear cells (corthesy and kraehenbuhl, ; kraehenbuhl and neutra, ; saif, ; totterdell et al., ) . the aim of this study was to assess the cellular immune response following natural infection with pedv and also after inoculation of an attenuated virus, and the contribution to the establishment of a protective response. virus-specific lymphoproliferative responses of systemic tissues (spleen and blood) and mesenteric lymph nodes were studied in conventional piglets after primary inoculation with the virulent, wild type, strain cv- of pedv or its cell culture attenuated form and after challenge, weeks later, with a high dose of the virulent virus. vero cells were grown with eagle's minimum essential medium (gibco, life technologies) buffered with bicarbonate and supplemented with % (v/v) fetal calf serum (gibco), . % (w/v) yeast extract (difco, mi, usa), streptomycin ( mg/l) and penicillin ( , ui/l) (penicillin-streptomycin, gibco). the cell culture adapted strain of pedv cv- , attenuated by many passages, was propagated in vero cells as described by hofmann and wyler ( ) , infecting confluent monolayers of cells after removing the growth medium and adding the viral inoculum diluted in medium without fetal calf serum but containing ml/ml of trypsin (difco). a pedv-infected cell lysate was used as attenuated pedv inoculum. the wild type isolated of the cv- strain of pedv, kindly provided by dr peansert (gent, belgium), was amplified by passages in conventional -week-old piglets without antibodies against pedv and prepared in pbs for use as virulent pedv inoculum. after oral inoculation, the animals were killed in the acute phase of diarrhoea, and the intestinal contents and the small intestine were collected at necropsy. the small intestine from each animal was macerated in pbs ( : (w/v)) and, like the intestinal contents, clarified by centrifugation at × g for min at °c. the richest fractions were pooled and stored at − °c. a total of conventional -day-old piglets, seronegative to pedv and from a herd with no previous history of the disease were assigned to three different experimental groups which were maintained in isolation facilities to prevent virus circulation. group (n = ) was inoculated with a low dose of the virulent isolate of pedv strain cv- that was adjusted in a previous experiment to produce a high morbidity without causing severe disease in the animals. pigs from group (n = ) were inoculated with . × fluorescent focus-forming units per pig of the attenuated isolate of the same pedv strain. the third group (n = ) was mock-inoculated and served as control. twenty-one days after inoculation, the three groups of pigs were challenged with the virulent pedv, using a two times higher dose than that used to inoculate the group . animals were observed daily for clinical symptoms and rectal swabs were taken for days after inoculation and for days after challenge. faecal scores were recorded as normal faeces, pasty faeces, semiliquid (moderate diarrhoea) and liquid (watery diarrhoea). at different postinoculation and postchallenge days (postinoculation days , and , and postinoculation/postchallenge days / , / and / ) subsets of each group of pigs (n= - ) were killed by injection of barbiturate overdose (eutalender, normon, madrid, spain). hundred millilitre of blood were collected from cardiac cavities in % (v/v) acid citrate glucose. spleen and mesenteric lymph nodes were also collected aseptically and placed in ice-cold wash medium (rpmi containing mm hepes and mg of gentamicin and mg ampicillin per ml). three pedv-seronegative, unexposed pigs served as negative control and were killed to obtain the background values for the lymphoproliferative assay. mononuclear cells from blood and tissues were isolated as described previously (de arriba et al., a) . briefly, peripheral blood lymphocytes were obtained by density gradient centrifugation in ficoll-paque (ficoll-paque research grade, pharmacia biotech, upsala, sweden). lymphocytes collected from the interface were washed twice in hanks' balanced salt solution and suspended in rpmi containing % fetal calf serum, mm l-glutamine, mm sodium pyruvate, . mm nonessential aminoacids, mm hepes and mg of ampicillin and mg of gentamicin per ml (enriched medium). mononuclear cells from spleen and mesenteric lymph nodes were obtained by pressing the tissues through stainless steel screens ( mesh) of a cell collector (cellecter; e-c apparatus corp., fla, usa). cell suspensions were centrifuged, and the mononuclear cells were removed from the pellet by continuous and discontinuous gradient centrifugation in percoll (pharmacia biotech.), washed twice with wash medium and resuspended in enriched medium. viability of all mononuclear cells preparations was confirmed by the trypan blue exclusion test, in every case being \ %. the lymphoproliferative assay for detection of pedv-specific t cells was adapted from methods published previously (brim et al., , ward et al., ) . semipurified pedv antigen for in vitro stimulation of mononuclear cells cultures was obtained from lysates of pedv-infected cell cultures that were concentrated times by ultracentrifugation at , ×g for h at °c and then semipurified by ultracentrifugation through % sucrose under the same conditions. the most favourable concentration of antigen for optimal antigenic stimulation of the mononuclear cells was established by dose-response curves. a pedvcontrol antigen was obtained giving the same treatment to mock-infected cultures. the t-cell mitogen phytohaemagglutinin (gibco) was used as positive control at a final concentration of ml/ml, following the manufacturer's instructions. optimal conditions of number of cells and duration of incubation were determined by preliminary studies. mononuclear cells at a concentration of × cells per ml and per well were placed in -well culture plates and stimulated in triplicate with the pedv antigen or the control antigen or the phytohaemagglutinin. cells were incubated for h at °c in % co and h prior to harvest each well was labelled by pulsing mci of [ h]thymidine (amersham pharmacia biotech). harvesting of cells was carried out on glass fiber filters (filtermat, skatron inc., va, usa) and [ h]thymidine incorporation was determined by liquid scintillation spectrophotometry. the lymphocyte proliferative responses for each mononuclear cells sample assayed was expressed as the stimulation index (si), calculated as si= mean cpm of pedv stimulated wells/mean cpm of control antigen stimulated wells, being cpm counts per minute. viral antigen was detected in faecal samples by the double antibody sandwich elisa described by carvajal et al. ( a) . this elisa is based on the use of two monoclonal antibodies (lelsytad cvi- . and lelystad cvi- . ) directed specifically against the s protein of the virus. a blocking step with rabbit-anti pedv hyperimmune serum was included to increase the specificity. twofold serial dilutions of the samples were assayed starting at : and titres were expressed as the inverse of the lowest positive dilution. for calculation of the geometric mean titre (gmt), negative samples were given a titre of . one-way analysis of variance followed by the paired student's t-test was used to determine the nature of differences observed in virus shedding and lymphocyte proliferation responses among inoculated groups, tissues and days. correlation between proliferation responses at challenge day and protection against the infection was established by spearman's correlation coefficient (z). significance was assessed at pb . . for the analysis the systat for windows v. . (sys-tat inc.) and the spreadsheet microsoft excel v. . (microsoft comp.) were used. a summary of faecal virus shedding and clinical disease after inoculation and challenge is given in table . after primary inoculation of pigs from group with virulent pedv, moderate to severe diarrhoea was observed in % of the animals and virus shedding in %. the onset of diarrhoea was observed between postinoculation days and and the average duration was . days. virus shedding was detected in some of the pigs at postinoculation day but most shed pedv in faeces from postinoculation day to . the average duration of the shedding period was . days. the gmt of viral antigen in faecal samples was measured using elisa; it increased strongly from postinoculation day and reached a peak at postinoculation day . conversely, pigs from group inoculated with the attenuated pedv did not show typical signs of the disease and only one pig had moderate diarrhoea for day and virus shedding was detected in only one sample at postinoculation day and with a low titre. differences between the gmt of antigen in the faeces of the two groups were significant statistically. on the challenge day, at postinoculation day , pigs from group were protected against infection and disease, none developed diarrhoea and not viral antigen was detected in the samples taken after challenge. diarrhoea was not observed in pigs from group after challenge either, but viral detection in rectal swab samples revealed that protection against the infection was only partial ( %), and the antigen was detected in % of the challenged animals ( out of ). in the control group moderate diarrhoea was seen in % of the pigs starting between postchallenge days and and with an average duration of . days. viral antigen was detected in faeces of all of the challenged pigs from this group in which the average duration of viral shedding ( . days) was significantly higher than in group ( . days). the gmt of viral antigen detected in the control group was also significantly higher than in group . in order to obtain the optimal secondary antigenic stimulation for the lymphoproliferative assay, two different doses of antigen were used, . ng of the semipurified antigen were added to each × mononuclear cells from spleen and blood, whereas in the mesenteric lymph nodes a minor dose, . ng, yielded the optimal stimulation of the cells. mean cpm obtained after stimulation of each tissue with the different antigens and the mitogen are shown in table . background cpm, obtained after stimulation of each mononuclear cells culture with the control antigen, were low in mesenteric lymph nodes and blood (usually b ) but not in spleen, where these counts sometimes reached values close to , . after inoculation of group , a specific lymphocyte proliferation response was detected for the first time in the mesenteric lymph nodes at postinoculation day (fig. ) and was maintained until challenge day (postinoculation day ), when the maximum value of si was found ( . ). si in this tissue was significantly higher from postinoculation day to than that observed in unexposed pigs. the lymphoproliferative responses in mesenteric lymph nodes of pigs from group increased significantly at postinoculation day compared with their responses in previous days ( fig. ) and si reached its peak that day at . . values of the si between postinoculation days and were significantly higher compared with responses for mononuclear cells from mesenteric lymph nodes in unexposed pigs. group si was greater than group at any pid, however statistical significance was only detected at postinoculation day . group was mock inoculated and served as control at challenge. statistically significant differences among groups are denoted by the letters: 'a' when differences are between groups and , 'b' when differences are between groups and and 'c' for differences between groups and . level of significance is defined by *p . , **p . . a pid: postinoculation days. b pcd: postchallenge days. c gmt: geometric mean titre of antigen detected in faeces by elisa. mononuclear cells were stimulated with positive, negative antigen or the t-cell mitogen phytohaemagglutinin (pha). background values correspond to pigs with no previous contact to pedv. pid, postinoculation day; pcd, postchallenge day. fig. . correlations between lymphocyte proliferative responses in mononuclear cells collected from mesenteric lymph nodes, blood and spleen from pigs inoculated with virulent or attenuated pedv or mock-inoculated and protection against challenge days later with virulent pedv. correlations were assessed by spearman rank correlation test. lymphocyte proliferative responses were expressed as mean cpm of pedv stimulated wells versus mean cpm of control antigen stimulated wells, being cpm counts per minute. fig. . course of the virus-specific lymphocyte proliferative responses represented by si for mononuclear cells from mesenteric lymph nodes, spleen and blood from pigs after inoculation with virulent (group ) or attenuated (group ) pedv or mock-inoculation (group ) and after challenge with virulent pedv. the si are the mean cpm of virus-specific stimulated wells versus mean cpm of control antigen stimulated wells, being cpm counts per minute. the mean value of the si obtained from the group of unexposed pigs is represented by a line crossing the y-axis. statistically significant differences (p . ) with values obtained in nonexposed pigs are noted as *. differences between groups are nodded as: 'a' when differences are between groups and , 'b' between groups and and 'c' for differences between groups and . mononuclear cells purified from blood of group showed a vigorous proliferative response after inoculation starting at postinoculation day with significant increases over the following days. the si obtained for this group in blood were significantly higher than values in blood of unexposed pigs between postinoculation days and and as in the mesenteric lymph nodes, the peak value occurred at postinoculation day (fig. ) . virusspecific lymphoproliferative responses in blood from group occurred at postinoculation day , the only day in which si value was significantly higher than that in unexposed animals (si= . , pb . ). likewise in mesenteric lymph nodes, the si of group were minor than the indexes of group , although the difference was statistical significant only at postinoculation day . the magnitude of the virus-specific proliferation in the spleen of group after inoculation was lower than in the other tissues, being also less regular. at postinoculation days and , the response of this group was low and similar to the proliferation shown by unexposed pigs. in the following days there was an increase in the si that peaked at postinoculation day , however, the value of si was not significantly higher than the background values at any postinoculation time. group did not show a virus-specific proliferative response in mononuclear cells from spleen after inoculation, with an si similar to that in unexposed pigs. after challenge at postinoculation day , the lymphoproliferative responses in mesenteric lymph nodes of group underwent an important increase and even though at postchallenge day (postinoculation day ) the si was lower than on challenge day (although not significantly), this value again reached its peak at postchallenge day (postinoculation day ) with a value of . (fig. ) . in group lymphocyte proliferation responses after challenge were low and only at postchallenge day (postinoculation day ) was the si significantly higher than that in unexposed animals. responses in group , the mock-inoculated control group, after challenge were similar to responses described in mesenteric lymph nodes of pigs from group after inoculation with virulent pedv, but showed a higher intensity. the si in this group was significantly higher than in unexposed animals from postchallenge day (postinoculation day ) and reached days later (at postchallenge day , postinoculation day ) the highest value detected in the mesenteric lymph nodes of all the groups. when the si of the three groups were compared, statistically significant differences were found at postchallenge day (postinoculation day ) between group and groups and and at postchallenge day (postinoculation day ) the si of groups and were significantly higher than index in group . lymphocyte proliferative responses in blood after challenge in group were significantly lower at postchallenge day (postinoculation day ) compared to the challenge day, however, from postchallenge day (postinoculation day ) there were significant increases, reaching maximum value at postchallenge day (postinoculation day ) (fig. ) , the only day that this value could be demonstrated significantly higher than that in unexposed pigs. similarly to mesenteric lymph nodes, the response detected after challenge in blood from group pigs was low, only the si at postchallenge day (postinoculation day ) was significantly higher than unexposed animals index. in the control group, lymphoproliferative responses were low up to postchallenge day (postinoculation day ) and there was not statistical significance in the differences observed with regard to the unexposed animals. comparisons between the si in the blood after challenge in the different groups showed a higher response in group , although statistical significance was only shown at postchallenge day (postinoculation day ). the si was lower in group than in group , but not significantly. in the spleen, responses after challenge of group increased significantly at postchallenge day (postinoculation day ) with regard to the previous day, similar to the mesenteric lymph nodes. group response after challenge was maximum at postchallenge day (postinoculation day ), this being the only time in which the si of mononuclear cells of spleen from this group was significantly higher than the si of unexposed animals. the control group underwent for the first time a specific proliferation response at postchallenge day (postinoculation day ). the si obtained in each group at each point in time were compared and no statistically significant differences were found. correlations between lymphoproliferative responses detected in each tissue and group at the challenge day and protection against challenge, represented by the protection rate against infection, were established by the spearman rank correlation test and are shown in fig. . the magnitude of the response in all tissues examined at postinoculation day correlated positively with protection against challenge, although statistical significance were not attained. the highest correlation was detected in the mesenteric lymph nodes (z= . , p= . ). protection in swine gastroenteric viral infections, as ped, has been related almost exclusively to the antibody immune responses. however, cell-mediated immunity must play an important role in protecting and recovery from infection, besides the control function of the b cell-humoral responses carried out by t cell populations (corthesy and kraehenbuhl, ; kraehenbuhl and neutra, ; mcghee et al., ; saif, ; totterdell et al., ) . thus, without any b cell population deficiency in humans the lack of antibody and specific t-cell responses, resulting in rotavirus persistent infection with viral excretion in faeces for months (totterdell et al., ) . moreover, welch et al. ( ) , in pigs inoculated with transmissible gastroenteritis virus, related peaks of lymphoproliferative responses ending up with final virus shedding in faeces and the beginning of recovery from the disease. in this study, an in vitro virus-specific proliferation assay was carried out as a method to estimate the cell-mediated immune response since this antigen-induced proliferation has been recognised as a property of cd +(t helper) cells in studies undertaken on pigs, mice and humans with rotavirus and coronavirus offit et al., ; ward et al., ) . the specific proliferative response after inoculation of pigs with virulent pedv was detected immediately in the mesenteric lymph nodes, the organs directly associated with the mucosal immune system. the maximum values were found around postinoculation day , just when a strong response of virus-specific antibody-secreting cells was detected in this organ and also in the duodenum and ileum lamina propria (de arriba et al., b) . in pigs inoculated with the attenuated strain of pedv this specific lymphoproliferative response was detected later, at postinoculation day and it was lower that in pigs inoculated with the wild virus. this minor response of group also corresponded to a low response of pedv-specific antibody-secreting cells (de arriba et al., b) . the difference observed between the lymphoproliferative responses of the two inoculated groups has also been described by other researchers (brim et al., , ward et al., ) in other gastroenteric viruses of swine, describing that the lymphocyte proliferative responses induced by attenuated strains of transmissible gastroenteritis and rotavirus were significantly lower than that induced by the homologous virulent virus. these results suggest that a protective antibody response to the virulent pedv could be associated with previous development of a strong specific cell-mediated immune response. this consideration could be reinforced by the fact that antibody production by specialised b cells requires t cell help (corthesy and kraehenbuhl, ) . the virus-specific lymphocyte proliferative response in the systemic lymph tissues (blood and spleen) was observed later than in mesenteric lymph nodes, as well as being considerably lower in the spleen than in other tissues. this delay could be explained if it is considered that the pedv-specific t cells located in blood and spleen originate in the inductive sites from the gut-associated lymphoid tissues, like the mesenteric lymph nodes, and its presence in systemic tissues is due to the homing process necessary for its maturation (corthesy and kraehenbuhl, ; kagnoff, ; kantele et al., ; salmi and jalkanen, ) . the specific cell proliferation response in the blood was more similar to that observed in the mesenteric lymph nodes than in the spleen, especially in the group , in spite of both, blood and spleen, being linked to the systemic immune system. however, the blood, together with the lymphatic system, is the main vehicle for lymphocyte migration (salmi and jalkanen, ) , and its lymphoid population may reflect primed t cells migrating to the gut for some time after an infection. the lymphocyte proliferative responses at the challenge day showed high correlation with protection against challenge. pigs from group inoculated initially with the virulent pedv, were % protected against infection days later with a higher dose of the same virus whereas protection in group , inoculated with the attenuated pedv, was just partial and only % of pigs were protected against infection with the virulent virus. the highest correlation was observed in mesenteric lymph nodes. this result again suggests that t-cell response, especially in the gut associated lymphoid tissues, contributes in an important way to the development of a protective immune response in pedv infections. the highly attenuated pedv conferred partial protection against challenge with virulent virus in conventional pigs, this protection is related to the inoculated dose and increases when a higher dose is used (de arriba et al., b) . kweon et al. ( ) also described the induction of protective immunity by a attenuated strain of pedv inoculated intramuscularly. after the challenge, there was an increase in the lymphocyte proliferative response in pigs from group , however this increase was not reflected either by an enhancement of the virus-specific antibody secreting cell response or the gmt of pedv-specific serum igg and iga (de arriba et al., b) . thus, ward et al. ( ) also reported that pigs inoculated and challenged with virulent rotavirus strains showed after challenge lymphoproliferative responses similar or poorer than after inoculation. although there is no clear explanation in this, the possibility remains that this secondary response after challenge could be related to the proliferation of cell clones involved in immune regulatory functions different to providing help for antibody production, such as t suppressor populations. in this study the development of cell-mediated immunity occurred in systemic and lymphoid tissues after inoculation with virulent and attenuated strains of the pedv. the results suggest that cell-mediated immune responses contribute significantly to the instauration of protective immune status against homologous virulent virus challenge. the lymphoproliferative responses both in gut associated lymphoid tissues and systemic tissues had a higher magnitude when virulent pedv was used versus attenuated pedv to inoculate pigs. however, higher doses and administration methods have to be assayed in order to develop vaccines. lymphocyte proliferation responses of transmissible gastroenteritis virus or porcine respiratory coronavirus cellular immune responses of pigs after primary inoculation with porcine respiratory coronavirus or transmissible gastroenteritis virus and challenge with transmissible gastroenteritis virus evaluation of a bloking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies seroprevalence of porcine epidemic diarrhea virus infection among different types of breeding swine farms in spain revision of the taxonomy of the coronavirus, torovirus and arterivirus genera antibody-mediated protection of mucosal surfaces isotype-specific antibody-secreting cells in systemic and mucosal associated lymphoid tissues and antibody responses in serum of conventional pigs inoculated with pedv mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) mucosal immunology: new frontiers homing potentials of circulating lymphocytes in humans depend on the site of activation molecular and cellular basis of immune protection of mucosal surfaces derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate the mucosal immune system: from fundamental concepts to vaccine development viral taxonomy and nomenclature rotavirus-specific helper t cell responses in newborns, infants, children and adults porcine epidemic diarrhea enteric viral infections of swine enteric viral infections of pigs and strategies for induction of mucosal immunity immunity to transmissible gastroenteritis virus and porcine respiratory coronavirus infections in swine transmissible gastroenteritis and porcine respiratory coronavirus how do lymphocytes know where to go: current concepts and enigmas of lymphocyte homing an immunohistochemical investigation of porcine epidemic diarrhoea serum and intestinal isotype antibody responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease systemic lymphoproliferative responses to rotavirus contribution of antibody-secreting cells induced in mucosal lymphoid tissues of pigs inoculated with respiratory or enteric strains of coronavirus to immunity against enteric coronavirus challenge prevalence of infections with enzootic respiratory and enteric viruses in feeder pigs entering fattening herds development of mucosal and systemic lymphoproliferative responses and protective immunity to human group a rotavirus in a gnotobiotic pig model cell-mediated immune responses of suckling pigs inoculated with attenuated or virulent transmissible gastroenteritis virus systemic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease we wish to thank dr m.b. pensaert for providing the wild type isolated of the pedv strain cv- and dr l.j. saif and dr l.a. ward for laboratory training. we also wish to thank g.f. bayó n and b. escudero for their excellent techni-cal assistance. this work was funded by the comisió n interministerial de ciencia y tecnología (cicyt) project no. agf- . salaries were provided by the excelentisima diputació n provincial de leó n. key: cord- -ytrkob h authors: chen, pan; wu, xing; mao, qunying; gao, fan; hao, xiaotian; bian, lianlian; zhu, fengcai; li, wenhui; xu, miao; liang, zhenglun title: a rapid and quantitative assay for measuring neutralizing antibodies of coxsackievirus b date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: ytrkob h coxsackievirus b (cvb ) infection has been found to account for an increasing proportion cases of hand, foot and mouth disease (hfmd) in recent epidemiology studies. cvb is a single stranded, non-enveloped rna virus and the infection can cause prominent health threat to pre-school children. here, by taking approaches of reverse genetics, we established a single-round infection system for cvb . the pseudovirus was produced by sequential transfection of cvb capsid expresser plasmid and cvb replicon rna bearing firefly luciferase as a reporter. the cvb pseudovirus system was used for quantifying neutralizing antibody (ntab) levels of human serum samples and showed superior specificity and sensitivity comparing traditional cytopathic effect (cpe) assay. furthermore, we compared the seroprevalence of cvb ntabs in pre-school children and healthy adults, and found that only . % of pre-school children were ntabs positive which suggested that most children were naive to cvb infection; while there is much higher positive rate in adults ( %) indicating that most adults have experienced cvb infection during childhood. this rapid and quantitative assay greatly facilitates evaluating the level of ntabs against cvb in populations and will help to advance cvb vaccine development. coxsackievirus b (cvb ), a member of coxsackie b viruses which belong to the genus enterovirus within the family picornaviridae, is an important pathogen causing aseptic meningitis, myocarditis and acute pancreatitis in severe cases. cvb -associated aseptic meningitis have been reported in china (tao et al., ; tseng et al., ; wong et al., ) , and cvb -associated hand foot and mouth disease (hfmd) cases have also been noticed in national notifiable diseases surveillance system (nndss) for hfmd. however there is still no effective cvb vaccine or antiviral drugs available. neutralizing antibodies (ntabs) elicited after viral infection or vaccination conveyed humoral protection to the population and are essential to viral defense. thus quantification of ntabs would be an important criterion in vaccine evaluation as well as the assessment of group protection in human populations before or after vaccination. there are traditional neutralization assay based on inhibition of cytopathic effect (cpe) for viruses that can cause cpe effect on susceptible cell lines in vitro including enterovirus (ev ) (chang et al., ) , coxsackievirus a (ca ) as well as cvb . however, cpe methods usually take - days of inoculation to let plaques develop and are labor-intensive. modified enzyme-linked immunosorbent spot assays which significantly reduced the duration time still have its shortcomings, such as biosafety concerns raised by live viruses, limited sensitivity, and semi quantitative results due to poor linearity (huang et al., ; yang et al., ) . however, pseudovirus based neutralization assays have distinct advantages, as single-round infection ensures superior biosafety and luciferase reporter enables sensitive quantification (bentley et al., ) . pseudovirus based neutralization assays have been widely used to measure neutralizing antibodies for enveloped viruses, such as human immunodeficiency virus (hiv) (montefiori, ) , influenza virus (tsai et al., ) , severe acute respiratory syndrome coronavirus (sars-cov) (li et al., ; sui et al., ) . most pseudoviruses of enveloped viruses are utilizing optimized lentivirus or retrovirus pseudotyping system, while there is no common pseudotyping system for nonenveloped viruses. application of reverse genetics in picornavirus has enable us to get full length infectious viral clones, some pseudoviruses have been made successfully by trans-encapsidation, such as polio virus (jia et al., ) , ev , ca (jin et al., ) . thereby pseudovirus based neutralizing assays have been successfully developed for ev (wu et al., ) , ca (jin et al., ) . however there no efficient pseudotyping system for cvb and current assays for detecting neutralizing antibodies against cvb are cpe based assays and newly reported elisa assay (yang et al., ) . in this work, we first established a robust pseudotyping system for cvb , then developed a neutralizing assay based on this system and verified its suitability in measuring neutralizing antibodies in human serum samples from clinical researches for cvb . besides, we used this assay to perform a small scale investigation on cvb seroprevalence in chinese population and evaluated potential threat caused by cvb to current hfmd disease control. human embryonic kidney cell (hek)- t, human rhabdomyosarcoma (porter et al.) anti-polio virus serum was who standard antisera against polio virus type (sabin), goat anti-g /ca serum (nt titer ) was a gift from the institute of medical biology, chinese academy of medical science; mouse anti-fy /ev serum was raised by immunization with inactivated ev virus fy , isolated from a patient in fuyang city during the hfmd epidemic of in china (genbank accession no. eu , subtype c ); mouse anti-coxsackievirus b (cvb ) serum was raised by immunization with inactivated cvb strain, isolated from a three-year-old patient diagnosed with herpangina in pizhou city in china; and mouse anti-hepatitis a virus (hav) was raised by immunization with hav vaccine produced by sinovac biotech co., ltd. serum samples were collected from pre-school children ( months to two years old) who were enrolled in a phase iii clinical trial on ev inactivated vaccine (clinicaltrials.gov identifier: nct ). written informed consent was obtained from parents or guardians of each subject. independent ethics committee approvals were obtained from the ethics committee of the jiangsu provincial center for disease prevention and control. serum samples were collected from healthy adults ( - years old) in a hepatitis a virus vaccination cohort, which were approved by china food and drug administration (clinical research document no. l ) and ethics committee of the jiangsu provincial center for disease prevention and control. written informed consent of each participant was obtained. . . cvb single round infection system . . . cvb sub-genomic replicon cvb replicon was constructed by replacing the capsid coding region with a firefly luciferase reporter gene on cvb -nancy, an infectious viral cdna clone kindly provided by dr. jeffrey m. bergelson (pan et al., ) . a a protease cleavage site (aittl) was inserted between luciferase gene and a. cvb replicon was linearized with mlui and g phenol-chloroform extracted dna was then used as template for cvb replicon rna synthesis with ribomax large scale rna production kit (promega) following the manufacturer's instructions. transcribed rna was purified by isopropanol precipitation and used for transfection or frozen at − • c until use. the cvb (nancy) capsid expresser was constructed on pcdna . a and expressed the capsid genes in trans. egfp gene was inserted upstream of the cvb (nancy) capsid gene, and was separated by a a protease self cleavage site (aittl). the egfp reporter is used for monitoring transfection efficiency and expression level of the structural genes. . . preparation of cvb pseudovirus-cvb (nancy)-luc cvb (nancy)-luc was produced by sequential transfection of cvb capsid expresser plasmid and cvb replicon rna into hek- t cells. briefly, cvb (nancy) capsid expresser was reverse transfected into hek- t cells at % confluence with jetprime ® (polyplus); h post capsid transfection, replicon rna was then transfected with lipofectamine tm (life technologies). cvb (nancy)-luc pseudovirus was harvested at h post rna transfection with rounds of freeze-thaw cycle. pseudovirus was stable at − • c for at least six months without significant infectivity decrease. viral rna was extracted from l pseudovirus sample with qiaamp viral rna mini kit (qiagen) and cdna was synthesized with primescript rt reagent kit (takara). viral titer was quantified by measuring the genome copy equivalents with qpcr using sybr premix ex taq ii (perfect real time) (takara). the primers targeted to firefly luciferase reporter gene were used for qpcr quantification (qluc-f: -caaatacgatttatctaatttacacga- ; qluc-r: -ccggtatccagatccacaac- ). equal amount of cvb (nancy) wild type virus and pseudovirus ( l) were mixed and loaded on a - % discontinuous sucrose gradient, followed by ultracentrifugation at , rpm for min at • c with beckman mls- rotor. l of each fraction samples (total fractions) were subjected to rt-qpcr. the copy number of viral genomic rna in each fraction was subsequently determined by rt-qpcr. cvb (nancy) virus was quantified with primers located in vp (qvp -f: -cagtgtgttttggaccgagg- ; qvp -r: -agcgtgcccatgttgtttag- ); specific primers for firefly luciferase reporter gene (qluc-f and qluc-r) were used for quantifying cvb (nancy)-luc pseudovirus. the pseudovirus titer was determined by measuring the % cell culture infective dose (ccid ) using a microtitration assay. l two fold series diluted cvb (nancy)-luc was added to well plate in duplicates and l hela cell suspension ( × per well) was added later. after incubation at • c for - h, the cells were lyzed in passive lysis buffer to measure luciferase activity following the manufacturer's instructions (promega). luciferase activity measured in relative light units (rlus) was determined and single round cvb infection system. (a) schematic map of cvb viral rna genome, cvb replicon and capsid expresser for single round cvb infection system. the cvb capsid expresser was used to express all the structural capsid genes in trans, an egfp gene was inserted upstream of the cvb (nancy) capsid gene and all other cvb viral genome were deleted, the egfp was separated by ev a self cleavage site (-aittl-) from the structural genes; cvb replicon was produced by replacing the capsid coding region with a firefly luciferase reporter gene in the full length cvb genome and a t promoter was placed at the end for transcription in vitro. (b, c) relative infectivity of cvb (nancy) wild type virus on hela, rd and vero cells. (b) immunostaining of cvb nancy strain wild type virus infected cells. cells with % confluence on plates were infected with nancy virus at tcid and stained with mouse anti- cd serum at h post infection (c) the infection ratio was quantified with columbus tm (perkinelmer). (d) relative infectivity of cvb (nancy)-luc virus on hela, rd, and vero cells. cells were infected with l cvb (nancy)-luc virus (∼ × copies), luciferase activity was measured at h post infection. cells which had rlus times above the background were considered positive of pseudovirus infection. the background control was cells without pseudovirus infection. . . immunostaining of cvb infection . . . purification of cvb (nancy) cd recombinant protein cdna coding full length nancy cd pro precursor was cloned to pet a. transformed bl (de ) cells were induced with m iptg at • c for h in lb (kan + ). recombinant protein was purified with ni-nta agarose beads (qiagen). the purified cd protein was then concentrated, aliquoted, and stored at − • c. cd proteins were emulsified with equal volume of complete freunds adjuvant (for first immunization) or incomplete freunds adjuvant (for booster) and - weeks-old balb/c mice were immunized at weekly intervals ( g/mouse). serum was collected at one week after the third booster. cells were fixed and permeabilized with ice-cold methanol for min at room temperature at h post cvb (nancy) infection, then washed with pbs and incubated with mouse anti- cd polyclonal antibodies diluted in % bsa/pbs for h at • c. after times of washes with pbs, the cells were incubated with donkey anti-mouse igg (h+l) secondary antibody, alexa fluor ® con-jugate ( : diluted in % bsa/pbs) for . h at • c. after times of washes with pbs, anti-fading reagent was added before observation under fluorescent microscope (nikon). fluorescent images were analyzed with columbus tm (perkinelmer). sera were heat-treated at • c for min to inactivate complements. two-fold serial dilutions of sera from : were made, l diluted serum was taken out and incubated with equal volume of diluted pseudovirus at • c for h in -well plates. then l hela cell suspension ( × per well) was added into the wells and incubated at co incubator. after incubation, medium supernatant was discarded and cells were lyzed in passive lysis buffer (promega) with two rounds of freeze-thaw cycles, and stored at − • c before measurement. luciferase activity measured in relative light units (rlus) was determined according to the luciferase assay system user's manual (berthold). inhibition ratio was calculated as: [ − (rlu virus incubated with serum − rlu background )/ (rlu virus control − rlu background )] × . and non-linear regression curve was plotted with logarithm- transformed dilution concentration against inhibition ratio by graphpad. the titer of neutralizing antibodies was determined as the reciprocal of the dilution at which % of the complete pseudovirus neutralization (pnt ). in brief, two-fold serial diluted sera were mixed with an equal volume ( l) of virus working solution containing tcid /well ( % of tissue culture infective dose) of cvb strain (dh g/js/ , genbank accession no. kp ) at • c for h in -well microtiter plates. l vero cells ( × per well) were added and incubated at • c for - days. sera were tested in duplicates. the neutralization titer was determined as the reciprocal of the highest dilution at which over % of wells showed complete inhibition of cpe. . statistical analysis results were obtained from at least duplicates and reported as the mean ± standard deviation (sd). all statistical analyses were performed with the graphpad prism software package. we first established a single-round cvb reporter virus system which could produce cvb pseudovirus with high titer and robust infectivity. similar strategies had been applied based on our experiences in previously established ev single round infection system . cvb sub-genomic replicon plasmid (cvb replicon) contains untranslated regions (utrs), firefly luciferase (luc) reporter gene and all nonstructural protein regions, with a t promoter sequence at the upstream of end used for in vitro transcription. the capsid expressing plasmid, which is designated as egfp cvb (nancy) capsid expresser, contains all the structural genes (vp - ) and an egfp reporter gene that are under control of a cmv promoter (fig. a) . a a protease cleavage sequence (aittl) which was from ev genome were introduced both in cvb replicon (between luciferase gene and nonstructural genes) and egfp cvb (nancy) capsid expresser (between egfp gene and capsid). efficient protease cleavage upon viral polyprotein translation in these two sites was required for viral proteins maturation and pseudovirus encapsidation. the single round pseudovirus was produced by sequential transfection of the capsid expressing plasmid and cvb sub-genomic rna transcribed from cvb replicon in vitro (see section ). the produced virus is designated as cvb (nancy)-luc. these pseudoviruses can only infect cells once as they lack structural genes and their capsid was provided in producing cell in trans. successful production of high titer cvb pseudovirus showed that inserted a sequence from ev could be efficiently recognized by cvb viral proteases. we next compared cvb (nancy)-luc with wildtype virus in respects of tropism, virion structure and antigenicity. the sensitivity to different cell lines was the same as wild type cvb nancy strain (fig. b-d) . in an ultracentrifugation analysis, cvb (nancy)-luc pseudoviruses migrated the same as the wild type virus in a discontinuous - % sucrose ( fig. a) , and this showed that pseudovirus particles had the same sediment coefficient as wild type virions. and cvb (nancy)-luc infection could be neutralized with mouse anti-cbv serum in a dose-dependent manner (fig. s ). we next verified that antiserum from mice immunized with inactivated nancy viruses could specifically neutralize the infection of cvb (nancy)-luc, while who standard antisera against polio virus, goat anti-g /ca serum, mouse anti-fy /ev serum, mouse anti-coxsackievirus b (cvb ) serum, mouse anti-hepatitis a virus (hav) could not neutralize either cvb (nancy)-luc (fig. b ) nor cvb (nancy) wild type virus (data not shown) infection in vitro. taken together, these results proved that the singe-round cvb (nancy)-luc pseudovirus system could be used as a surrogate for wild type virus. this pseudovirus not only provides the excellent tool for studying cvb entry process in basic research by avoiding any ambiguity that might be caused by post entry events, but also has some other clinical applications. previous neutralization result showed good prospect in utilizing cvb (nancy)-luc to detect neutralizing antibodies in clinical sera, which is a significant indicator for vaccine efficacy. we next explored the feasibility to develop an in vitro neutralization assay based on this cvb pseudovirus expressing firefly luciferase. time course experiment was done to find optimal incubation time post cvb (nancy)-luc infection and showed that rlus was rather stable between h and h post infection, while significantly decreased after h post infection (fig. a) . linearity between relative light units (rlus) and cvb (nancy)-luc input (showed as genome equivalent) was analyzed and the result was shown to be quantitative, with linear correspondence between luciferase activity and the amount of input virus over a broad range (fig. b ). ccid of cvb (nancy)-luc had also been determined, a dose of ccid pseudoviruses per well which had been in the linear correspondence were used for the following experiments. reproducibility is an important criterion to be considered. for the lack of cvb national antibody standard, we used a mouse serum collected from mice immunized with formalin inactivated cvb virus strain and quantified its neutralizing activity against cvb (nancy)-luc pseudovirus in duplicate on the same plate in six independent tests. pearson correlation analysis of neutralizing results from six independent tests showed high degree of reproducibility (r > . ), while the pnt of this mouse serum was . ± . (fig. s ). previously we have measured neutralizing antibodies titers of serum samples collected from pre-school participants ( a b fig. . characterization of cvb (nancy)-luc pseudovirus. (a) ultracentrifugation analysis of cvb (nancy)-luc pseudovirus. both cvb (nancy) wild type virus and pseudovirus were mixed and applied on a sucrose gradient. after ultracentrifugation at , rpm for min at • c with beckman mls- rotor, the copy number of viral genomic rna in each fraction was subsequently determined by quantitative rt-pcr. wild type virus was quantified with primers located in vp , while cvb (nancy)-luc pseudovirus with primer located in firefly luciferase reporter gene. (b) specific neutralization of cvb (nancy)-luc with antisera. antisera including mouse anti-coxsackievirus b (cvb ) serum, mouse anti-hepatitis a virus (hav), mouse anti-fy /ev serum, goat anti-g /ca serum, who standard antisera against polio virus, mouse anticoxsackievirus b (cvb ) serum were serially diluted and incubated with cvb (nancy)-luc at • c for h prior to infection of hela cells in -well plate in a total volume of l in quadruplicates. luciferase activity was measured at h post infection. months to two years old) in a phase iii clinical trial on inactivated ev vaccine using traditional cpe assay. then we applied our newly established pseudovirus assay to re-analyze the titers of these samples, and compared the results with the cpe data. we used capsid protein of cvb prototype nancy strain to encapsidate pseudovirus. although sequence alignment of capsid proteins revealed several mutations in strain compared with nancy strain (fig. s ) , the neutralizing data using both assays were highly consistent, no obvious divergence in antigenicity of and nancy strains had been observed. as there is no antibody standard reference with known concentration (units) for cvb , we could not determine antibody concentration with units; instead we used the reciprocal of the dilution at which % of the complete pseudovirus neutralization (pnt ). as for the traditional cpe assay, neutralization titer was determined as the reciprocal of the highest dilution at which over % of wells showed complete inhibition of cpe (ccid ), samples with titer ≥ were considered positive, otherwise were negative. two sets of data were then underwent statistical analysis. . % ( / ) of the samples were positive in pseudovirus assay compared with . % ( / ) were positive in cpe assay. spearman correlation analysis showed that there was good correlation between the results from these two assays (spearman r = . , p < . ) (fig. c) . bland-altman method comparison analysis further showed that these two methods were highly consistent. the average of logarithmic difference for quantitative results of the two methods was . , and the standard deviation was . (fig. d) . we next used this pseudovirus assay to measure cvb neutralizing antibodies titers in serum samples collected from health adults ( - years old). % of serum samples ( / ) were cvb seropositive. infant participants, whose serum samples were collected within the same period as the adult participants, were picked out for seroprevalence analysis. we found the pre-school group has significantly lower positive ratio ( . %, / ) than adults. the geometric mean of neutralizing antibodies titer in preschool children was . ( % ci: . - . ), while in adults it was . ( % ci: . - . ). although unpaired t test analysis showed that the difference in titer of these two groups was not statistically significant, the geometric mean titers were slightly higher in adults group (fig. ) . more detailed seroprevalence pattern would be discovered if the population can be further stratified into more specific groups according to their ages or gender. trans-encapsidation methods have been used to produce pseudoviruses for picornaviruses. poliovirus replicon was able to pseudotype some picornaviruses when their capsid proteins were provided in trans, though with variable efficiency; among these viruses, cvb was the most efficient in trans-encapsidation of the poliovirus replicon (jia et al., ; porter et al., ) . in our previous work, we found ca could be pseudotyped with ev replicon with moderate efficiency; however cvb failed to be pseudotyped (fig. s ) . so we established a cvb single round infection system with high titers using cvb replicon, and found that neither ev nor ca could be pseudotyped with cvb replicon (fig. s ). different compatibility among picornaviruses suggested that there might be encapsidation signal either on replicon rna or in trans provided capsid proteins or both, and common determinants might be found if viruses were classified into categories according to their pseudotyping compatibility. our work also developed a quantitative pseudovirus luciferase assay for detecting neutralizing antibodies in clinical serum samples. this assay was proven to be safe, fast, sensitive and showed good correlation with traditional cpe based assay. this assay would greatly facilitate cvb vaccine development and evaluation as well as seroprevalence survey. besides the above application, this assay could also be used in screening for anti cvb human monoclonal antibodies and entry inhibitors which would be potent antiviral drugs against cvb infection. although only capsid of nancy strain was tested in this work, other subtypes of cvb are ready to be pseudotyped with their capsid proteins. cross neutralizing activity of antibodies could be easy to be analyzed and broad neutralizing antibodies with therapeutic value could thus be identified, as well as potential differences in antigenicity could be analyzed which would contribute to vaccine candidate selection. increasing cvb infection cases have been reported in hfmd surveillance and changes in hfmd pathogen spectrum have been noticed. after successful development of effective inactivated whole-virus ev vaccines, ev infection cases would drop significantly in the future, while cvb might be a prominent enterovirus replacing ev . serological investigation of neutralizing antibodies in the population would allow us to assess the protection level against viral infections. in this work, we also did a small scale serological survey of cvb in health adults, and compared cvb seroprevalence in pre-school children and adults. the existence of neutralizing antibodies reflects previous infections. the higher positive rate of cvb neutralizing antibodies in adults indicates that there have been cvb infections during childhood. despite the different geographic sampling, the serological pattern is the same as the investigation in yantai city of china (tao et al., ) . low positive rate of cvb neutralizing antibodies in infants indicated these infants were naïve to cvb and with higher risk in potential cvb outbreaks. not only cvb , but also many enteroviruses including ev , ca , ca have been reported with higher positive rate and titers of neutralizing antibodies in adults, while low positive rate in infants or pre-school children which makes them susceptible to enteroviruses infection (ji et al., ; ang et al., ) . we found that there was no cross neutralization between cvb and selected enteroviruses (ev , ca , cbv , pv, hev), using sera collected from mice immunized with inactivated viruses (fig. b) ; moreover, we previously measured neutralizing antibodies titers against ev in the serum samples collected from pre-school children, and found no statistically significant correlation of neutralizing antibodies titers against ev and cvb (data not shown), and this observation indicated that there were also no cross neutralization between cvb and ev in human. taken together, we propose that neutralizing antibodies against ev and cvb can be measured simultaneously. we next would substitute firefly luciferase reporter in cvb replicon with renilla luciferase reporter, and use both our previously developed ev pseudovirus expressing firefly luciferase and cvb pseudovirus expressing renilla luciferase to measure neutralizing antibodies against each virus in the same well (duel pseudovirus expressing luciferase reporter system). this would further minimize the cost and required clinical serum volume in practice. many enteroviruses have been found to be associated with hfmd in previous hfmd surveillance; multivalent vaccine may be needed for more effective hfmd control . assays with the capacity to evaluate neutralizing antibodies to multiple pathogens simultaneously would be preferred in the future. in summary, we established a single round infection system of cvb and developed an in vitro assay for detecting neutralizing antibodies in clinical serum samples, and it was a superior surrogate of the assays using wild type viruses including traditional cpe assay and enzyme-linked immunosorbent spot assay. we also analyzed the differences in cvb seroprevalence in two representative population groups and found that most pre-school children were naive to cvb . although more serum samples from diverse geographic areas and different ages are needed to get a more detailed view of cvb seroprevalence, our work provided evidences that cvb might impose potential public health threats and further efforts are needed for cvb infection control. seroepidemiology of coxsackievirus a , coxsackievirus a , and enterovirus infections among children and adolescents in the use of pseudotypes to study viruses, virus sero-epidemiology and vaccination risk factors of enterovirus infection and associated hand, foot, and mouth disease/herpangina in children during an epidemic in taiwan molecular determinants of enterovirus viral entry: cleft around gln- on vp protein interacts with variable region on scavenge receptor b development of a high-throughput assay for measuring serum neutralizing antibody against enterovirus trans-encapsidation of a poliovirus replicon by different picornavirus capsid proteins development of a coxsackievirus a neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum angiotensin-converting enzyme is a functional receptor for the sars coronavirus prospect and challenges for the development of multivalent vaccines against hand, foot and mouth diseases evaluating neutralizing antibodies against hiv, siv, and shiv in luciferase reporter gene assays single amino acid changes in the virus capsid permit coxsackievirus b to bind decay-accelerating factor demonstration of the specificity of poliovirus encapsidation using a novel replicon which encodes enzymatically active firefly luciferase potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s protein that blocks receptor association seroprevalence of coxsackievirus b in yantai measurement of neutralizing antibody responses against h n clades in immunized mice and ferrets using pseudotypes expressing influenza hemagglutinin and neuraminidase epidemiological survey of enterovirus infections occurring in taiwan between and : analysis of sentinel physician surveillance data coxsackievirus b -associated aseptic meningitis: an emerging infection in hong kong development and evaluation of a pseudovirus-luciferase assay for rapid and quantitative detection of neutralizing antibodies against enterovirus development of an enzyme-linked immunosorbent spot assay to measure serum-neutralizing antibodies against coxsackievirus b this work was supported by national high technology research and development program (" " program, no. aa a ) from the ministry of science and technology of the people's republic of china supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . key: cord- - k oox authors: sharma, vikrant; chaudhry, dhruva; kaushik, samander title: evaluation of clinical applicability of reverse transcription-loop-mediated isothermal amplification assay for detection and subtyping of influenza a viruses date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k oox background: influenza a viruses (iavs) have always remain a serious concern for the global economy and public health. a rapid, specific and sensitive detection method is always needed to control the influenza in its early stages by timely intervention of therapy and early clinical management. objectives: to develop rt-lamp assays for detection of influenza a viruses, their further subtyping into seasonal (h n , h n ) and novel pandemic h n viruses and to evaluate clinical applicability of optimized rt-lamp assays on patients’ samples. study design: in this study, we optimized rt-lamp assay to detect iavs by using primers against matrix gene and subtyping of iavs was done by using primers against hemagglutinin gene. optimized rt-lamp assays were applied on clinical samples from patients having influenza like illness and results were compared with conventional one-step rt-pcr and real-time rt-pcr. results: rt-lamp assays successfully detected and differentiated iavs into h n , h n and pdm /h n subtypes. one hundred and sixty seven clinical swab samples from influenza suspected patients were taken and tested with rt-lamp assay, detecting ( . %) samples positive for influenza a virus. out of samples, , and were found positive for pdm /h n , h n and seasonal h respectively. conventional one-step rt-pcr detected a total of ( . %) samples for influenza a and further subtyping showed and samples positive for pdm /h n and h n virus respectively whereas none was found positive for seasonal h n . rt-lamp assay demonstrated higher sensitivity ( . %) than conventional rt-pcr ( . %) for influenza a viruses detection in clinical samples. conclusions: rt-lamp assay is rapid, sensitive, specific and cost effective method for detection of influenza a viruses than conventional one-step rt-pcr and it can serve as a good alternate for diagnosis and surveillance studies during influenza outbreaks in resource-limited setups of developing countries. influenza a viruses (iavs) pose a serious threat to the world and are responsible for severe morbidity and mortality among human population globally. seasonal outbreaks, epidemics and pandemics have been witnessed for iavs and have resulted in immense loss to public health and economy (wright and neumann, ) . the devastating potential of influenza is shown by the facts that annually there are estimated - million cases of severe illness and , - , deaths around the globe (who, ) . influenza viruses belong to the family orthomyxoviridae, characterized by segmented, negative-sense, single stranded rna genome (shaw and palese, ) . the family contains seven genera viz., influenzavirus a, influenzavirus b, influenzavirus c, influenzavirus d, isavirus, quaranjavirus and thogotovirus (ictv, ) . out of the four genera of influenza viruses, influenza a and b are the main cause of seasonal epidemics while influenza c causes mild reparatory illness and influenza d affects cattle and not known to cause any infection in humans (hause et al., ; cdc, ; ferguson et al., ) . influenza or "flu" is a respiratory illness characterized by high grade fever, myalgia, headache, malaise, sore throat, rhinitis and cough (cox and subbarao, ). generally influenza is a self limiting disease but may become life-threatening in high risk group individuals including young children, pregnant women, aged people and people with compromised immunity (cox and subbarao, ; cox and subbarao, ) . influenza vaccines are updated annually according to the circulating strains and this information is provided by the surveillance system of who's global influenza surveillance and response system (gisrs) for both the northern and southern hemispheres (who, ; webster and govorkova, ) . antivirals treatment proved effective against influenza if given within - h of the onset of disease (moscona, ) . hence, early and accurate diagnosis of influenza plays a crucial role for timely intervention of therapy, clinical management and thus controlling the spread of disease. clinical symptoms of influenza and other respiratory infections can be very similar, making it hard to differentiate. only laboratory diagnosis can provide an accurate influenza diagnosis. these include conventional virus isolation, antigen/antibody detection and molecular methods like reverse transcription polymerase chain reaction (rt-pcr) (poddar, ) and real time rt-pcr (rrt-pcr) (spackman and suarez, ; carr et al., ; wang and taubenberger, ) . virus isolation and serological assays are time consuming and take days to weeks to get results making them unsuitable during epidemics and also these methods require highly sophisticated biosafety laboratories. molecular methods such as rt-pcr and rrt-pcr are although rapid, sensitive and specific but need costly equipments and trained laboratory persons making these assays hard to use in resource-limited laboratories of developing countries. loop-mediated isothermal amplification (lamp) is a specific, efficient and rapid technique that is similar to pcr amplification but the target dna amplification is under isothermal conditions. this method makes use of a dna polymerase with strand displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences on the target dna (notomi et al., ) . the method is applicable to amplification of rna templates when combined with a reverse transcription reaction (rt-lamp). rt-lamp assay has been successfully applied for the detection of various rna viruses including chikungunya (parida et al., ; lu et al., ) , dengue (lu et al., ; parida et al., ) , japanese encephalitis (parida et al., ; toriniwa and komiya, ) , severe acute respiratory syndrome (thai et al., ) and west nile virus . influenza viruses and their novel subtypes like h , h , h and h , have been effectively detected by rt-lamp assay (poon et al., ; ito et al., ; imai et al., ; chen et al., ; zhang et al., ; luo et al., ) . the specificity of rt-lamp is extremely high due to use of four primers that recognize six distinct regions on the target dna. it is faster than pcr because of single temperature is needed for amplification and also cost effective as costly equipments are not required. the amplification results can be viewed directly by using fluorescent dye like sybr green, reducing the time of the assay. the objectives of the current study were to ( ) optimize rt-lamp assay for detection of influenza a viruses and their subtypes (h n , h n and pdm /h n ); ( ) determine sensitivity and specificity of rt-lamp assay; ( ) clinical evaluation of rt-lamp assay and conventional one-step rt-pcr in comparison to who recommended rrt-pcr taken as standard. viral rna was extracted from an aliquot of each reference strain of influenza viruses, using the genejet viral dna/rna purification kit (thermo fisher scientific, usa) by following the manufacturer's instructions. rna was eluted in a final volume of μl in elution buffer (provided with kit) and divided into aliquots of μl each and stored at − °c until used. rna extraction was performed in class ii biosafety cabinets. for detection of influenza a viruses, published primers were taken from matrix (m) gene (poon et al., ) . for subtyping of iavs, primers were selected from hemagglutinin (ha) gene. published primers were used to detect pandemic influenza h n virus (pdm /h n ) (nakauchi et al., ) . for seasonal h n and h n virus, ha gene sequence of the respective reference strain was retrieved from genbank database (genebank accession numbers cy and kc ) and primers were designed by using primer explorer version (https:// primerexplorer.jp/e/). four primers consisting of two internal primers (forward internal primer or fip and backward internal primer or bip) and two external primers (f and b ) were designed for the study. all primers were chemically synthesized by sigma-aldrich (sigma-aldrich, bangalore, india) and were of hplc grade purity (table ) . twenty five microliters of rt-lamp reaction cocktail was prepared in . ml tubes using . μm of each outer primers (f and b ); . μm of each inner primers (fip and bip); . mm of each deoxynucleotides (dntps); mm tris-hcl (ph . ); mm (nh ) so ; mm kcl; mm mgso ; . m betaine; . % tween ; u of bst dna polymerase (new england biolabs); . u of amv reverse transcriptase (invitrogen, usa) and μl of sybr green i (thermo fisher scientific, usa). two microliters of each reference strain's rna was added to their respective tubes (containing strain specific primers) making final volume μl. no template or negative controls were also included in the assay. the reaction tubes were incubated at °c for h either in a water bath or a heating block. the tubes were heated at °c for min to stop the reaction. amplified products were visualized both by electrophoresis using . % agarose gel and by visual detection of green fluorescence due to added sybr green i stain (thermo fisher scientific, usa) under ultra violet (uv) light. conventional one-step rt-pcr was performed on rna samples extracted from the reference strains of iavs. for detection of iavs, primer pair was taken from the matrix (m) gene while subtyping primer pairs were taken from hemagglutinin (ha) gene. for diagnosis of pandemic influenza virus, primer pair was selected from the conserved region of ha gene (genebank accession number ku ) using primer blast software (https://www.ncbi.nlm.nih.gov/tools/primerblast/). details of primer sets were given in table . one-step rt-pcr reaction was carried out in final reaction volume of μl by using verso -step rt-pcr kit (thermo fisher scientific, usa) by following manufacturer's instructions. briefly, reaction mixture was comprised of . μl of x rt-pcr buffers; μl of rt enhancer; . μm of each forward and reverse primer; . μl of enzyme mix and μl of extracted rna. the protocol for optimization of one-step rt-pcr was as follows: °c for min, °c for min, followed by cycles of °c for s, °c- °c for s, °c for s and final extension at °c for min. pcr products were separated by electrophoresis using % agarose gel and visualized by uv transilluminator. influenza virus a/california/ / (h n ) strain with known virus titre as plaque forming units per microliters (pfu/μl) was used to compare sensitivities of rt-lamp and conventional rt-pcr with who recommended taqman real-time rt-pcr (rrt-pcr). ten-fold serial dilutions were made from virus strain in separate tubes ranging from to − pfu per reaction using molecular grade water. rna was extracted from each dilution and used separately for rt-lamp, conventional rt-pcr and rrt-pcr. matrix gene specific primers and μl of rna from each dilution were used in all assays. likewise, sensitivities of rt-lamp, conventional rt-pcr and rrt-pcr were also assessed for subtyping of influenza viruses using ha gene specific primers on h n and h n reference strains. for specificity evaluation, each individual rt-lamp assay was applied on influenza a viruses (pdm /h n , a/ h n and a/h n ), influenza b virus along with other additional respiratory viruses including respiratory syncytial virus (rsv) and human metapneumovirus (hmpv), and cross-reactivity of the assay was checked. a total of clinical samples were collected from patients with influenza like illness (high grade fever (> . °c), cough, sore throat and myalgia) from post graduate institute of medical sciences (pgims), rohtak, haryana, india from november to december . one throat and one nasal swab was collected in ml of viral transport medium (hanks' balanced salt solution supplemented with % bsa, u/ml penicillin and μg/ml streptomycin) from each patient and transported to the laboratory under cold conditions at °c. the samples were processed and rna was extracted in class ii biosafety cabinets. clinical samples were evaluated by using optimized rt-lamp and conventional one-step rt-pcr and detection results were compared with who recommended taqman real-time rt-pcr (rrt-pcr) considering the later as standard. standardized rt-lamp and conventional one-step rt-pcr were applied on clinical samples and amplified products were analyzed by gel electrophoresis. taqman rrt-pcr assay was applied on extracted rnas from clinical samples for detection of influenza a viruses and further subtyping into h n , h n and pdm / h n subtypes. taqman rrt-pcr assay was performed in a μl reaction volume using who recommended protocol and primers/probes sequences (who, ). all three reference strains of influenza a virus were detected by matrix gene specific rt-lamp reaction (fig. a) . on further subtyping, each respective subtype was also detected by ha gene specific rt-lamp assay (fig. a) . optimum amplification temperature and incubation time were found to be °c and h respectively. amplification results were visualized by both gel electrophoresis and green fluorescence detection under uv light ( fig. b and b) . conventional one-step rt-pcr was able to detect all three influenza a virus strains using matrix gene primers. optimum annealing temperature and time was found to be °c for s. amplicons of bp were visualized using % agarose gel electrophoresis (fig. c) . for subtyping conventional rt-pcr, annealing at °c for s was found to be optimum for both pdm /h n and h n subtypes whereas for seasonal h n subtype, annealing temperature of °c for s was optimized. amplicons of bp, bp and bp were detected for pdm /h n , h n and h n respectively, after % agarose gel electrophoresis (fig. c) . sensitivity of rt-lamp assays were assessed for detection of influenza a viruses using m gene primers and for detection of three subtypes i.e. h n , h n and pdm /h n , using ha gene primers (table ) . the results were compared with that of conventional one-step rt-pcr and rrt-pcr. the detection limit of m gene specific rt-lamp was found to be ten times higher ( . pfu/reaction) than conventional one-step rt-pcr ( . pfu/reaction) (fig. a) and ten times lower than rrt-pcr ( . pfu/reaction) ( table ). the detection limits of ha gene specific rt-lamp assays for pdm /h n , h n and h n were . , . and . pfu per reaction respectively. the detection limits of conventional one-step rt-pcr for pdm /h n , h n and h n were , . and pfu per reaction respectively, making it - times less sensitive than rt-lamp. rt-lamp demonstrated similar sensitivities as that of rrt-pcr for the detection of pdm /h n and h n while times less sensitivity for detection of h n subtype (table ) . specificity of each rt-lamp primer set was evaluated against different influenza viruses and other respiratory viruses. all the rt-lamp primer sets were found to be highly specific for their respective influenza viruses as there was no amplification with other influenza viruses or different respiratory viruses. as shown in fig. b , pdm /h n rt-lamp (ha gene) was found to be highly specific for detecting pandemic iavs as only pdm /h n reaction showed positive amplification while there was no amplification with other viruses. all clinical samples were analyzed for presence of iavs and their subtypes (seasonal h n , h n and pdm /h n ) using who recommended taqman real-time rt-pcr, rt-lamp assay and conventional rt-pcr assay. real-time rt-pcr results showed that out of swab samples, ( . %) were positive for influenza a viruses. further subtyping of (n = ) positive samples by real-time rt-pcr showed that ( . %) samples were positive for pdm /h n , ( . %) were positive for h n and ( . %) were positive for h n virus. matrix gene specific rt-lamp assay detected ( . %) samples positive for influenza a viruses which were further sub-typed using ha gene specific rt-lamp. results showed that out of ( %) samples were positive for pdm /h n , ( . %) samples were positive for h n , and ( . %) samples were positive for h n virus. these results showed the comparable sensitivity and specificity of real-time rt-pcr with that of rt-lamp assay (table ). conventional rt-pcr detected ( . %) samples positive for influenza a viruses out of which ( . %) were found positive for pdm /h n and ( . %) were positive for h n virus where as no sample was detected positive for h n virus. all samples which were found to be negative by realtime rt-pcr also come negative by both rt-lamp and conventional rt-pcr assay. influenza surveillance has importance not only in diagnosis of circulating strains of the virus but also to upgrade the available vaccine on annual basis to provide protection against influenza. control of novel influenza a viruses mainly depends of early and accurate identification of virus strain followed by early intervention of antiviral therapy. so, a rapid, sensitive, specific and cost effective method of detection and subtyping of influenza a viruses is always seems fruitful in this scenario. virus isolation using egg embryo culture or cell culture is considered as 'gold standard' for influenza virus detection but the whole process is labour intensive and lengthy taking - days for obtaining results (ellis and zambon, ) . serological methods are also not very reliable for influenza surveillance as these are time consuming and require analysis of serum samples from both acute phase and recovering phase. rapid detection kit based assays are although fast but are not very consistent, have low sensitivity and may produce false results (wang and taubenberger, ; ellis and zambon, ; kim and poudel, ) . molecular detection methods have emerged as very table limit of detection of rt-lamp, conventional one-step rt-pcr and real-time rt-pcr assay using virus titre as plaque forming units (pfu). useful tools in comparison to time consuming conventional virus isolation and serological methods. molecular methods based on conventional rt-pcr and real-time rt-pcr have been proved useful in diagnosis and surveillance of influenza viruses, but most of these assays require highly sophisticated instruments and are not feasible to conduct in resource-limited settings of developing countries. therefore, present study was focused on rt-lamp assay for detection of influenza a viruses and further subtyping into seasonal influenza (h and h ) and novel pandemic influenza a virus (pdm /h n ). rt-lamp assay has been successfully applied to detect influenza a viruses like swine flu (h n ), pdmh n , avian influenza h , h , h and h subtypes (poon et al., ; ito et al., ; imai et al., ; chen et al., ; luo et al., ; kubo et al., ; parida et al., ; bao et al., ) . in this study, we used matrix gene based primers for detection of influenza a viruses (poon et al., ) and hemagglutinin gene based primers for their further subtyping. matrix gene based rt-lamp assay was able to amplify all three reference strains of influenza a virus and hemagglutinin gene based rt-lamp assay successfully detected the respective subtypes. no cross-reactivity was observed for other related viruses and influenza subtypes making the reaction highly specific. sensitivity of a detection assay is an important deciding factor when it comes to applicability of the assay. in this study we have compared rt-lamp with conventional rt-pcr and taqman real-time rt-pcr. in our study, detection limit of taqman rrt-pcr was found to be ten times more than rt-lamp for influenza a and its subtype h n whereas for detection of pdm /h n and h n both assays showed similar values. sensitivity comparison of rt-lamp with conventional rt-pcr showed that rt-lamp was - times more sensitive than conventional rt-pcr. previous studies have also established that rt-lamp is more sensitive than conventional rt-pcr and has comparable sensitivity with real-time rt-pcr for detection of influenza viruses (poon et al., ; kubo et al., ; parida et al., ; bao et al., ; sharma and kaushik, ) . in the present study, a total of clinical samples were analyzed for the presence of iavs and their further subtyping was done. we have showed that rt-lamp assay can be applied successfully for the diagnostic and surveillance studies of influenza viruses. detection time is a crucial factor of any diagnostic assay. rt-lamp assays were completed in h and results were shown after min of agarose gel electrophoresis. so rt-lamp assay gave efficient results within one and a half hours of sample processing (rna extraction) making this assay fast when compared with conventional rt-pcr and real time rt-pcr which gave results within - h (gu et al., ) . previous studies have reported that by using two accessory loop primers the amplification time can be reduced from min to - min (luo et al., ; kubo et al., ; gu et al., ; imai et al., ; peng et al., ) . for the sake of simplicity and making our reaction more cost effective we stuck to the basic protocol of rt-lamp assay and use only four primers for amplification which didn't compromise with sensitivity of the assay. rt-lamp assay can be modified to detect samples in real-time by using turbidity measurement by turbidimeter as turbidity of reaction increases due to release of pyrophosphates during amplification. moreover, addition of a florescent dye like calcein or sybr green in the reaction can be used for direct visualization of the amplification products under uv light thus eliminating the need of gel electrophoresis making the reaction more rapid and less prone to crosscontaminations (luo et al., ; kubo et al., ; bao et al., ; gu et al., ; peng et al., ) .we used sybr green i dye (sigma-aldrich, usa) for direct visualization under uv light and both visual detection and agarose gel electrophoresis produced similar results. this showed that rt-lamp assay can be applied as a point-of-care test during influenza outbreaks due to its simplicity, high sensitivity and less turn-around time. in conclusion, this study showed that rt-lamp assay can be used for detection and surveillance studies during the time of influenza outbreaks as a good alternative for conventional rt-pcr and real-time rt-pcr. rt-lamp assay is highly cost effective and easy to perform as sophisticated instruments like a thermocycler and fluorescent data analyzing computers (as in real-time rt-pcr) are not needed and the whole reaction can be done in a controlled temperature water bath or dry heating block. the results of the study clearly showed that rt-lamp assay can be applied successfully to the clinical samples and the results were compatible with that of the who's recommended real-time rt-pcr. the rt-lamp assay is a simple, rapid, specific, sensitive and cost effective method for detection and subtyping of influenza a viruses and may prove useful in the resource-poor laboratories of the developing countries during influenza outbreaks. vs has designed the study, performed the experiments, analyses and writing, dc participated in designing the study, sk designed the study, performed analyses and writing. this work was funded by a major research project grant ( - / ) of university grants commission (ugc), new delhi, india. this study was approved by the human ethical committee (letter no. phy/ / ), maharshi dayanand university, rohtak, haryana, india and detailed informed consent was taken from each patient before taking swab samples. development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype h types of influenza viruses development of a real-time rt-pcr for the detection of swine-lineage influenza a (h n ) virus infections development of reverse transcription loop-mediated isothermal amplification for rapid detection of h avian influenza virus global epidemiology of influenza: past and present molecular diagnosis of influenza pathogenesis of influenza d virus in cattle rapid and specific detection of h swine influenza virus using reverse transcription loop-mediated isothermal amplification method characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family virus taxonomy development of h -rt-lamp (loop-mediated isothermal amplification) system for rapid diagnosis of h avian influenza virus infection rapid diagnosis of h n avian influenza virus infection by newly developed influenza h hemagglutinin gene-specific loop-mediated isothermal amplification method rapid detection and typing of influenza a and b by loop-mediated isothermal amplification: comparison with immunochromatography and virus isolation tools to detect influenza virus development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (h n ) virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings rapid identification of chikungunya and dengue virus by a real-time reverse transcription-loop-mediated isothermal amplification method reverse-transcription, loopmediated isothermal amplification assay for the sensitive and rapid detection of h subtype avian influenza viruses neuraminidase inhibitors for influenza evaluation of reverse transcription loop-mediated isothermal amplification assays for rapid diagnosis of pandemic influenza a/h n virus loop-mediated isothermal amplification of dna 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 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 the swine-origin influenza a h n virus visual detection of h subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay evaluation of a single step multiplex rt-pcr for influenza virus type and subtype detection in respiratory samples detection of human influenza a viruses by loop-mediated isothermal amplification comparative analysis of molecular methods for detection of influenza viruses orthomyxoviridae type a influenza virus detection and quantitation by real-time rt-pcr development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus rapid detection and quantification of japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification vaccines against influenza, who position paper who information for molecular diagnosis of influenza virus-update influenza (seasonal) fact sheet methods for molecular surveillance of influenza continuing challenges in influenza orthomyxoviruses rapid and sensitive detection of h n avian influenza virus by use of reverse transcription-loop-mediated isothermal amplification none declared. key: cord- -o ep b authors: carolan, louise a.; butler, jeff; rockman, steve; guarnaccia, teagan; hurt, aeron c.; reading, patrick; kelso, anne; barr, ian; laurie, karen l. title: taqman real time rt-pcr assays for detecting ferret innate and adaptive immune responses date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: o ep b the ferret is an excellent model for many human infectious diseases including influenza, sars-cov, henipavirus and pneumococcal infections. the ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. however, the range of reagents available to measure the ferret immune response is very limited. to address this deficiency, high-throughput real time rt-pcr taqman assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (ifnα, ifnβ, ifnγ, il α, il β, il , il , il , il , il , il p , il , granzyme a, mcp , tnfα), as well as four endogenous housekeeping genes (atf , hprt, gapdh, l ). these assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to ng rna per real time rt-pcr reaction) and to select the most appropriate housekeeping genes. using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. these new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states. ferrets are an outbred population widely used to study influenza virus infection (belser et al., ; laurie et al., ; rockman et al., ; hurt et al., ) as well as a range of other diseases, including sars-coronavirus (cov) (reviewed in (roberts et al., ) ) and henipaviruses, such as infection with hendra virus and nipah virus (bossart et al., ; pallister et al., pallister et al., , geisbert et al., ) . the anatomical and physiological similarity between human and ferret lungs also enables ferrets to be used as a model to study lung carcinomas (reviewed in baric et al., ) . recently, absence of the cystic fibrosis transmembrane conductance regulator (cftr) was associated with spontaneous disease induction in the lung and pancreas in ferrets, showing similar pathology to that of cystic fibrosis in humans (sun et al., , reviewed in keiser and engelhardt, ) . the broad utility of this model is highlighted by the use of ferrets to study pneumococcal transmission, reproductive biology and human fetal brain development (reviewed in baric et al., ) . while the ferret is a good model for human respiratory virus infections, reagents to identify ferret leukocytes and immune mediators are limited. studies have identified cross-reactive antibodies that recognize populations of ferret leukocytes, such as cd , cd ␤, cd and cd , and cytokines ifn␥, tnf␣, il and il (rutigliano et al., ; martel and aasted, ; pillet et al., ) . cloning and sequencing of ferret cytokine genes have enabled molecular approaches targeting the corresponding mrnas (von messling et al., ; danesh et al., ; nakata et al., ; ochi et al., ; qin et al., ) . expression of cytokine and chemokine genes has been assessed in ex vivo samples following infection of naïve or vaccinated ferrets with influenza virus or sars-cov by microarray analysis fang et al., ; rowe et al., ) . cytokine and chemokine gene profiles have also been assessed ex vivo and in in vitro epithelial cultures using sybr green real time rt-pcr assays (svitek and von messling, ; cameron et al., ; danesh et al., danesh et al., , svitek et al., ; kim et al., ; fang et al., ; hamelin et al., ; kobinger et al., ; rowe et al., ; kang et al., ; von messling, , ; pillet et al., ; huang et al., ; maines et al., ; belser et al., ; zeng et al., ) . taqman chemistry incorporates target-specific fluorescent labeled probes enabling multiple genes can be assessed in a single real time pcr reaction (giulietti et al., ) . to date, taqman real time rt-pcr assays have only been developed for a smaller number of ferretspecific gene targets (nakata et al., ; suguitan et al., ) . to enable a broader characterization of the immune response in the ferret model, we developed a panel of taqman assays to detect mrna of fifteen ferret cytokines, chemokines and immune mediators (ifn␣, ifn␤, ifn␥, il ␣, il ␤, il , il , il , il , il , il p , il , granzyme a, mcp- , tnf␣) and four housekeeping genes (atf , gapdh, l and hprt). the cytokine and chemokine profile induced by stimulation of ferret leukocytes with mitogens or influenza virus was also assessed to investigate the relevance of the ferret immune response to human infection studies. sequences for cytokine, chemokine and housekeeping genes of multiple species were obtained from genbank (http://www. ncbi.nlm.nih.gov/genbank) and aligned. regions of conservation were identified and primers were designed using primerselect (dnastar lasergene , madison, usa) or primerexpress (applied biosystems, california, usa) to amplify the region from ferret cdna. cloned genes were sequenced and taqman real time pcr primers and probes designed using primerexpress. all oligonucleotide primers and probes used in this study, including those previously published, are listed in table . primers for ifn␣ were designed to amplify multiple subtypes ( - ) (easlick et al., ; hillyer et al., ) . lyophilized oligonucleotide primers were synthesized by geneworks (adelaide, australia) and dissolved in nuclease-free water (promega, madison, usa) at m. all taqman ® mgb tm probes were synthesized by applied biosystems with a reporter dye (either fam, ned or vic) and a non-fluorescent quencher (nfq). adult male and female ferrets (weight - g) were purchased from independent breeders and housed at csl limited (victoria, australia) using services provided under a support services agreement. serum samples were tested by hemagglutination inhibition assay to ensure seronegativity (titer < ) to currently circulating influenza strains before use. experiments using ferrets were conducted with approval from the csl limited/zoetis australia animal ethics committee, in accordance with the australian government national health and medical research council australian code of practice for the care and use of animals for scientific purposes (nhmrc, ). a/tasmania/ / (a(h n )pdm ) influenza virus was passaged in the allantoic cavity of embryonated hen's eggs and stored in aliquots at − • c. to heat inactivate, virus was incubated at • c for min. retropharyngeal lymph nodes were collected from naïve ferrets and placed in rpmi- aq media tm (sigma-aldrich, new south wales, australia) supplemented with % (v/v) fetal calf serum (interpath services, victoria, australia), mm l-glutamine (safc biosciences, usa), u/ml penicillin/ g/ml streptomycin (sigma-aldrich) (complete-rpmi). single cell suspensions were made by mashing the tissue and passing through a sterile m cell strainer (bd, san jose, usa). cell suspensions were washed twice then resuspended in complete-rpmi. lymph node cells from each ferret ( × per well) were plated in a -well plate in ml complete-rpmi with or without l of live or heat-inactivated virus ( tcid ) or g/ml concanavalin a (cona), phytohaemagglutinin (pha-p), lipopolysaccharide (lps), ionomycin (iono) or phorbol -myristate -acetate (pma) (all from sigma-aldrich) in duplicate or triplicate. cell cultures were incubated at • c in % co in a humidified incubator for the indicated periods. total rna was extracted from cultured cells using the rneasy ® mini kit (qiagen, victoria, australia) according to the manufacturer's instructions. briefly, cells from a single well were pelleted and resuspended in l rlt buffer. the sample was vortexed and then run through a qiashredder column. rna was extracted from the supernatant using the animal cells spin protocol, without on-column dnase digestion and eluted with a l volume. rna purity was assessed (a /a ) using a nanodrop spectrophotometer (thermo scientific, massachusetts, usa). rna was stored at − • c. for removal of genomic dna, ng rna was incubated with units dnase i (rnase-free) (new england biolabs, massachusetts, usa) in dnase reaction buffer (final volume ) at • c for min. the reaction was terminated by the addition of edta (final concentration mm, sigma-aldrich) and incubation at • c for min. cdna was generated using the superscript iii first strand synthesis system for rt-pcr (invitrogen, california, usa) with random hexamer primers, according to the manufacturer's instructions. rnaseh treatment was performed. a simultaneous reaction without the reverse transcription enzyme was performed in parallel to generate a '-rt' control. the reaction volume resulted in ng initial rna/l final cdna preparation for culture samples, except at points indicated in the text, where ng initial rna/l final cdna preparation was used. cdna standards were generated in parallel with cdna test samples for each experiment. cdna standards were prepared using rna pooled from a range of test samples within each experiment, with at least l of ng initial rna/l final cdna standard prepared. ten-fold and two-fold serial dilutions (five dilutions of each) were prepared and used for standard curve generation for efficiency calculations. cdna was stored at − • c. table oligonucleotide primer and probe sequences used in this study. primers and probes developed for the taqman real time rt-pcr assay in this study are highlighted in bold italics. primers (and probes) from other published real time rt-pcr studies, taqman and sybr green, are indicated. primers used to clone inserts for plasmid controls are also indicated, and referenced as appropriate. amplification of gene-specific products was performed using platinum ® taq dna polymerase high fidelity (invitrogen) according to the manufacturer's instructions. reactions were run on a s tm thermal cycler (bio-rad laboratories pty ltd., new south wales, australia). total rna was extracted from stimulated ferret cell cultures, reverse transcribed and ferret genes amplified by conventional and taqman real time pcr, using primers indicated in table . both cdna and 'no rt' control samples were run to ensure the specificity of the amplification. the matrix influenza gene was amplified from rna extracted from virus isolate a/california/ / (a(h n )pdm ). the products were agarose gel-purified using the qiaquick gel extraction kit (qiagen) according to the manufacturer's instructions. purified dna was quantified and ligated into pgem ® -t easy vector (promega) (ifn␥, il , il , il , il , il , il p , il , mcp , tnf␣, atf , hprt, gapdh, l , matrix) or pcr tm blunt-topo ® vector (invitrogen) (ifn␣, ifn␤, il ␣, il ␤, granzyme a) according to manufacturer's instructions. ligation reactions were transformed into one shot ® top chemically competent e. coli (invitrogen) by heat shock according to the manufacturer's protocol. positive transformants were identified by pcr using m primers (promega) and gene-specific primers. plasmid dna was isolated from bacterial cultures using the qiaprep spin miniprep kit (qiagen) according to the manufacturer's instructions. the generation of oligonucleotide dimers for each taqman primer pair was assessed using power sybr ® green pcr master-mix (applied biosystems) with melting curve analysis, according to the manufacturer's instructions. primers which resulted in oligonucleotide dimer generation were redesigned and retested. a comparison between primer pairs was also performed using power sybr ® green pcr mastermix without a melting curve, according to the manufacturer's instructions. all other real time pcr assays were performed using the taqman ® fast universal pcr master mix ( ×), no amperase ung (applied biosystems), according to the manufacturer's instructions. one microliter cdna sample was assayed per reaction. each reaction consisted of cycle of • c for s, followed by cycles of • c for s and • c for s. real time pcr runs for each gene included cdna standards ( -fold and -fold dilutions, in duplicate), dna plasmid controls ( -fold dilutions; dilutions), no template control and test samples. when test samples were run over multiple plates for a single gene, dna plasmid controls were included on each plate and the same mastermix preparation was used for all pcr plates. dna plasmid controls were reproducible (< ct difference) between plates. all real time pcr reactions were run on an applied biosystems fast real-time pcr system using the fast system software, version . . . (applied biosystems). data were analyzed using fast system software, version . . . except for plasmid dna efficiency calculations which were determined using fast system software version . . . the efficiency of each gene amplification was calculated by plotting the average ct (y-axis) against the logarithm of the input amount of rna/l cdna (x-axis). both a -fold dilution series and a -fold dilution series were used for each gene and rna table ferret genes amplified in this study and sequence similarly to other published ferret experimental and predicted sequences by blastn. features of the blastn alignments are indicated. note that the aligned sequences are divided into sequences submitted from laboratory-derived experimental data as well as sequences from the ferret genome which have been predicted and designated using the gene prediction tool. genbank accession # generated for this study the efficiency of each reaction was determined using a -fold dilution standard curve. cytokine and chemokine (black circles) and housekeeping (white circles) genes are shown. the mean efficiency for all genes is indicated by the horizontal line. the reaction efficiency (e) is indicated with the corresponding % efficiency, with the ideal value 'e = ' and the acceptable range ( . - . ), indicated. (c) one or two primer/probe sets were combined in a real time pcr reaction and assayed against each of the single target genes. (d) one primer/probe set was assayed against the target gene in a pool (four to seven plasmids) or alone. (e) one or two primer/probe sets were combined in a real time pcr reaction and assayed against the target gene in a paired pool. (c-e) all samples were run in triplicate, with mean and standard deviation indicated. *p < . . set. real time pcr efficiency (e) = ( − /slope ) for -fold dilution series (pfaffl, ) and ( − /slope ) for -fold dilution series. % real time pcr efficiency = (e − ) × . if the standard deviation for the efficiencies determined using -fold and -fold dilution series fell within %, the average efficiency was used in all calculations. if the standard deviation was > %, the efficiency calculated using -fold dilutions was used. the geometric mean of the efficiencies for the indicated genes was used for the housekeeping gene efficiency. the gene stability of housekeeping genes was calculated using genorm in qbase+ version . (biogazelle) (vandesompele et al., ) . the fold change of expression of a gene was calculated using relative quantitation with kinetic pcr correction (pfaffl, ) . fold change = (e target ) ct target (control−test) /(e hkp ) ct hkp (control−test) where 'hkp' was the geometric mean of all housekeeping genes for each data point as indicated in figure legends and ' ct' = ct control -ct test. "undetermined' data points in cytokine gene expression were assigned a ct of " " to enable calculation of fold change and are indicated in figure legends. the control sample for cultures was unstimulated cells cultured in complete-rpmi. the ability to perform duplex real time pcr was assessed using two tailed t-test. data were analyzed using r software (version . . ( - - )) (team, ) . expression of target genes in cultured cells was compared using one-way anova and tukey's post hoc test (only differences to unstimulated cells are indicated). the mean of duplicate or triplicate wells was calculated and used as a single value for each ferret. *p < . , **p < . , ***p < . . . . development of a two-step taqman real time pcr assay to measure expression of ferret immune mediator and housekeeping genes cytokine, chemokine and housekeeping genes were amplified from total mrna generated from cultures of ferret lymph node cells using primers targeting conserved regions of each gene. all table comparison of the sensitivity of taqman and sybr green real time pcr assays. all reactions were performed with the same plasmid standard, except in cases where the appropriate plasmid standard encompassing both primer sets was not available, and a cdna sample was used instead. the same samples were used to compare the taqman assays in this study with either previously published taqman assays or previously published sybr green assays for an individual gene. genes cloned in this study were sequenced and the sequences run in blastn whereby they matched other published and predicted sequences for the corresponding ferret gene on genbank, especially those derived from the ferret genome project (di palma et al., ) ( table ) . sequences generated in this study were also translated and all sequences aligned to known sequences of the corresponding proteins from other species in genbank (data not shown). all primer/probe sets for the taqman real time pcr assay (table ) were tested to ensure specificity and optimum efficiency using plasmid dna standards. the absence of non-specific amplicons was verified for all primer/probe sets (fig. a) . the % efficiency of each primer/probe set was consistent and between and % which is equivalent to an efficiency of . - . (fig. b) , indicating that the template doubled after each cycle during exponential amplification. the correlation co-efficient for all samples was between . and . (data not shown). the sensitivity of the primer/probe sets for each gene was consistent across the different taqman assays, with all genes detected down to . pg dna plasmid (average . rna copies), except gapdh, which was detected down to . pg dna ( . rna copies) ( table ). the limits of detection were consistent for previously published taqman primer/probes sets (nakata et al., ) (table ) . there was no change in the sensitivity of the assay when primer/probes were mixed in the indicated duplex real time pcr reactions (fig. c) , or when plasmid samples were prepared in pools (fig. d) , or a combination of both duplex real time pcr reactions and sample pools (fig. e) , except for l , which was less sensitive when assayed with other genes (fig. e) . to enable comparison between samples from multiple ferrets, the expression of all genes was determined as a fold change, using the calculation for relative quantification to housekeeping gene(s), with kinetic pcr efficiency adjustment (pfaffl, ) . as this equation relies on consistency between samples in the rna quantity assayed, and the optimum reaction efficiency, as well as minimal fluctuation in the expression of housekeeping genes (endogenous controls) (peters et al., ; mane et al., ; bruder et al., ) , these parameters were optimized using rna extracted from cultured ferret lymph node cells stimulated with mitogens or with influenza virus. as the rna quantity varied between samples (range . - . g/well, fig. a ), we first determined the minimum amount of rna for use in the taqman real time rt-pcr assay. the reproducibility of both reverse transcription (fig. b) and real time pcr (fig. c ) steps was assessed separately for samples with different amounts of rna. a minimum of ng total rna produced highly reproducible results for both reverse transcription and real time pcr steps (standard deviation < . ct) and the ct value was able to detect gene expression ( fig. b and c) where ct (cycle threshold) is the cycle number at which the fluorescent signal of the reaction crosses the threshold to exceed background level. a set of cdna standards was generated from the mitogenand influenza virus-stimulated ferret lymph node cells and the reaction efficiency for all genes determined (examples of graphs and calculations for efficiency are shown in fig. a , compilation of efficiencies for all genes is shown in fig. b and c) . although the reaction efficiencies clustered for each of the ferret lymph node cdna preparations (mitogen or virus stimulation in fig. b and c, respectively), the efficiencies using the -fold dilutions of cdna samples were more broadly spread than for the plasmid preparations (compare -fold dilution in fig. b and c with fig. b) . furthermore, analysis of the -fold dilutions indicated some variability due to low level cytokine mrna expression (such as for il ␣, ifn␣). use of -fold dilutions of the cdna standards enabled the efficiency to be determined and data points overlaid with the data points generated by the -fold dilution series, suggesting this approach is acceptable to determine cytokine levels (fig. a) . to minimize the potential effect of initial sampling variability, a calculation of gene stability was performed for housekeeping genes (vandesompele et al., ) . assessment of the stability of expression of housekeeping genes following stimulation with different mitogens (fig. a ) or influenza virus (fig. b ) demonstrated that expression of some housekeeping genes, such as gapdh, was affected more than others and this effect was specific to different stimuli (compare fig. a to b). as gapdh was more variable in expression than the other housekeeping genes; atf , l and hprt following mitogen stimulation, gapdh was excluded and a combination housekeeping genes was used as a reference (fig. c ). in contrast, following virus stimulation, all housekeeping genes were relatively stable and thus all housekeeping genes were able to be used as references (fig. d) . these data suggests that reaction efficiencies and the most stable housekeeping genes should be determined for a type of sample set of cdna under analysis to be able to accurately calculate gene expression. to test the ability of ferret leukocytes to produce mrna cytokines and chemokines, lymph node cells from naïve ferrets were cultured with various mitogens known to activate t and b lymphocyte and macrophage/monocyte responses (fig. ) and with live or heat inactivated influenza virus (fig. ) and the cytokine and chemokine expression profiles were determined (summarized in (cona) or phytohaemagglutinin (pha), which act by cross linking t cell receptors via sugars on the surface of human t lymphocytes (chilson and kelly-chilson, ) , induced similar cytokine profiles, increasing expression of il , il , il , il , il , granzyme a, tnf␣ and ifn␥, most with high fold changes, consistent with effective stimulation of t lymphocytes (fig. ) . pha also induced expression of il p mrna. the addition of con a or pha significantly reduced the expression of mcp- and ifn␣ in their respective cultures (fig. ) . ionomycin and phorbol -myristate -acetate (pma) are mitogenic for lymphocytes as they bypass surface receptors and activate cellular responses by increasing intracellular calcium and directly activating protein kinase c, respectively (nishezuka, ; al wabel et al., ) . culture with ionomycin significantly increased mrna expression of il , il and ifn␥. pma significantly increased levels of tnf␣ and il ␤. expression of ifn␣ and il p mrnas was significantly reduced upon culture with ionomycin or pma (fig. ) . lipopolysaccharide (lps) is a potent activator of naïve and mature b lymphocytes (andersson et al., ; smith et al., ) , monocytes and macrophages. stimulation with lps induced significant levels of il and a small increase in expression of il . no expression of ifn␤ was detected in any culture stimulated with mitogen. real time pcr assays performed as duplex pcrs with the same primer pairs as shown in fig. e detected upand down-regulation of the same cytokines as assays using single primer/probe sets (data not shown). real time assays run with (vandesompele et al., ) . the least stable gene is shown on the left, the most stable gene on the right. -fold less cdna ( ng rna/l cdna) for il , ifn␥, l and atf detected the same fold differences (data not shown). culture of ferret leukocytes with live or heat inactivated a(h n )pdm virus for h induced different cytokine profiles from those stimulated by mitogens (fig. ) . live a(h n )pdm virus induced a significant reduction in expression of il ␣, il ␤, il and mcp , as well as large increases in expression of granzyme a, ifn␣ and ifn␥, whereas heat inactivated virus induced negligible effect on cytokine expression. note that cultures assayed at and h also showed similar results (data not shown). fig. . gene expression in mitogen-stimulated ferret lymph node cells. lymph node cells from four naïve ferrets were stimulated with the indicated mitogens for h, then rna was isolated, reverse transcribed and assayed by real time pcr using ng initial rna/reaction. each data point represents the average fold change of duplicate or triplicate culture wells compared to the geometric mean of l , hprt and atf housekeeping genes. ifn␤ was not detected. horizontal bars indicate the mean for each group; dotted line indicates no fold change. statistical difference in fold-change compared to cells with no stimulation is indicated. in this study, we describe a series of real time taqman rt-pcr assays that can be used to characterize the expression of cytokines and chemokines of the innate and adaptive immune response in ferrets. the sequences of the ferret genes cloned in this study and used for design of the primers and probes, aligned with other previously published sequences as well as with the predicted sequences from the ferret genome project (di palma et al., ) . previous studies have predominantly utilized sybr green real time rt-pcr and required a minimum quantity of ng initial rna per reaction for detection of a single ferret cytokine or chemokine (svitek et al., ; . here we have demonstrated that ng total rna is sufficient in a taq-man real time rt-pcr assay to detect most cytokine and chemokine mrnas. by multiplexing reactions with probes using different fluorochromes, samples with low amounts of rna, such as ferret respiratory samples (suguitan et al., ) , may still be analyzed using this assay without loss of sensitivity, providing a significant sample and cost reduction. in our study we showed that a minimum of duplex assays could be used, mixing primers and probes specific for a housekeeping gene and a cytokine/chemokine gene or two cytokine/chemokines genes. with further optimization, a higher number of targets may be able to be multiplexed. real time pcr results can be reported in copy number or as fold change both compared to a control, depending on whether absolute or relative quantification is required (giulietti et al., ; pfaffl, ) . absolute quantification requires dna or in vitro transcribed rna standards to be included in each reaction, whereas relative quantification can be achieved by including a set of cdna standards generated from the sample of interest, or no standards at all, if the efficiency of each reaction is identical (pfaffl, ) . as it is difficult to maintain the stability of a large number of rna standards (giulietti et al., ) , absolute quantification was not used in this study. furthermore, the necessity for a standard curve on each assay plate would increase the number of plates required for an experiment, which may become impractical when assaying a large number of test samples. calculation of fold change relative to housekeeping gene(s), with kinetic pcr efficiency adjustment, incorporates corrections for reverse transcription efficiency and pcr efficiency, as well as individual sample addition, by using normalizer housekeeping genes. in our study, although initial assays using dna plasmids demonstrated that the efficiencies of all primer/probe sets in the taqman real time pcr assays were consistent, data from cultures of ferret lymph nodes and our preliminary data with ferret respiratory samples (not shown) indicate that the efficiency of reactions clusters for each set of cdnas and appropriate housekeeping genes need to be determined for each sample type. the importance of careful assessment of suitable housekeeping genes for real time pcr has also been demonstrated for human, canine and ferret tissues (peters et al., ; mane et al., ; bruder et al., ) . as real time pcr measures the ct in the exponential phase of the pcr, it has been argued that the effect of differences in pcr efficiency is minor (giulietti et al., ) . however, we anticipate this taqman assay will be useful for various ferret samples, particularly nasal washes or bronchoalveolar lavages, and respiratory tissues in virus-infected ferrets, which may have low amounts of rna, and variable levels of expression of cytokine and chemokine genes. given the variability in the cellular composition of samples from different sites (e.g. lung tissue compared to lymph node), incorporation of corrections for reverse transcription and pcr efficiency and housekeeping gene variability are necessary in these samples. the cytokine profiles induced by stimulation of lymph node cells from naïve ferrets with mitogens were consistent with those reported in studies using human and other animal leukocytes. stimulation of ferret lymph node cells with cona induced similar profiles to cultures of mouse splenocytes (candolfi et al., ) , human pbmcs (al wabel et al., ; yaqoob and calder, ; radke et al., ) , woodchuck pmbcs (menne et al., ) and calf cd + and cd + pbmcs (tanaka et al., ) stimulated with cona. of interest, cona has been shown to induce expression of il ␤ and low levels of il ␣ in human pbmcs (yaqoob and calder, ), but we did not detect increases in either cytokine in ferret lymph node cells. we also used the other published primers for detecting il ␤ rowe et al., ) with our samples but could not detect expression (data not shown). increased levels of tnf␣, il and ifn␥ were reported following culture of human pbmcs with pha (godoy-ramirez et al., ; anderson and teuber, ) , and we obtained similar results using ferrets cells. ionomycin and pma have been shown to increase production of il , il and ifn␥ in human pbmcs (jung et al., ) and il , il , ifn␥ and tnf␣ in ferret bal, splenocytes or pbls (rutigliano et al., ; martel and aasted, ); all of these genes were also upregulated in this current study following stimulation of ferret leukocytes with either ionomycin or pma. lps is a potent stimulator of il ␣ and ␤, il , and tnf␣ from human pbmcs (al wabel et al., ; yaqoob and calder, ; matera et al., ; coch et al., ) and il and tnf␣ from mouse pbmcs and macrophages (kawai et al., ) . similarly, il ␣ and ␤, il , and il , but not tnf␣, were increased in lps-stimulated ferret lymph node cultures. a similar study in which ferret pbmcs were cultured with lps detected expression of il , ifn␥, il and tnf␣ by taqman real time rt-pcr at earlier timepoints, suggesting the kinetics of the response are important (nakata et al., ) . we also assessed tnf␣ expression using previously published taqman primers and probe (nakata et al., ) and sybr green primers rowe et al., ) with our samples and the profiles were consistent with those obtained with the taqman assay designed in this study (data not shown). stimulation of ferret lymph node cultures with live influenza virus resulted in upregulated expression of ifn␥ and granzyme a, and these markers of t lymphocyte (and nk cell) activation, are also upregulated following stimulation of human pbmcs with influenza virus (forbes et al., ; vanders et al., ) . il was not detected in ferret cells, although it has been detected by flow cytometry in human cells following in vitro influenza virus stimulation (scheible et al., ; guérin-el khourouj et al., ) . increased expression of type i interferons (also seen upon virus stimulation in human pbmcs (forbes et al., ) ) and a corresponding decrease in mcp expression was induced in ferret cells following exposure to influenza virus. this inverse relationship has also been reported in mice co-infected with bacteria and influenza virus and is suggested to be due to type i interferon-mediated suppression of macrophages (nakamura et al., ) . this may also explain the reductions in il and il in ferret lymph node cells cultured with virus as both these mediators would be typically produced by macrophages in the lymph node. the significant down-regulation of ifn␣ and variable expression of mcp- and il p in cultures stimulated with mitogens may also be due to altered activation of cells of the innate immune system. the magnitude and differential patterns of gene expression detected here indicate 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differential sensitivity to glutamine availability tropism and infectivity of influenza virus, including highly pathogenic avian h n virus, in ferret tracheal differentiated primary epithelial cell cultures the authors are grateful for advice provided by dr wa-chin boon, dr john roiniotis, professor paul hertzog and mr steve vander hoorn. the authors are also grateful for technical assistance provided by biocsl limited animal house staff. the authors also acknowledge the contribution of gifts of reagents from dr liyen loh and ms sarina camuglia. the melbourne who collaborating centre for reference and research on influenza is supported by the australian government department of health. key: cord- -ruuzr l authors: garnett, lauren; bello, alexander; tran, kaylie n.; audet, jonathan; leung, anders; schiffman, zachary; griffin, bryan d.; tailor, nikesh; kobasa, darwyn; strong, james e. title: comparison analysis of different swabs and transport mediums suitable for sars-cov- testing following shortages date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: ruuzr l on march , , the world health organization (who) assessed covid- , caused by sars-cov- , as a pandemic. as of june , , sars-cov- has had a documented effect of over million cases world-wide, amounting to over , deaths (world health organization, . novel coronavirus (covid- ) situation. http://https://covid .who.int/). consequently, the high demand for testing has resulted in a depletion of commercially available consumables, including the recommended swabs and viral transport media (vtm) required for nasopharyngeal sampling. therefore, the potential use of unvalidated alternatives must be explored to address the global shortage of testing supplies. to tackle this issue, we evaluated the utility of different swabs and transport mediums for the molecular detection of sars-cov- . this study compared the performance of six swabs commonly found in primary and tertiary health care settings (purflock ultra, floqswab, puritan pur-wraps cotton tipped applicators, puritan polyester tipped applicators, medpro ” cotton tipped applicators, and hologic aptima) for their efficacy in testing for sars-cov- . separately, the molecular detection of sars-cov- was completed from different transport mediums (dmem, pbs, % ethanol, . % normal saline and vtm), which were kept up to three days at room temperature (rt). the results indicate that there is no meaningful difference in viral yield from different swabs and most transport mediums for the collection and detection of sars-cov- , indicating swab and medium alternatives could be used if supplies run out. in december , a cluster of acute respiratory illnesses were reported in wuhan, hubei province, china, initially classified as 'pneumonia of unknown etiology' (lu et al., ) . the etiology has since been attributed to a novel positive-sense rna virus from the coronaviridae family, named severe acute respiratory syndrome coronavirus (sars-cov- ), the causative agent of coronavirus disease . the clinical spectrum of sars-cov- infections can range from asymptomatic/mild, moderate, severe to critical (lipsitch et al., ; wang et al., ) . mild patients present minimal symptoms with no radiographic features; moderate patients exhibit fever, respiratory symptoms and radiographic features; whereas severe patients demonstrate dyspnea, reduced oxygen saturation (< %), or reduced pao /fio (< mmhg); and critical patient meet one of the following criteria: respiratory failure, septic shock or multi-system organ failure . an important finding related to the global spread of sars-cov- is that people can be highly infectious and shed virus during the asymptomatic phase . due to fast global spread and high morbidity and mortality rates, the world health organization (who) declared covid- a pandemic. data provided by the who health emergency dashboard (june , ) shows more than million reported cases and over , associated deaths have occurred since the beginning of the pandemic, high-lighting the highly infectious nature of sars-cov- (world health organization., ). most countries established clinical and epidemiological screening criteria to determine if testing a patient for sars-cov- is warranted. screening criteria are implemented to ensure diagnostic testing is available for patients who have high pretest probability and for which the results will impact public health management. if screening criteria for testing is met, the who recommends collecting samples from the upper respiratory tract, including naso-(np) or oropharyngeal (op) swabs. if np or op swabs cannot be obtained, lower respiratory tract samples (endotracheal aspirate, expectorated sputum or bronchoalveolar lavage) can be submitted (centre for disease control and prevention, ) . in terms of np collection, the center for disease control and prevention (cdc) has stated that np swabs must be completed using synthetic fiber swabs with plastic shafts, due to the deleterious effect that calcium alginate swabs or swabs with wooden shaft may have on virus inactivation or polymerase chain reaction (pcr). the cdc also recommends that all swabs are to be transported in - ml of viral transport media (vtm) (centre for disease control and prevention, ). more recently, countries with more liberal testing paradigms, have noted better success in managing this pandemic. while unlimited testing is unrealistic, expanded testing, not limited to individuals with symptoms, those in hospitals, long-term care facilities, travelers, and health workers, have made early recognition, contact tracing, isolation and management more effective. this high demand for testing has depleted key supply stocks, including recommended testing swabs and transport medium. this study aimed to examine the efficacy of six different swabs that are commonly found in hospital settings (purflock ultra, floqswab, puritan pur-wraps cotton tipped applicators, puritan polyester tipped applicators, medpro " cotton tipped applicators, and hologic aptima), along with more readily available alternative transport mediums (dmem, pbs, % ethanol, . % normal saline and vtm) for their use in molecular detection of sars-cov- . to compare the amount of fluid each swab could retain after being dipped and rotated in dmem, µl of medium was placed into a ml cryovial and weighed on an analytical scale. each swab, in its own cryovial, was dipped and rotated making sure all sides of the tip were coated. the swab was then taken out and remaining medium was weighed. each swab was tested separate times and the mean volume of media retained (µl) was used to help determine virus recovery efficiency (table ) . to test the efficacy of each swab to detect sars-cov- , virus was serially diluted -fold in dmem for concentrations of . x down to . x - pfu/ ml. l of each virus dilution was then dispensed into separate sterile reagent reservoir trays (corning inc, ). each swab tip was submerged into the separate virus dilutions and rotated, ensuring the tip was entirely coated. the swabs were then placed into ml cryovials containing µl plain dmem until the remainder of the swabs and dilutions were completed and virus inactivation initiated following the protocol outlined below. all swab j o u r n a l p r e -p r o o f testing was completed in two biological replicates. as a comparison control, samples from each virus dilution were inactivated and extracted without the use of a swab. to test sars-cov- viral yield from different transport mediums over time, sars-cov- was serially diluted into plain dmem for concentrations of . x and . x . subsequently, µl of each virus dilution was then placed into separate sterile reagent reservoir trays (corning incorporated, ). using a medpro " cotton tipped applicator (a.m.g medical inc, canada), the tip of the swab was immersed into the virus dilution aliquot and rotated to absorb as much virus dilution as possible. the swab was then placed into a ml cryovial containing l of either dmem, pbs, . % normal saline, % ethanol or vtm and the shaft was broken off to allow capping of the ml cryovial tube. the tips were left in the various transport mediums at rt until time of virus inactivation at , , , and hours postinoculation. each transport medium time point was completed in two biological replicates. the vtm used in this study was prepared following protocols from lennette et al. (lennette, . as a control comparison for all mediums, l of the virus dilution was placed into ml cryovials and inactivated and extracted at the same time-points as the swab samples. all experiments with live sars-cov- were performed in a biosafety level (bsl) laboratory. to remove samples from bsl for further analysis, µl of sample was inactivated in µl buffer avl for minutes, then the contents were transferred to a tube containing µl % ethanol for an additional . rt-qpcr was performed using the lightcycler rna master hydolysis probes kit and run on a quantstudio rt-qpcr system to measure the quantification cycle (cq). the data were analyzed using two models. a -parameter logistic curve was fit to the dose-cq data for the different swabs. a linear model was fit to the time-cq data for the recovery media. the swab absorption volumes are modeled as student t-distributed, with each swab having its own mean and standard deviation, with shared degrees of freedom. in all cases, the models were fit using a bayesian framework and sampled using r (stan development team, ). the details of each model are provided in the supplementary statistical analysis. each swab was dipped in serially diluted sars-cov- and then broken off into cryovial tubes containing l dmem. the viral medium was then inactivated, and the rna was extracted for analysis by rt-qpcr. our data shows that all swab types (purflock ultra, floqswab, puritan pur-wraps cotton tipped applicators, puritan polyester tipped applicators, medpro " cotton tipped applicators, and hologic aptima) give similar yields of sars-cov- rna when compared to each other at all virus dilutions ( figure ). we also determined how much volume of media each swab could retain (table ) and used this information to calculate the efficacy at which the swabs are able to pick up virus from the dilutions and elute into the media. although each swab gave similar levels of rna at each dilution (figure ), the volume that each swab was able to retain and elute varied giving a range in percentage of virus recovery (table ) . comparison of the different transport media was done at two virus dilutions, . x , . x pfu/ml respectively. these concentrations were chosen as they span the upper and lower cycle threshold limits of the assay. sars-cov- was diluted in dmem, medpro " cotton tipped applicators were dipped in each dilution and then broken off into ml cryovials containing either dmem, % ethanol, pbs, j o u r n a l p r e -p r o o f . % normal saline or vtm and left at rt for up to hours ( days). our results indicate that dmem, % ethanol, pbs, and vtm eluted and preserved similar amounts of viral rna for molecular diagnostics at both concentrations ( figure ). however, . % normal saline did show substantial loss of detectable rna over time. molecular based assays such as rt-qpcr have supplanted viral culture as the reference assay and the most widely used modality for diagnostics of viral respiratory pathogens such as sars-cov- . however, effective specimen collection and transport to centralized laboratories is required, and as exemplified by the current covid- pandemic, recommended materials may be limited. this study addressed the issue of the shortage of swabs and transport mediums by evaluating unvalidated alternatives. we found little variation between different swabs and media for the molecular detection of sars-cov- . we were unable to find other studies comparing different types of swabs and transport media for the efficacy of detecting for sars-cov- under controlled conditions. comparison of different swab types have been completed in the past for other respiratory viruses. np and op testing using nylon swabs or rayon, a material similar to cotton, has shown that nylon swabs show better viral genome yield compared to rayon swabs (daley et al., ; hernes et al., ) . another study assessing cotton versus flocked swabs found that both retained virus at room tempreature for days, allowing for successful extraction and detection; however, the flocked swab did allow for consistently higher yields (moore et al., ) . for all respiratory sampling, including sars-cov- , the cdc does not recommend the use of cotton or wood shafted swabs as it is difficult to elute virus and the wooden shaft can absorb elution buffer, thereby inhibiting pcr (centre for disease control and prevention, , ) . we, however, did not observe any significant decrease in sars-cov- yield with the use of wooden shafted cotton swabs compared to a synthetic fiber swab (figure ) . therefore, our results suggest that the cotton and wood for the portion of the study focusing on alternative transport media, we assessed the ability of dmem, pbs, . % normal saline, and % ethanol compared to vtm to be used as medium for the preservation and recovery of viral rna to be quantified by molecular detection. our results indicate that all media, with the exception of . % saline, preserved similar amounts of sars-cov- rna for extraction and detection over hours (figure ). although our study focused on if virus yield decreased over time, it should be noted that there are slight differences in the virus recovery when comparing the different mediums to each other. from this data it seems that % ethanol has the best virus recovery while still maintaining stable viral rna levels over hours. this is possibly due to its denaturing effects on the virus, changing the way it interacts with the fibers of the swab allowing it to elute into the transport medium at a higher efficiency. these results indicate the % ethanol may be the best recommended transport medium in the event that viral transport medium stocks are depleted. supporting the results of our study, another study compared vtm to saline, universal transport medium (utm), liquid amies medium and amies gel containing charcoal, finding slight differences in yield but resulting in all media able to give amplifiable product over days (druce et al., ) . additionally, ethanol-based transport medium (cymol) has been studied, finding that it preserved influenza viral rna for at least days at temperatures from c to rt (luinstra et al., ) . to ensure virus-spiked fluid is acceptable to use to recapitulate actual human samples (for retention and release as well as for the presence of pcr inhibitors), we also tested the most relevant biological fluid (np swab sample obtained from a volunteer). this was chosen since, np samples are widely regarded as the 'gold-standard' in most labs/testing facilities. to this end, we have tested control np swab samples spiked with sars-cov- and found that the addition of the np swab sample made no difference to the rt-qpcr results when compared to similarly spiked control samples lacking the human np swab sample (unpublished data). it should also be noted that this study did not test the viability of the different swab collections and transport media alternatives to retain culturable virus. while % ethanol precludes culture, the other recovery media may not be able to deal with potential bacterial, fungal, protease and nuclease contamination that may be associated with samples from the respiratory tract. this may be an important consideration as this outbreak moves into a different phase that may rely on determining infectious risks of patients and timing of disease onset/recovery (bullard et al., ) . despite finding similar levels of viral rna collected using different swabs and transport media, there is variation when evaluating different respiratory clinical samples while testing for sars-cov- . comparing np swabs, op swabs and sputum at different points in infection, studies have found that sputum samples generally showed the highest viral loads (lin et al., ; pan et al., ) . these conclusions were also seen from a detailed study which found that / bronchoalveolar lavage (bal) and / sputum samples were positive for sars-cov- , compared to / nasal swabs, / brush biopsies and / pharyngeal swabs (kaul, ) . another study found that sputum induction was more sensitive in detection of sars-cov- rna when compared to throat swabs in convalescent patients (han et al., ) . however, it should be noted that most jurisdictions are discouraging induced sputum due to the generation of aerosols and potential associated spread. comparing np and op swabs for sars-cov- molecular diagnostics, it has been found that np swabs gave significantly stronger positive tests than op swabs from the same patient from day to day + of symptom onset . alternatively, a study evaluating sars-cov- patients in germany found that both np and op swabs gave similar pcrpositive results on days - post symptom onset (woelfel et al., ) . it is of paramount importance to identify different swab and transport medium alternatives for the molecular detection of sars-cov- as supplies are limited during this unprecedented pandemic. our observations suggest that, with the exception off . % saline, the collection swabs and transport medium types we tested are appropriate options for collection, transport and preservation of sars-cov- rna for molecular detection. it should be noted that a major limitation of this study is not testing these alternatives on actual patients or human volunteers. our artificial swabbing environment compared to swabbing j o u r n a l p r e -p r o o f predicting infectious sars-cov- from diagnostic samples interim guidelines for collecting, handling, and testing clinical specimens from persons for coronavirus disease (covid- ) [www document collecting specimens for varicella zoster virus (vzv) testing detection of novel coronavirus ( -ncov) by realtime rt-pcr comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients evaluation of swabs, transport media, and specimen transport conditions for optimal detection of viruses by pcr sars-cov- rna more readily detected in induced sputum than in throat swabs of convalescent covid- patients swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs pharyngeal and nasal swabs may not have adequate sensitivity for sars-cov- . new engl collection and preparation of specimens for virological examination comparison of throat swabs and sputum specimens for viral nucleic acid detection in cases of novel coronavirus (sars-cov- ) infected pneumonia defining the epidemiology of covid- -studies needed outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle mahony, inactivation of respiratory viruses dry cotton or flocked respiratory swabs as a simple collection technique for the molecular detection of respiratory viruses using real-time nasba viral load of sars-cov- in clinical samples unique epidemiological and clinical features of the emerging novel coronavirus pneumonia (covid- ) implicate special control measures presymptomatic transmission of sars-cov- -singapore virological assessment of hospitalized cases of coronavirus disease novel coronavirus (covid- ) situation [www document evaluating the accuracy of different respiratory specimens in the laboratory j o u r n a l p r e -p r o o f diagnosis and monitoring the viral shedding of -ncov infections key: cord- -fvdq yes authors: wang, jinfeng; wang, jianchang; zhang, ruoxi; liu, libing; shi, ruihan; han, qingan; yuan, wanzhe title: rapid detection of transmissible gastroenteritis virus in swine small intestine samples using real-time reverse transcription recombinase polymerase amplification date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: fvdq yes a rapid and specific real-time reverse-transcription recombinase polymerase amplification assay (rt-rpa) was developed to detect the transmissible gastroenteritis virus (tgev) in this study. the primers and exo probe were designed to be specific for a portion of spike (s) gene conserved in tgev, but absent in the closely related porcine respiratory coronavirus (prcv). the amplification was performed at °c for min. the assay could only detect the tgev, and there was no cross-reaction with other pathogens tested. using the in vitro transcribed tgev rna as template, the limit of detection of the developed rt-rpa was copies per reaction. the assay performance was evaluated by testing clinical samples by rt-rpa and a real-time rt-pcr. fourteen samples were tgev rna positive in rt-rpa ( . %, / ), which were also positive in the real-time rt-pcr. the diagnostic agreement between the two assays was % ( / ). the r( ) value of rt-rpa and real-time rt-pcr was . by linear regression analysis. the developed rt-rpa assay provides a useful alternative tool for rapid, simple and reliable detection of tgev in resource-limited diagnostic laboratories and on-site facilities. transmissible gastroenteritis (tge) is an acute enteric viral disease of pigs resulting in vomiting and diarrhea in all ages of pigs and with high mortality rate in piglets (garwes, ) . the disease is caused by tge virus (tgev), which is an enveloped, single-stranded, positivesense rna virus belonging to the genus alphacoronavirus of the family coronaviridae (saif and wesley, ) . tge was first reported in in the united states, and tgev was first isolated and identified in (doyle and hutchings, ) . since then, tgev has spread throughout the world including america, europe and asia, and has caused significant economic loss in the pig industry (kim et al., a; stevenson et al., ) . since the mid s, a variant respiratory form of the tgev known as porcine respiratory coronavirus (prcv) has become common in pigs (laude et al., ) . compared to tgev, prcv has a deletion of between and nucleotides near the ′ end of the spike (s) gene (laude et al., ) . although prcv replicates predominantly in the respiratory tract, some pigs infected with prcv could also shed the virus in their feces (costantini et al., ; saif and wesley, ) . therefore, any diagnostic assay for tgev should be able to differentiate it from prcv. rapid and specific detection of tgev would be extremely important in the prevention and control of tge. the conventional methods for detection of tgev are time-consuming, laborious, and requiring welltrained technicians, such as virus isolation, immunofluorescence assay, electron microscopy and elisa (carman et al., ; dulac et al., ; van nieuwstadt et al., ) . a series of developed rt-pcr assays for pedv require the high-precision, sophisticated and expensive instruments, well-trained technicians and good laboratory circumstance, thus unsuitable for being applied in under-equipped laboratories and on-site applications, such as rt-pcr (paton et al., ) , nanoparticle-assisted rt-pcr (zhu et al., ) and real-time rt-pcr (vemulapalli et al., ) . recently, rt-lamp assays have been developed for tgev detection (chen et al., ; li and ren, ) . six primers were needed in the rt-lamp assays for tgev, the reaction time was or min, and the results were visualized by agarose gel electrophoresis (chen et al., ; li and ren, t recombinase polymerase amplification (rpa) is an isothermal gene amplification technique that has been demonstrated to be a rapid, specific, sensitive, and cost-effective molecular diagnostic method (daher et al., ; piepenburg et al., ) . rpa employs three core enzymes: a recombinase, a single-stranded dna-binding protein (ssb) and a strand-displacing polymerase. the recombinase forms a nucleoprotein filament with primers and probes. this filament scans the double-stranded dna (dsdna) target searching for homologous sequences and invades the dsdna once homology is found. then a d-loop structure is formed, which is a local separation of dna strands in which the complementary strand is stabilized by ssb and the target strand is hybridized with primer. recombinase disassembly from the nucleoprotein filament, the strand-displacing dna polymerase adds bases to the ′-end of the primer and extension occurs (daher et al., ; piepenburg et al., ) . real time detection of rpa amplicons could be performed through adding exonuclease iii and exo probes to the reaction mixture. the exo probe typically consist of an oligonucleotide backbone that contains an abasic nucleotide analogue (tetrahydrofuran residue or thf) flanked by a dt-fluorophore and a corresponding dtquencher group. in a double-stranded context the thf residue presents a substrate for exonuclease iii, which will cleave the probe at the thf position, thereby separating the fluorophore and the quencher and generating a fluorescent signal. real-time rpa assays had been developed for the detection of a series of important viruses in swine (wang et al., a,b) . in this study, we developed and evaluated the userfriendly on-site detection platform integrating the real-time rpa technology and a field-deployable device (genie iii tube scanner) for the rapid detection of tgev. the genomic rna or dna of tgev (strain hb-yx), procine epidemic diarrhea virus (pedv, strain jscz ), porcine rotavirus (porv, strain hb-bd/ ), prcv, porcine deltacoronavirus (pdcov), lawsonia intracellularis (strain lx ), porcine circovirus (pcv , strain hb-mc ) and porcine parvovirus (ppv, strain bj- ) were kept in our laboratory. seventy-six small intestinal samples from the piglets with signs of severe watery diarrhea, dehydration were collected from eleven pig farms in hebei province between april and february . the piglets were - days old. the samples were homogenized with phosphate-buffered saline (pbs, ph . ) as a % (w/v) suspension and centrifuged for min at g at °c. two hundreds microliter of the supernatant was used for rna extraction using the trizol reagent. all the sample rna were quantified using nd- c spectrophotometer (nanodrop, wilmington, usa), and were stored at − °c until use. the bp rt-pcr product encompassing the tgev-specific s gene was generated from viral genomic rna extracted from strain hb-yx using f and r primers (paton et al., ) . the resulting fragment was ligated into a pgem-t easy vector and transformed into e.coli dh α chemically competent cells according to standard procedures. the in vitro transcribed tgev standard rna was produced with ribomax large scale rna production system-t (promega, madison, usa). the length from the t promoter region to the ndei cut site of the pgem-t easy vector is nucleotides, generating a total transcript of nucleotides in length. the in vitro transcripts were quantified using nd- c and the copy number of rna molecules was calculated by the following formula: amount (copies/μl) = [rna concentration (g/ μl) /(transcript length in nucleotides × )] × . × . the in vitro transcribed rna was diluted in ten-fold serial dilutions to achieve rna concentrations ranging from . × to . × °copies/μl, which were used as the standard rna for tgev rt-rpa assay. according to the s gene nucleotides of different tgevs (accession numbers: af ; af ; af ; ay ; af ; af ; hq ; m ; m ; s ; z ) and prcvs (accession numbers: kr ; m ; m ; m ; m ) available in genbank, the rt-rpa primers and exo probe were designed to be specific to a portion of the s gene sequences conserved in tgev but absent in prcv. the forward primer was: ´-ttc agaggcaaattgtggtaatatgctgtatggc- ´; the exo probe was: ´-gcagatgaggttgttgcttatttacatgg(rox-dt)g(thf)(bhq -dt)agttaccgtattag-c spacer- ´; the reverse primer was ´-acg catatcaccaaatgtgacagtgccagacca- ´. the primers and probe were synthesized by a commercial company (sangon, shanghai, china), and the length of the amplicon was bp. rt-rpa reactions were performed in a total volume of μl containing . μl rehydration buffer and . μl magnesium acetate ( mm) from the twistamp tm rt exo kit (twistdx, cambridge, uk). other components included nm each rpa primer, nm exo probe, and μl of viral rna or μl sample rna. the rt-rpa reactions were performed at °c for min in the genie iii scanner device. ten nanograms of the different pathogen genomic rna or dna were used as template in the rt-rpa, and only the tgev rna were detected by rt-rpa while the other pathogens were not detected (fig. ) . no cross detections were observed in five independent reactions, demonstrating the high specificity of rt-rpa assay for the detection of tgev. in the analytical sensitivity analysis, the limit of detection (lod) of the rt-rpa was . × copies per reaction, which was the same as the realtime rt-pcr (fig. ) . the two-fold serial dilutions of the in vitro transcribed tgev rna were made from . × to . × copies, and from . × to . × copies, respectively. the above two-fold serial dilutions of the tgev rna, and . × copies in vitro transcribed tgev rna were further used in the rt-rpa, and the lod of the assay still was copies per reaction. the threshold time (tt) values were . min and . min for copies and copies, while there were no fluorescence amplification curves for . copies and copies. the rt-rpa assay was performed five times on the quantitative rna with similar results. for evaluating the potential applicability of the developed rt-rpa assay, clinical samples had rna extracted and were tested by the rt-rpa and real-time rt-pcr (vemulapalli et al., ) . fourteen samples ( . %, / ) were tgev rna positive in the rt-rpa, which were also positive in the real-time rt-pcr. the overall agreement between the rt-rpa and the real-time rt-pcr was % ( / ). the basic rpa assay were also performed for the tgev rna positive samples using the twistamptm rt basic kit (twistdx, cambridge, uk), and the reaction system was the same as rt-rpa except the exo porbe was not used. the rpa amplification products were sequenced by a commercial company (sangon, shanghai, china), then were blasted in the genbank. the blast results demonstrated that all the sequences showed more than . % homology to tgev strains in genbank, which confirmed the good specificity of the rt-rpa assay for tgev. for the positive samples, it took − min in the rt-rpa to see amplification, while it took - min in the real-time rt-pcr with the ct values ranging from . to . . the performance of the rt-rpa was comparable to real-time rt-pcr, and the former is much faster. the threshold time (tt) and cycle threshold (ct) values of the two assays were respectively well at an r value of . (fig. ) . the molecular diagnostic assays, which could be suitable for deployment closer to suspect cases of tge, would be of significant importance in the tge controlling. recent advances of the rpa technology on the portable tube scanner device provide a simple, convenient and inexpensive method for the point-of-care (poc) detection of pathogens in clinical samples (abd el wahed et al., a,b) . in this study, we developed a rt-rpa platform integrating the real-time rpa technology and a tube scanner genie iii for the rapid and sensitive detection of tgev. considering the close relationship between tgev and prcv, multiple alignment of the tgev and prcv s gene sequences were made, and a nucleotide-long region corresponding to sequence between nucleotides and of the tgev s gene orf, which is absent from all prcv strains, was set as the target of rt-rpa. other tgev strains were not included in the assay except for the isolated field strain hb-yx, which is the deficiency of this study. the rpa is tolerant to - mismatches in primer and probe showing no influence on the performance of the assay (abd el wahed et al., ; daher et al., ) , and there were only - mismatches in this study with other circulating tgev strains available in genbank. it is assumed the assay would detect all the circulating strains of tgev, based on the facts that the rt-rpa assay targeted the conserved s gene of tgev. the tube scanner genie iii weighs only . kg and incorporates a rechargeable battery that could support operation for a whole day. it is especially important for tgev detection in under-equipped laboratories and in the field. the user-friendly on-site detection platform offers an excellent tool for initial screening of clinical specimens for tgev, which should make valuable contributions to the rapid diagnosis of tge. the real-time rt-pcr required the expensive thermal cycler (vemulapalli et al., ) , and the rt-lamp were inconvenient to perform due to the agarose gel electrophoresis (chen et al., ; li and ren, ) . the developed rt-rpa assay needed no complicated apparatus, was performed easily and completed within min, showing distinct advantages in terms of equipment requirements, convenience and detection time. in conclusion, a user-friendly on-site detection platform for tgev was developed. the good analytical specificity and sensitivity, simple and easy operation protocol make the platform ideal for the rapid detection of tgev in under-equipped laboratories and the promising tool for poc detection of tgev, especially in the resource-limited settings. all authors declare that they have no conflicts of interest. a portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus recombinase polymerase amplification assay for rapid diagnostics of dengue infection diagnostics-in-a-suitcase: development of a portable and rapid assay for the detection of the emerging avian influenza a (h n ) virus field 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interactions reverse transcription loop-mediated isothermal amplification for rapid detection of transmissible gastroenteritis virus detection of transmissible gastroenteritis virus by rt-pcr and differentiation from porcine respiratory coronavirus dna detection using recombination proteins transmissible gastroenteritis and porcine respiratory coronavirus emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences solid phase immune electron microscopy for diagnosis of transmissible gastroenteritis in pigs a real-time taqman rt-pcr assay with an internal amplification control for rapid detection of transmissible gastroenteritis virus in swine fecal samples an exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus reverse transcription recombinase polymerase amplification assay for the rapid detection of type porcine reproductive and respiratory syndrome virus a sensitive duplex nanoparticle-assisted pcr assay for identifying porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus from clinical specimens this work was supported by natural science foundation youth project of hebei province (c ) and science and technology project foundation of hebei province ( d) and partially funded by the fund for one-hundred outstanding innovative talents from hebei institution of higher learning (slrc ). key: cord- -zlah u s authors: günther, sonja; felten, sandra; wess, gerhard; hartmann, katrin; weber, karin title: detection of feline coronavirus in effusions of cats with and without feline infectious peritonitis using loop-mediated isothermal amplification date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zlah u s feline infectious peritonitis (fip) is a fatal disease in cats worldwide. the aim of this study was to test two commercially available reaction mixtures in a reverse transcription loop-mediated isothermal amplification (rt-lamp) assay to detect feline coronavirus (fcov) in body cavity effusions of cats with and without fip, in order to minimize the time from sampling to obtaining results. rna was extracted from body cavity effusion samples of cats, including samples from cats with a definitive diagnosis of fip, and samples of control cats with similar clinical signs but other confirmed diseases. two reaction mixtures (isothermal mastermix, optigene ltd.and pcrun™ molecular detection mix, biogal) were tested using the same primers, which were designed to bind to a conserved region of the fcov membrane protein gene. both assays were conducted under isothermal conditions ( °c– °c). using the isothermal mastermix of optigene ltd., amplification times ranged from and min with a sensitivity of . % and a specificity of . % for the reported sample group. using the pcrun™ molecular detection mix of biogal, amplification times ranged from to min with a sensitivity of . % and a specificity of . %. although the rt-lamp assay is less sensitive than real time reverse transcription pcr (rt-pcr), it can be performed without the need of expensive equipment and with less hands-on time. further modifications of primers might lead to a suitable in-house test and accelerate the diagnosis of fip. feline coronavirus (fcov), a member of the genus alphacoronavirus of the subfamily coronavirinae, family coronaviridae within the order nidovirales (de groot et al., ) , belongs to a group of enveloped, positive-sense rna viruses that cause diseases in several species, such as severe acute respiratory syndrome (sars) in humans or transmissible gastroenteritis (tge) in pigs. despite the high prevalence of fcov infections in the cat population worldwide, only - % of fcov-infected cats develop the fatal disease feline infectious peritonitis (fip) (addie and jarrett, ) . this change of virulence of a harmless fcov biotype that usually causes no clinical signs into the pathogenic variant is thought to be caused by mutations in the fcov spike protein gene (chang et al., ; vennema et al., ) . these mutations cause a change in tropism from enterocytes to macrophages, giving fcov the ability to infect and effectively replicate within cells of the macrophage lineage and cause a lethal systemic disease with multi-organ involvement (pedersen, ) . the median survival time of cats with effusive fip is only a few days (ritz et al., ) , and the diagnosis of fip commonly leads to euthanasia, since to date, no treatment has been proven to be effective. cats with fip show nonspecific clinical signs such as fever, weight loss and anorexia, often accompanied by body cavity effusions and/or ocular and neurological signs. a definitive diagnosis of fip ante mortem remains challenging, especially when no body cavity effusions can be detected (hartmann et al., ) . presently, the gold standard for the diagnosis of fip is considered to be immunostaining of fcov antigen in macrophages within tissue lesions, a technique that requires invasive tissue collection (kipar and meli, ) . in cats with fip, fcov can be detected by rt-pcr in cell-free body cavity effusions in more than % of the cases, while serum or blood samples often are negative (doenges et al., ) . for both immunostaining and rt-pcr, samples have to be sent to specialized laboratories, resulting in the delay of diagnostic results. this leads to further unnecessary testing for other diseases, to withholding necessary therapy of other treatable diseases, or to delayed euthanasia in cats suffering from severe signs of fip. therefore, a fast and simple point of care test would be very beneficial in the diagnostic process. loop-mediated isothermal amplification (lamp) is a simple, rapid, and cost-effective nucleic acid amplification method (notomi et al., ) and is already used for the detection of coronaviruses in humans and several animal species (hong et al., ; nemoto et al., ) . a set of four to six primers is used, that form products with self-hybridizing loop structures. by using a dna polymerase with strand displacement activity, no melting or annealing steps are required, and amplification products of different lengths are formed at a constant temperature of - °c (nagamine et al., ) . since lamp reactions only require a simple heat block with constant temperature, and dna amplification can be detected by fluorescence or color change, the method can be applied for point-of-care diagnostics (surabattula et al., ) . the aim of this study was to test specificity and sensitivity of two commercially available reaction mixtures in a reverse transcription lamp (rt-lamp) to detect fcov in body cavity effusions of cats with and without fip, and to minimize the time from sampling to obtaining results. this study included cats that were presented to the clinic of small animal internal medicine, lmu munich, germany. all cats included had body cavity effusions. in every cat presenting with body cavity effusions, fip is a potential differential diagnosis. an earlier study showed that fip is responsible for about % of effusions, while most of the remaining cases were caused by malignomas, cardiac insufficiency or purulent serositis (hirschberger et al., ) . the fip group (n = ) included cats with a definitive diagnosis of fip by one or more methods: all effusions of cats with fip tested positive for fcov by rt-pcr by a commercial laboratory, and in / samples putative disease-causing mutations could be detected. the rt-pcr detection method has been described previously (felten et al., ) . in / cats fip diagnosis was achieved by post-mortem examination, including full body necropsy with histopathological examination. diagnosis of fip was confirmed when typical histologic lesions where detected (surfacebound multi-systemic pyogranulomatous and fibrinonecrotic disease with venulitis with or without high-protein exudate). in / cats with full body necropsy immunohistological staining for fcov-antibody was done on tissue sections and returned a positive result. immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in / cats, and all samples returned a positive result. a summary of the cases in the fip group can be found in the supplementary table . cats were included in the control group (n = ) if they were definitively diagnosed with a disease other than fip that explained the effusion. cats of the control group suffered from neoplasia (n = ), decompensated cardiac diseases (n = ), inflammatory diseases (n = ), such as bacterial peritonitis and pleurisy, or other diseases (n = ). one cat had chronic thoracic chylous effusion of unknown origin and secondary fibroplastic pleurisy. in another cat, an end stage kidney disease caused effusion, and one cat had thoracic effusion after subcutaneous urethral bypass placement, which resolved after treatment. the diseases of the cats of the control group (n = ) were definitively confirmed ante-mortem (n = ) or at necropsy with histopathological examination (n = ). ante-mortem diagnosis was established by echocardiography for cardiac diseases (n = ), and by cytology for neoplasia (n = ). immunofluorescent staining of fcov antigen in macrophages of thoracic or abdominal effusion was done in / cats, with three positive and eight negative results. all effusions of the cats in the control group were tested for fcov by rt-pcr, and all results were negative. the rt-pcr detection method has been described previously (felten et al., ) . a summary of the cases in the control group can be found in supplementary table . body cavity effusion samples of all cats were obtained ante mortem with ultrasound guidance for diagnostic purposes. the use of samples for this study was approved by the institutional animal care and use committee ('ethikkommission des zentrums für klinische tiermedizin'), permission number - - - . samples were stored at - °c in a . ml eppendorf safe-lock microcentrifuge tube until assayed. all samples were centrifuged for s at , × g. the supernatant of centrifuged thoracic and abdominal fluids was used for rna extraction. when using fresh fluid samples, omission of the centrifugation step should be considered to include intact cells with a high viral burden (pedersen et al., ) . in thawed samples, cell integrity is lost and cell debris can be removed. viral rna was isolated using the commercial zr viral rna kit™ (zymo research corp.) following the manufacturer's instructions. briefly, μl aliquots of samples were mixed with a buffer that facilitates viral particle lysis and allows for rna adsorption onto the matrix of the zymo-spin™ column. then the rna was washed and eluted with μl of rnase free water. extracted rna aliquots were stored at − °c in an eppendorf . ml safe-lock microcentrifuge tube until further processing. the rt-lamp primer design was assisted by the software primerexplorer (https://primerexplorer.jp/e/). based on sequence analysis, the gene for the membrane protein (m) was selected as a target because it is highly conserved among fcov strains. the dna sequence from position , to , of the fcov strain black (genbank accession number: eu . ) was used to design the rt-lamp primers used in this study. a set of six primers, including two outer primers (forward primer f and backward primer b ), two inner primers (forward inner primer fip and backward inner primer bip), and two loop primers (forward loop primer loopf and backward loop primer loopb) were selected as the target sequence ( fig. and table ). detection of fcov was performed using rt-lamp. two different commercial reaction mixtures (isothermal mastermix by optigene ltd., uk, pcrun™ molecular detection mix by biogal, israel,) were compared using the same set of primers. for the amplification following the isothermal mastermix protocol, the total volume of μl per reaction tube included μl isothermal master mix, μl template, μl primer mix and . μl superscript® iii reverse transcriptase (thermo scientific). the primer mix consisted of pmol each of f and b primers, pmol each of fip and bip primers and pmol each of loopf and loopb primers. for negative control, μl water were added instead of μl template. the reaction mix was incubated at °c for min in a real-time pcr system (applied biosystems). during rt-lamp, fluorescence of dna products was measured once every minute (fam detection channel, λ max nm), and the time to threshold crossing was analyzed. a positive sample (positive in rt-pcr and sequenced for mutations) was included in every run. all samples run in the realtime pcr system were subjected to a melt curve analysis after the run. a single sharp peak in the melt curve analysis demonstrates amplification of a single pcr product. the positive samples all showed a single peak and the same melting temperature as the positive control sample. following the pcrun™ molecular detection protocol the reaction mix contained . μl of luminescent reagent, μl of template, μl of primer mix and additional . μl superscript iii reverse transcriptase (thermo scientific). the primer mix consisted of . pmol each of f and b primers, pmol each of fip and bip primers and pmol each of loopf and loopb primers. for negative control, μl water were added instead of μl template. the reaction mix was incubated at °c for min in a pcrun™ reader (biogal). amplification was detected by measuring bioluminescence twice every minute (fig. ) . pcr products of both methods were verified on an agarose gel showing a typical pattern of multiple bands of different molecular weights (fig. ) . the results of the samples in the fcov rt-lamp assays using two different commercial reaction mixtures are shown in table . two samples tested false positive using the isothermal mastermix and one sample tested false positive with the pcrun™ molecular detection mix. sensitivity and specificity for the reported sample groups of both rt-lamp assays are shown in table . the pcrun™ molecular detection mix performed better both in sensitivity and specifity than the isothermal mastermix. the amplification times for the isothermal mastermix positives ranged between and min and for the pcrun™ molecular detection mix positives between - min. in the present study, rt-lamp assays were evaluated as a diagnostic tool for detection of fcov, in order to distinguish cats with and without fip that are presented with body cavity effusions. time from sample to result was kept to a minimum by isolating rna in about to min using a simple rna extraction kit. both commercially available reaction mixtures allowed an easy and fast preparation of the amplification reaction. with lamp assays, direct detection methods built into the amplification device are preferred, since opening lamp reaction tubes after amplification is not advisable to decrease the risk of carry-over contamination (parida et al., ; zanoli and spoto, ) . in the present study, a portable device results was used for the pcrun™ molecular detection mix. positive and negative amplification reactions were indicated by the device with '+' and '-', making it compatible as a point-of-care instrument. the isothermal mastermix is intended for use with a portable device, which was not part of this study. the machine used instead replicates the reaction conditions with a constant block temperature and uses a comparable fluorescence detection system. both assays tested have similar demands concerning handling skills and preparation time. detection of positive samples took about half the time with isothermal mastermix compared to the pcrun™ molecular detection mix, yet both tests require less than min. the specificity of both methods for the reported sample group was comparable, with . % and . %, respectively. however, false positive results in a test that diagnoses a fatal disease are very critical and should not occur. a cross-reaction of the lamp-primers or carry-over contamination might be the cause of these false positive results. however, the negative controls without sample material did not show any indication for carryover contamination and stayed negative in the lamp assays. another possibility for false positive results is the detection of fcov in cats without fip, since systemic spread of fcov does occur, but does not inadvertently result in fip (porter et al., ) . this explanation is not very likely for the three samples of cats without fip that were positive by rt-lamp, since all three were negative by rt-pcr. the sensitivity of the pcrun™ molecular detection mix was superior for the reported sample group with . % compared to . % of the isothermal mastermix, using the same primers and pcr conditions. since the samples for the rt-pcr and for the rt-lamp had the same preanalytical treatment (frozen, thawed, and centrifuged), the results can be directly compared and showed that the rt-pcr for fcov performed much better than the rt-lamp in our sample group. this is in agreement with a study on other coronaviruses, where rt-lamp also exhibited a lower analytical sensitivity compared to rt-pcr (bhadra et al., ) . false negative results can occur in samples with a lower viral burden. the fcov viral load determined by rt-pcr in effusions has been found to be quite low in some samples of fip-suspected cats (lorusso et al., ) . in our study, the results for rt-pcr were only returned as 'positive' or 'negative' without quantification, leaving open the question whether only effusions with a high viral load resulted in a positive rt-lamp detection. another possible reason for the lack of sensitivity might be that sequences of current fcov strains show sequence variations compared to the sequences deposited in the genbank database, which were used to design the rt-lamp primers. although the primers were chosen to bind in highly conserved regions, variations can occur, which might impair binding and eventually lead to low or no amplification, resulting in poor sensitivity. reliable primers for rt-pcr target the ′ utr of the fcov sequence, but the rt-lamp primers that were suggested by the primerexplorer software in this region included more sequence differences than the m region that we selected. modifications of the primers might enhance binding and improve sensitivity. two studies on lamp-based identification of fcov have been published to date. a study from thailand tested samples of body cavity effusions from cats that were suspected to have fip both by rt-pcr and by rt-lamp. more samples tested positive with the rt-lamp than with the rt-pcr ( % vs. %) (techangamsuwan et al., ) . however, the inclusion criteria for cats to be suspected of having fip were not described in that study. their control samples consisted of plasma and fecal samples from healthy cats without any contact to other cats. the samples from this healthy control group also had more positive results by rt-lamp than by rt-pcr ( % vs %). the authors mention high rates of false positives in the negative controls (no template controls) when using rt-lamp, so it remains unclear whether their rt-pcr was less sensitive or their rt-lamp was prone to unspecific amplification. in the second study, different sample types of cats with a clinical suspicion of fip and fecal samples for screening for fcov-shedding cats were tested (stranieri et al., ) . in most sample types, including effusions, their rt-pcr had about twice as many positive results as their rt-lamp, and none of the rt-pcr-negative samples was positive in the rt-lamp method. in agreement with our findings, the sensitivity of the rt-lamp appears to be inferior to the rt-pcr. for detection of amplification, both studies used gel electrophoresis and one study (stranieri et al., ) additionally used detection of color change from violet to blue with hydroxynaphtol blue. while gel electrophoresis is quite time-consuming, the color change can be difficult to detect in samples with low amplification. amplification detection with fluorescence or luminescence as used in the present study is preferable, since the results can be read immediately and could be easily converted to a quantitative format with a standard curve. as a perspective for the future, rt-lamp reactions for virus detection could be run on new devices that integrate nucleic acid extraction, amplification and detection in a miniature format to achieve a true point-ofcare diagnosis (stumpf et al., ) . in conclusion, the rt-lamp in the present study was relatively specific but not very sensitive. the sample type was restricted to effusions of cats unequivocally diagnosed with fip and of cats without fip but with clinical signs indicative of fip. this is a realistic setting in which a veterinarian would use a test for detection of fcov in the effusion sample. the rt-lamp assay with the pcrun™ molecular detection mix can be used in a clinical setting to a certain extent. however, before it can replace conventional rt-pcr methods, sensitivity and specificity have to be enhanced by optimizing primers and amplification conditions. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the pcrun™ molecular detection mix and the pcrun™ reader were provided by biogal free of charge. biogal was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. the fcov rt-pcr and subsequent mutation detection was done by idexx free of charge. idexx was not involved in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. a study of naturally occurring feline coronavirus infections in kittens real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east 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transcriptase loopmediated isothermal amplification for the detection of feline coronavirus labdisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza a h n virus simple, rapid, inexpensive platform for the diagnosis of malaria by loop mediated isothermal amplification (lamp) development and application of reverse transcription loop-mediated isothermal amplification (rt-lamp) for feline coronavirus detection feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses isothermal amplification methods for the detection of nucleic acids in microfluidic devices we thank biogal for providing the pcrun™ molecular detection mix and the pcrun™ reader for this study. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jviromet. . . . key: cord- - s sy authors: banerjee, arinjay; rapin, noreen; miller, megan; griebel, philip; zhou, yan; munster, vincent; misra, vikram title: generation and characterization of eptesicus fuscus (big brown bat) kidney cell lines immortalized using the myotis polyomavirus large t-antigen date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: s sy it is speculated that bats are important reservoir hosts for numerous viruses, with viral families reportedly detected in bats. majority of these viruses have not been isolated and there is little information regarding their biology in bats. establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. currently, few bat cell lines have been developed and only tb -lu, derived from tadarida brasiliensis is commercially available. here we describe a method to establish and immortalize big brown bat (eptesicus fuscus) kidney (efk ) cells using the myotis polyomavirus t-antigen. subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. cell clones expressed interferon beta in response to poly(i:c) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (vsv), porcine epidemic diarrhea coronavirus (ped-cov), middle-east respiratory syndrome coronavirus (mers-cov) and herpes simplex virus (hsv). to our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. the cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture. it is speculated that bats are an important reservoir host for several viruses, such as ebola virus (family filoviridae, genus ebolavirus), marburg virus (family filoviridae, genus marburgvirus), severe acute respiratory syndrome coronavirus (sars-cov; family coronaviridae, subfamily coronavirinae, genus betacoronavirus), middle-east respiratory syndrome coronavirus (mers-cov; family coronaviridae, subfamily coronavirinae, genus betacoronavirus), porcine epidemic diarrhea coronavirus (ped-cov; family coronaviridae, subfamily coronavirinae, genus alphacoronavirus) and hendra and nipah viruses (family paramyxoviridae, genus henipavirus). there is evidence that many of these viruses have been transmitted from bats to other hosts where they caused serious dis- * corresponding author. e-mail address: vikram.misra@usask.ca (v. misra). ease (drexler et al., ; changula et al., ; wacharapluesadee et al., ) . over different viruses from families have been detected in bats [reviewed in (moratelli and calisher, ) ] but most of these viruses have yet to be isolated and there is scant information regarding the biology of these viruses in bats. bats are genetically diverse and are found dispersed across much of the planet. with over species, bats display major differences in their behavior, feeding habits and the viruses they harbor [reviewed in (moratelli and calisher, ) ]. very little is known, however, about bat immune responses and if these differ across genera and species. currently a single bat cell line (tb -lu, atcc number ccl- , derived from the lung of tadarida brasiliensis) is available through the american type culture collection. research groups have established other bat cell lines, from fruit and insectivorous bats using established techniques such as using the sv t-antigen and expressing human telomerase reverse transcriptase (htert), but these are not commercially available yet (crameri et al., ; jordan et al., ; maruyama et al., ) . bats are the only mammals capable of true flight and as such they may have unique physiological adaptations. for example, they display unique strategies for neutralizing the dna-damaging byproducts of oxidative metabolism produced as a result of increased metabolic activity (shen et al., ) . zhang et al. hypothesize that bats have evolved and accumulated genetic changes as a result of their adaptation to flight. this is to limit collateral damage caused by by-products of an elevated metabolic rate (zhang et al., ) . these genetic changes may be important in the expansion and contraction of important gene families, including genes involved in the innate response pathway (zhang et al., ) . north american bat species are at risk of drastic population depletion due to white-nose syndrome (knudsen et al., ; alves et al., ) and conducting terminal in vivo experiments might not be entirely possible in future. establishing stable bat cell lines would provide an alternative for conducting in vitro host-pathogen studies. experiments using cultured bat cells could provide useful preliminary information on bat innate immune defense responses, virus-cell interactions and cellular physiology. there are several established methods for immortalizing primary cells. the first involves the introduction and stable expression of genes coding for the simian virus (sv ) large t antigen (sv tag). the large t antigen binds to and attenuates the tumor suppressor protein p and proteins of the retinoblastoma tumor suppressor family (prb, p and p ). this promotes dna replication and cell division. this method has been used to immortalize cells from a number of species including human (mayne et al., ) , rabbit (scott et al., ) and rat (lechardeur et al., ) . the second method involves the introduction and stable expression of the catalytic subunit of the human telomerase reverse transcriptase (htert). ectopic expression of htert has been successfully used to immortalize primary cells in a range of mammalian species such as goat mammary epithelial cells ) and canine schwann cells (techangamsuwan et al., ). this enzyme subunit prevents the shortening of telomeres with repeated cell divisions and thus prevents cellular senescence. here we describe a method for establishing and characterizing a kidney cell line (efk ) from eptesicus fuscus (the n. american big brown bat) using the myotis polyomavirus t antigen (mypvtag). we characterized the capacity of mypvtag to enhance dna replication in vero cells and found that it significantly increased their dna content. we then transfected mypvtag into primary bat kidney cells and sub-cloned several cell lines. we characterized the lineage of these clones and tested their expression of the interferon beta (ifn beta) gene in response to polyinosinic-polycytidylic acid (poly(i:c)) stimulation. we further tested three cloned kidney cell lines for their ability to support the replication of viruses from the families coronaviridae, herpesviridae and rhabdoviridae. the parental cell line and clones were capable of expressing ifn beta and supported the replication of viruses such as vesicular stomatitis virus (vsv; family rhabdoviridae, genus vesiculovirus), herpes simplex virus (hsv; family herpesviridae, subfamily alphaherpesvirinae, genus herpesvirus), ped-cov and mers-cov. ped-cov and mers-cov are viruses for which transmission from bats, either directly or via an intermediate reservoir, has resulted in high mortality in pigs (lee, ) and humans (coleman and frieman, ) , respectively. vsv and hsv are members of viral families that have previously been detected in bat species [reviewed in (moratelli and calisher, ) ]. although e. fuscus primary embryonic cells have been described before (qian et al., ) , to our knowledge, this is the first cell line established from a northern latitude insectivorous bat that was transformed by using a viral element (mypvtag) selected from a known bat virus. furthermore, the established kidney cell lines were able to support the replication of selected viruses from three different virus families. all procedures related to the handling and euthanasia of bats were submitted to and approved by the committee on animal care and supply of the university of saskatchewan animal research ethics board (protocol # ) and were in accordance with regulations approved by the canadian council on animal care. a moribund male e. fuscus bat submitted to the laboratory was humanely euthanized. brain, liver, lungs, spleen and kidney were harvested. each organ was finely minced, and incubated at room temperature in . % trypsin-edta (gibco, usa) with agitation. periodically cells were recovered after neutralizing trypsin with fetal bovine serum (fbs; seradigm, usa) added to %. cells were resuspended in dulbecco's minimal essential medium (dmem; corning, usa) containing penicillin (gibco, usa), streptomycin (gibco, usa) and amphotericin b (sigma, usa), placed in cm flasks (cellstar, germany) and incubated at • c in an atmosphere of % co . only kidney cells grew to form a monolayer. these cells were recovered by trypsinization, diluted / and re-plated. cell samples at various passages were cryopreserved in dmem containing % fetal bovine serum (fbs) and % dimethyl sulfoxide (emd chemicals, usa). bat kidney cells were immortalized by using viafect (promega, usa) to transfect cells with either . g of pcdna (invitrogen, usa) empty vector or plasmids expressing either sv large t-antigen (sv tag) or myotis polyomavirus large t-antigen (mypvtag). transfected cells were cultivated in dmem containing % fbs and geneticin reagent (invivogen, usa). only cells transfected with mypvtag continued to replicate. cells were confirmed to be e. fuscus cells by amplifying and sequencing a segment of mitochondrial cytochrome b transcripts (parson et al., ) . mrc cells (atcc ccl- ) were cultured in mem medium (corning, usa) supplemented with % fbs (seradigm usa), / non-essential amino acids (neaa; gibco), / ( -( -hydroxyethyl)- -piperazineethanesulfonic acid (hepes; gibco) and / gentamycin (gibco, usa). vero cells (elaine van moorlehem, vaccine and infectious disease organization -international vaccine center (vido-intervac)) were cultured in dmem with glutagro (corning, usa) supplemented with % fbs (seradigm, usa) and penicillin/streptomycin. all cell lines were checked and controlled for mycoplasma by a semi-nested pcr (described below). efk- b cells were seeded at a concentration of × in a t- flask. the cells were grown up to % confluency and treated with . g/ml colcemid (roche, usa) as mentioned previously (howe et al., ) . the cells were processed, spread on slides and chromosomes were stained with giemsa staining solution as mentioned previously (howe et al., ) . the mypvtag was amplified from the myotis polyomavirus whole genome (national centre for biotechnology information (ncbi), accession number nc . ) cloned in a topo vector (invitrogen, usa) and sub-cloned into a pcdna (invitrogen, usa) backbone. sv tag (a generous gift from ivan sadowski, university of british columbia) was also sub-cloned in pcdna . the pcdna plasmids encoding the t-antigens were used for transfection studies. to quantify dna in t-antigen (t-ag) transfected vero cells, the cells were seeded at a concentration of × cells/well in -well plates. the cells were transfected with . g of either sv tag (sv tag in pcdna ), mypvtag (mypvtag in pcdna ) or pcdna using lipofectamine (thermofisher scientific, usa). the cells were harvested and prepared for flow cytometry twenty-four hours after transfection. briefly, cells were harvested and re-suspended in dulbecco's phosphate-buffered saline (dpbs) (thermofisher scientific, usa). cells were fixed in % ice-cold ethanol for mins and stained for the respective t-antigens using . g/ml mouse anti-sv tag (cross-reactive for sv tag and mypvtag) (molecular probes, usa). the secondary antibody cocktail contained . mg/ml propidium iodide (molecular probes), . mg/ml rnase a (sigma, usa), . g/ml goat anti-mouse immunoglobulin-alexa conjugate (molecular probes) and . % triton x- (sigma, usa) in dpbs. the cells were filtered through m nylon mesh prior to analyses. for analyzing the cell lineage of the clones, intracellular staining using the commercial bd fixative (bd biosciences, usa) was carried out following the manufacturer's recommendation. murine monoclonal antibodies against vimentin ( / dilution of monoclonal anti-vimentin, clone vim . , mouse ascites fluid igm) (sigma-aldrich, usa) and cytokeratin were used ( / dilution of monoclonal anti-cytokeratin . , clone k . , mouse ascites fluid igg a isotype) (sigma, usa). secondary antibodies used were . g/ml goat anti-mouse igm ()-fitc conjugate (caltag/invitrogen, usa) and . g/ml goat anti-mouse igg a-fitc conjugate (caltag/invitrogen, usa). cells were analysed using facscalibur (bd biosciences, usa) with forward scatter detection using a photodiode with / nm bandpass filter and side scatter detection pmt with brewster-angle beam splitter. fitc was detected with a nm laser and / nm band pass filter. for each sample , events were accumulated and analyzed with cellquest pro (bd biosciences, usa). all rna extractions were performed using the rneasy plus mini kit (qiagen, germany) as per manufacturer's instructions. cdna was prepared using the quantitect reverse transcription kit (qiagen) as per manufacturer's instructions. one g of rna was used for cdna preparation. cdna was used as a template for the quantification of target genes. conventional pcr (polymerase chain reaction) to determine the cell lineage of the clones was performed using primers specific for e. fuscus vimentin (bbb-vimentin-f-tcaagaatacccgcaccaacg and bbb-vimentin-r-actgctgacggacgtcgcgc) and cytokeratin (bbb-cytoker-f-gaagacctacaaggtgtccac and bbb-cytoker-r-ccatctcgggtctcaatcttc). primers were designed using the annotated e. fuscus genome on ncbi (accession no. vimentin -xm . ; cytokeratin -xm . ). after initial denaturation for min at • c, the remaining pcr cycles were at • c/ s, • c/ s and • c/ min. the final extension was at • c for min. conventional pcr for the detection and identification of e. fuscus cytochrome b was performed using primers cytb us -cccchcchcayatyaarccmgartgata and cytb ds -tcracdg-gntgycctccdattcatgtta. after initial denaturation for min at • c, the remaining pcr cycles were at • c/ s, • c/ s and • c/ min. the final extension was at • c for min. semi-nested pcr using primers specific to the s rrna gene of mollicutes was performed for the detection of mycoplasma in cell lines. primers were designed as mentioned previously (kong et al., ; yoshida et al., ) . briefly, primers my- -acggcccadactyctacggraggcagcagta and my- -ccrtgcaccayttgtcwhhhbgwwaacctc were used for the first pcr. after initial denaturation for min at • c, the remaining pcr cycles were at • c/ s, • c/ s and • c/ min. the final extension was at • c for min. primers my- and my- -gtaatacatagctcgcaagcgttatc were used for the second pcr. after initial denaturation for min at • c, the remaining pcr cycles were at • c/ s, • c/ s and • c/ min. the final extension was at • c for min. for ifn beta quantification, qpcr assays targeting the ifn beta transcripts and the normalizer (gapdh, glyceraldehyde- phosphate) were performed for the clones. stratagene's mx p pcr (stratagene, usa) cycler was used in conjunction with quantifast sybr green pcr kit (qiagen). primers used were interferon beta (bbb ifnbeta-f-gctccgattccgacagagaagca and bbb ifnbeta-r-atgcatgaccaccatggcttc) and gapdh (bbb gapdh-f-ggagcgagatcccgccaacat and bbb gapdh-r-gggagttgtcatacttgtcatgg). primers were designed using the annotated e. fuscus genome (ncbi, accession no. interferon beta: xm . and gapdh: xm . ). samples were prepared as previously mentioned (rapin et al., ) . the products were quantified based on the amount of relative ifn beta expression. briefly, cells were either transfected with ng/ml poly(i:c) (invivogen, usa) using lipofectamine (thermofisher scientific, usa) or mock transfected. for quantifying ped-cov transcripts, primers were designed to amplify the ped-cov nucleocapsid (n) gene (genbank accession number kf ), (pedv-s gcaacaacaggtccagatctc) and (pedv-r ctccacgaccctggttatttc). for qpcr, after the initial denaturation step of • c for min, the remaining cycles were at • c/ s, • c/ min and • c/ min. the absorbance reading was taken after the • c step. relative fold change in gene expression between the two groups of cells was calculated and plotted after normalizing the ct values for ifn beta using gapdh. three housekeeping genes were tested (gapdh, beta-actin and beta- microglobulin) and none showed variation between treated and mock treated samples. thus gapdh was used for normalizing the data. difference of one ct indicates a two-fold difference in gene expression. pcr and qrt-pcr products were confirmed on a gel and sequenced (macrogen, south korea). reaction efficiencies for qrt-pcr primers were calculated to be between and %. total number of viable cells was determined by using a hemocytometer to count viable cells by trypan blue exclusion method. cells were cultivated in -well plates, trypsinized and re-suspended in media at every time point. efk parental cell line and three subclones were inoculated with vsv-indiana strain (dr. ellis' lab at the university of saskatchewan), hsv, ped-cov (dr. zhou's lab at vaccine and infectious disease organization -international vaccine center (vido-intervac)), and mers-cov (strain emc/ , dr. fouchier at erasmus medical center in the netherlands). for vsv inoculations, wst concentration of the virus was used to infect the cells, which were seeded in well plates at a concentration of × cells/well. wst was determined as that dilution of virus that produced % cell death as measured using the wst- assay. wst- assay is similar to the mtt assay (heldt et al., ) . briefly, cells were seeded in well plates at a density of × /well and analyzed with the wst assay at the indicated time points. l wst- reagent (roche, usa) was added to each well and incubated at • c for h. colour developed was measured at nm with a reference wavelength of nm using molecular devices vmax spectrophotometer. intensity of the colour developed is directly proportional to the number of viable cells in the wells. cells cultured in -well plates were inoculated with l virus (vsv) for hr, rinsed with sterile pbs and medium replaced with dmem containing % fbs. cells and the supernatant were harvested at and h post infection and frozen at − • c. after freeze-thawing the supernatant and cells three times, virus from efk parental cell line and the subclones was titrated in vero cells using the wst assay. virus was quantified using the wst assay and formula as described by heldt et al. (heldt et al., ) . for hsv titration, mrc , efk b, efk f and efk b subclones were seeded in triplicates at a concentration of . × cells/well in well plates. the cells were inoculated with hsv at a multiplicity of infection (moi) of . viral inoculum ( . ml) was replaced with complete medium and plates frozen at and h post inoculation. plates were freeze-thawed x, transferred to ml tubes and centrifuged at rpm for min. supernatant was collected and serially diluted : down to − and titrated in vero cells ( × cells/well in -well plates). for quantifying the virus, ul of diluted virus was added to the wells and incubated for an hour. the inoculum was replaced with . ml of dmem + % pooled human serum (mp biomedical cellect). plaques were counted under the microscope days later. multistep replication kinetics were determined by inoculating wells of cells in triplicate with mers-cov (strain emc/ ) with a moi of . , % tissue culture infectious dose (tcid ) per cell. one hour after inoculation, cells were washed once with dmem and culture medium replaced. culture supernatants were sampled at , , , , and h after inoculation. mers-cov was titrated by end-point titration performed in quadruplicate using vero e cells cultured in dmem supplemented with % fetal calf serum, mm l-glutamine (lonza, usa), u/ml penicillin and g/ml streptomycin. cells were inoculated with ten-fold serial dilutions of virus, and scored for cytopathic effect days later. the tcid was calculated by the method of spearman-karber (hamilton et al., ) . for ped-cov infection, cells were seeded in -well plate at a density of . × /well and cultured overnight. cells were inoculated with ped-cov at an moi of . cells were harvested and total rna extracted at , , , and h. virus replication was quantified by qrt-pcr. significance between t-ag data was calculated by mann whitney u test for two independent samples using ibm spss (version ). the sv large t-antigen is well characterized and is known to enhance dna replication in cells and immortalize primary cells (crameri et al., ) . our laboratory has previously detected a novel polyomavirus in m. lucifugus (misra et al., ) . to determine if the myotis polyomavirus t-ag shared the ability of its sv homologue to induce dna replication, we transfected vero cells with plasmids encoding genes for the two t-antigens. we then confirmed that cells expressed t antigen by immunostaining and compared the dna content of t antigen expressing cells with cells transfected with the pcdna null plasmid. fig. shows cells expressing myotis polyomavirus and sv t antigen contained more dna vero cells were transfected with plasmids expressing either sv tag, mypvtag or empty vector (pcdna). twenty-four hr after transfection, cells were immunestained for cytoplasmic t-antigen and with propidium iodide to quantify dna. the dna content of t-antigen and pcdna transfected cells was determined by flow cytometry and expressed as the fold increase in dna content relative to pcdna transfected cells. the ratio for cells transfected with pcdna was taken as ' . experiments were done in triplicate and mean values plotted. error bars represent standard deviation. statistical difference was calculated using mann-whitney u test for two independent samples. * < . . than cells transfected with pcdna plasmid. sv tag expressing vero cells showed the highest increase in dna content. we attempted to immortalize primary cells derived from the kidney of e. fuscus by transfection with plasmids expressing sv tag, mypvtag or empty vector (pcdna). we observed clusters of cells in cultures transfected with either t-ag but after several passages, only the mypvtag transfected primary cells (efk) continued to replicate. the immortalized efk cell line was cloned by limiting dilution to generate three clones (efk , and ). clones were further isolated by end point dilution of efk , efk and efk cells. we established the clones as separate cell lines and characterized representative clones from each of the three clones i.e. efk , efk and efk , along with the parental efk cells for their cell type markers, interferon beta response, virus susceptibility and cell division rates. we determined the number of chromosomes in efk- b to rule out the possibility of chromosome number abnormality in immortalized cells (supplementary fig. ). efk- b had n = chromosomes, which is normal for genus eptesicus (fedyk and ruprecht, ) . we screened the clones for cytokeratin, a lineage-specific marker for epithelial cells (xie et al., ) and vimentin, a lineage marker for fibroblasts (zschemisch et al., ) . since specific antibodies are not available for bat cytokeratin and vimentin, we used antibodies specific to the human proteins. no positive staining of the bat clones was observed with either anti-vimentin or anti-cytokeratin antibodies when cells were analyzed with flow cytometry (data not shown). we then screened the cell lines for expression of vimentin and cytokeratin transcript by conventional pcr. five of the eight clones analyzed contained detectable transcripts for both cytokeratin and vimentin. in contrast, only vimentin transcripts were detected in primary efk cells at passage (table ) . we characterized the efk clones for their capacity to respond to polyinosinic-polycytidylic acid (poly(i:c)), a synthetic analogue of double-stranded rna (mian et al., ) , through analysis of table transcripts for cell lineage markers vimentin (fibroblast) and cytokeratin (epithelial) are expressed by the efk clones. the ability of the cells to respond to poly(i:c) treatment with increased ifn beta gene expression was detected by qrt-pcr. along with the clones, a parental cell line (efk ) and primary kidney cells (efk; not transfected with either t-ag) were compared. + = pcr product detected, − = no pcr product detected. interferon beta transcription. poly(i:c)-treated primary efk cells displayed an average of , -fold increase in interferon beta transcripts when compared to mock transfected cells. all clones displayed increased interferon beta transcription following poly(i:c) treatment, with the level of increase ranging from six thousand to over sixty thousand. (table ) . all clones displayed similar multiplication rates when assayed with the wst- reagent (data not shown). we determined the cell division rates of three subclones ( b, f and b) and the parental efk cell line by counting viable cells using a hemocytometer at various time points after seeding (fig. ) . the three clones and parental cell line (efk ) did not differ from one another in their multiplication rates. we evaluated the efk parental cell line and three clones ( b, f and b) for their competence in supporting vsv, hsv, ped-cov and mers-cov replication. as a positive control, vero cells were infected with mers-cov and ped-cov and mrc cells were infected with hsv and vsv. vsv caused rounding, sloughing and detachment of mrc cells in culture, ped-cov caused syncytia in vero cells and mers-cov caused rounding and sloughing off of vero cells. cytopathic effects (cpe) such as plaques or rounding and sloughing off of cells following infection with vsv and mers-cov was observed with all efk clones. two of the three clones responded to ped-cov infection with cytopathology. in contrast, efk- f did not exhibit noticeable cpe h.p.i. with ped-cov (fig. ) . the positive control cell lines exhibited cpe h.p.i. following infection with the respective viruses. the evolutionary pressures of flight are thought to have conferred upon bats unique physiological adaptations. most viruses that have been transmitted from bats to other species have been studied in animal models of human disease or in cell lines of non-bat origin. in addition, most in vitro studies of mammalian innate immune and anti-viral responses have been performed in human and rodent cell lines. the results from these studies may not accurately represent pathogen-host interactions that occur in bats. establishing bat cell lines enable researchers to study relevant virus-host interactions in a system that more closely resembles the reservoir host. cell lines have been established from fruit bats (crameri et al., ; virtue et al., ) , myotis myotis (he et al., ) , tadarida brasiliensis and other insectivorous bats (maruyama et al., ) . primary embryonic cells have been developed from e. fuscus (qian et al., ) but an immortalized e. fuscus cell line capable of supporting the replication of viruses from three diverse viral families is not commercially available yet. historically, cells have been immortalized by either using the large t-antigen from sv , which is a monkey virus or by the ectopic expression of htert. we characterized the mypvtag and sv tag's ability to enhance dna replication in cells. both mypvtag and the sv tag significantly increased dna content in vero cells. sv t-ag is known to enhance dna content in cells (ohkubo et al., ; ahuja et al., ) and interestingly the bat polyomavirus tantigen shared similar properties. myotis polyomavirus belongs to the same family as sv . large t-ags from both these viruses were transfected to immortalize the e. fuscus kidney cells, but only cells expressing mypvtag gave rise to stable cell lines. we do not know why sv tag failed to immortalize bat cells. however, it might be possible that the expression of mypvtag, derived from a virus found in this bat, could have been favored by the cellular machinery over the sv tag derived from sv , which has not been detected in big brown bats yet. clones b, a, e, f, b expressed transcripts for lineagespecific proteins of both epithelial and fibroblast cells and clones h, a and b expressed mrna for vimentin. since primary bats cells at passage had detectable transcript for vimentin alone, it is possible that the immortalization procedure may have altered transcription in some of the clones. there is evidence, however, that tumor cells can co-express both epithelial and fibroblast markers (viale et al., ) . it is also possible that mypvtag immortalized a mixture of both epithelial and fibroblast cells and the epithelial cells had a replication advantage during the process of limiting dilution cloning. when cells were being passaged, some cells were more strongly adhered to the plastic and were not removed by trypsiniza- tion. this process could have selected for particular cell types based on their adherent properties. relatively little is known about the innate immune response of insectivorous bats to viral infection. we were able to generate an insectivorous bat cell line capable of upregulating ifn beta gene expression in response to a known innate immune stimulus. an early interferon response is known to inhibit replication of some viruses (katze et al., ) . interferon response in terms of interferon beta transcript upregulation by bat cell lines, mostly cell lines from fruit bats, has been demonstrated before (hagmaier et al., ; crameri et al., ; biesold et al., ; virtue et al., ) . we analyzed the capacity of the efk clones to respond to poly(i:c) stimulation by monitoring interferon beta gene expression. this synthetic analogue of dsrna is usually used as a pathogen-associated molecular pattern (pamp) to stimulate interferon responses (crameri et al., ) . the clones responded to poly(i:c), as we observed a several thousand-fold increase in interferon beta transcript when compared to mock treated cells. immortalization of cells is sometimes known to compromise the ability of the cells to transcribe the interferon beta gene (biesold et al., ) . the efk clones, however, retained a remarkable but variable capacity to respond to poly(i:c). west nile virus, eptesipox virus, novel group i coronavirus and american bat vesiculovirus are examples of viruses that have been detected in eptesicus fuscus (donaldson et al., ; moratelli and calisher ) . the ability of this cell line to support the replication of viruses from three different viral families demonstrates the potential application of this cell line to isolate and study viruses in bats that have previously only been detected using pcr and sequencing. further work can be done to identify receptors specific to these viruses. vsv, hsv, ped-cov and mers-cov grew to varying levels in the efk clones. clone f did not exhibit any visual cytopathology on infection with ped-cov although virus replication was detected by qrt-pcr. this could be due to a lower level of virus replication in f (fig. d ) as the images (fig. ) were taken h post-infection. at this point we do not know if f mounts a more robust interferon response or if it lacks other factors required for ped-cov replication during the initial h post-infection. in conclusion, we established a stable kidney cell line from a northern latitude bat, which has the capacity to respond to a known innate immune stimulus with transcription of ifn beta. furthermore, this cell line supported the replication of viruses from three virus families known to be harbored by bats. not much is known about innate immune responses in bats and how they are activated during viral infections. establishing well-characterized cell lines from relevant bat reservoir species is the first step in addressing the many questions that researchers have about innate immunity in bats moratelli and calisher ) . this cell line will help us better understand the bat innate responses and how they may contribute to the absence of overt disease symptoms when bats are infected with these viruses. the e. fuscus kidney cell line developed here is now available for research through kerafast, usa. the goal of this study was to generate a cell line that would allow researchers to potentially study viruses derived from bats in a reservoir in vitro model. sv large t antigen targets multiple cellular pathways to elicit cellular transformation the potential impact of white-nose syndrome on the conservation status of north american bats large t antigens of polyomaviruses: amazing molecular machines antiviral immune responses of bats: a review type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum ebola and marburg virus diseases in africa: increased risk of outbreaks in previously unaffected areas? coronaviruses: important emerging human pathogens establishment, immortalisation and characterisation of pteropid bat cell lines metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat bats host major mammalian paramyxoviruses chromosome of some species of vespertilionid bats.i. banding patterns of eptesicus serotinus chromosomes mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading stats trimmed spearman-karber method for estimating median lethal concentrations in toxicity bioassays an immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (htert) gene transfer establishment of myotis myotis cell lines -model for investigation of host-pathogen interaction in a natural host for emerging viruses a colorimetric assay for viral agents that produce cytopathic effects chromosome preparation from cultured cells authentication of the r e fruit bat cell line viruses and interferon: a fight for supremacy potential spread of white-nose syndrome of bats to the northwest: epidemiological considerations species-specific pcr for identification of common contaminant mollicutes in cell culture induction of blood-brain barrier differentiation in a rat brain-derived endothelial cell line porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus characterization of the envelope glycoprotein of a novel filovirus, lloviu virus efficient immortalization and morphological transformation of human fibroblasts by transfection with sv dna linked to a dominant marker length of dsrna (poly i:c) drives distinct innate immune responses, depending on the cell type detection of polyoma and corona viruses in bats of canada bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? sv large t antigen reinduces the cell cycle in terminally differentiated myotubes through inducing cdk , cdc , and their partner cyclins species identification by means of the cytochrome b gene role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation activation of innate immune-response genes in little brown bats (myotis lucifugus) infected with the fungus pseudogymnoascus destructans maintenance of expression of differentiated function of kidney cells following transformation by sv early region dna adaptive evolution of energy metabolism genes and the origin of flight in bats transfection of adult canine schwann cells and olfactory ensheathing cells at early and late passage with human tert differentially affects growth factor responsiveness and in vitro growth coexpression of cytokeratins and vimentin in common epithelial tumours of the ovary: an immunocytochemical study of eighty-three cases interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines diversity of coronavirus in bats from eastern thailand establishment and characterization of a telomerase-immortalized canine bronchiolar epithelial cell line rapid detection of mycoplasma genitalium, mycoplasma hominis, ureaplasma parvum, and ureaplasma urealyticum organisms in genitourinary samples by pcr-microtiter plate hybridization assay comparative analysis of bat genomes provides insight into the evolution of flight and immunity immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain wky/ztm we thank nathalie berube supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . key: cord- - aclcc authors: liu, jianbo; gao, ran; shi, hongyan; cong, guangyi; chen, jianfei; zhang, xin; shi, da; cao, liyan; wang, xiaobo; zhang, jialin; ji, zhaoyang; jing, zhaoyang; feng, li title: development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific siga in colostrum date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: aclcc porcine epidemic diarrhea virus (pedv) causes very high mortality in newborn piglets. the mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. antibodies derived from the secretory immunoglobulin a (siga) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. therefore, the measurement of siga levels is an important index in evaluating pedv infections and immune status. a simple and rapid method for the detection of pedv-specific siga using an immunochromatographic test strip has been developed; incorporating a colloidal gold-labeled anti-siga secretory component (sc) mab probe for the detection of anti-pedv-specific siga in swine. on the strip, a gold-labeled anti-siga sc mab was applied to a conjugate pad; purified pedv particles and goat anti-mouse antibodies were blotted onto a nitrocellulose membrane to form the test and control lines, respectively. results showed that the immunochromatographic test strip had high sensitivity and specificity. when compared with enzyme-linked immunosorbent assay, kappa value suggesting that the strip could be used to detect pedv specific siga in colostrum samples. furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. we found that the immunochromatographic test strip was a rapid, sensitive, and reliable method for the identification of pedv specific siga, indicating its suitability for epidemiological surveillance as well as vaccine immunity when studying pedv. porcine epidemic diarrhea virus (pedv) belongs to the family coronaviridae, first recorded in pigs in england in (pensaert and de bouck, ) , and subsequently spread to other european and asian countries (song and park, ) and north america (huang et al., ) . porcine epidemic diarrhea is a global infectious disease and is characterized by high morbidity and mortality in pre-weaned piglets, and causes serious economic losses to the swine industry in china (gao et al., ; li et al., ) . pedv is an enveloped coronavirus with a kb positive-stranded rna genome, containing at least seven open reading frames (kocherhans et al., ) . these mainly encode four major structural proteins: the spike (s), nucleocapsid, membrane, and envelope proteins. the s protein is a glycoprotein and can form homotrimers to mediate membrane fusion and gain entry into host cells (bosch et al., ) . it is known that the s protein of coronavirus plays a crucial role in the induction of neutralizing antibodies, and it has been used to prepare effective vaccines (brian and baric, ; tuboly and nagy, ) . the amino-terminal portion of the s protein of several coronaviruses has been shown to contain key antigenic sites that are responsible for eliciting humoral and cellular immune responses (gebauer et al., ) . the mucosal immune system in the gut faces the formidable task of eliminating potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. antibodies of the secretory immunoglobulin a (siga) class act as the first line of antigenspecific immunity in the gut, and can recognize both pathogens and commensals (song and park, ) . in contrast to serum iga, which is derived from plasma cells in the bone marrow, siga is generated locally by plasma cells in the lamina propria, which lies beneath the intestinal epithelium (kaetzel, ) . the protective action of siga in the infant gut is a result of many processes, including intracellular neutralization t and viral particle excretion, immune exclusion, whereby siga agglutinates bacteria and viruses, as well as prevents the binding of pathogens to mucosal surfaces, and interference with bacterial motility (van egmond et al., v) . at high endogenous concentrations of siga, infants are less likely to have experienced illness in the preceding and subsequent months (breakey et al., ) . pedv can affect the intestinal tract, and based on the above results, we can infer that the siga concentration in the colostrum and rectal swab is a better marker than iga of protection and survival rate after virulent pedv challenge. tools to monitor pedv mucosal immunity and colostral immunity could be useful for the development of preventative programs on affected farms. therefore, the aim of this study was to develop a simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled anti-siga sc mab probe for the detection of anti-pedv-specific siga in swine, and to compare its performance with an indirect siga elisa based on the whole pedv virus (cong et al., ) . vero e cells (american type culture collection no. crl- ) were used to propagate pedv (genbank accession no. kt ). virus was propagated according the method described by liu et al. ( ) with minor modifications. briefly, growth medium was removed (containing % fbs) when the cells formed a confluent monolayer and were then washed with phosphate-buffered saline (pbs, ph . ). then, pedv containing × plaque forming units (pfu) were diluted in ml of dmem (dmem, gibco brl life technologies, usa) (containing μg trypsin), and added to each culture bottle and incubated at °c. virus was harvested and purified according to the methods described by liu et al. ( ) . the purified viruses were negatively stained with % phosphotungstic acid (ph . ) for s according to previously described procedures (chen et al., ; jung et al., ) . and then identified by h- electron microscopy (hitachi, japan). spf swine were used to produce colostrum. a total of four specific pathogen free swine (supplied by experimental animal base, harbin veterinary research institute of the chinese academy of agricultural sciences) were used in this study. three swine were immunized with pedv (lnct strain), transmissible gastroenteritis virus (tgev) strain h (genbank accession no. fj ), and porcine rotavirus (porv) a (genbank accession no. jf ), respectively. all the viruses were inactivated by . % methanol solution (pbs, ph . ) at °c for h, and then emulsified with freund's complete adjuvant (sigma-aldrich, st. louis, mo, usa). and they were performed by neck intramuscular injection. the fourth animal was immunized with dmem as control and each swine was immunized twice. the first immunization was days before delivery whereas the second immunization was days before delivery. after delivery, colostrum from the four swine was collected and store at − °c. the protocols involving animal immunity in this study were approved by our institute's animal care and use committee and performed in accordance with standard ethical guidelines. the hybridoma cell line f , stably secreting mab against siga sc protein (gao et al., ) was cultured in the peritoneal cavities with pristane (sigma-aldrich, st. louis, mo, usa) primed balb/c mice to obtain ascites fluid. the ascites fluid was purified by hitrap protein g hp (ge healthcare) according the manufacturer's instruction and the purified antibody was identified by sds-page. the gel was stained by coomassie blue. colloidal gold with an average particle diameter of approximately nm was purchased from shanghai kinbio tech. co. ltd. (china). the colloidal gold-labeled mab was prepared according to the methods described by li et al. ( ) with some modifications. briefly, . ml ( mg/ml) of purified mab was incubated with ml of colloidal gold solution (ph . ) for min at °c. after addition of ml of a % casein solution, the mixture was incubated at °c for min. then, the mixture was centrifuged at , × g for min at °c. the supernatant was discarded and the pellet of the colloidal gold-labeled mab was suspended in ml of . m sodium borate buffer (containing . % nan , % bovine serum albumin (bsa), % sucrose) and stored at °c. the colloidal gold-based lateral flow test strips were generated according to the procedures described by liang et al. ( ) , with some modifications. briefly, × -mm pieces of glass fiber was immersed in phosphate-buffered saline ( . m, ph . , containing mm edta, % tween- , and % bsa) for h at °c. next, the pieces were dried for h at °c and stored for use as the sample pad. glass fiber was also immersed in phosphate-buffered saline ( . m, ph . , containing . % mycose, % bsa, % tween- ) for h at °c and then dried for h at °c and stored for use as the conjugate pad. the conjugate pad was covered with an appropriate volume of colloidal gold-labeled anti-siga sc mabs ( f ) using a xyz dispense platform (biodot, inc., irvine, ca, usa), and then dried at °c for h and stored dry. purified pedv particles were dispensed onto the nitrocellulose (nc) membrane to serve as the test line, and goat antimouse igg polyclonal antibody (pab) was dispensed onto the nc membrane, (being separated by a distance of mm) to serve as the control line. the pedv particles and goat anti-mouse igg pab were diluted with coating buffer to a final concentration of . and . mg/ ml, respectively. then, the nc membrane was dried at °c for h and stored in the dry at room temperature until use. the test strips were assembled and used as described by liang et al. ( ) . the sample pad, conjugate pad, nc membrane, absorbent pad and backing plate were assembled in sequence. the sample pad and the conjugate pad were overlapped by mm with one end of the conjugate pad; the other end of the conjugate pad was overlapped ( mm) with one end of the nc membrane underneath the conjugate pad; the absorbent pad was stuck to the other side of the nc membrane. after this, the whole plate was cut into mm-wide strips, which were assembled in the strip cassettes with desiccant. to test for colostrum, we diluted the samples : in sample buffer and mixed thoroughly then, μl of the solution was dispensed onto the sample pad well of the strip apparatus to determine the presence of pedv specific siga, as described above. with this system, the liquid migrates towards the absorption pad by capillary action. the siga in the colostrum samples will form a "gold-labeled antibody-siga" complex with the gold-labeled antibody on the conjugate pad. if the colostrum sample has pedv specific siga present, the pedv specific "gold-labeled antibody-siga" complex, while migrating to the nc membrane, will bind to pedv, thereby displaying a red line in the t line area. additional complex migrates further to react with the goat antimouse igg pab, showing another red line in the c line area. negative samples do not produce a red line in the t area, whereas the c line will always show a red line. the results can be seen within eight minutes at room temperature. . . validation of specificity and sensitivity of the immunochromatographic strip test pedv siga positive colostrum, tgev siga positive colostrum, porv siga positive colostrum, and pedv siga negative colostrum were used to test the specificity of the strip. the sensitivity of the strip was evaluated by comparing the detection of the same samples with pedv siga elisa kit. the detection limit of the strip was evaluated by using pedv siga positive-colostrum and colostrum titers determined by using an elisa kit (cong et al., ) as the reference standard with minor modifications. in brief, sample buffer was used to dilute colostrum from : to : , and μl of the solution was added into microtiter plates (costar, corning, ny, usa) incubated at °c for h. after a washing step, a horseradish peroxidase mouse anti-pig siga sc antibody : dilution was added and incubated at °c for min. after three times wash with pbst, the peroxidase reaction was visualized using , ′-azino-bis ( -ethylbenzothiazoline- -sulfonic acid) diammonium salt (bbi, shanghai, china) as the substrate for min at °c , and the reaction was stopped by adding μl of . m sulfuric acid to each well. optical densities (ods) were measured at nm using an elisa plate reader (biotek instruments, winooski, vt, usa). for the sensitivity of the elisa kit, one pedv specific siga positive colostrum was still positive when diluted at : . for the specificity, the elisa kit has no cross-reaction with tgev as well as porv specific siga positive colostrum (cong et al., ) . sixty three colostrum field samples from hebei, shandong and heilongjaing province were assayed by the strip and the pedv specific siga elisa (cong et al., ) . by comparing results obtained by the two methods, kappa statistic value was used to evaluate the effectiveness of the test strip. after removing sucrose, the bands between the % and % and % and % sucrose solutions were identified by electron microscopy. in the % and % sucrose solution band, pedv particles were observed and their diameter was approximately nm (fig. ) . ascites fluid containing mab f was purified by hitrap protein g hp and the purified antibody was identified by sds-page. as the results show (fig. ) , the purified antibody had two brands, a light chain and heavy chain. the purified antibody was stored until further use. the assay specificity was determined using anti-pedv specific siga positive colostrum, anti-tgev specific siga positive colostrum, anti-porv specific siga positive colostrum, and anti-pedv specific siga negative colostrum. as shown by the strips listed in fig. , the anti-pedv specific siga positive colostrum was positive at the test line; the other colostrum samples were negative, suggesting that the immunochromatographic strip had good specificity. anti-pedv specific siga positive colostrum was diluted to determine the sensitivity of the strip. according to the results shown in fig. , the strip had a sensitivity of up to : , while the detection limits of the elisa kit reached : dilution (table ) . all of the colostrum samples were detected by the strip and elisa mentioned above. as shown in table , colostrum samples were anti-pedv specific siga positive as determined by the strip assay, whereas colostrum samples were anti-pedv specific siga positive as detected by the elisa. the kappa statistic value for the comparison of the strip versus the elisa kit, was . . for the interpretation of agreement of the kappa statistic, a value of . to . is considered fair, fig. . identification of purified pedv by electron microscopy. the band between the % and % sucrose solutions was collected with a syringe, and after removing the sucrose the purified viruses were identified by electron microscopy. scale bar = nm. . to . is moderate, . to . is good, and . to . is very good (altman, ) . the kappa statistic value was . , suggesting that the strip could be used to detect pedv specific siga in colostrum samples. antiviral antibody detection is key for vaccine immunity surveillance and in determining pig exposure to pedv. currently, indirect elisas used to assay igg, iga, siga, have been established to monitor antibodies against pedv li et al., ; cong et al., ) . also, reverse transcription-polymerase chain reaction (rt-pcr) (ishikawa et al., ) , duplex rt-pcr (kim et al., ) , and other methods have been used to monitor pedv. although the presence of antibodies in serum is not directly related to protection of sows or piglets, serological examinations facilitate the assessment of the humoral responses to pedv, elicited either through vaccination or natural infection. however, the above-mentioned methods are time-consuming and require professional/technical personnel and are mainly limited to laboratory use. the immunochromatographic test strip used to detect anti-pedv specific siga described here is easy to operate, and sensitive. it is known that pedv is mainly transmitted by the fecal-oral route and disease is initiated following interaction with the mucosal surface lining the digestive tract. the primary defense used by this tissue is the mucosal immune system. antibodies from the siga class act as the first line of antigen-specific immunity in the gut. they prevent pathogens from binding to mucosal surfaces, and agglutinate bacteria and viruses (van egmond et al., v) . therefore, siga levels are an important index by which to evaluate pedv infection and immune states. in this study, siga, as opposed to igg, detection was developed to assess pedv infections and vaccination. here a pedv siga-specific immunochromatographic test strip was developed. the purified pedv-lnct particles, which belongs to the g genotype, were used as capture antigen. the shared amino acid identity of the pedv lnct s protein and the pedv cv (g genotype) s protein (genbank accession number kt ) is . %, whereas it is only . % for the pedv lnct s protein and the tgev s protein (purdue strain, genbank accession number dq ). reactivity using the polyclonal antibodies (pabs) revealed significant cross-reactivity between the two pedv subtypes, although there was a two-fold difference in the antigenic responses based on pab titers in the elisa and ifa (wang et al., ) . therefore, the elisa antigen based on the pedv-lnct particles could detect both g and g genotype strains. additionally, the pedv particles used in the present study had no cross-reactivity with anti-tgev positive specific colostrum or anti-porv positive specific colostrum (fig. ) . furthermore, the strip had a sensitivity of up to : , whereas the elisa kit limits for the same sample was : (table ) . based on these results, the anti-pedv specific siga immunochromatographic test strip could be used to effectively monitor siga levels. previously, researchers have used elisas to study the effect of pedv on mucosal immunity; an indirect elisa based on the s protein of pedv was used to detect iga levels in colostrum and fecal samples to evaluate pedv colostral immunity (gerber et al., and . iga plays an important role in providing protection at mucosal surfaces, and it is effective in neutralizing bacterial toxins. furthermore, polymeric iga has been shown to be more effective at neutralizing exotoxins from clostridium difficile when compared to either monomeric iga or igg with the same variable regions (stubbe et al., ) . siga consists of the sc, two iga molecules, and a linking chain. sc protects siga from proteolytic degradation (kaetzel, ) , making the siga more stable. it has been estimated that approximately g of siga is transported daily fig. . specificity of the immunochromatographic strip. lanes to : strip detection of the anti-pedv specific siga positive colostrum, anti-tgev specific siga positive colostrum, anti-porv specific siga positive colostrum, anti-pedv specific siga negative colostrum, water, milk. the samples were diluted at : in sample buffer and mixed thoroughly. then, μl of the solution was dispensed onto the sample pad. c stands for control line, t stands for test line. into the intestines of the average adult (mestecky et al., ; conley and delacroix, ) , where siga forms the first line of antigen-specific immune protection against ingested, inhaled, or sexually transmitted pathogens and antigens at mucosal surfaces (kaetzel, ) , making it more important than iga. in this study, we investigated the sc portion to detect siga in colostrum samples, as this can eliminate the effect of monomeric iga. after infection with coronaviruses, such as pedv or tgev, or after vaccination, siga is generated locally by plasma cells located in the lamina propria, which underlies the epithelium (kaetzel, ) . therefore, the anti-pedv specific siga immunochromatographic test strip established in this study could be used to detect infections and immune states. by determining the siga level, we could assess whether pig herds (unvaccinated) have been infected by pedv, as well as pigs (vaccinated) could been protected against pedv, highlighting the versatility of our method. therefore, monitoring siga titers in colostrum and milk samples could be performed to ensure that piglets receive adequate passive immunity. the anti-pedv specific siga immunochromatographic test strip for the detection of siga antibodies developed in our study provides a simple, sensitive and specific tool for monitoring passive immunity and pedv infection. null stands for not done. the cut off value of od value was . . the colostrum samples with an od value ≥ . were considered to be positive while < . were considered to be negative. practical statistics for medical research the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex illness in breastfeeding infants relates to concentration of lactoferrin and secretory immunoglobulin a in mother's milk coronavirus genome structure and replication isolation and characterization of porcine epidemic diarrhea viruses associated with the disease outbreak among swine in the united states establishment and application of indirect elisa for detection of porcine epidemic diarrhea virus specific siga intravascular and mucosal immunoglobulin a: two separate but related systems of immune defense? development of an enzyme-linked immunosorbent assay for themonitoring and surveillance of antibodies to porcine epidemicdiarrhea virus based on a recombinant membrane protein phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in china preparation and identification of monoclonal antibodies against porcine siga sc fragment residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein detection of immunoglobulin (ig) a antibodies against porcine epidemic diarrhea virus (pedv) in fecal and serum samples detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect elisa origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states direct and rapid detection of porcine epidemic diarrhea virus by rt-pcr pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex rt-pcr completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field strains in central china based on the orf gene and the main neutralization epitopes development of an indirect elisa based on a truncated s protein of the porcine epidemic diarrhea virus development of an immunochromatographic strip for detection of antibodies against porcine reproductive and respiratory syndrome virus rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (iii) chelate microparticles-based lateral flow test strips neutralization of genotype porcine epidemic diarrhea virus strains by a novel monoclonal antibody the human iga system: a reassessment a new coronavirus-like particles associated with diarrhea in swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines polymeric iga is superior to monomeric iga and igg carrying the same variable domain in preventing clostridium difficile toxin a damaging of t monolayers construction and characterization of recombinant porcine adenovirus serotype expressing the transmissible gastroenteritis virus spike gene iga and the iga fc receptor immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes g and g this work was supported by grants from the national key r & d plan for the th five year plan ( yfd ), national natural science foundation of china (no. ), the natural science foundation of heilongjiang (no. qc ). all authors have carefully revised the manuscript point-by-point according to the reviewers' comments. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. none of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. key: cord- -iqwojhq authors: dedkov, vladimir g.; magassouba, n.’faly; safonova, marina v.; naydenova, ekaterina v.; ayginin, andrey a.; soropogui, barre; kourouma, fode; camara, amara b.; camara, jacob; kritzkiy, andrey a.; tuchkov, igor v.; shchelkanov, mikhail yu.; maleev, victor v. title: development and evaluation of a one-step quantitative rt-pcr assay for detection of lassa virus date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: iqwojhq lassa fever is a severe viral hemorrhagic illness caused by lassa virus. based on estimates, the number of lasv infections ranges from , to , cases in endemic areas with a fatality rate of %. development of fast and sensitive tools for the control and prevention of lassa virus infection as well as for clinical diagnostics of lassa fever are crucial. here we reported development and evaluation of a one-step quantitative rt-qpcr assay for the lassa virus detection – lasv-fl. this assay is suitable for the detection of lineages i-iv of lassa virus. the limit of detection of the assay ranged from ( ) copies/ml to ( ) copies/ml and has . % diagnostic sensitivity, whereas analytical and diagnostic specificities both were %. serum, whole blood and tissue are suitable for use with the assay. the assay contains all the necessary components to perform the analysis, including an armored positive control (arc+) and an armored internal control (ic). the study was done during the mission of specialized anti-epidemic team of the russian federation (saet) in the republic of guinea in - . based on sequencing data, lasv-specific assay was developed using synthetic ms -phage-based armored rna particles, rna from lassa virus strain josiah, and further, evaluated in field conditions using samples from patients and mastomys natalensis rodents. lassa virus (lasv) is a single-stranded ambisense rna virus belonging to the arenavirus genus in the arenaviridae family (radoshitzky et al., ) . lasv causes an acute hemorrhagic illness in humans-lassa fever disease (lfd), which is characterized by fever, muscle aches, sore throat, nausea, vomiting, chest, and abdominal pain (ogbu et al., ) . however, the mild manifestation of lfd and asymptomatic infection with lasv is common (frame et al., , ajayi et al., . the natural host of lasv is the multimammate mouse (mastomys natalensis, person-to-person transmission was reported, particularly in nosocomial settings). the role of m. erythroleucus as a natural host of lasv is under discussion. these rodents are ubiquitous and highly commensal in africa (keenlyside et al., , monath et al., . despite the wide distribution of m. natalensis, lasv is endemic only in west african countries, including nigeria, sierra leone, liberia, guinea, benin, mali, and côte d'ivoire (frame et al., , safronetz et al., . based on estimates, the number of lasv infections ranges from , to , cases in endemic areas (siddle et al., ) with a fatality rate of %. (ogbu et al., ) . humans primarily become infected with lasv through inhalation or ingestion of infected rodent excreta. in addition, the infection can occur due to handling and preparation of infected m. natalensis for grilling, as they are considered delicious in the west african region (ter meulen et al., ) . person-to-person transmission was also reported, especially in nosocomial settings (monath et al., ) . given the high incidence and fatality, lfd is a severe burden for regional health care. moreover, lfd is one of the most prominent exotic viral hemorrhagic fever disease in africa (macher and wolfe, ) . therefore, fast and sensitive tools to control and prevent lasv infection, as well as for clinical diagnosis of lfd, are crucial. traditionally, the diagnosis of lfd is done by real-time polymerase chain reaction (rt-pcr), lateral flow immunoassays, and enzymelinked immunosorbent assay (elisa). however, rt-pcr is most suitable for the detection of lasv, particularly in the early stage of illness, because of its high sensitivity and ease of implementation in the study (asogun et al., ) . therefore, the development of rt-pcr assays, particularly based on quantitative rt-pcr (rt-qpcr) technique, is highly crucial for improving lfd diagnosis and for lasv surveillance and epidemiological control. this study aimed to develop and evaluate a one-step rt-qpcr assay for the detection of lasv-lasv-fl, targeting the l gene. this assay may be suitable for the detection of various lineages of lasv. the lasv strain josiah provided by the virology and biotechnology centre "vector," was propagated using vero e cell culture, which is commonly available in russian cell culture collections; the concentration of viral particles was evaluated and inactivation was performed at the virology and biotechnology centre "vector," novosibirsk, russia under bsl conditions. armored rna particles (arps) were synthesized by the biotechnology branch at the central research institute for epidemiology, moscow, russia. both the lasv strain josiah and arps were used to assess the limit of detection (lod) of the assay. whole blood samples from m. natalensis (n = from individuals and n = pools of three animals) were provided by the virology laboratory of hemorrhagic fevers research project of gamal abdel nasser university of conakry, guinea. viral rna was extracted from μl of the whole blood of m. natalensis using the qiamp viral rna kit (qiagen, germany, in accordance with the manufacturer's instructions. another part of biological samples (the lung and spleen tissue) from m. natalensis (n = ) and serum from humans (n = ) were collected by the staff of the centre de recherché en epidemiologie, microbiologie et soins medicaux (crems) in kindia, guinea. the lung and spleen tissue samples were homogenized in hanks' buffered salt solution using a tissue lyser lt (qiagen, germany). suspensions were centrifuged at rpm for min to precipitate debris, and μl of the supernatant was then used for viral rna extraction. the extraction of nucleic acids from the samples collected by crems (including samples from m. natalensis and serum from humans) was performed using the ribo-prep extraction kit (amplisens, russia) as per the manufacturer's instructions. viral rnas were examined for lassa immediately after extraction by the staff of the virology laboratory of hemorrhagic fevers research project of gamal abdel nasser university of conakry, guinea and were then used to assess diagnostic sensitivity and specificity. one-step rt-pcr assay targeting gp, with primers ows- -fwd ( gcgcaccggggatcctaggc) and ows- -rev (agcatgtcacaa-aaytcytcatcatg) was used for lasv detection (ehichioya et al., ) . all sequences of lasv l gene available in genbank (ncbi) were aligned to identify conserved sites. the alignment was performed using the bioedit . . software package (ibis biosciences, usa). a -nt fragment of the l gene (nt positions - in the reference sequence of lasv, strain josiah, genbank accession number jn ) was selected as a target for amplification using plotcon (http://emboss. bioinformatics.nl/cgi-bin/emboss/plotcon). the primers and probes were designed in accordance with the current guidelines, regarding taqman primers and probes for rt-pcr techniques (tyagi and kramer, , van pelt-verkuil et al., ) . the melting temperature for the primers was calculated using the oligonucleotide properties calculator (http://biotools.nubic.northwestern.edu/ oligocalc.html) (kibbe, ) . in addition, the oligonucleotide properties calculator and mfold were used to assess the thermodynamic characteristics of the probes and the probability of the appearance of secondary structures (http://unafold.rna.albany.edu/?q=mfold/ download-mfold). specifically, the lasv-specific probes were covalently attached to the fluorescent reporter dye rhodamine g and black hole quencher at the ′ and ′ ends, respectively. the primers and probes were synthesized by the branch associated with bioorganic synthesis at the central research institute for epidemiology, moscow, russia. the total volume of the rt-qpcr reaction mix was μl, including the following: μl of the rna sample, . μm of each primer and probe (lvl-forw , lvl-forw , lvl-rev , lvl-rev , lvl-prb a, lvl-prb b, and lvl-prb c) and . μm of each ic-detection primer and the probe (ic-forwa, ic-reva, and ic-prba), . μl of dntps ( . mm; amplisens, russia), μl of rt-pcr-mix fep/frt (amplisens, russia), . μl of mmlv reverse transcriptase (amplisens, russia), . μl of rtg-mix (amplisens, russia), and . μl of taqf polymerase (amplisens, russia). the thermal cycling parameters were as follows: °c for min, °c for min, and then cycles of °c for s and °c for s. fluorescence was observed at °c in a rotor-gene (qiagen, germany) in the yellow and green channels for specific signals and ic signals, respectively. the threshold value of fluorescence was chosen as the middle of the linear increase in the positive-control fluorescence expressed in the logarithmic units. amplification results were considered positive if the level of fluorescence crossed the threshold. to control all stages of the rt-qpcr reaction, an external positive control for pcr (c+) and an armored recombinant positive control for reverse transcription (arc+) were developed. the commercially available ic (ic-fl, amplisens, russia) was used to monitor rna extraction; for this purpose, ic-fl-specific primers and probe were added to the reaction mixture. in addition, a negative control for extraction (ec−) and pcr (c−) were used to exclude false-positive results due to possible or inadvertent cross-contamination. the cdna region ( bp) equivalent to l gene of lasv (strain josiah) that included the primer and probe target sequences was generated using step-out amplification, as previously described (dedkov et al., ) . the final pcr product was purified using a minelute gel extraction kit (qiagen, germany), ligated into the pgem-t plasmid vector (promega, usa), and transformed into escherichia coli (xl -blue strain) (maniatis et al., ) . recombinant plasmids from individual clones were purified using a plasmid miniprep kit (axygen, usa), and the orientation and absence of mutations in the cloned pcr fragment were evaluated by sanger sequencing using an abi-prism xl (applied biosystems, usa). the diluted plasmid solutions of known concentrations were used as c+. furthermore, the same cdna region was used to prepare arc + based on a previously described procedure for ms -phage-based arps (cheng et al., , pasloske et al., with certain minor modifications. in brief, the pcr fragment containing the target region and additional flanking nucleotides (see above) was ligated into a linearized in-house plasmid vector containing the ms coat protein gene. after verifying by dna sequencing, the generated recombinant plasmid was transformed into e. coli (strain b ) and protein expression was induced with isopropyl-l-thio-d-galactopyranoside. after induction, the cells were collected, lysed using a method combining lysozyme and freeze-thawing, and treated with dnase i (fermentas, usa) and rnase a (fermentas, usa). the derivate was then purified using cscl gradient centrifugation, quantified, and diluted in rnalater stabilization solution (life technologies, usa). the absence of residual dna in the treated sample was verified using the developed qpcr assay without the reverse transcription step. the c + and arc + concentrations were measured with a qx system (bio-rad) using a pcr supermix for probes kit (bio-rad), a one-step ddpcr supermix for probes kit (bio-rad), specific primers, and suitable probes as per the manufacturer's instructions (table ) . to assess the efficiency of rna extraction, an ic-fl (amplisens, russia) exogenous ic was added to the reagent mixture. the ic-fl is specifically an artificial rna sequence ( - nt, gc content %) mixture, surrounded by an ms -derived protective protein coat. the lod of the lasv-fl assay was determined using lasv strain josiah, which was provided by the center of virology and biotechnology "vector" as a part of their collection. in addition, lod was assessed using a series of -fold dilutions of arps. for this purpose, eight lasv sequences of a maximal number of mismatches in the targeting region of l gene were selected (including a sequence of the strain josiah, which was also used for the generation of the positive controls) and generated for the production of arps as described above (table ) . moreover, the concentrations of arps were measured with a qx system (bio-rad) using a one-step ddpcr supermix for probes kit (bio-rad), specific primers, and suitable probes as per the manufacturer's instructions. lod was assessed using a series of -fold dilutions of arps and lasv strain josiah. in brief, arps and lasv particles of known concentrations were diluted -fold using rnase-free elution buffer (amplisens, russia), added to intact human serum to make the final volume of μl, extracted using the ribo-prep extraction kit (amplisens, russia, in accordance with the manufacturer's instructions), and then tested using the lasv-fl assay to establish the standard curves and limit of detection (lod). the lod was set as the minimal dilution detected in three replicates (cherpillod et al., ) . potential cross-reactivity was assessed using the high-titer solutions (more than copies/ml) of viral rna and dna from viral species belonging to viral families, which are part of the collection of central research institute for epidemiology. the summary of rna or dna used in the study is shown in table . the assay sensitivity and specificity were determined using serum samples from patients with the clinical symptoms of fever who were found lassa positive (n = ) and lassa negative (n = ) and using whole blood and tissue samples from m. natalensis (n = positive and n = negative). the % confidence interval for a proportion was calculated according to r. newcombe derived from a procedure outlined by e. wilson (newcombe, , wilson . the primers and probes were designed in accordance with the current guidelines, regarding taqman primers and probes for rt-pcr techniques the lasv-specific probes were covalently attached to the fluorescent reporter dye rhodamine g and black hole quencher at the ′ and ′ ends, respectively. eight lasv sequences of a maximal number of mismatches in the targeting region of l gene were selected and generated for the production of arps. the lod assessed using arp dilutions was between copies/ml and copies/ml (depending on the arp). the multiple alignments (suppl. fig. ) of the sequences of lasv available in the genbank at the beginning of the study allowed the identification of highly conserved regions required for the designing of the lasv-specific primers and respective probes (table ) . on the basis of the sequencing data, oligonucleotide primers and fluorescent probes were designed and synthesized, and the lasv-specific assay was developed. the developed assay included all components required for rt-qpcr. the advantage of this assay is that it allows the verification of all steps of the analysis, including extraction, reverse transcription and pcr. in addition, using ec − and c−, the risk of false-positive results because of cross-contamination was minimized. the lod assessed using arp dilutions was between copies/ml and copies/ml (depending on the arp), and the lod measured using lasv strain josiah was pfu/ml (table ). standard detection was linear ranging from copies/ml (c t = . - . ) to × - copies/ml (c t = . - . ) of the lasv arps (r = . - . ; fig. ). the potential for cross-reactivity was assessed using high-titer rna or dna from viral species. none of the different viral species showed a positive reaction with the lasv rt-qpcr. consequently, the evaluated analytical specificity was %. in addition, a total of biological samples (positive n = and negative n = ) examined for lassa previously by the virology laboratory of hemorrhagic fevers research project of gamal abdel nasser university of conakry, guinea were tested using the lasv rt-pcr assay, with samples testing positive and negative (tables and ). discordance in the two samples (negative by the lasv-fl assay and positive by vlhv) was observed. the ct − values of the positive samples ranged from . - . cycles. thus, the diagnostic sensitivity and specificity of lasv-fl rt-qpcr assay were . % ( / ) ( % ci, . - . %) and % ( / ), respectively. lasv antigen can be detected by elisa using lasv-specific antibodies , niklasson et al., , jahrling et al., . however, this method has a relatively low sensitivity (ranging from - pfu/ml) (jahrling et al., ) . this peculiarity limits its usage, particularly at an early stage of lfd because of the low viral load in the biological fluids of patients. in addition, serological methods based on specific antibody detection in the case of lfd cannot be used during the initial days of the onset of the disease because a considerable concentration of lasv-specific igm appears during - days (salvato et al., ) . however, a lack of an antibody response has been reported in some fatal cases of lassa fever (wulff and johnson, ) . moreover, differing antibody responses depending on the virus strain used (emmerich et al., ) . therefore, rt-qpcr technique is most suitable for the diagnosis of lasv (salvato et al., ) , particularly during the initial days after lfd onset and is the preferred method for the rapid and early diagnosis of lassa fever because of its high sensitivity and easy implementation (gunther and lenz, ) . in this paradigm, a number of rt-pcr assays for lasv detection were developed and evaluated. most of them targeted the s-segment encoding the glycoprotein precursor and nucleoprotein as limited information is available regarding lasv sequences (drosten et al., , trappier potential cross-reactivity was assessed using the high-titer of viral rna and dna from viral species belonging to viral families, which are part of the collection of central research institute for epidemiology. lod was assessed using a series of -fold dilutions of lasv strain josiah, which was provided by the center of virology and biotechnology "vector" as a part of their collection. et al., , demby et al., , trombley et al., . however, some lasv strains were not detected using the developed rt-pcrs (trappier et al., ) . from the new information regarding the sequences of the s-segment, lasv is a virus of high genetic variability and designing reliable lasv-specific primers and probes is problematic . the appearance of a sufficient number of the l-gene sequences facilitated in designing an assay that could be more reliable for lasv detection because rna polymerases that are encoded by the l-gene share conserved amino acid motifs even between different virus families (poch et al., , poch et al., . during the past years, several lasv genomes have been completely sequenced, and the l-gene sequences of lasv also demonstrate high genetic variability even within one genetic lineage. therefore, the genetic diversity of lasv is a natural peculiarity, which most likely will be a limitation in the usage of rt-qpcr in the diagnosis of lasv. in this study, we developed an assay that is suitable, in equal measure, to detect the broad range of lassa strains. however, the lod of the assay considerably differs and depends on the lasv strain (table ) . two discordant samples of m. natalensis (negative by the lasv-fl assay and positive by vlhv) found in the assessment of diagnostic sensitivity supporting this assumption. however, we successfully used it in our routine practice to detect lasv in guinea using various types of samples, including whole blood and tissue of m. natalensis, as well as the serum samples from humans collected at - days after the onset of lfd. thus, our assay can be used for lfd diagnosis and surveillance in the case of lasv endemic in fig. . the assay lod and assay standard curves assessed using arps. standard detection was linear ranging from copies/ml to × - copies/ml of the lasv arps (depending on the arp). the lod was set as the minimal dilution detected in three replicates. guinea. the developed assay may be used for lasv strains circulated in other endemic territories. however, this suggestion needs to be confirmed in further studies. the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. the study has been evaluated and approved by local ethics committees of the pasteur institute, saint-petersburg, russia and comité national d'ethique pour la recherche en santé, guinea the authors declare that they have no competing interests the authors express deep gratitude to dr. elisabeth fichet-calvet from bernhard-nocht institute of tropical medicine (hamburg, germany), who advised dr. barre soropogui 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key: cord- -irji owi authors: britton, paul; evans, sharon; dove, brian; davies, marc; casais, rosa; cavanagh, dave title: generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: irji owi a reverse genetics system for the avian coronavirus infectious bronchitis virus (ibv) has been described in which a full-length cdna, corresponding to the ibv (beaudette-ck) genome, was inserted into the vaccinia virus genome following in vitro assembly of three contiguous cdnas [casais, r., thiel, v., siddell, s.g., cavanagh, d., britton, p., . reverse genetics system for the avian coronavirus infectious bronchitis virus. j. virol. , – ]. the method has subsequently been used to generate a recombinant ibv expressing a chimaeric s gene [casais, r., dove, b., cavanagh, d., britton, p., . recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. j. virol. , – ]. use of vaccinia virus as a vector for the full-length cdna of the ibv genome has the advantage that modifications can be made to the ibv cdna using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. we describe the use of homologous recombination as a method for modifying the beaudette full-length cdna, within the vaccinia virus genome, without the requirement for in vitro assembly of the ibv cdna. to demonstrate the feasibility of the method we exchanged the ectodomain of the beaudette spike gene for the corresponding region from ibv m and generated two recombinant infectious bronchitis viruses (ribvs) expressing the chimaeric s protein, validating the method as an alternative way for generating ribvs. avian infectious bronchitis virus (ibv), a group three member of the genus coronavirus (order nidovirales, family coronaviridae), is a highly infectious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney and oviduct (cavanagh, ; cavanagh and naqi, ; cook et al., ) . genetically very similar coronaviruses cause disease in turkeys and pheasants (cavanagh et al., , . coronaviruses are enveloped viruses that replicate in the cell cytoplasm and contain an unsegmented, -capped and -polyadenylated, single-stranded, positive-sense rna genome of - kb (de vries et al., ; lai and cavanagh, ; siddell, ) . all coronavirus envelopes contain at least three membrane proteins, the spike glycoprotein (s), a small membrane protein (e) and an integral membrane protein (m). in addition, the coronavirus virion also contains a nucleocapsid protein (n) that interacts with the grna. molecular analysis of the structure and the role of individual genes in pathogenesis of rna viruses has been advanced by the availability of full-length cdnas, for the generation of infectious rna transcripts that can replicate and result in infectious viruses from permissive cell lines (boyer and haenni, ) . such reverse genetics systems have resulted in the recovery of a number of infectious positive-stranded rna viruses including, picornaviruses, caliciviruses, alphaviruses, flaviviruses, arteriviruses and closterovirus, whose rna genomes range in size from to kb in length (agapov davis et al., ; racaniello and baltimore, ; rice et al., rice et al., , satyanarayana et al., ; sosnovtsev and green, ; sumiyoshi et al., ; van dinten et al., ) . recently full-length cdnas capable of producing infectious rna transcripts for several coronaviruses, viruses with the largest rna genomes, transmissible gastroenteritis virus (tgev; (almazán et al., ; yount et al., ) ), human coronavirus e (hcov; ), ibv (casais et al., ) , murine hepatitis virus (mhv; (yount et al., ) ), and severe acute respiratory syndrome coronavirus (sars-cov; yount et al., ) have been produced. the assembly of full-length cdnas corresponding to the coronavirus genomes was hampered due to some cdnas, derived from regions of the replicase gene, being unstable in bacteria. however, despite this, three methods have been described for the assembly of full-length cdnas of the genomic rna of coronaviruses. the first method involved the assembly of a tgev full-length cdna in a bacterial artificial chromosome (bac), immediately downstream of a viral rna polymerase ii promoter. transfection of the bac dna into susceptible cells resulted in synthesis of infectious rna and the recovery of infectious virus (almazán et al., ) . the second system involved the in vitro assembly of full-length cdnas using a series of contiguous cdnas with engineered specific restriction sites. bacteriophage t -rna polymerase-derived rna transcripts of the cdnas were used to generate infectious virus (yount et al., (yount et al., , (yount et al., , . the third strategy involved the in vitro assembly of contiguous cdnas followed by direct cloning into the genome of vaccinia virus. infectious rna transcripts were synthesised in vitro from the vaccinia virus dna, using bacteriophage t rna polymerase, and transfected into cells resulting in the recovery of infectious virus . in an alternative strategy infectious ibv was recovered following transfection of restricted vaccinia virus dna, containing the ibv full-length cdna, into cells infected with recombinant fowlpox virus expressing t rna polymerase (casais et al., ) . the vaccinia virus system has the advantage that the coronavirus full-length cdna is present in a non-bacterial system and offers the opportunity of modifying the coronavirus cdna by homologous recombination. falkner and moss ( ) devised a general method for modifying the vaccinia virus genome, involving insertions, deletions and specific mutations, termed transient dominant selection (tds). the method relies on a two-step procedure. in the first step, a complete plasmid sequence is introduced into the vaccinia virus genome as a result of a single cross-over event involving homologous recombination between vaccinia virus sequences in the plasmid dna and virus genome. the plasmid sequence contains the sequence being modified and a dominant selectable marker gene, escherichia coli guanine xanthine phosphoribosyltransferase (ecogpt) (mulligan and berg, ) , under the control of the vaccinia virus p . k promoter. recombinant viruses expressing the gpt gene are selected for resistance against mycophenolic acid (mpa) in the presence of xanthine and hypoxanthine. in the second step, the mpa-resistant viruses are grown in the absence of selection, resulting in loss of the ecogpt gene due to a single homologous recombination event between duplicated sequences, present as a result of the integration of the plasmid dna. the second step results in one of two recombination events; return to the original input virus sequence, without modification to the virus or introduction of the modified sequences into the vaccinia virus genome. both events should occur with equivalent efficiencies if the homology regions are of equal length. we have utilised the in vitro ligation and direct cloning into the vaccinia virus genome method of generating ribvs for the recovery of a ribv expressing a chimaeric s gene (casais et al., ) . the ribv expressed an s gene in which the ectodomain of the protein was derived from a different strain of ibv. having demonstrated that it was possible to produce a ribv expressing a modified s gene we decided to investigate an alternative, quicker and simpler, procedure for modifying the full-length ibv cdna within the vaccinia virus genome for the production of ribvs containing the chimaeric s gene. in this paper, we describe the generation of two homologous ribvs, ribv-m s-a and ribv-m s-b, in which the ectodomain sequence of the beaudette s gene in vaccinia virus vnoti-ibv fl (casais et al., ) was exchanged with the corresponding region from the m strain of ibv using the tds method. our results demonstrated that ribvs can be generated following direct modification of the full-length ibv cdna by homologous recombination within the vaccinia virus genome. the growth of ibv in either -day-old embryonated domestic fowl eggs or chick kidney (ck) cells was as described previously (pénzes et al., stirrups et al., ) . the ibv isolates used were: ( ) beaudette-ck (beau-ck; (cavanagh et al., ) ), a virus adapted for growth in ck cells that can grow on, but has not been adapted for growth on, vero cells an african green monkey cell line; ( ) beau-r, a recombinant ibv (ribv) produced from an infectious rna transcribed from a full-length cdna of beau-ck (casais et al., ) ; ( ) m -ck, a virus adapted from m (darbyshire et al., ) for growth on ck cells but not vero cells; and ( ) beaur-m (s), a ribv expressing a m -ck/beau-ck chimaeric s gene, produced following in vitro assembly of a full-length ibv cdna in vaccinia virus, that grows on ck but not vero cells (casais et al., ) . beaudette and m belong to the same, massachusetts, serotype. vaccinia viruses were titrated on monkey kidney fibroblast cells (cv- ) grown in dulbecco's-modified eagle medium (d-mem) supplemented with . % (w/v) sodium bicarbonate, l-glutamine, % foetal calf serum and an-tibiotics. baby hamster kidney cells (bhk- ) cells were grown in glasgow medium supplemented with . % (w/v) sodium bicarbonate, tryptose phosphate broth, l-glutamine, % foetal calf serum and antibiotics and used for the propagation of vaccinia viruses for isolation of virus dna. recombinant fowlpox virus rfpv-t (fpeflt pol; , expressing the bacteriophage t rna polymerase under the direction of the vaccinia virus p . early/late promoter, was grown in chick embryo fibroblast (cef) cells (casais et al., ) . recombinant dna techniques used standard procedures (ausubel et al., ; sambrook et al., ) or were used according to the manufacturers' instructions. all ibv-related nucleotide and amino acid residue numbers refer to the positions in the ibv beau-r genome (casais et al., ) , accession no. aj . the complete beau-ck s gene, within a . kb stui and bamhi fragment, was removed from pfrag- (casais et al., ) and inserted into hincii and bamhi digested pgpt-neb (a gift from dr. m. skinner iah) (boulanger et al., ) , resulting in pgpt-ibv-stui-bamhi ( fig. ) . plasmid pgptneb contains the e. coli guanine xanthine phosphoribosyltransferase (ecogpt) gene under the control of the vaccinia virus p . early/late promoter and a separate multiple cloning site. most ( %) of the beau-ck s gene was removed from pgpt-ibv-stui-bamhi by digestion with paci and styi followed by end repair of the dna and ligation, resulting in pgpt-ibv-s (fig. ) . the m -ck/beau-ck chimaeric s gene, within a . kb paci and bamhi fragment, was removed from pfrag- -m s (casais et al., ) and used to replace the beau-ck s gene sequence in pgpt-ibv-stui-bamhi, resulting in pgpt-m s (fig. ) . recombinant vaccinia viruses were generated as a result of transient dominant selection (tds; (falkner and moss, ) ) using the ecogpt gene as the transient selectable marker. cv- cells were infected with vaccinia virus vnoti-ibv fl , containing the full-length ibv cdna derived from beau-ck, at a multiplicity of infection (moi) equivalent to . plaque-forming units (pfu) per cell, and subsequently transfected with pgpt-ibv-s in the presence of lipofectin (invitrogen). recombinant viruses expressing gpt, from the ecogpt gene, were selected by three rounds of plaque purification in cv- cells in the presence of selection medium, cv- growth medium containing g/ml mpa, g/ml xanthine and g/ml hypoxanthine (fig. ) . several mpa-resistant vaccinia viruses were subsequently plaque purified (x ) in the absence of mpa resulting in the spontaneous loss of the ecogpt gene (fig. ) . several recombinant vaccinia viruses that had a gpt-negative phenotype were analysed by pcr and found not only to lack the ecogpt gene sequence but also, as expected, to have the beau-ck s gene sequence deleted from the ibv cdna. one such recombinant vaccinia virus, vnoti-ibv-s fl , was used in subsequent experiments. the tds method was used to insert the m -ck/beau-r chimaeric s gene into the ibv cdna within vnoti-ibv-s fl using pgpt-m s and involving ecogpt selection in the presence of mpa followed by loss of the ecogpt gene in the absence of mpa (fig. ) . recombinant vaccinia viruses that had a gpt-negative phenotype were analysed by pcr and found to lack the ecogpt gene sequence. further analysis identified a recombinant vaccinia virus, vnoti-tdsr-m s fl , that contained the m -ck/beaudette-ck chimaeric s gene sequence within the full-length ibv cdna. vaccinia virus vnoti-tdsr-m s fl was grown in t flasks of bhk- cells until a cpe was evident. the infected cells were harvested, resuspended in mm tris-hcl ph . , mm edta lysed by three cycles of freeze thawing, sonicated and the cell nuclei pelleted by centrifugation at × g for min at • c. virus particles were partially purified by centrifugation, through a cushion of % sucrose in mm tris-hcl, ph . , at , rpm for h at • c, using a sw c rotor in a sorvall otd b ultracentrifuge and resuspended in mm tris-hcl, ph . , mm edta. the semi-purified virus was incubated in mm tris-hcl ph . , mm edta, . % sds, mm nacl containing . mg/ml proteinase k, incubated at • c for h, extracted with phenol/chloroform and the dna ethanol precipitated. the vaccinia virus dna was digested with asci ( u/g of dna) and analysed by pulse field gel electrophoresis using % agarose gels (casais et al., ) . ck cells, grown to % confluence (approximately × cells) in mm diameter plates, were infected with rfpv-t at an moi of . after - min, the cells were transfected with g of ascirestricted vnoti-tdsr-m s fl dna and g of pci-nuc (hiscox et al., ) using g of lipofectin (invitrogen) (casais et al., (casais et al., , . the transfected cells (p cells) were incubated at • c for h after which the transfection medium was replaced with fresh maintenance medium (pénzes et al., ) . at . days post transfection, the culture medium, potentially containing ribv (v ), was removed, centrifuged for min at rpm, filtered through a . m filter, to remove any rfpv-t , and used for serial passage on ck cells. two independently rescued the recombination vector is based on a gpt expressing plasmid, pgptneb , in which a segment of the ibv genome, corresponding to the ibv s gene and flanking sequences of approximately equal length, was introduced. the majority of the s gene was then removed, leaving only the transmembrane and c-terminal domains within the recombination vector, pgpt-ibv-s. the flanking regions were included to allow homologous recombination events to occur between the ibv sequences in pgpt-ibv-s and the corresponding sequences in the ibv full-length cdna in the vaccinia virus (vnoti-ibv fl ) genome. the flanking regions were of approximate equal length to allow unbiased homologous recombination events. the various ibv gene sequences and relevant restriction sites are indicated; s represents the spike glycoprotein gene, a, b, c (e) represent the ibv gene products; m represents the integral membrane protein; a, b represent the ibv gene products; n represents the nucleocapsid protein; utr represents the ibv untranslated region; h␦r represents the hepatitis delta ribozyme sequence; t term represents the t termination sequence; ecogpt represents the e. coli gpt gene and vv p . represents the vaccinia virus early/late p . promoter. both types of recombination events have an equal chance of occurring. the various ibv genes are as described in the legend to fig. rep represents the end of the ibv replicase gene, s indicates that the signal sequence and ectodomain are deleted with the transmembrane and c-terminal domain sequences remaining in the partial s gene sequence in the ibv cdna; and represent the ibv gene and sequences, respectively. ribvs, ribv-m s-a and ribv-m s-b, were obtained and characterised. oligonucleotides gpt-forw ( -atgagcgaaaaat-acatcgtc- ) and gpt-rev ( -ttagcgaccgga-gattgg- ) were used to determine the presence and absence of the ecogpt gene sequence in the intermediary vaccinia virus recombinants and in the vaccinia virus recombinants, potentially containing the modified ibv sequences; a bp product was indicative of the ecogpt gene. oligonucleotides bg- ( - gctggtggacctata-act - ) and bg- ( - agcaattgaaactg-aaagtg - ) were used to analyse vnoti-ibv-s fl and vnoti-tdsr-m s fl dna to determine the absence of the beau-r or presence of the m -ck s gene sequences, respectively; a bp product was indicative of the m -ck s gene sequence. sequence analysis was used to confirm that the pcr products were derived from the m -ck s gene sequence. total cellular rna was extracted from ck cells infected with the ribvs, ribv-m s-a and ribv-m s-b, by the rneasy method (qiagen) and analysed by rt-pcrs, using ready-to-go tm rt-pcr beads (amersham pharmacia biotech) and a variety of oligonucleotide pairs. the rt-pcr products were sequenced to confirm the sequence of the s gene present in the two ribvs. confluent monolayers of ck and vero cells in mm dishes were infected with . × pfu of m -ck, beaur-m (s), ribv-m s-a and ribv-m s-b or × pfu of beau-r. a lower multiplicity of infection for beau-r was used to further enhance the difference of growth on vero cells of ibvs expressing either a beaudette-derived or m derived s protein ectodomain. following adsorption, for h at • c, the cells were washed three times with phosphatebuffered saline (pbs) to remove residual virus and incubated at • c in ml of ck media. samples of media were, at selected times over a h period, analysed in triplicate for progeny virus by plaque assay in ck cells. sequence analysis of plasmid dna, pcr products from the ibv cdna sequences within vnoti-ibv-s fl and vnoti-tdsr-m s fl and from rt-pcr products generated from rna isolated from ribv-m s-a and ribv-m s-b infected ck cells, was done using an abi prism bigdye terminator cycle sequencing ready reaction kit (applied biosystems) or ceq tm dtcs quick start kit (beckman coulter). oligonucleotide primers used for the sequencing reactions were derived from the beau-ck sequence (boursnell et al., ) . sequences were determined on an applied biosystems dna sequencer or a ceq tm capillary sequencer. pregap and gp of the staden sequence software programs (bonfield et al., ) were used for sequence entry, assembly and editing. we have developed a reverse genetics system for the avian coronavirus ibv in which the full-length ibv cdna was assembled by in vitro ligation followed by insertion into the vaccinia virus genome (casais et al., ) . we decided to investigate an alternative procedure, utilising homologous recombination by tds (falkner and moss, ) , for modifying the full-length ibv cdna within the vaccinia virus genome to circumvent the necessity of assembling the ibv cdna in vitro and subsequent insertion into the vaccinia virus genome. to test the tds-based method we proposed to replace the ectodomain of the beau-ck s gene, within the full-length ibv cdna in vnoti-ibv fl (casais et al., ) , with the ectodomain of the m -ck s gene. however, to replace the . kb beau-ck s sequence with the corresponding m -ck s sequence an intermediary recombinant vaccinia virus, vnoti-ibv-s fl , containing the beau-ck cdna with the s gene deleted had to be produced. this was to avoid re-combination events occurring within the s gene sequence if attempts were made to directly exchange the two sequences. plasmid pgpt-ibv-s ( fig. ) with % of the beau-ck s gene sequence deleted and beaudette-derived sequences, bp proximal and bp distal to the deleted region of the s gene sequence, for homologous recombination with the ibv cdna in vnoti-ibv fl , was used for the tds process for generation of vnoti-ibv-s fl (fig. ) . pcr and sequence analysis of the ibv cdna within vnoti-ibv-s fl confirmed that the beau-ck s gene had been deleted (data not shown). the m -ck/beau-ck chimaeric s gene sequence for insertion into the ibv cdna within vnoti-ibv-s fl was identical to the sequence we had previously used for the generation of ribv beaur-m (s) (casais et al., ) . the chimaeric s gene consisted of the signal sequence, ectodomain and transmembrane regions derived from ibv m -ck and the cytoplasmic tail domain from beau-ck. the transmembrane domains are identical between the two sequences but the cytoplasmic tail domain of the m -ck s protein is truncated by nine amino acids when compared to the beau-ck s protein. we had previously indicated that we retained the beau-ck cytoplasmic tail domain because this region of the s protein has been demonstrated to interact with other virus proteins (godeke et al., ) . the chimaeric s gene sequence, from pfrag- -m s (casais et al., ) , was used to replace the corresponding beau-ck s gene sequence in pgpt-ibv-stui-bamhi, resulting in pgpt-m s (fig. ) . plasmid pgpt-m s, contained the chimaeric s gene with beaudette-derived sequences, bp proximal and bp distal to the chimaeric s sequence, for homologous recombination with the beau-ck-derived ibv cdna in vnoti-ibv-s fl , to insert the chimaeric s gene sequence into vnoti-ibv-s fl (fig. ) . a recombinant vaccinia virus, vnoti-tdsr-m s fl , was isolated and pcr and sequence analysis confirmed that the ibv cdna within the vaccinia virus genome contained the chimaeric s gene sequence. two ribvs, ribv-m s-a and ribv-m s-b, were independently recovered from ck cells, previously infected with rfpv/t , to provide t rna polymerase, and co-transfected with asci-restricted vnoti-tdsr-m s fl dna and pci-nuc (casais et al., (casais et al., , . the vnoti-tdsr-m s fl dna was prepared from semi-purified vaccinia virus and pci-nuc, a plasmid expressing the ibv n protein under control of both the t and cmv promoters, was required for the successful recovery of ribv (casais et al., ) . the transfected ck cells (p ) were incubated for . days post transfection, until they showed a cytopathic effect (cpe), the medium was filtered to remove any rfpv/t and any potential ibv passaged on fresh ck cells (p ). the two independently rescued ribvs, isolated from the p cells, were partially analysed and found to contain the chimaeric s gene and subsequently virus rna derived from p ck cells, was sequenced to confirm the presence of the chimaeric s gene. the ibv strains, beau-ck and m -ck, have different cell tropisms, both viruses replicate to similar titres in ck cells but only beau-ck produces infectious virus on vero cells (casais et al., ) . previous results using beaur-m (s), containing the chimaeric s gene following assembly of the full-length ibv cdna in the vaccinia virus genome, showed that the ribv had a phenotype like that of m -ck rather than beau-ck for growth on vero cells (casais et al., ) . therefore, we analysed the growth characteristics of ribv-m s-a and ribv-m s-b, containing the chimaeric s gene generated as a result of the tds method, on ck and vero cells for comparison with beaur-m (s). beau-r, m -ck, beaur-m (s), ribv-m s-a and ribv-m s-b were used to infect ck and vero cells and the titre of progeny virus determined over a h period. all five viruses displayed similar growth profiles on ck cells; progeny viruses were detectable h post infection with peak titres between × and × pfu/ml h post infection (fig. a ). all the viruses caused observable cpe within h on the ck cells. analysis of the growth profiles of the five viruses on vero cells (fig. b) showed that only beau-r replicated, with a maximum titre of pfu/ml by h post infection. in contrast, both ribv-m s-a and ribv-m s-b together with m -ck and beaur-m (s) showed significantly lower titres on vero cells in comparison to beau-r (fig. b) . overall, our results showed that ribv-m s-a and ribv-m s-b had similar phenotypes to those observed for 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a this work was supported by the department of environment, food and rural affairs (defra) project code od , european communities specific rtd program quality of life and management of living resources qlk -ct- - and the biotechnology and biological sciences research council (bbsrc). key: cord- -yuqc dk authors: tang, mengjun; wang, hongning; zhou, sheng; tian, guobao title: enhancement of the immunogenicity of an infectious bronchitis virus dna vaccine by a bicistronic plasmid encoding nucleocapsid protein and interleukin- date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: yuqc dk a dna vaccine against infectious bronchitis virus (ibv) can induce specific humoral and cell-mediated immunity. however, compared to conventional vaccines, dna vaccines usually induce poor antibody responses. to develop a more potent ibv dna vaccine formulations, a monocistronic vector encoding the nucleocapsid protein of ibv and a bicistronic vector separately encoding the nucleocapsid protein and immune-stimulatory interleukin- were constructed. when the dna vaccines were administered to the quadriceps muscle of chickens, the induced humoral and cellular responses were evaluated. there was a significant difference in elisa antibody levels elicited by either monocistronic or bicistronic dna vaccines. the percentage of cd (+), cd (+)cd (+) and cd (+)cd (+) subgroups of peripheral blood t-lymphocytes in chickens immunized with bicistronic dna vaccine were higher than those in chickens immunized with monocistronic dna vaccine. when chickens were challenged with a virulent strain of ibv, the protective efficacy could be enhanced significantly after immunization with bicistronic dna vaccine. these results demonstrated that bicistronic dna vaccine is an effective approach to increase ibv dna vaccine immunogenicity. infectious bronchitis (ib) is an acute and highly contagious respiratory disease of chickens. it is still a major health problem in the chicken industry in the world. vaccination to control ib has been practiced for over half a century (bijlenga et al., ; cavanagh and naqi, ; cavanagh, ) . such conventional vaccines, although generally effective, do have some disadvantages. attenuated vaccines, which generally induce long-lasting immunity, have a risk of insufficient attenuation and/or genetic instability (cook et al., ) . the limitations of inactivated vaccines include high cost for manufacturing and lack of long-term immunity. therefore a new generation of ib vaccines is called for. infectious bronchitis virus (ibv) is an enveloped coronavirus that contains an unsegmented, single-stranded, positive-sense rna genome. in addition to the internally localized nucleocapsid (n) protein, the virion is composed of spike protein, membrane protein and a small envelope protein. n protein carries epitopes inducing cross-reactive antibodies and is the most abundant virus-derived protein produced throughout infection (seah et al., ; boots et al., ; yu et al., ) . the carboxyl end of n protein has ctl epitopes for mhc compatible chickens . chickens inoculated with dna plasmids expressing the carboxyl end of n were protected from ibv infection . in recent years, the development of dna vaccines as a potentially safe alternative has been explored. dna immunization is an important vaccination strategy that has many characters desirable for an ideal vaccine, including induction of broad immune responses, long-lasting immunity and simple and cheap production. experimental dna vaccines against viral, bacterial, and parasitic disease have been described (tacket et al., ; strugnell et al., ; kalinna, ) and dna vaccines have been licensed for two nonhuman applications: one for west nile virus for horses (powell, ) and the other for infectious hematopoietic necrosis virus for salmon (lorenzen and lapatra, ) . hence dna vaccines appear to be a useful technology. it is believed that delivery of only a single dna plasmid of antigen is not optimal to protect against infection. adjuvants are used widely in various vaccine formulations for the enhancement of immune responses. among the adjuvants, cytokines have been explored extensively to augment the potency of dna vaccines (gurunathan et al., ; reyes-sandoval and ertl, ; scheerlinck, ; barouch et al., ; calarota and weiner, ; stevenson, ; barouch, ) . interleukin- (il- ), initially known as t-cell growth factor, is a powerful immunoregulatory lymphokine which produced by lectin-or antigen-activated t-cells (morgan et al., ) . it is secreted by mature t-lymphocytes upon stimulation and certain t-cell lymphoma cell lines constitutively. plasmid il- has been investigated as a potential vaccine adjuvant in several studies and has been shown to increase dna vaccine protective immunity against pathogens (diane and carlos, ; rompato et al., ; bu et al., ) . in the present study, a bicistronic plasmid encoding the nucleocapsid protein and immune-stimulatory interleukin- has been constructed and its immunogenicity and protective effect in chickens has been evaluated. it has been shown that the delivery of a bicistronic plasmid containing n gene and il- can accelerate specific antibody induction with an increase t-cell responses. the use of bicistronic vector may enable more efficient delivery of both antigen and cytokine in dna vaccination and promote synergistic responses. the specific-pathogen-free (spf) chicken embryos were purchased from shangdong institute of poultry science, shandong, pr china. chickens were hatched and housed in a specificpathogen-free environment at the laboratory animal and resources facility, sichuan university. the nephropathogenic strain of ibv, saibk strain was propagated in the allantoic cavities of -day-old spf embryonated chicken eggs, and then harvested allantoic fluid h post-inoculation. the % chicken infection dose (eid ) was determined by inoculating serial fold dilutions of virus into -day-old spf embryonated chicken eggs. vero cells were cultured in dulbecco's modified eagle's medium (gibco) supplemented with % fbs, u/ml penicillin and g/ml streptomycin at ph . and were kept at • c with % carbon dioxide. the pires-egfp/dsred bicistronic plasmid which was constructed and identified previously, was used to construct monocistronic and bicistronic dna vaccines. this plasmid contained two fluorescent protein genes which were inserted into the multiple cloning sites (mcs) located on either side of the internal ribosome entry site from the encephalomyocarditis virus (ecmv). the ibv n gene was subcloned from recombinant plasmid pibvn into egfp of pires-egfp/dsred between nhei and xhoi restriction sites, and generated the plasmid pires-n/dsred. the il- gene was amplified by pcr from the plasmid pdnail- as a template. the forward primer ( -ccaggatccaccatgatgtg aag- ) and reverse primer ( -gaagcggccgcagattagttagc- ) specific for the il- gene were employed. the pcr product of il- was digested with bamhi and noti and ligated into similarly digested pires-n/dsred, then the bicistronic plasmid pires-n/il was created. the plasmid pires-n/dsred was digested with bamhi and noti, gel purified and blunt ended to create monocistronic pires-n. identification of the recombinant plasmids was performed by double enzymes digestion and dna sequencing. six-well tissue culture plates were seeded with vero cells ( /well), and the cells were grown until they were about % confluent. the purified plasmids, pires-n, pires-n/il- and pires were transfected respectively into the vero cells with lipofectamine according to the manufacturer's instructions (invitrogen, ca, usa). the expression products were identified after - h. the transfected cells were harvested after h and total cellular rna was prepared from the transfected cells by trizol reagent (gibco brl, usa). the transcription products were detected by reverse-transcription polymerase chain reaction (rt-pcr) with specific primer sets for the n gene and il- as listed. n forward primer: -catctcgagtctt-ttatcatggcaagc- , reverse primer: -ggcgaattca-ttagagttcattttcac- ; il- forward primer: -ccaggatccaccatgatgtgcaaag- , reverse primer: -gaagcggccgcagattagttagc- . the medium was aspirated h after transfection, and the cells were washed once with phosphate-buffered-saline (pbs), fixed with % acetone for min at − • c, then washed three times for min each with pbs. thereafter, transfected cells were incubated at • c for h with the antibody, which was antiserum of rabbit to ibv. then the cells were washed twice for min each with pbs and incubated for a further h at • c with the secondary fitc-conjugated goat-anti-rabbit igg antibody that included . % azovan blue (purpose of using azovan blue was to identify the positive vero cells and negative vero cells). the positive vero cells expressing n protein were stained green; the negative vero cells were stained red. the cells were washed twice with pbs and analyzed the expression of the recombinant plasmid by fluorescence microscopy. the plasmids pires-n/il , pires-n, pires were amplified in escherichia coli jm and extracted using the alkaline lysis method as described previously (sambrook et al., ) . after purification by peg precipitation, the plasmids were resuspended in phosphate-buffered saline (pbs, ph . ) and kept at − • c until used for immunization. for vaccination, the chickens were randomly divided into four groups (n = each). the -day-old chickens were injected intramuscularly into the quadriceps muscle with g of plasmid pires-n/il (group ). a second group was given g of plasmid pires-n (group ). other groups included chickens administered with g of empty vector pires (group ), and chickens injected with . ml pbs only (group ). all groups were boosted with an equivalent dose at days after the initial inoculation. pre-vaccination sera were collected from all vaccinated chickens. blood was also collected before booster vaccination as well as before challenge. sera were stored at − • c for serologic analysis. total serum immunoglobulin g (igg) specific for ibv was measured by indirect enzyme-linked immunosorbent assay (elisa) as described previously, with brief modifications: elisa plates were coated with ibv lysate at g/ml in carbonate buffer, ph . , for overnight at • c and blocked with % non-fat dried milk in pbs at • c for h. serum samples were tested in : dilution in % dried milk in pbst. igg against ibv was revealed with horseradish peroxidaselabeled goat-anti-chicken conjugate diluted : in pbst. the substrate solution used was tmb microwell peroxidase. after min of incubation in the dark, the reaction was stopped by the addition of l of m h so , and the optical density at nm was measured in an elisa microplate reader. sera were run in duplicate. negative and positive control sera were included in each assay. total serum immunoglobulin g (igg) specific for ibv are represented as the optical density. peripheral blood samples from immunized chicken were collected from the jugular vein in . ml syringes preloaded with . ml of sodium heparin to prevent clotting on day after the boosting vaccination. peripheral blood mononuclear cells were isolated from each blood sample by ficoll-hypaque density gradient centrifugation. pbmc were adjusted to × cells/ml. the l of samples ( × cells) were incubated for h at room temperature with antibody as follows: mouse anti-chicken cd -pe, mouse anti-chicken cd -fitc, mouse anti-chicken cd -sprd (bd biosciences pharmingen). leukocyte samples were triply labeled with cd , cd and cd antibodies. the samples were processed on fluorescence activated cell sorter. all of the chickens were challenged with eid of the ibv saibk strain in . ml by the nasal-ocular route at days after the boosting immunization. the challenged chickens were examined daily for signs of clinical illness such as coughing, sneezing, ataxia, dyspnea or death for weeks. dead chickens were necropsied to confirm death by ibv infection. the challenged chickens generally began to show clinical signs from to days after challenge. chickens in each group were euthanized at days post-infection. necropsies were performed immediately postmortem and kidney tissues were collected for further detection of virus. the kidney tissues were incised individually from either the dead or euthanized chickens at days post-challenged. the virus in the kidney tissues of the challenged chicken was detected by rt-pcr. total rna was extracted using trizol ls reagent and subjected to rt-pcr using primers directed to the untranslated region (forward primer: -gatgaggagaggaacaatgc- ; reverse primer: -tgggcgtcctagtgctgt- ). total protection was defined as negative for the presence of virus in the kidney. data were analyzed using the one-sided student's t test. differences were considered statistically significant with p < . . the monocistronic and bicistronic dna vaccine plasmids encoding ibv n protein and il- were constructed using bicistronic plasmid pires-egfp/dsred as shown in fig. . to confirm the transcription of constructs pires-n/il , pires-n in a eukaryotic system, the plasmids were transfected respectively into vero cells. total rna was extracted from transfected cells at h and analyzed by rt-pcr for the presence of each corresponding mrna. the predicted rt-pcr products were of . kb in size for n and . kb for il , all of which were confirmed by gel electrophoresis. no specific band of a similar size was seen in any of the mrna samples in the absence of reverse transcription (fig. ) . this result showed that constructs encoding n and il- gene can be transcribed successfully in the eukaryotic system. the expression of pires-n/il , pires-n was demonstrated by indirect immunofluorescence assay. after transfection with lipofectamine, the transfected cells displayed positive signals for the protein and located cytoplasm, where there was green fluorescence. expression of n protein was not detected in transfected empty plasmid control cells (fig. ) . this result shows that constructs encoding n protein can be successfully expressed in the eukaryotic system. the plasmids pires-n/il , pires-n induced detectable antibodies to ibv ag in chickens one week after injection and the levels increased with subsequent vaccination. there was no specific antibody response in the group of chickens receiving pbs and pires plasmid. there was a significant difference in elisa antibody levels (p < . ) elicited by either monocistronic or bicistronic dna vaccines since the th-day after first inoculation. the result suggests that the bicistronic pires-n/il can enhance humoral responses (fig. ) . peripheral blood lymphocytes were analyzed by flow cytometry on day after the boosting immunization. the percentage of cd + and cd + cd + t-lymphocytes significantly higher (p < . ) was observed from chickens immunized with pires-n/il than the pires-n group. the percentage numbers of cd + cd + t-lymphocytes subgroups of the pires-n/il vaccinated group were higher than the pires-n-vaccinated group but no significant difference (p > . ) (fig. ) . mortality, kidney infection and percent protection after challenge of chickens were summarized in table . chickens that started to show clinical signs or died from viral infection did so beginning on day after challenge. the chickens immunized with either control vector pires or pbs were not protected and developed coughing, nasal discharge, dyspnea. the death rate of the pires and pbs immunized chickens was and % at days after challenge, respectively. the death rate of the pires-n/il dna vaccine immunized chickens was only %, lower than that of the chickens injected with the pires-n dna vaccine. to evaluate the level of protective response after challenge, the collected kidney samples were analyzed by rt-pcr. pcr results indicated that and % of birds vaccinated with the pires-n/il and pires-n plasmids were positive for the presence of virus in the kidney, respectively. all chickens immunized with either control vector pires or pbs were positive in rt-pcr test. the protection percent of group that vaccinated with the pires-n alone was higher than that of the empty vector or pbs. the group vaccinated with the pires-n/il dna vaccine ( %) had the highest protection rate in all vaccinated groups. this suggests that the plasmid expressing both n protein and il- offers enhanced resistance against a virulent ibv challenge. in order to increase the efficiency of this immunization procedure, cytokine was co-expressed with the viral protein. this strategy has already been used successfully to enhance the effect of dna vaccination procedures (li et al., ; chow et al., ; henke et al., ) . the pires-egfp/dsred bicistronic plasmid which was constructed and identified previously was used to construct monocistronic and bicistronic dna vaccines. the plasmid pires-egfp/dsred enabled the simultaneous translation of two genes of interest from the same rna transcript. each gene is cloned into one of the multiple cloning sites on either side of the internal ribosomal entry site of the encephalomyocarditis virus (emcv). the entire construct is under control of the cytomegalovirus (cmv) immediate-early promoter allowing the expression of two individual proteins from one plasmid. in this study, an experimental immunization strategy was developed and tested against ibv. two recombinant plasmids pires-n/il , pires-n were constructed. these recombinant plasmids were inoculated in chickens and tested in a protection-challenge experiment, demonstrating that vaccination with the co-expression plasmid pires-n/il can induce stronger immune response than vaccination with pires-n. thus, it seems that vaccination with a bicistronic dna vaccine expressing both ibv n protein and il- may elicit potent immune response. in this study, the level of specific antibodies developed in pires-n/il group was higher than that of pires-n group. however, the precise role of antibodies for the control of ibv infection remains controversial. some reports have shown that circulating antibody titer did not correlate with protection from ibv infection (gelb et al., ; gough and alexander, ; raggi and lee, ) . other studies demonstrated that humoral immunity plays an important role in disease recovery and virus clearance (cook et al., ; thompson et al., ; toro and fernandez, ) . the antibody-dependence of the mechanism of protection against the disease remains unclear, suggesting an important role of the t-cell response in efficacy protection. in order to evaluate recombinant plasmids induced t-cell response, peripheral blood lymphocytes were analyzed by flow cytometry. results of t-lymphocytes subgroup detection indicated, that the percentage numbers of cd + , cd + cd + and cd + cd + t-lymphocytes subgroups in pires-n/il vaccinated chickens group were higher than those in the pires-n vaccinated chickens. this demonstrated that il- has the ability to stimulate t-cell growth. it has been shown that cellmediated immunity to ibv is also induced and is believed to be a protective mechanism in ibv infection. cd + ctl are critical in the control of infectious bronchitis in poultry dhinakar and jones, ; seo et al., ) . cd + t-cell responses may increase the proliferation, maturation and functional activity of cd + ctl, providing increased help for b-cells and directly producing antiviral cytokines. by increasing the number of t-cells at the same site being able to respond to n antigen, there might have been a limiting effect on viral replication leading to better protection. to investigate the level of protection elicited by pires-n/ il , vaccinated chickens were challenged with a nephropathogenic strain of ibv. chickens that received the pires-n/il plasmid dna were better protected than that administered with the plasmid pires-n. mortality of pires-n/il group was lower than that of pires-n group. the protection rate of pires-n/il group was the highest in all the vaccination groups, possibly indicating protective immunity overcome by virus aggressiveness. these results suggested that vaccination with the co-expression plasmid of n protein gene and il- may have increased the protection rate against challenge. results of immune response and viral challenge showed that the group inoculated with pires-n/il provided stronger immune response and better protection rate than pires-n vaccinated group. this indicated that il- might effectively enhanced humoral-mediated immune and cell-mediated immune responses to some extent. this was consistent with the result of previous reports (li et al., ; chow et al., ; henke et al., ) . these results demonstrated that bicistronic dna vaccine is an effective approach to increase ibv dna vaccine immunogenicity. our results showing the induction of both antibody and t-cell responses against the ibv challenge in chickens demonstrate that the delivery of antigens and cytokines via bicistronic vectors is feasible in the chicken model. rational design of gene-based vaccines the role of cytokine dnas as vaccine adjuvants for optimizing cellular immune responses development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review induction of anti-viral immune responses by immunization with recombinant-dna encoded avian coronavirus nucleocapsid protein co-delivery of il- or liposomes augment the responses of mice to a dna vaccine for pseudorabies virus ie enhancement of human immunodeficiency virus type -dna vaccine potency through incorporation of t-helper molecular adjuvants severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus infectious bronchitis improvement of hepatitis b virus dna vaccines by plasmids coexpressing hepatitis b surface antigen and interleukin- cytotoxic t lymphocytes are critical in the control of infectious 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the immunization effect of a dna vaccine expressing herpes simplex glycoprotein d dna vaccines for aquacultured fish selective in vitro growth of t lymphocytes from normal human bone marrows dna vaccines: back in the saddle again? lack of correlation between infectivity, serologic response and challenge results in immunization with an avian infectious bronchitis vaccine dna vaccines positive inductive effect of il- on virus-specific cellular responses elicited by a prrsv-orf dna vaccine in swine molecular cloning-a laboratory manual genetic adjuvants for dna vaccines location of linear b-cell epitopes on infectious bronchitis virus nucleocapsid protein specific cytotoxic t lymphocytes are involved in in vivo clearance of infectious bronchitis virus the carboxylterminal -residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protects chickens from acute infection adoptive transfer of infectious bronchitis virus primed alpha beta t cells bearing cd antigen protects chicks from acute infection dna vaccines and adjuvants dna vaccine for bacterial infections phase i safety and immune response studies of a dna vaccine encoding hepatitis b surface antigen delivered by a gene delivery device systemic and local antibody responses to infectious bronchitis virus in chickens inoculated with infectious bursal disease virus and control chickens avian infectious bronchitis: specific lachrymal iga level and resistance against challenge study of protection by recombinant fowl poxvirus expressing c-terminal nucleocapsid protein of infectious bronchitis virus against challenge this work was supported by the foundation of chinese national programs for high technology research and development (project number aa a ). key: cord- -c e h u authors: hosmillo, myra d.t.; jeong, young-ju; kim, hyun-jeong; collantes, therese marie; alfajaro, mia madel; park, jun-gyu; kim, ha-hyun; kwon, hyung-jun; park, su-jin; kang, mun-il; park, sang-ik; cho, kyoung-oh title: development of universal sybr green real-time rt-pcr for the rapid detection and quantitation of bovine and porcine toroviruses date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: c e h u toroviruses (tovs) are a group of emerging viruses that cause gastroenteritis in domestic animals and humans. currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time rt-pcr) have not yet been developed for the rapid detection and quantitation of bovine (btov) and porcine (ptov) toroviruses. using btov and ptov rna standards generated by in vitro transcription, the detection limit of the sybr green real-time rt-pcr assay was . × ( ) btov and . × ( ) ptov copies/reaction (correlation coefficiency = . and . , respectively), whereas those of rt-pcr and nested pcr were . × ( ) and . × ( ) (btov) and . × ( ) and . × ( ) (ptov) crna viral copies/reaction, respectively. archived diarrhea specimens of calves (n = ) and piglets (n = ) were subjected to rt-pcr, nested pcr and sybr green real-time rt-pcr. by conventional rt-pcr, ( . %) bovine and ( . %) porcine samples tested positive to btov and ptov, respectively. with nested pcr, ( . %) bovine and ( . %) porcine samples tested positive. sybr green real-time rt-pcr assay detected btov and ptov in of ( . %) bovine and of ( . %) porcine samples. these results indicate that sybr green real-time rt-pcr (p < . ) is a more sensitive assay, which can be reproduced as a reliable, sensitive, and rapid tool for the detection and quantitation of toroviruses. toroviruses (tovs) are enveloped, positive-stranded polyadenylated rna viruses and are classified as the second genus in the family of coronaviridae, order nidovirales that contains many important pathogens. toroviruses infect both animals and humans and are associated predominantly with enteric diseases (koopmans and horzinek, ) . the first report of tovs described the identification of the berne virus in from a rectal swab of a horse with diarrhea in berne, switzerland (weiss et al., ) . a decade later, a virus that was related morphologically and antigenically to the berne virus, which was designated the breda virus (btov) and breda virus-like particles, was described in the stools of neonatal calves and children (beards et al., ; woode et al., ) . a porcine torovirus (ptov) has been identified and characterized in the feces of swine (kroneman et al., ) . toroviruses have been established as infectious gastrointestinal agents in cat-tle and as a predominant cause of acute enteric infections in piglets (ito et al., ; kroneman et al., ) . significantly, zoonosis was possibly implicated when tov-like particles detected in patients with gastroenteritis exhibited similar morphology and cross-reacted with btov, which supports the need for continuous monitoring of potential zoonotic infection with tovs (hoet and saif, ) . detection of btovs and ptovs usually involves a crosshybridization assay, electron microscopy (em), enzyme-linked immunosorbent assay (elisa) and reverse transcriptionpolymerase chain reaction (rt-pcr) (durham et al., ; koopmans and horzinek, ; matiz et al., ; smits et al., ) . real-time rt-pcr is a more recent method that is more sensitive than em and conventional rt-pcr for quantitating virus load, pathogenesis studies and identification of virus latency (glass et al., ; park et al., ). real-time rt-pcr is relatively easy to perform and has a high-throughput capacity as reported with other viruses using sybr green chemistry and/or the taqman assay (chun et al., ; park et al., ) . as far as we know, there is only one report describing the use of real-time rt-pcr for the detection of ptov (pignatelli et al., ) . there is no report of the use of real-time rt-pcr for btov. table list of primers used for conventional rt-pcr, nested pcr and sybr green real-time rt-pcr assay for the detection and quantitation of bovine and porcine toroviruses in the fecal specimens from diarrheic calves and piglets. rt-pcr. b all the procedure of rna extraction, conventional rt-pcr and nested pcr were performed as described previously (cho et al., ; park et al., ) . c position as counted from the start codon of the complete genome of bovine breda strain. d position as counted from the start codon of the porcine torovirus markelo strain m gene. e universal primer pair for sybr green real-time rt-pcr is designed from the m gene of the bovine and porcine torovirus strains reported in genbank database as follows; ay , aj , dq , ab , dq , aj , dq , dq , aj , aj , aj , aj , gu , gu , gu , gu , dq . the present study developed, optimized and validated a sybr green real-time rt-pcr assay using a universal primer pair for the detection and quantitation of both btovs and ptovs in archived stool samples. from the same samples evaluated previously by conventional rt-pcr and nested pcr assays, sybr green real-time rt-pcr proved to be a more specific and sensitive detection and quantitation method. bovine (n = ) and porcine (n = ) stool specimens were selected from archived fecal samples submitted to the laboratory of animal diseases, college of veterinary medicine, chonnam national university, by local veterinary clinicians in korea during - and . the stool specimens were stored at − • c until used. rna was extracted using trizol-ls (gibco-brl, grand island, ny) from a l starting volume of a centrifuged % fecal suspension of each sample. total recovered rna was suspended in l of rnase-free water and stored at − • c until analysis. the oligonucleotide primers, and conventional rt-pcr and nested pcr conditions for the detection of btov and ptov (table ) were described previously (park et al., ; shin et al., ) . a one-step real-time rt-pcr was developed based on the detection of sybr green. the primers were designed based on the published sequence of the membrane (m) gene (table ). all reactions were performed using a corbett research rotor-gene series real-time amplification system (corbett research, mortlake, australia) and sensimix one-step rt-pcr kit with sybr green (quantace, london, uk). optimization of primer concentration was achieved using rna from positive fecal samples. reactions were run using primer concentrations from . to . m. a concentration of . m of each primer was found to give the highest sensitivity. real-time rt-pcr was performed with a final volume of l containing l of rna template, . l of sensimix one-step mixture, l each of . m forward and reverse primers (final concentration of each primer: nm), . l of × sybr green solution (final concentration: ×), . l of rnase inhibitor (final concentration: units), . l of mgcl (final concentration: . mm) and l of rnase-free water. reverse transcription was carried out at • c for min, followed by the activation of the hot-start dna polymerase at • c for min and three-step cycles: • c for s, • c for s and • c for s. to perform the melting curve analysis, after reaction cycles, the temperature ramp was programmed from to • c in increments of • c, waiting s before each acquisition. samples were considered positive if both an exponential increase of fluorescence and a btov-or ptov-specific melting peak were observed. quantitative detection of tovs was averaged from three independent runs with duplicate reactions. the targeted bp m gene fragment in btov and the bp fragment in ptov obtained by rt-pcr were used as the source dna for the preparation of in vitro rna transcripts (table ). the amplicons were inserted into a yt&a cloning vector (yeastern biotech, intron biotechnology, taipei, taiwan) and, after cloning, a clone was selected based on correct sequence of the insert. plasmid dna of the recombinant clone was digested with the restriction enzyme psti, electrophoresed using a . % agarose gel, and the purified digested plasmid was recovered from the gel using the qiaquick gel-extraction kit (qiagen, valencia, ca). the gel-purified linearized dna clone served as the template for the in vitro transcription. reverse transcription reaction was performed using t rna polymerase in a mmessage mmachine t kit (ambion, austin, tx). after h of incubation at • c, the dna template was removed by digestion with dnase using the turbo dna free kit (ambion). crna was then purified with the rneasy mini kit (qiagen). after dilution, the concentrations were calculated by measuring the absorbance at nm with nanodropnd (nanodrop technologies, wilmington, de). crna samples were stored at − • c until used. the regression lines between the logarithms of the input amounts of crnas and the corresponding mean threshold cycle (ct) values were calculated using the rotor-gene software version . . (corbett research). to check the reproducibility of the new real-time rt-pcr method, -fold serial dilutions of the obtained crna were assayed in triplicate by real-time rt-pcr. the intra-assay coefficient variation (cv) was computed from the results obtained with three replicates of each dilution sample tested at the same time. the inter-assay cv was computed for each nine diluted samples carried out by three independent assays on different days. both cvs were calculated by dividing the standard deviation of each tested sample by its mean and multiplying that result by (pignatelli et al., ) . statistical analyses were performed by spss version . . for windows (spss, chicago, il). the two-tailed fisher's exact test was used to assess the statistical significance of the detection rate between real-time rt-pcr, conventional rt-pcr and nested pcr. a p-value of < . was considered significant statistically. real-time rt-pcr was standardized using in vitro transcribed crna of btov and ptov. the standard rna was diluted in a -fold dilution series ranging from . × to . × − , and was tested for each run within the sybr green real-time rt-pcr assay. quantities used for each dilution corresponded to . × - . × and . × - . × viral copies per reaction of btov and ptov, respectively. the sybr green real-time rt-pcr assay detected as little as . × copies of btov crna and . × copies of ptov crna per reaction, and displayed linearity over a wide dynamic range of copy numbers ( fig. a and d) . amplicons of the expected size by sybr green real-time rt-pcr were visualized by gel electrophoresis (fig. c and f) . standard curves with a higher correlation coefficient (r > . ) and slope values of . and . ( fig. b and e) were generated using a serial dilution of in vitro btov and ptov rna transcripts, respectively. the standard rna subjected to sybr green real-time rt-pcr presented a specific fluorescence signal and ct values between . and . for btov and between . and . for ptov (data not shown). the intra-assay reproducibility showed that at the highest dilution where crna was detected, both replicates were found positive for btov or ptov. at the − dilution containing crna copies, % in the standard curve of these dilutions, each dot represents the result of duplicate amplification of each dilution. the coefficient of determination (r ) and the slope(s) of the regression curve are indicated. (c) sybr green real-time rt-pcr products using serially diluted in vitro transcripts. m, molecular marker; lanes - : . × , . × , . × , . × , . × , . × , . × , . × , . × and . × viral copies/reaction; n, negative control. (d) amplification of , − , − , − , − , − , − , − , − and − dilutions of crna standard used in parallel with each sybr green-based real-time rt-pcr assay. (e) standard curves of the real-time rt-pcr based on serial dilutions of ptov crna standards. in the standard curve of these dilutions each dot represents the result of duplicate amplification of each dilution. the coefficient of determination (r ) and the slope (s) of the regression curve are indicated. (f) sybr green real-time rt-pcr products using serially diluted in vitro transcripts. m, molecular marker; lanes - : . × , . × , . × , . × , . × , . × , . × , . × and . × viral copies/reaction; n, negative control. comparison of the detection rates of btov by real-time rt-pcr, conventional rt-pcr, and nested pcr assay. reproducibility was achieved for the two viruses. the intra-assay coefficient variation of ct values ranged from . % to . % and . % to . % for standard dilutions from to − btov and ptov crna copies per reaction, respectively. the inter-assay coefficient variation of ct values ranged from . % to . % and . % to . % for standard dilutions from to − btov and ptov crna copies per reaction, respectively. these results indicate that real-time rt-pcr was successful in detecting and quantifying crna for btov and ptov. for the comparison and assessment of sybr green real-time rt-pcr sensitivity and its end point with two conventional assays, -fold serial dilutions of the in vitro transcripts described above were tested. the lowest detection limits of conventional rt-pcr and nested pcr using the serial dilutions were, respectively, . × and . × viral copies/reaction for btov and . × and . × viral copies/reaction for ptov. analysis of the dilution series showed that the sybr green real-time rt-pcr assay could detect dilutions times lower than nested rt-pcr for both tovs, however, it could detect and , times lower than rt-pcr for btov and ptov, respectively. non-specific reactions were not evident with any of the three pcr approaches (data not shown). the prevalence of btovs and ptovs and their genetic properties in the diarrhea specimens of calves (n = ) and piglets (n = ) was reported previously (park et al., ; shin et al., ) . using the fecal samples, the detection rates of btov and ptov by real-time rt-pcr were compared with those of conventional rt-pcr and nested pcr (tables and ). one ( . %) bovine and ( . %) porcine samples were positive by both conventional rt-pcr and real-time rt-pcr, ( . %) bovine and ( . %) porcine samples were positive by real-time rt-pcr and negative by the conventional rt-pcr. no samples that were negative by real-time rt-pcr were positive by conventional rt-pcr. ninety-nine bovine and porcine samples were negative by both tests (tables and ) . the percentage agreements between these two assays were . % for btov and . % for ptov detection. the sensitivity and specificity of real-time rt-pcr compared with conventional pcr for btov were % and . %, respectively, and for ptov were % and . %, respectively. the agreement beyond chance was calculated with a kappa statistic of . for btov and . for ptov, meaning a statistically fair agreement. the detection rate of real-time pcr was markedly higher than that of conventional rt-pcr (p < . ). comparing nested pcr and real-time rt-pcr, ( . %) bovine and ( . %) porcine samples were positive in both methods, nine ( . %) bovine and ( . %) porcine samples were positive by realtime rt-pcr and negative by nested pcr. no samples were negative by real-time rt-pcr and positive by nested pcr, and bovine and porcine samples were negative by both tests (tables and ). the percentage agreements between the two assays were . % for btov and . % for ptov detection. the sensitivity and specificity of real-time rt-pcr compared with nested pcr were, respectively, % and . % for btov, and % and . % for ptov. the agreement beyond chance was calculated with a kappa statistic of . in btov and . in ptov, meaning that it was statistically significant. the detection rate of real-time pcr was significantly higher in btov and moderately higher in ptov than that of nested pcr (p < . ). to confirm whether the samples positive by the sybr green real-time rt-pcr assay but negative by both rt-pcr and nested pcr assays were true positive, bovine and porcine amplicons positive by the real-time rt-pcr assay were sequenced directly. the nucleotide sequence of all amplicons matched that of the bovine and porcine m gene (data not shown), indicating that the positive reaction by the real-time rt-pcr assay was true positive. the efficacy and reliability of the sybr green real-time rt-pcr assay for the detection and quantitation of btov and ptov in the bovine and porcine stool samples were evaluated. all positive samples subjected to sybr green real-time rt-pcr presented a fluorescence signal and ct values between . and . for btov and . and . for ptov (data not shown). btov and ptov amplicons displayed melting temperatures (t m ) between and • c (data not shown). the range of t m table comparison of the detection rates of ptov by real -time rt-pcr, conventional rt-pcr, and nested pcr assay. values for btov and ptov using real-time rt-pcr sybr green chemistry of in vitro transcripts showed . - . • c for btov and . - . • c for ptov-positive samples. however, the nontemplate controls were • c. the viral copy numbers of positive samples per assay as measured by sybr green real-time rt-pcr ranged from . × to . × for btov, and . × to . × for ptov. one btov and seven ptov samples that tested positive in both real-time rt-pcr and conventional rt-pcr contained . × and . × - . × viral copies per reaction, respectively. thirteen bovine and porcine samples that tested positive by real-time rt-pcr and nested pcr had . × - . × and . × - . × viral copies per reaction, respectively. however, the nine bovine and porcine fecal samples that tested positive only by real-time rt-pcr possessed comparatively lower viral copies, ranging from . × to . × and . × to . × viral copies per reaction, respectively. the specificity of btov by real-time rt-pcr was assessed using bovine fecal samples that had tested positive for other enteric pathogens including groups a, b and c bovine rotaviruses (brv a-c), bovine coronavirus (bcov), bovine norovirus, bovine viral diarrhea virus (bvdv), salmonella spp., clostridium spp., campylobacter spp., shiga-toxin-producing escherichia coli, coccidium spp. and cryptosporidium spp. (asakura et al., ; park et al., ) . the specificity to ptov by real-time rt-pcr was assessed using porcine diarrhea specimens that had tested positive for porcine rotaviruses a, b, c (prv a-c), and porcine sapovirus. there was no positive signal recorded for any of these pathogens. in this study, an optimized real-time rt-pcr was developed using sybr green chemistry to detect btov or ptov in bovine or porcine fecal samples for the efficient and rapid screening of samples on a large scale. the primer pair for the sybr greenbased real-time rt-pcr assay was designed to target sequences centered on the conserved m gene. the conserved stretches in a portion of the m gene have shown high homology among the published btov and ptov isolates and, with the nucleocapsid gene, was thought to be the most sensitive and specific detection genes for both viruses in fecal samples (ito et al., ; pignatelli et al., ) . using both target genes, the detection of btov or ptov by real-time rt-pcr was demonstrated to be specific and sensitive in this study and in another study (pignatelli et al., ) . the findings of the present study proved the application and efficiency of using one primer pair for the detection of two viruses. however, the results showed a higher analytical sensitivity of btovs over ptovs. the detection of a larger amplicon size that was > bp ( bp), and the positive weaker amplicons in the case of ptovs could have possibly affected the melt rate and phenomenon of dye translocation, which consequently affected sybr green binding and detection (varga and james, ) . to refine further the method developed, it was first intended to find ways of distinguishing the two viruses according to the melt curve analysis. the m gene region has different gc/at ratios in btov and ptov. accordingly, the functions of the gc/at ratio, length and sequence can be demonstrated by melting curve analysis and can be used to differentiate amplification products with < • c intervals in melting temperature (ririe et al., ) . melting curve analysis of the in vitro transcripts showed a difference of only • c between btov and ptov, and, using the field samples, melting curve analysis showed overlapping temperature ranges for btov ( . - . • c) and ptov ( . - . • c). from this, it can be inferred that the differentiation between btov and ptov by the cur-rent real-time rt-pcr assay becomes more difficult to determine and insignificant. nonetheless, the desired products can be distinguished from undesirable products, and in many cases the need for gel electrophoresis is eliminated (ririe et al., ) . the samples that showed close melting temperature with the no-template control were reconsidered positive as they showed a good level of viral copies and a clear band after electrophoresis. in this study, all of the real-time rt-pcr samples tested were verified by electrophoresis and sequencing. the ideal method for the detection of btov or ptov in stool samples should have a high degree of sensitivity and specificity, low risk of contamination (koopmans and horzinek, ) , and consistency of performance in the laboratory. at present, the sybr green rt-pcr assay is and , times more sensitive than btov and ptov detection by conventional rt-pcr, respectively, and times more sensitive than nested pcr for both viruses when using crna. therefore, the one-step sybr green quantitative real-time rt-pcr assay is significantly superior (p < . ) to the other pcr assays in terms of sensitivity, specificity and quantitative linearity. the short amplicons in the real-time pcr assays used in this study likely resulted in more efficient amplification and higher analytical sensitivity. despite the shorter amplicon size in ptov nested pcr, the real-time pcr reproduced amplicons with greater analytical sensitivity. taken together, these observations indicate that the sybr green-based rt-pcr assay is a useful and reliable method for the detection of btov and ptov. the reported fecal prevalence of btovs in bovine diarrhea specimens by conventional rt-pcr or nested pcr was . % in canada (duckmanton et al., ) , . % in austria (haschek et al., ) , . % in the united states (hoet et al., ) , . % in hungary (matiz et al., ) , . - . % in japan (kiriwasa et al., ) and . % in south korea (park et al., ) . porcine toroviruses were detected in % of the diarrhea specimens of piglets in hungary (matiz et al., ) , . % in japan (kiriwasa et al., ) , and . % in south korea . using the same south korean bovine and porcine fecal samples (park et al., ; shin et al., ) , sybr green realtime rt-pcr assay detected btovs in . % and ptovs in . % of the samples. these findings support the above suggestion that sybr green real-time rt-pcr is more sensitive than conventional rt-pcr and nested pcr. in addition, these observations indicate that the fecal prevalence of btov and ptov in calves and piglets might be higher than previously reported in south korea (park et al., ; shin et al., ) . toroviruses in calves and piglets have been identified in a number of outbreaks in bovine and porcine herds with severe and recurring diarrhea negative for routine virologic, bacteriologic and parasitologic agents (hoet et al., ; kiriwasa et al., ; park et al., ; shin et al., ) . interestingly, those fecal samples that tested positive only by sybr green real-time rt-pcr contained lower numbers of btov and ptov rna ( . × - . × and . × - . × viral copies per reaction, respectively). these diarrhea specimens with lower number of tovs were co-infected with other enteric pathogens, including bcov, brva and/or bvdv in the bovine fecal samples, and prv a-c in the porcine fecal samples (data not shown). therefore, it can be presumed that btov or ptov can either induce diarrhea alone, or can play a role in accelerating the clinical and pathological presentation of diarrhea with the other pathogens (hoet et al., ; kiriwasa et al., ; park et al., ; shin et al., ) . in conclusion, a rapid, sensitive, specific and reproducible onestep sybr green real-time rt-pcr was developed for the detection and quantitation of btov and ptov in bovine and porcine stool samples. this high-throughput assay may be useful for the establishment of a surveillance system and diagnostic purposes for btov and ptov infections worldwide. detection and genetical characterization of shiga toxin-producing escherichia coli from wild deer an enveloped virus in stools of children and adults with gastroenteritis resembles the breda virus of calves cross protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and rt-pcr and nested pcr for their detection development of one-step real-time reverse transcription polymerase chain reaction assays for rapid detection of porcine group c rotaviruses detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms viruses and virus-like particles detected during examination of faeces from calves and piglets with diarrhea the epidemiology of enteric caliciviruses from humans: a reassessment using new diagnostics detection of bovine torovirus in neonatal calf diarrhea in lower austria and styria (austria) detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle bovine torovirus (breda virus) revisited. anim epidemiological analysis of bovine torovirus in japan detection of bovine torovirus in fecal specimens of calves with diarrhea in japan toroviruses of animals and human: a review identification and characterization of a porcine torovirus torovirus detection in faecal specimens of calves and pigs in hungary: short communication molecular epidemiology of bovine toroviruses circulating in south korea development of sybr green real-time rt-pcr for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus detection of porcine torovirus by real time rt-pcr in piglets from a spanish farm molecular characterization of a new ptov strain. evolutionary implications product differentiation by analysis of dna melting curves during the polymerase chain reaction detection and molecular characterization of porcine toroviruses in korea phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events j. real-time rt-pcr and sybr green i melting curve analysis for the identification of plum pox virus strains c, ea, and w: effect of amplicon size, melt rate, and dye translocation purification and partial characterization of a new enveloped rna virus (berne virus) studies with an unclassified virus isolated from diarrheic calves this study was supported by a grant ( - ) from the korea science and engineering foundation (kosef) and the regional technology innovation program of the ministry of commerce, industry and energy (mocie), republic of korea. the authors would like to acknowledge a graduate fellowship from the korean ministry of education and human resources development through the brain korea project. 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 - 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- journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: bh uugwy culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. primary rhesus monkey kidney (pmk) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (ifa). kinetic studies of coxsackievirus b and echovirus infection of pmk cells were performed on days – after inoculation. flow cytometry results for echovirus , , , and and coxsackievirus b correlated with ifa in all cases. coxsackievirus b and echovirus infections were detected day earlier by flow cytometry than ifa. flow cytometry can be effectively used for detecting enterovirus-infected cells in a clinical laboratory with the advantages of better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems. human enteroviruses, members of the picornaviridae family, are among the most common human pathogens. they cause a broad spectrum of human disease, including myocarditis, hand-footand-mouth disease, meningoencephalitis, and paralytic myelitis (romero, ) . at least serotypes have been defined. periodic enterovirus outbreaks occur and are a major public health concern (khetsuriani et al., ) . while most enteroviral infections are not life-threatening, certain serotypes (e.g., polioviruses, enterovirus ) can cause devastating neurological damage and death. poliovirus outbreaks still occur in developing nations despite eradication efforts. oral poliovirus vaccine-derived strains are an increasing cause of viral paralytic syndrome and must be differentiated from wild-type poliovirus and other causes of paralytic myelitis such as west nile virus, coxsackievirus a , and enterovirus / (muir et al., ) . hence, the capacity to classify human enteroviruses in clinical specimens is imperative to disease prevention, treatment, and prognosis. historically, serotyping enteroviruses has been accomplished by a combination of indirect fluorescence antibody testing (ifa) and viral neutralization assays using anti-sera pools provided by the world health organization (who). these techniques are timeconsuming, labor-intensive, and technically demanding. the who no longer produces anti-sera for viral neutralization studies, which now must be obtained from the few remaining sources such as the national institute of public health and the environment (rivm, the netherlands). flow cytometry is a potential alternative to ifa for serotyping clinical isolates and provides quantitative information with less inter-observer variability. with flow cytometry, hundreds of cells can be analyzed in a short period of time (seconds) with increased sensitivity to detection of fluorescence signals due to the use of lasers and photomultiplier tubes, and has the ability for automation. despite these advantages over ifa, no studies have focused on the use of flow cytometry for serotyping enterovirus prior to this study. isolates were obtained from a frozen repository and had been previously cultured and serotyped using serum neutralization studies in the clinical virology laboratory at associated regional and university pathologists (arup) laboratories as previously described (she et al., ) . all enteroviral isolates were stored at − • c prior to use in this study. for each serotype, - shell vials containing a confluent layer of primary rhesus monkey kidney (pmk) (viromed laboratories, minnetonka, mn) were inoculated with l echovirus , echovirus , echovirus , echovirus , coxsackievirus b , or rhinovirus in ml minimum essential medium (mem) with % fetal bovine serum (fbs) and centrifuged for min at × g. specific viral inocula were not determined. uninoculated cells were included as negative controls in each study. cells were incubated at • c and were harvested for staining and analysis as follows. the media were aspirated and cells were washed three times in phosphate buffered saline (pbs). cells were overlaid with l tryple trypsin replacement (invitrogen, carlsbad, ca) for s before addi-tion of ml mem with % fbs. the cells were then transferred to a . ml microcentrifuge tube and centrifuged for min at × g. if cells were off the monolayer due to cytopathic effect (cpe), the overlying media was additionally saved and centrifuged. the cell pellet was washed in l pbs, centrifuged for min at × g, and resuspended in l pbs. the fix & perm cell permeabilization kit (invitrogen) was used for viral staining following the manufactures directions. briefly, cells were transferred to a ml tube and incubated with l reagent a for min at room temperature. the cells were then washed with ml pbs with % fbs, centrifuged for min at × g, and resuspended in l reagent b (permeabilization medium) and one drop (approximately l) of primary antibody which included pan-enterovirus (diagnostic hybrids inc. (dhi), athens, oh), normal mouse igg (isotype control), coxsackievirus b blend, poliovirus blend, enterovirus blend, and echovirus blend antibodies (millipore, billerica, ma) in all cases. for echovirus , echovirus antibody was tested and for echovirus , individual antibod- ies directed against echovirus , , , , and were tested. for coxsackievirus b , individual antibodies directed against coxsackievirus b through b were tested. after min incubation at room temperature in the dark, the cells were washed with ml pbs with % fbs, centrifuged for min at × g, and resuspended in l reagent b and one drop of secondary antibody (fluorescein isothiocyanate (fitc)-conjugated mouse monoclonal antibody; dhi or millipore). the cells were incubated for min at room temperature in the dark and washed and centrifuged as above. the cells were resuspended in l pbs containing % paraformaldehyde and analyzed by flow cytometry immediately. all antibodies used were monoclonal antibodies or blends of monoclonal antibodies. flow cytometer instrument setup was performed daily using flow-check and flow-set fluorospheres (beckman coulter, miami, fl). the stained samples were analyzed using fc flow cytometer (beckman coulter, miami, fl), creating listmode files containing at least events. listmode files were analyzed using cxp (beckman coulter, miami, fl) and flowjo (tree star, ashland, or) software. cells were prepared for traditional ifa staining using the trypsinized and washed cells prepared as described above. the l cell suspension was spotted to -or -well slides, dried on a slide warmer set at • c, then fixed in cold acetone for min. one drop of primary antibody was placed on each well and slides were incubated at • c in the dark for min. slides were washed with pbs and distilled water and air-dried. one drop of fitc-conjugated secondary antibody was placed on each well and slides were incubated at • c in the dark for min. after a rinse with pbs and distilled water, a coverslip was placed on each slide using mounting media (dhi) and examined under a fluorescent microscope for positive, fluorescent green staining of the cells. uninoculated cells stained with enterovirus-specific antibodies and enterovirusinfected cells stained with normal mouse antibody were included as controls. for initial feasibility studies, flow cytometric analysis was performed on pmk cells that showed cpe following infection with echovirus , echovirus , echovirus , echovirus , or coxsackievirus b . the percentages of infected cells were quantified by first gating on the forward and side scatter signals to help eliminate cell debris and non-cellular events that may be present. negative control levels were determined using isotype control antibodies, which gave similar results to unstained cells or cells stained with antibodies directed against other (irrelevant) enterovirus serotypes. the infected cells represented those gated cellular events staining above negative control levels using virus specific antibodies. as shown in fig. , coxsackievirus b infected cells demonstrated positive staining for pan-enterovirus, coxsackievirus b blend, and coxsackievirus b antibodies and were negative for isotype control, echovirus blend, enterovirus blend, poliovirus blend, and coxsackievirus b , b , b , b , and b antibodies. representative data shown in fig. demonstrate that echovirus -infected cells were positive for echoblend and echovirus -specific antibodies and negative for echovirus , , , and individual specific antibodies. the thresholds used in figs. and were based on subjective criteria in which a natural break between positive and negative cells could be illustrated, allowing for easy comparison between infected cells and negative controls. pmk cells infected with echovirus , echovirus , or echovirus demonstrated positive staining for pan-enterovirus and echovirus blend antibodies and were negative for isotype control, enterovirus blend, coxsackievirus b blend, and poliovirus blend antibodies (not shown). echovirus -infected cells were additionally positive for echovirus -specific antibody. rhinovirus -infected cells stained similarly to uninoculated cells for panenterovirus, echovirus blend, coxsackievirus b blend, and isotype antibodies. in all cases, ifa demonstrated qualitatively similar results compared to the flow cytometry determined percentages. pmk cell shell vials were inoculated with echovirus or coxsackievirus b and analyzed by flow cytometry at , , , and h after inoculation. cells were examined for cpe and by ifa in parallel to flow cytometric testing. for coxsackievirus b , flow cytometry and ifa detected pan-enterovirus, coxsackievirus blend, and coxsackievirus b antibody-stained cells day after inoculation, while ifa became positive days after inoculation ( fig. and table ). the thresholds used in fig. were set to allow for one or no positive events in the uninfected cell control. positive events were considered those staining above the threshold level. similar results were obtained with echovirus , in that pmk cells with detectable antibody staining for pan-enterovirus or echovirus blend were found by flow cytometry days after cells were inoculated, whereas ifa detected positive-staining cells by the same antibodies days after inoculation. in both echovirus -and coxsackievirus b -infected cells, cpe became evident at the same time ifa was positive. different antibodies (pan-enterovirus, coxsackievirus b blend, and coxsackievirus b antibodies for coxsackievirus b -infected cells and pan-enterovirus and echovirus blend antibodies for echovirus -infected cells) were used to monitor over time enterovirus infection in pmk shell vial cells and all gave similar results. while the applications of flow cytometry are many and varied, it has not been well-studied for detection of enterovirus infection from culture. previous studies have focused on using flow cytometry to quantitate poliovirus infection in neuronal cells (daley et al., ) , characterize enterovirus binding to host cell surfaces (freistadt and eberle, ; mbida et al., ; triantafilou et al., ) , confirm cytomegalovirus infection of tissue culture cells with a genetically engineered fluorescence reporter system (kung et al., ) , and serotype human immunodeficiency virus type (zolla-pazner et al., ) . this is the first report of a clini- table kinetic study of coxsackievirus b -infected pmk cells as demonstrated by flow cytometric analysis, ifa, and observation of cpe. data for flow cytometry and ifa are from cells stained with coxsackievirus b blend antibody. flow cytometry detected positively staining, infected cells day prior to ifa and appearance of cpe. cal application for flow cytometry in detecting enterovirus from culture. in this study, flow cytometry proved to be a sensitive method for detecting fluorescently stained enterovirus-infected cells. for both echovirus -and coxsackievirus b -infected pmk cells, flow cytometry was able to detect infection day before the viral infection became detectable by ifa. this is likely due to the ability of flow cytometry to quickly analyze a larger number of cells than is routinely examined by ifa. it was also able to quantitate the level of infection even when there are low numbers of virus-infected cells. the capacity to quantitate allowed for monitoring enterovirus infection in pmk cells over a -day period using different antibodies which gave similar results. the coxsackievirus b strain used in this study was highly efficient at infecting pmk cells compared to the echovirus strain. as the predilection of different enterovirus serotypes and even strains within a serotype for different cells lines is well-known (she et al., ) , it is not surprising that a different rate of infection between the two serotypes used in this study was observed. false positives did not occur by flow cytometric analysis in that infected cells were negative after staining with isotype control antibodies and antibodies directed against other enterovirus serotypes. this is not surprising as the specificity of flow cytometry in general is related to the particular antibodies employed, and therefore, should be similar to microscopy based detection methods using the same antibodies. also, uninoculated cells did not demonstrate positive staining with any of the antibodies used. study of additional human enterovirus serotypes using this method is warranted. typing of enteroviruses by sequence analysis has emerged as an accurate surrogate for conventional serotyping methods (oberste et al., ) . because highly variable regions are targeted by genotyping studies, degenerate primer sequences and/or nested designs are necessary to ensure adequate sensitivity. use of an antibody-based method, such as flow cytometry and ifa, may have the advantage of better consistency in identifying enterovirus serotypes. flow cytometry can also be potentially faster and less expensive than molecular methods for viral typing. although flow cytometric analysis was shown to be highly sensitive, the current method was cumbersome and not optimized for performance in a clinical laboratory. shell vials were used to facilitate infection of cells with centrifugation, as is commonly done in the clinical virology laboratory. it is possible that a more efficient tissue culture system could be employed, such as use of multiple well plates (e.g., -well plate format). in this way the need for handling numerous shell vials per specimen is obviated. others have found the use of centrifuged multiple well plates combined with blind immunoperoxidase staining for enterovirus to be both rapid and sensitive (bourlet et al., ; terletskaia-ladwig et al., ) and this method can potentially be performed using fluorescent antibody staining followed by flow cytometric analysis instead. because the pmk cells grow as a surface monolayer, trypsinization was used to create an optimal single cell suspension (grogan and collins, ) . another potential option is the use of lymphocytic or monocytic cell lines which have been described for cultivation of a limited number of enterovirus serotypes (okada et al., ; skarsvik et al., ; vuorinen et al., vuorinen et al., , . cultivation of enterovirus in such a manner would facilitate interrogation by flow cytometry and may be studied in the future. other cell lines that are routinely used in the clinical laboratory for isolation of enterovirus, e.g., rd, mrc- , and bgm, should also be investigated. directly labeled antibodies are not commercially available for enteroviruses but would be useful in streamlining the protocol described. multicolor flow cytometry could be used to simultaneously monitor infections of different cell types and/or viruses from a single culture. thus, modifications of the method described in this study could make the use of flow cytometry considerably more practical and automated for routine use in clinical microbiology laboratories. flow cytometry can be effectively used for detecting enterovirus-infected cells in a laboratory setting. its potential advantages over fluorescent microscopy include better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems. these could lead to faster laboratory identification of pathogenic viruses. the use of flow cytometry on direct clinical specimens was not explored but could be studied in the future on cellular specimens such as those from the respiratory tract. prospective application of this method to patient isolates is still needed to confirm its validity. the analyses in this study were limited to enterovirus, but the methods that were tested and discussed can be applied to a wide array of other clinically important viruses. adenovirus, emerging coronaviruses, and influenza virus, including highly pathogenic strains, affect the public health and in each case, knowledge of serotype provides important strain information and epidemiologic data. additional studies are still needed to extend the methods presented to other viruses and optimize detection strategies. comparison of a rapid culture method combining an immunoperoxidase test and a group specific anti-vp monoclonal antibody with conventional virus isolation techniques for routine detection of enteroviruses in stools poliovirus replication and spread in primary neuron cultures fluorescent poliovirus for flow cytometric cell surface binding studies guide to flow cytometry methods. marcel dekker enterovirus surveillance-united states rapid diagnosis and quantification of herpes simplex virus with a green fluorescent protein reporter system competition binding studies with biotinylated echovirus in cytofluorimetry analysis molecular typing of enteroviruses: current status and future requirements. the european union concerted action on virus meningitis and encephalitis comparison of classic and molecular approaches for the identification of untypeable enteroviruses poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility of cell killing enteroviruses and parechoviruses comparison of multiple shell vial cell lines for isolation of enteroviruses: a national perspective decreased in vitro type immune response against coxsackie virus b in children with type diabetes a convenient rapid culture assay for the detection of enteroviruses in clinical samples: comparison with conventional cell culture and rt-pcr identification of echovirus and coxsackievirus a receptor molecules via a novel flow cytometric quantification method enterovirus receptors and virus replication in human leukocytes coxsackievirus b infection in human leukocytes and lymphoid cell lines serotyping of primary human immunodeficiency virus type isolates from diverse geographic locations by flow cytometry this work was supported by a grant from the cap foundation, nih grant r de (dwb), and the arup institute for clinical and experimental pathology. key: cord- -c hfh dq authors: gunson, rory; maclean, alasdair; davies, eleri; bennett, susan; miller, rhona; carman, w.f. title: development of a multiplex real-time rt-pcr that allows universal detection of influenza a viruses and simultaneous typing of influenza a/h n / virus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: c hfh dq on june , , the world health organization declared that the influenza a/h n / virus had become the first influenza pandemic of the st century. rapid detection and differentiation from seasonal and avian influenza would be beneficial for patient management and infection control. it was the aim of this study to develop a real-time rt-pcr that can detect all influenza a viruses and offer simultaneous typing for influenza a/h n / . this would be a useful addition to existing diagnostic protocols for influenza a. its routine use would allow laboratories to screen out influenza a/h n / positive samples rapidly and would reduce overall testing costs. on april and , , a novel swine-lineage influenza a (influenza a/h n / ) infection was reported to the world health organisation (who) by the centers for disease control and prevention (cdc) in atlanta in two children presenting with febrile respiratory illness from adjacent counties in southern california (cdc, ; novel swine-origin influenza a (h n ) virus investigation team, ). these cases were not epidemiologically linked and neither child had exposure to swine. since the original identification of influenza a/h n / in the united states and mexico, sustained human-to-human transmission has been seen in other countries and on june , , the world health organization declared that the virus had become the first influenza pandemic of the st century. although the influenza a/h n / virus is likely to become the predominant influenza a type encountered in most countries, seasonal influenza a types may also co-circulate (kelly et al., ) and in some countries sporadic h n infections may still occur (who report of avian influenza, ). determining the subtype of influenza virus is important as it has implications for patient management and infection control (meijer et al., ; beigel and bray, ; hall et al., ) doses of oseltamivir are necessary for successful treatment of infection (lackenby et al., ; white et al., ) . in order to detect and then type influenza a viruses most laboratories use a two tier testing system comprising of a universal influenza a screening assay complemented with a suite of subtyping assays that determine whether the sample is seasonal influenza a (human h n and h n ), avian h n or the influenza a/h n / virus. although useful for the reasons outlined above, the testing structure prolongs the time it takes to complete testing and is costly as most influenza a positive samples will have to be tested using the individual typing assays. this article describes the development of a multiplex real-time reverse transcription polymerase chain reaction (rtpcr) that allows universal detection of all influenza a viruses and simultaneously subtypes all that are influenza a/h n / . an internal control rtpcr assay was also incorporated in order to detect pcr inhibition, failed extraction/pcr and technical error. use of this assay will allow laboratories to screen respiratory samples for influenza a/h n / virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. the influenza a/h n / rtpcr assay was designed to target segment of the na using published sequence data table ) amplify a -bp amplicon and bioinformatics analysis using blast and clustal alignments showed no significant homology to human or other known influenza a gene sequences (fig. ) . the highest homology is % to classical swine flu, e.g. a/swine/england/wvl / (h n ). with this strain there were mismatches in the probe sequence, which would be expected to prevent hybridisation although this was not experimentally tested. in addition we would not expect to be screening humans infected with classical swine flu strains. the chosen primers and probe do not span any of the variable antigenic sites minimising the possibility of point mutations occurring (abed et al., ) . a widely used universal influenza a rtpcr was used as part of the multiplex rtpcr. the assay targets the matrix region of the virus. participation in various eqa schemes has shown this assay to detect influenza a viruses from humans and animals with high sensitivity (carr et al., ). the rtpcr assay used for the internal control targets equine arteritis virus (eav) and was taken from an existing publication (scheltinga et al., ) . the primer and probe concentrations for each assay were individually optimised using in house protocols. all assays, singleton or multiplex, used the primers at an optimised concentration of m and the probe at . m in a l reaction volume (all primers and probes are shown in table ) . for singleton assays, one-step rtpcr was performed on l of rna extract with the platinum one-step qrt-pcr kit (invitrogen) on an abi prism sds real-time platform (applied biosystems). the following thermal profile was used: a single cycle of reverse transcription for min at • c, min at • c for reverse transcriptase inactivation and dna polymerase activation followed by amplification cycles of s at • c and s at • c each (annealing-extension step). for the multiplex rtpcr, one-step rtpcr was performed on l of rna extract with the qiagen quantifast mulitiplex rt-pcr kit (qiagen, crawley, united kingdom) on an abi prism sds real-time platform. the following thermal conditions were used: a single cycle of reverse transcription for min at • c, min at • c for reverse transcriptase inactivation and dna polymerase activation followed by amplification cycles of s at • c and s at • c each (annealing-extension step). data acquisition occurred at the annealing step of each cycle and the threshold cycle (ct) for each sample was calculated by deter- sequences had % homology with the primers and probe, whereas there were a significant number of differences between both seasonal hin and swine and avian n sequences. all reference sequences were obtained from the influenza sequence database (isd) at los alamos. sequences were aligned using bioedit. mining the point at which the fluorescence exceeded the threshold limit. please note that the qiagen quantifast multiplex rt-pcr kit was used for the multiplex rtpcr only as, unlike the invitrogen rt-pcr kit, it is specially designed to prevent competition from occurring between the pcr tests within a multiplex. such competition often results in false negative reactions or strange traces which can impede result interpretation. previous comparisons between this kit and the invitrogen kit have shown it to be superior for multiplex rtpcr (gunson et al., ) . the specificity of the multiplex assay was assessed using a recent world health organisation (who) panel. this panel contained examples of seasonal influenza a, h n and h n , the influenza a/h n / and numerous avian a/h n viruses. a pool containing the following commonly encountered respiratory pathogens was also tested: influenza b, influenza c, parainfluenza - , human metapneumovirus, respiratory syncytial virus, mycoplasma pneumoniae, rhinovirus (untyped) and coronaviruses e, oc and nl . the end point detection limit of the new multiplex rtpcr was directly compared to each rtpcr in singleton format using a dilution series of an influenza a/h n / clinical sample. this was carried out to ensure that multiplexing did not result in a reduction in end point detection limit. the final assessment comprised of assessing the multiplex rtpcr on clinical samples. of the samples, were seasonal influenza a viruses ( subtyped as h n and as h n ) that had been submitted to the wossvc during previous respiratory seasons. the remaining were samples that had been sent to the laboratory in and found to be influenza a/h n / positive using an alternative rtpcr (carr et al., ) . all samples contained h n at varying concentrations (ct values range from to ). total nucleic acid was extracted from the aforementioned samples using qiaamp viral rna kit (qiagen, crawley, united kingdom) on the qiagen mdx according to the manufacturer's instructions. please note that eav was added to the lysis buffer prior to extraction in order to detect pcr inhibition, extraction failure or technical error ( l × − (ct ) of bhk tissue culture grown eav was added to the lysis buffer to extract samples). internal control was also added to ensure that its presence did not compete with the diagnostic components of the assay and inhibit their performance. the multiplex rtpcr was evaluated using a recent panel distributed by the who comprising seasonal influenza a (h n and h n ), avian (h n ) and influenza a/h n / influenza a viruses ( table ). the universal influenza a component detected all samples as positive. the na assay was positive in only two cases (samples and ) and did not detect the seasonal influenza a (h n and h n ) viruses or avian influenza viruses. the multiplex assay also did not detect any of the commonly encountered respiratory pathogens as positive. the end point detection limit of both singleton rtpcr assays were compared using a dilution series of a influenza a/h n / clinical sample. the universal influenza a test detected the - dilution in one out of two occasions (table ) whereas the na assay was slightly less sensitive detecting the - dilution. comparing the end point detection limit of each of the test components in singleton and multiplex form showed that multiplexing had no effect on test performance. the final comparison assessed the multiplex assay on clinical samples. the multiplex rtpcr detected all samples as influenza a positive. the na assay detected all but of the influenza a/h n / samples as positive. the false negative sample had a ct value > in the universal influenza and was confirmed as positive using an alternative influenza a/h n / assay (carr et al., ) . none of the samples containing seasonal influenza a were positive by na assay. this article describes the development of a rapid, specific and sensitive multiplex rtpcr assay that detects all influenza a types and simultaneously identifies samples that contain the pandemic influenza a/h n / virus. the assay also incorporates an internal control and thus is useful for detecting pcr inhibition or technical error. multiplexing was shown to have no effect on the performance of the individual test components and, because the assay utilised the qiagen quantifast multiplex rt-pcr kit, the presence of internal control within the samples did not compete with the other diagnostic components. the universal influenza a assay has been described elsewhere and the results shown here confirm it to be sensitive and able to detect a wide range of influenza a types. the na assay was specific as it did not cross-react with seasonal influenza a strains currently circulating (subtypes h n and h n ) or other commonly encountered respiratory pathogens. in addition, the n assay did not detect as positive any of the influenza a, subtype h n viruses. close homology was observed between amplicon and classical swine influenza a (h n ). the assay was not assessed against this virus and therefore we cannot be sure as to whether the test will detect this virus. however, four mismatches in the probe were observed which may reduce the likelihood of detecting this virus. the sensitivity of the na assay was slightly less than the frontline universal influenza a test. this was shown using the dilution series and was confirmed when it was assessed using the influenza a/h n / positive clinical samples where the na assay failed to detect one sample which was positive using the universal influenza a test and an alternative influenza a/h n / assay. the ct value of the sample was > in the universal influenza a assay which, based on the results from the dilution series, was likely to be beyond the detection limit of the h n assay. based on the results shown here, the multiplex rtpcr is a useful addition to existing diagnostic protocols for influenza a. its routine use would allow laboratories to screen out pandemic influenza a/h n / positive samples rapidly. such samples are likely to be in the majority and therefore only a small number of samples would require to be tested using seasonal or h n specific assays. use of this test would provide clinicians with complete results in rapid fashion and would reduce costs as far fewer samples will be sent for seasonal influenza or h n typing. divergent evolution of hemagglutinin and neuraminidase genes in recent influenza a:h n viruses isolated in canada current and future antiviral therapy of severe seasonal and avian influenza development of a real-time rt-pcr for the detection of swine-lineage influenza a (h n ) virus infections update: infections with a swine-origin influenza a (h n ) virus-united states and other countries using multiplex real time pcr in order to streamline a routine diagnostic service pandemic influenza a (h n )v viruses currently circulating in new zealand are sensitive to oseltamivir epidemiological characteristics of pandemic influenza h n and seasonal influenza infection european influenza surveillance scheme. oseltamivir-resistant influenza virus a (h n ) emergence of a novel swine-origin influenza a (h n ) virus in humans diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time rna pcr what is the optimal therapy for patients with h n influenza key: cord- - hvi el authors: rodrigues, r.; telles, j.-n.; essere, k.; ducournau, c.; roqueplo, c.; levieuge, a.; davoust, b.; parola, p.; paranhos-baccalà, g.; peyrefitte, c.n. title: development of a one step real time rt-pcr assay to detect and quantify dugbe virus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: hvi el a one-step real time quantitative rt-pcr (qrt-pcr) assay was developed to detect all published dugbe virus (dugv) genomes of the nairovirus genus. primers and probes were designed to detect specific sequences on the most conserved regions of the s segment. the limit of detection of the assay was copies per reaction which is an improvement of log( ) ffu/ml over the sensitivity of conventional rt-pcr. the specificity of the primers and probe was confirmed with the closely related nairoviruses cchfv and hazara virus, and on the non-related viruses coronavirus and influenza a virus. this qrt-pcr assay was used to screen nucleic acids extracted from ticks collected in the republic of chad. one sample was found positive suggesting that dugv is present in this part of the world. the molecular assay developed in this study is sensitive, specific and rapid and can be used for research and epidemiological studies. dugbe virus (dugv), a member of the genus nairovirus of the bunyaviridae family, was first isolated in from the amblyomma variegatum tick in nigeria (causey, ) . frequently detected in tick-borne virus surveys in africa (guilherme et al., ) , dugv is a tri-segmented single-stranded negative rna enveloped virus and is considered endemic in arid regions (burt et al., ) . dugv is also one of the most common tick-borne viruses found throughout africa (david-west and porterfield, ) . for example, a study in kenya documented frequent isolation of dugv from ticks infesting market livestock (sang et al., ) . among the serogroups characterized in the nairovirus genus (elliott et al., ) , dugv belongs to the nairobi sheep disease group which includes the nairobi sheep disease virus, pathogenic to sheep and goats (davies, ) and kupe virus, the most closely related virus (crabtree et al., ) . the genetically and serologically related crimean-congo hemorrhagic fever (cchf) group, which also includes hazara virus (begum et al., ) , is the other group of importance because of the very high pathogenicity of cchf virus in humans, leading to fatal hemorrhagic disease in % of patients (swanepoel et al., ) . in contrast, the pathogenicity of dugv is relatively low in humans -a thrombocytopenia case was once described (burt et al., ) -but in newborn mice the virus is neuroinvasive and lethal (boyd et al., ) . detection of most nairovirus is currently dependant on the time consuming virus isolation in cell culture or intra-cerebral inoculation in newborn mice from infected samples. an elisa has also been established (burt et al., ) as well as a conventional rt-pcr (ward et al., ) for the detection of dugv. to date, this conventional rt-pcr method alone has been used for the study of dugv replication in supernatants, clinical samples or for epidemiological studies (bridgen et al., ; marriott et al., ; marriott and nuttall, ; sang et al., ) . the aim of this study was to develop a sensitive, specific and rapid one-step quantitative real time rt-pcr assay (qrt-pcr) to detect dugv in infected cell supernatants, ticks or serum samples. . . qrt-pcr development . . . virus preparation, titration and rna extraction dugv (ibh ), cchf (ibar ), hazara (jc ), coronavirus (atcc vr- ), influenza a (atcc vr- ), were used in this study. all infectious work was carried out in a biosafety-level and . the batches of viral inocula used in this study were prepared by passages in bhk cells (atcc ccl- ) grown at + • c, % co in dmem medium (invitrogen, karlsruhe, germany), supplemented with % fetal calf serum (fcs) (pan, biotech, gmbh, aidenbach, germany), × u penicillin and mg streptomycin (invitrogen, karlsruhe, germany). briefly, cells were incubated for h at + • c, % co in the presence of the initial virus inoculum at a multiplicity of infection (moi) of , in dmem medium without fcs. medium containing % of fcs was then added and cells were maintained for days. absence of mycoplasma was confirmed using the mycoalert kit (lonza, verviers, belgium). cell culture supernatants were centrifuged ( min, × g, + • c), aliquoted and frozen (− • c) until used for viral rna extraction. viral titers, expressed in focus forming units per ml (ffu/ml) were estimated by the immunoperoxydase assay on vero cells seeded in a flat bottom -well plate (bd, franklin lakes, nj, usa), according to the dilution methods reported previously using an in-house hyperimmunized mouse ascitic fluid specific to dugv (peyrefitte et al., ) . all viral rnas were extracted from l of cell supernatants using the qiamp viral rna mini kit (qiagen, courtaboeuf, france) according to the manufacturers' instructions. to evaluate the influence of the serum, virus from infected cells supernatant was serially diluted in human serum (invitrogen, karlsruhe, germany). sequences retrieved from genbank database (accession numbers: af , af , af , af , and af ) were aligned using clustal w . . software (thompson et al., ) to select the most genetically conserved regions. primers and probe ( table ) were chosen in the highest conserved s segment region and according to the specifications needed for a real-time rt-pcr detection system (bustin, ) . the aligned sequences were identical in the region used for the primers and probe design. the specific dugv probe was labeled at the -end with the reporter -carboxy-fluorescein (fam) and at the -end with the quencher carboxytetrametyl-rhodamine (tamra) (eurogentec s.a., herstal, france). the qrt-pcr was performed in a final volume of l, containing l of extracted rna, l of × one step rt-pcr buffer (qiagen), l of dntp mm, pmol of the probe, pmol of each primer (eurogentec s.a., herstal, france), u of rnase inhibitor (rnasin, promega) and l of the one step hot start enzyme mix (qiagen). assays were carried out in a biorad ® cfx instrument with the following steps: reverse transcription at + • c for min, hot start and denaturation at + • c for min, and cycles with + • c for s, + • c for s, + • c for s, followed by a + • c for s cooling step. in the conditions described in section . . , except for the thermocycler used (geneamp pcr system , applied biosystems, courtaboeuf, villebon sur yvette, france) and the absence of probe, a rt-pcr product was obtained using dugv isolate ibh (genbank accession number af ). the bp amplicon was cloned into the t polymerase expression vector pbluescript ii sk (±) (stratagene, ca, usa). the plasmid was digested with the xhoi endonuclease (roche, heidelberg, germany), then purified and in vitro transcribed using the kit-ribomax tm large scale rna production system t (promega, wi, usa). residual dna was removed by dnase i treatments (roche). the rna was quantified using the genesys bio spectrophotometer (thermo electron corporation, oh, usa). absence of residual dna was assessed by pcr using the expand high fidelity enzyme mix (roche). the quality of the synthetic dugv transcripts was evaluated by controlling the absence of residual dna. no amplification was observed when synthetic rna was subjected to pcr, showing no dna contamination. the rna was quantified on the spectrophotometer, then converted to copy number based on base composition and length of the rna fragment. the sensitivity and reproducibility of the qrt-pcr dugv assay was determined using -fold dilutions of in vitro transcribed dugv ( to copies per reaction) in triplicate. after a -fold serial dilution of titrated virus supernatant, rna was extracted and tested in triplicate ( . × to . × − ffu/ml). the specificity was evaluated by using rna extracted from supernatants from cchfv, hazara virus, coronavirus and influenza a virus infected cells. to mimic infected human sera, viral supernatants were -fold diluted in human serum (sigma-aldrich, saint quentin fallavier, france) . to compare the qrt-pcr we used the conventional rt-pcr described by ward et al. ( ) . total rna was extracted from ticks collected from zebu and sheep, in the slaughter house of n'djamena, in chad in . the attached ticks were pulled off manually and placed in sterile plastic tubes containing % camphorated ethanol, then stored at + • c, until identification. after a washing step in sterile pbs, ticks were identified to the species level by using the identification key (walker et al., ) . each tick was individually triturated by using a fast-prep fp cell disrupter (qbiogene, illkirch, france) in l of sterile pbs. the mixture was centrifuged ( min, × g, + • c), aliquoted and frozen (− • c) until used for viral rna extraction. the sequence of the dugv transcript was confirmed by sequencing (data not shown). the primers-probe set selected to detect potentially all dugv known strains was able to detect the tested dugv rna. optimal qrt-pcr conditions were determined using virus supernatant. the standard curve was obtained using the qrt-pcr amplification of the quantified rna transcript. results of the sensitivity and specificity of the qrt-pcr assay are summarized in table . the correlation coefficient of the standard curve ranged from . to . . the limit of detection of the assay was synthetic copies per reaction and . × − ffu/ml of viral supernatant. the specificity of the assay was evaluated by testing the closely related cchfv and hazara virus which were not detected. kupe virus was not assayed because it was not in our virus collection. despite the closest phylogenetical relationship between kupe and dugbe viruses, a nucleotide difference was observed when the primers and probe were aligned (genebank accession number eu ). no cross reactivity was detected with the unrelated viruses coronavirus and influenza a virus (table ) . the qrt-pcr assay was first compared with the conventional rt-pcr (ward et al., ) using extracted viral rna. the presented dugv assay was log ffu/ml more sensitive than the conventional rt-pcr (fig. ) . the qrt-pcr assay detected as low as rna copies/assay while the conventional rt-pcr detected . × copies/assay, in our hands. furthermore, influence of the serum before the nucleic acids extraction step, was evaluated in the assay sensitivity. no significant difference was observed between the qrt-pcr dugv assay carried out with water or serum (data not shown). in our hands it took us ∼ h to carry out the conventional rt-pcr, including gel migration, versus ∼ h for the qrt-pcr. among the captured ticks, one dugbe virus rna was detected in one tick using the qrt-pcr assay ( . %). the ct value was . . the sequence of this amplicon confirmed the presence of dugv genome. this dugv genome was retrieved from a hyalomma impeltatum female adult tick (z- ) collected on a year old male zebu. dugv, a member of the bunyaviridae family is detected frequently in tick-borne virus surveys in africa (guilherme et al., ) , however little data are available in the literature that provides a comprehensive view of virus circulation. thus, the detection of dugv in field samples of arthropods and animal sera is needed to provide greater understanding of the virus epidemiology. also, as described for other arboviruses, a rapid, specific and sensitive test is necessary for effective surveillance of new dugv circulating strains. currently dugv identification is based on virus isolation. however, a conventional rt-pcr (ward et al., ) and elisas (burt et al., ; ward et al., ) have also been described for specific detection of dugv in samples. to date, rna dugv quantification is not available; therefore a qrt-pcr assay would be useful. the specificity of the assay was also important to discriminate the closely related dugv, hazara virus and cchfv. nevertheless, since kupe virus was not sought, a genomic amplification of kupe virus cannot be excluded. the out of nucleotide differences displayed when the primers and probe were aligned with the kupe virus sequence suggested that they are not specific for kupe virus. of note the nucleotide difference was out of for both hazara and cchfv. furthermore, by comparison with the conventional rt-pcr (ward et al., ) , the dugv real-time detection system described above was more sensitive (increasing the sensitivity of log ffu/ml), rapid (approximately h vs. h) and minimized the potential for cross-contamination by using a one tube detection system. our field study highlights the value of increased sensitivity because the single positive sample (z- ) was detected at a ct of . . such a low ct would not have been detected using the conventional rt-pcr. even if the value was out of the rna in vitro transcript range, the amplicon amount was sufficient for sequencing. the prevalence of dugv positive tick in our study ( . %) was comparable to previous observations of tick pools obtained from africa (sang et al., ) . the detection of dugv genome in hyalomma impeltatum tick confirms the presence of dugv in the chad sub-region. hyalomma ticks are known vectors of both dugv (booth et al., ) and cchfv (bridgen et al., ) suggesting the potential of tick-borne infection including cchfv in slaughter houses in africa. in conclusion, a sensitive and specific qrt-pcr assay was developed to detect and quantify dugv rnas in infected cell supernatants, extracts from ticks and potentially sera. in this study the presence of dugv genome in ticks captured in this region is described for the first time. the method described in this study might be useful for dugv epidemiological survey in animals and humans. tick-borne viruses of west pakistan. ii. hazara virus, a new agent isolated from ixodes redikorzevi ticks from the kaghan valley, w. pakistan structure and morphogenesis of dugbe virus (bunyaviridae nairovirus) studied by immunogold electron microscopy of ultrathin cryosection pathogenesis of dugbe virus infection in wild-type and interferon-deficient mice dugbe nairovirus s segment: correction of published sequence and comparison of five isolates investigation of tick-borne viruses as pathogens of humans in south africa and evidence of dugbe virus infection in a patient with prolonged thrombocytopenia absolute quantification of mrna using real-time reverse transcription polymerase chain reaction assays supplement to the catalogue of arthropod-borne viruses kupe virus, a new virus in the family bunyaviridae, genus nairovirus dugbe virus: a tick-borne arbovirus from nigeria nairobi sheep disease bunyaviridae seroprevalence of five arboviruses in zebu cattle in the central african republic dugbe nairovirus m rna:nucleotide sequence and coding strategy large rna segment of dugbe nairovirus encodes the putative rna polymerase differential activation profiles of crimean-congo hemorrhagic fever virus versus dugbe virus infected antigen presenting cells tickborne arbovirus surveillance in marquet livestock epidemiologic and clinical features of crimean-congo hemorrhagic fever in southern africa clustalw: improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position specific gap penalties and weight matrix choice ticks of domestic animals in africa: a guide to identification of species detection of an arbovirus in an invertebrate and a vertebrate host using the polymerase chain reaction expression of the nucleocapsid protein of dugbe virus and antigenic crossreactions with other nairoviruses this work was partly funded by irba and fondation mérieux. we thank m. beassem, k. matsumoto and j.-l. marié for their helpful collaboration in this study. we also thank jon davis for reviewing the manuscript. key: cord- -rjp d u authors: chutinimitkul, salin; suwannakarn, kamol; chieochansin, thaweesak; mai, le quynh; damrongwatanapokin, sudarat; chaisingh, arunee; amonsin, alongkorn; landt, olfert; songserm, thaweesak; theamboonlers, apiradee; poovorawan, yong title: h n oseltamivir-resistance detection by real-time pcr using two high sensitivity labeled taqman probes date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: rjp d u a single amino acid substitution, from histidine to tyrosine at position of the neuraminidase gene has converted oseltamivir sensitive h n influenza a virus into a resistant strain. currently, oseltamivir is being stockpiled in many countries potentially affected by the influenza a virus subtype h n epidemic. to identify this change in oseltamivir-treated patients, a method based on real-time pcr using two labeled taqman probes was developed for its rapid detection. in order to validate the method, oseltamivir specimen from treated (oseltamivir-resistant strain from a vietnamese patient, two oseltamivir-treated tigers) and untreated subjects have been used for this study. the results thus obtained as well as those derived from clone selection and sequencing showed that taqman probes could clearly discriminate wild type h from the mutant y variant. the sensitivity of this assay was as low as copies/μl and allowed the detection of the mutation in a mixture of wild type and mutant. overall, the assay based on real-time pcr with two labeled taqman probes described here should be useful for detecting oseltamivir-resistant h y h n influenza a virus in many species and various sources of specimens with high sensitivity and specificity. such studies can address potential differences in the diagnostic outcomes between patients who develop detectable oseltamivir resistance and those who retain only the wild type strain of h n . influenza virus is a rna virus from the orthomyxoviridae family. annually, influenza viruses may develop symptomatic influenza in % of children and % of adults worldwide (turner et al., ) . from the three types a-c only a and b cause widespread outbreaks. further subtyping of influenza a virus is based on the antigenic differences between two surface glycoproteins: haemagglutinin (ha) and neuraminidase (na). nowadays, ha (h -h ) and na (n -n ) subtypes have been described (fouchier et al., ; nicholson et al., ) . since , the influenza a virus subtype h n has been the cause of severe disease in various poultry and mammals. the clinical spectrum of avian influenza (h n ) in humans comprises initial symptoms of high fever (exceeding • c), lower respiratory tract symptoms, clinically significant lymphopenia, abnormalities on chest radiography, and in some cases diarrhea, vomiting (tran et al., ) and encephalitis. the overall fatality rate among hospitalized patients with avian influenza a (h n ) infection has amounted to % (who, ) . two groups of antiviral agents are currently available for the treatment of influenza infection. the adamantanes (amantadine and rimantadine) block the function of the m protein; however, drug resistance in patients has increased to % (hayden and hay, ) . the more recently developed class of viral neuraminidase inhibitors includes zanamivir (relenza) and oseltamivir (tamiflu) . an important antiviral medication used against all strains of influenza a virus is oseltamivir, which is the first orally active neuraminidase inhibitor in the form of a capsule or powder for liquid suspension (kim et al., ) . the neuraminidase inhibitor (nai) oseltamivir imitates natural neuraminidase substrate molecules and binds to the active site of the enzyme in addition to interfering with the release of progeny influenza virus from infected host cells (moscona, a) . therefore, neuraminidase (na) cannot cleave a terminal n-acetylneuraminic acid residue from an oligosaccharide chain and thus, the virions self-aggregate and bind to the surface of infected cells. oseltamivir resistance due to neuraminidase mutations have arisen both in challenge studies and in patients with naturally acquired infections. rates of resistance are estimated at around % in the adult population and % in pediatric patients (jackson et al., ; whitley et al., ) . in , kiso et al. ( ) analyzed influenza a viruses (h n ) collected from children before and during treatment with oseltamivir. eighteen percent of the children (n = / ) had neuraminidase mutations at arg lys (n = / ) or glu val (n = / ) or asn ser (n = / ). volunteers experimentally infected with influenza a/texas/ / (h n ) virus and treated with oseltamivir have shown an h y substitution at the neuraminidase active site (gubareva et al., ) . this mutation in response to oseltamivir phosphate treatment leaves the virus severely compromised both in vitro and in vivo (ives et al., ) and confers about - fold resistance (wetherall et al., ) . the mutation at position can influence the sensitivity of influenza n na yet not of n na to oseltamivir carboxylate by rearranging the shape of the active site to create a pocket for oseltamivir (gubareva, ; moscona, b; wang et al., ) . safeguarding against a potential influenza a virus subtype h n epidemic, many countries now stockpile oseltamivir. it has recently been reported that oseltamivir-resistant influenza a (h n ) viruses with the h y mutation have been isolated from three patients. h n viruses with pronounced oseltamivir resistance were isolated from two of eight vietnamese patients during oseltamivir treatment. both patients died in january and another resistant case died in february (beigel et al., ; de jong et al., ; le et al., ) . as an increase in oseltamivir-resistant viruses seems likely, a method aimed at rapidly identifying resistant h n strains applying real-time pcr using taqman probes was designed. the assay enables to identify the h y mutation from samples originated directly from infected tissue and plasma. oseltamivir-treated and non-treated specimens of several species infected with avian influenza a subtype h n , previously detected using the method described by payungporn et al. ( ) , were used. the oseltamivir-treated specimens were: h n oseltamivir-resistant strain in a vietnamese patient (n = ), oseltamivir-treated tiger cu-t ; panthera tigris tigris (n = ), white tiger ku- ; p. tigris tigris (n = ). the oseltamivir untreated specimens were: plasma of h n infected human (n = ) (chutinimitkul et al., ) , several tissues from different organs of tiger, lung (n = ), spleen (n = ), kidney (n = ), liver (n = ), brain of leopard (panthera pardus) (n = ), allantoic fluid of embryonated chicken eggs inoculated with the virus according to the method described by the office international des epizooties (oie) originating from a cat; felis catus (n = ), a dog; canis familiaris (n = ), a quail (n = ), an ostrich (n = ) and chicken (n = ). these specimens were isolated and provided by: ( ) the faculty of veterinary science, chulalongkorn university, bangkok, thailand; ( ) the department of livestock development, bangkok, thailand; ( ) faculty of veterinary science, kasetsart university, kampaengsaen campus, nakorn pathom, thailand; ( ) department of pediatrics, faculty of medicine, srinakharinwirot university, nakhon nayok, thailand; ( ) national institute of hygiene and epidemiology, hanoi, vietnam. the nucleotide sequences (n = ) of the neuraminidase gene of influenza a virus (h n ) were taken from the genbank database going back as far as - and hence, comprising entries isolated from various species, such as avian, cats, dog, tigers, swine and humans, including dq , the sequence of one vietnamese oseltamivir-resistant patient (a/vietnam/cl / (h n )). the alignments were performed using clustal x (version . from ftp://ftpigbmc.u-strasbg.fr/pub/clustalx) and bioedit sequence alignment software version . . (http://www.mbio.ncsu.edu/ bioedit/bioedit.html). assay target regions were first identified by visual inspection of the sequence alignment. primers were chosen from constant regions of all sequences specific for the neuraminidase gene n of influenza a virus most closely related to the probes. mgb taqman probes were chosen from the region covering the drug resistant area (h y) and designed to be specific for both wild type and mutant. both primers and probes were analyzed using the primer design software (oligos version . by ruslan kalendar, institute of biotechnology, university of helsinki, finland) and primer express software version . (applied biosystems, ca). the wild type (h) and mutant (y) mgb taq-man probes were labeled with fam and vic with emission wavelengths at and nm, respectively. the primers and probes used in this study are shown in table . an rna sample extracted from embryonated chicken eggs and previously identified as influenza a virus subtype h n (a/chicken/nakorn-patom/thailand/cu-k / (h n )) was applied to design a series of mutagenesis. the oligonucleotides depicted in table were used to generate the series of mutagenesis at amino acid position of the neuraminidase n gene which served as a control for all possible patterns of nucleotide change in the area of probe binding. the primers n muf and n mur served as the outer primers of each mutagenesis group, which paired with the mutagenesis primers. mutagenesis primer group for h is n mu f and n mu r. mutagenesis primer group for y is n mu f and n mu r. mutagenesis primer groups and were designed for possible varied strains occasionally found. the mutagenesis products of groups and were used to construct mutagenesis groups and . mutagenesis group for h is n mu f and n mu r. mutagenesis group for y is n mu f and n mu r. the primary mutagenesis pcr reaction mixture comprised . l of cdna cu-k , . m forward primer (outer or mutagenesis primer), . m reverse primer (outer or mutagenesis primer), l of . × mastermix (eppendorf, hamburg, germany) and nuclease-free water to a final volume of l. the secondary mutagenesis pcr reaction mixture comprised . l pcr product representative for each mutagenesis group, . m n muf, . m n mur, l . × mastermix (eppendorf) and nuclease-free water to a final volume of l. both amplification reactions were performed in a mastercycler personal (eppendorf) under the following conditions: predenaturation at • c for min followed by amplification cycles consisting of s denaturation at • c, s annealing at • c and min extension at • c and concluded by a final min extension at • c. four groups of mutagenesis pcr products were separated by % agarose gel electrophoresis and purified using the gel extraction kit (perfectprep gel cleanup, eppendorf, hamburg, germany). these purified products were inserted into the pgem-t easy vector system (promega, madison, wi) and plasmids were purified by using the high pure plasmid isolation kit (roche, gmbh, germany) according to the manufacturer's specifications. the series of h y mutations were sequenced and used as controls. viral rna was extracted from l samples of the allantoic fluid of inoculated embryonated eggs, plasma and the supernatant resulting from tissue extraction using the qiamp viral rna mini kit (qiagen, gmbh, germany) according to the man-ufacturer's specifications. reverse transcription was performed on l of each rna sample at • c for h using units of m-mlv reverse-trancriptase (promega), l of × m-mlv reaction buffer (promega), l of mm dntp (promega), units of rrnasin ® ribonuclease inhibitor (promega), m universal primer as described by hoffmann et al. ( ) . twelve microliters of rna from the rna extraction kit were heated to • c for min and cooled on ice before adding nuclease-free water to a final volume of l. real-time pcr was performed using the biotools quantimix easy probes kit (biotools, madrid, spain). both probes and primer pairs depicted in table were used in multiple formats, each primer and probe at a final concentration of . m and . m, respectively. a combination of . l cdna from embryonated eggs or l cdna from tissue, serum or plasma with a reaction mixture containing l of quantiprobes, . mm mgcl and nuclease-free water was adjusted to a final volume of l. real-time pcr amplification was carried out in a rotor-gene instrument (corbett research, sydney, australia). the amplification reaction consisted of a preincubation step at • c for min to activate the hotstartaq dna polymerase. this was followed by cycles of amplification including denaturation at • c for s, annealing at • c for s and extension at • c for s. two fluorescent signals were obtained once per cycle at the end of the extension step with detectors corresponding to the fam ( nm) and vic ( nm) channels, respectively. data acquisition and analysis of the real-time pcr assay were performed using the rotor-gene data analysis software, version . (corbett research supporting program). cdnas were amplified by pcr in a reaction mixture containing l of . × mastermix (eppendorf) . m primer f: -atactgagaactcaagagtc- , . m primer r: -ttatccctgcacacacatg- and nuclease-free water to a final volume of l. the amplification reaction was performed in a mastercycler personal (eppendorf) under the following conditions: predenaturation at • c for min followed by amplification cycles comprising denaturation at • c for s, annealing at • c for s and extension at • c for s and concluded by a final extension at • c for min. the pcr products were separated by % agarose gel electrophoresis and purified using the gel extraction kit (perfectprep gel cleanup, eppendorf, hamburg). the purified products were inserted into pgem-t easy vector system (promega), according to the manufacturer's protocol. ten clones were randomly selected from the resultant white colonies and plasmids were purified using the high pure plasmid isolation kit (roche, gmbh) according to the manufacturer's specifications. for automated dna sequencing, all plasmids were amplified using the gene amp pcr system (perkin-elmer, ma). the sequenced products were subjected to a perkin-elmer sequence analyzer (perkin-elmer). the specificity of the dual probe real-time pcr was evaluated by cross-reaction tests carried out between rna extracts from isolates or clinical specimens expressing the entire spectrum of na subtypes (n -n ) of who reference strain influenza and other viral pathogens, such as newcastle disease virus (ndv), respiratory syncytial virus (rsv) subgroups a and b, human metapneumovirus (hmpv), coronavirus oc , coronavirus e, infectious bursal disease virus (ibdv) and infectious bronchitis virus (ibv). the sensitivity was established with plasmids containing a copy of the wild type h and mutant y serving as reference. the wild type h and mutant y plasmids were used to determine the capacity of the real-time pcr assay to detect wild type and oseltamivir-resistant codon mutants in a single sample. dna concentration was determined by measuring absorbance at and nm. the two control plasmids were diluted to , , and copies/l and each resulting concentration was mixed at : , : , : , : and : wild type-to-variant ratios. real-time pcr analysis of potential codon variants was performed on each ratio at each concentration under the conditions described above. the result was obtained by using two taqman probes labeled with the fam and vic fluorescent signal for wild type and mutant detection, respectively. the fluorescent signal resulting from real-time pcr can be interpreted as shown in fig. . a sample containing only the wild type strain will emit the fluorescent signal exclusively via the fam channel ( nm) whereas a sample containing the oseltamivir-resistant variant with a nucleotide alteration at position of the neuraminidase gene will emit oseltamivir-treated vietnamese patient (le et al., ) showing both fluorescence signals indicative of a combination between wild type and resistant strain, ku- : oseltamivir-treated white tiger, cu-t : oseltamivir-treated tiger showing only the wild type signal. the fluorescent signal via the vic channel ( nm). in order to develop and optimize the assay these probes were tested on four sets of mutagenesis and obtained clearly discernible results irrespective of the concentrations of wild type and mutant plasmids or the ratio in which they had been mixed. the clinical samples tested in this assay were isolated from humans, tigers, leopard, cat, dog and various avian species previously infected with h n . two human specimens in this experiment were investigated. the first one isolated from human plasma and collected in nakhon-nayok province showed a positive result for the wild type only. in contrast, the second one obtained from a vietnamese patient previously reported by le et al. ( ) emitted signals specific for both wild type and mutant. the remaining specimens originating from tiger lung, spleen, kidney and liver, leopard brain and the allantoic fluid of various avian species, cat and dog displayed positive results specific for the wild type only. three specimens, treated with oseltamivir, were chosen to be cloned into plasmids for confirmation. the first specimen was from a tiger (a/tiger/thailand/cu-t / ) isolated from zoo tigers that had perished during the mid-october h n influenza outbreak. cu-t was isolated from a nasal swab of a tiger that eventually perished but had been treated with oseltamivir at mg/ kg twice daily for days prior to specimen collection (amonsin et al., ; thanawongnuwech et al., ) and inoculated into san-fowl eggs according to the method described by the office international des epizooties (oie). ten clones of cu-t were randomly selected and sequenced. all the clones were sensitive to oseltamivir. the second specimen was white tiger (a/tiger/thailand/ku- / ) was isolated from a sick white tiger found positive for h n by nasal swab and subsequently treated with oseltamivir at mg/ kg twice daily for days. this tiger survived and a rectal swab was taken. this specimen was inoculated into sanfowl eggs. ten clones of ku- were randomly selected and sequenced. all the clones were sensitive to oseltamivir. the third specimen was cdna from a vietnamese patient (le et al., ) . ten clones of this strain were randomly selected and sequenced. nine of the clones were resistant to oseltamivir and only clone was sensitive. the specificity of the assay by cross-contamination tests were evaluated and found no cross-reactivity to total human dna, any of the different na subtypes of influenza a virus, newcastle disease virus (ndv), respiratory syncytial virus (rsv) subgroups a and b, human metapneumovirus (hmpv), coronavirus oc , coronavirus e, infectious bursal disease virus (ibdv) and infectious bronchitis virus (ibv). likewise, any significant false positive or non-specific signal in any of the samples tested was not observed. overall, the results obtained on oseltamivir resistance with the two labeled probes indicate a high specificity of both primers and probes used for amplification. as for the sensitivity of real-time pcr, the threshold concentration for detecting both wild type (h ) and mutant ( y) was copies/l. furthermore, in order to establish the limits of real-time pcr to detect both wild type and mutant in the same sample, wild type and mutant plasmids were mixed in various ratios and diluted them over a range of concentrations : , : , : , : and : ratios of wild type and mutant. the result showed the high sensitivity of the detection that can be detected although the reaction had . : . plasmid copies/l of wild type and mutant. at present, there is a substantial risk of a global influenza a virus subtype h n epidemic not only affecting poultry but also mammalian species including humans. a medication capable of preventing the spread to humans is the neuraminidase inhibitor oseltamivir. yet, influenza a virus subtype h n has already developed drug resistance by mutations in the neuraminidase gene leading to amino acid substitutions predominantly at positions , , and (n numbering system) of the enzyme's active site (gubareva et al., ) . the amino acid substitution at position identified in mutants selected in the presence of na inhibitors both in vivo and in vitro has exclusively been found in n (gubareva et al., ) . in , h n virus resistant to oseltamivir due to an amino acid change from histidine (h) to tyrosine (y) based on a single nucleotide alteration at position has been isolated from three vietnamese patients one of whom, a years old, recovered (le et al., ) while the remaining two, a and years old, succumbed to the infection (de jong et al., ) . hence, patients found positive for h n infection ought to be monitored for the nucleotide change at position causing resistance to oseltamivir before the onset of treatment. with the mutation detected in time, alternative treatment applying, for example zanamivir might save the patient's life. in this experiment, both probes and primers were specifically designed to detect the nucleotide change causing oseltamivir resistance. the amino acid substitution of histidine (h) with tyrosine (y) is the consequence of a single nucleotide in the first codon of this amino acid change from c to t. the alignment of theh n neuraminidase gene sequence with more than sequences stored in the genbank database showed a nucleotide change in the specific probe area yet at a position not triggering the critical amino acid alteration. hence, the probe was designed to allow for this inconsequential mutation by using a degenerate nucleotide at that position thus closely mimicking the natural situation. moreover, since the conserved area of the gene restricted the probe's length to nucleotides taqman mgb was chosen. the probe was coupled with a minor groove binder enhancing its tm and had a non-fluorescent quencher attached to the end, which does not interfere with fluorescent signal detection. after having tested the probes with the mutagenesis control, the result showed high sensitivity and correct distinction between wild type and mutant upon mixing different ratios of wild type and mutant plasmid at low concentrations. in conclusion, real-time pcr using two labeled taqman probes provides a highly specific and sensitive method to detect the amino acid alteration at position of the influenza a subtype h n neuraminidase gene causing oseltamivir resistance. studies as the one described here could address the potential differences in diagnostic outcomes between patients who develop detectable oseltamivir resistance and patients who retain only the wild type strain of h n . however, the other point mutations of oseltamivir resistance in h n infected mammalian species need for the further investigation. genetic characterization of h n influenza a viruses isolated from zoo tigers in thailand writing committee of the world health organization (who) consultation on human influenza a/h , . avian influenza a (h n ) infection in humans h n influenza a virus and infected human plasma oseltamivir resistance during treatment of influenza a (h n ) infection characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gulls molecular mechanisms of influenza virus resistance to neuraminidase inhibitors influenza virus neuraminidase inhibitors selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir detection of influenza virus resistance to neuraminidase inhibitors by an enzyme inhibition assay. antiviral res emergence and transmission of influenza a viruses resistant to amantadine and rimantadine universal primer set for the full-length amplification of all influenza a viruses the h y mutation in the influenza a/h n neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo management of influenza: use of new antivirals and resistance in perspective influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent antiinfluenza activity resistant influenza a viruses in children treated with oseltamivir: descriptive study avian flu: isolation of drug-resistant h n virus neuraminidase inhibitors for influenza oseltamivir resistance-disabling our influenza defenses single step multiplex real-time rt-pcr for h n influenza a virus detection probable tiger-to-tiger transmission of avian influenza h n world health organization international avian influenza investigative team systematic review and economic decision modelling for the prevention and treatment of influenza a and b mechanism by which mutations at his alter sensitivity of influenza a virus n neuraminidase to oseltamivir carboxylate and zanamivir evaluation of neuraminidase enzyme assays using different substrates to measure susceptibility of influenza virus clinical isolates to neuraminidase inhibitors: report of the neuraminidase inhibitor susceptibility network oral oseltamivir treatment of influenza in children cumulative number of confirmed human cases of avian influenza a/(h n ) reported to who we would like to express our deep gratitude to the thailand research fund (senior research scholar), royal golden jubilee ph.d. program, center of excellence in viral hepatitis research, chulalongkorn university for supporting this study and roche diagnostics, thailand for providing mutagenesis primers, plasmid purification kit. we also would like to thank dr. surangrat srisuratanon, department of pediatrics, faculty of medicine, srinakharinwirot university, nakhon nayok, for her assistance and ms. petra hirsch for critically reviewing the manuscript. key: cord- -jc ckqy authors: chen, yu t.; lin, chi h.; ji, wen t.; li, shu k.; liu, hung j. title: proteasome inhibition reduces avian reovirus replication and apoptosis induction in cultured cells date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: jc ckqy the interplay between avian reovirus (arv) replication and apoptosis and proteasome pathway was studied in cultured cells. it is shown that inhibition of the proteasome did not affect viral entry and host cell translation but had influence on arv replication and arv-induced apoptosis. evidence is provided to demonstrate that ubiquitin-proteasome blocked arv replication at an early step in viral life cycle. however, viral transcription and protein translation were also reduced markedly after addition of proteasome inhibitor mg . treatment of bhk- cells with the mg markedly decreased virus titer as well as prevented virus-induced apoptosis. the expression of arv proteins σc, σa, and σns was also reduced markedly, suggesting that suppression of virus replication is due to down-regulation of these arv proteins by ubiquitin-proteasome system. mg was also shown to suppress arv σc-induced phosphrylation of p on serine , caspase activities, and dna fragmentation leading to complete inhibition of arv-induced apoptosis. avian reovirus (arv) causing arthritis, chronic respiratory diseases, and malabsorption syndrome leads to considerable economic losses (kibenege and wilcox, ) . arv-encoded proteins, including at least structural proteins and non-structural proteins have been demonstrated (varela and benavente, ; bodelon et al., ) . in the -class proteins of arv, protein c, is a minor outer-capsid protein of arv that is encoded by the largest open reading frame of the s segment (varela and benavente, ; bodelon et al., ) . it is not only an attachment protein (martinez-costas et al., ) but also an apoptosis inducer . some studies have suggested that c is the target for type-specific neutralizing antibodies while b is target for groupspecific antibodies (wickramasinghe et al., ) . protein p is a viroporin (bodelon et al., ) responsible for arv-induced cell fusion (bodelon et al., ) . a recent report suggests that p retards cell growth by activation of p pathway . arv a, encoded by the genome segment s (yin et al., ) , has been identified as a double-stranded rna (dsrna) binding protein (yin et al., ) and is an inhibitor of the double-stranded rnadependent protein kinas (gonzalez-lopez et al., ) . another arv protein, ns, encoded by the genome segment s (chiu and lee, ) , has been reported for its single-stranded rna (ssrna) binding activity (yin and lee, ) . the ubiquitin-proteasome pathway, a major intracellular protein degradation pathway in eukaryotic cells (myung et al., ) , plays an important role in a wide variety of cellular functions, including signal transduction, antigen processing, cell cycle regulation, transcription regulation, dna repair, and apoptosis (ciechanover, ; glickman and ciechanover, ) . in the present study, attempts were made to explore the role of proteasome inhibition in arv infectivity and the mechanisms involved in the proteasome inhibitor suppression of arv replication and apoptosis induction in cultured cells. it was shown previously that arv c is an apoptosis inducer that causes apoptosis by linking src kinase to p -mitochondrial pathway in cultured cells lin et al., ) . using the proteasome inhibitor mg to inhibit the cellular proteasome pathway, it was found that mg could reduce arv-induced apoptosis, cytopathic effect (cpe), virus titer, and protein expression. inhibition of the ubiquitin-proteasome pathway did not affect viral entry and host cell translation. to our knowledge, this is the first finding demonstrating that arv replication and arv-induced apoptosis could be suppressed by mg . two monoclonal antibodies against arv a and ns were kindly provided by dr. long h. lee (hou et al., ; pai et al., ; huang et al., ) . the monoclonal antibody against arv c was a laboratory stock . the monoclonal antibody, which detects ser -phosphorylated p was purchased from r&d systems inc. (minneapolis, mn, usa). the polyclonal antibodies against gfp and actin were from invitrogen (carlsbad, ca, usa) and chemicon (temecula, ca, usa), respectively. a modified , -( -dimethylthiazol- -yl)- -( -carboxymethoxy phenyl)- -( -sulfophenyl)- h-tetrazolium salt (mtt) assay, which measures mitochondrial function, was used to determine cell viability according to the manufacturer's instruction (promega, madison, wi, usa). bhk- cells were pretreated with various concentrations of mg and then infected with arv s (moi = ) for h. cell viability was assessed by mtt assay at h post-infection (h.p.i.). arv s propagated in bhk- cells was described previously . to study the effect of mg on arv replication and virus internalization, bhk- cells were infected with arv at various mois or treated with various concentrations of mg . virus titers were determined at h.p.i. by a plaque assay and western blot for confirmation of expression of a, ns, and c proteins. representative morphological changes of bhk- cells treated with mg or dmso were observed by a phase-contrast microscopy at h.p.i. to explore the effect of proteasome inactivation during the course of arv infection, bhk- cells were treated with mg at different times. the cells were washed to remove the drug and further incubated until h. whole cell lysates were collected at the indicted time points and a plaque assay was done for each lysate to determine the virus yield during preceding -h period. the gfp gene was derived from the commercial vector pib-gfp (invitrogen). following respective treatments with bam hi, klenow, and not i, the gfp fragment separated from pib-gfp was cloned into pcdna . (−) vector. the construct was named pcdna-gfp. in addition, the construction of pcdna-c has been described previously . to investigate the effect of mg on arv production was through the reduction of arv rna levels, rna extracted from bhk- cells h.p.i. in the presence or absence of mg was amplified by rt-pcr. the primers for amplification of a and gapdh were as follows: a forward primer, -ggcttctacttctcctcgaag actc- (identical to nucleotides - ) and a reverse primer, -agaagtcatta gcctcctgcgt ta- (complementary to nucleotides - ); gapdh forward primer, -cattgacctcaactacatgg- , gapdh reverse primer, -ttgccca cagccttggcagc- . reverse transcription was carried out at • c for min. pcr reactions were subjected to cycles consisting of denaturation for min at • c, annealing for min at • c and, extension for s at • c, and one final extension cycle at • c for min. cells in mm dishes were infected with arv at moi or transfected with g of plasmid dna using superfect reagent (qiagen, valencia, usa). after incubation for h, the medium was removed and cells were grown for an additional h. cells were harvested, washed, and lysed in . ml ripa lysis buffer ( mm tris, ph . , mm nacl, % nonidet p- , mm pmsf). both the supernatant and pellet were collected, mixed with laemmli sample buffer and boiled for min. after resolving the samples on a % sds-page, the proteins were transferred to pvdf membranes. the gfp and arv c, a, and ns proteins were detected using their respective antibodies described above. the signal was detected by ecl reagent (amersham pharmacia biotech, hong kong) and visualized by autoradiography. for ifa assay, arv s -infected bhk- cells were fixed in a mixture of % acetone and % methanol for min, and then incubated with an anti-c monoclonal antibody . the bound antibody was visualized by immunostaining with fitcconjugated second antibody raised against mouse igg. to study whether arv-induced apoptosis could be blocked in bhk- cells by mg , western blot detection of the level of ser -phosphorylated p , active caspase staining, and dna fragmentation analysis were carried out. bhk- cells were either pre-incubated min before infection or h.p.i. with mg . cell lysates were collected and assayed from infected cells h after treatment. active caspase in living cells was examined as described previously (chulu et al., ) . it was found that different doses of mg caused only slight cytotoxicity to bhk- cells (data not shown). arv replication in untreated cells resulted in the generation of a viral titer of . pfu/ml after h.p.i., wherease proteasome inhibition reduced the viral titer in a dose-dependent manner (fig. a) . the results indicated that the proteasome inhibitor significantly blocked arv production, suggesting that the proteasome may play an important role in arv replication. in this study, viral titers in the whole cell lysates harvested at h.p.i. were reduced dramatically by mg (fig. a) , suggesting that the effect is due to the reduction in virus growth rather than to a delay in virus release. different amounts of input virus change the absolute amount of progeny virus but do not affect significantly the percentage of reduction in viral titers, suggesting that the antiviral properties of mg are independent of the multiplicity of infection (fig. b) . in addition, arv-induced cpe is characterized by cell fusion, detachment of infected cells from culture flask, cell lysis and death. arv-induced cpe and cell death were reduced dramatically after the addition of mg (fig. c) . fig. a shows the experimental design for defining the major target of ubiquitin-proteasome system by following the mg treatment of arv-infected cells at different time points in the viral life cycle. fig. b shows that virus titer of the group treated for - h was . log units lower than the titer of the untreated sample. the virus titer of the groups treated for - h or - h was and . log units lower than the titer of the non-treated sample. the kinetic study suggested that the ubiquitin-proteasome system is involved most likely at an early step in the virus life cycle, since the inhibitory effect of mg was seen primarily in the - h treatment group (fig. b) . as describe above, the mg has an inhibitory effect on arv during early infection times. to dissect further each step in the presence of the mg to reveal the possible mechanism of inhibition, the virus internalization assay was carried out. there was no difference in virus titer in the presence or absence of mg (fig. c ). viral proteins a and ns are important for arv replication while c is an apoptosis inducer. to determine whether the inhibitory effects of mg ( um) on arv replication and apoptosis induc-tion depend on their activities, we also examined their expression levels in arv-infected bhk- cells. the results indicated that the expression of a, c, and ns was reduced significantly in bhk- cells either pre-incubated min before infection or h.p.i. with mg (fig. d) , suggesting that the ubiquitin-proteasome system is not involved in virus internalization. rt-pcr was carried out to investigate whether the effect of mg on arv production was through the reduction in arv rna levels. the viral mrna levels were dramatically decreased in arvinfected bhk- cells treated with the mg (fig. a) . the arv life-cycle consists of several steps. to determine which step(s) in the virus life cycle, the mg targeted, the reduction of viral titers was tested from whole cell lysates treated with proteasome inhibitor either min before virus infection or h.p.i. the results showed that exposure to mg inhibited significantly viral protein production h.p.i. (fig. d) , suggesting that inhibition of viral replication by mg is likely not dependent on the blockage of viral entry into host cells. cells positive for c expression were examined at h.p.i. by ifa and western blot assay in the presence of different concentrations of mg . the mg -treated cells showed significant reduction in amount of cells positive for arv c expression (fig. b ). in the present study, treatment with mg decreased c expression in a dose-dependent manner (fig. c) . the pcdna-gfp construct was transfected into cells infected with different mois to understand whether mg can influence protein translation in bhk- cells. fig. d shows that the expression of gfp protein was not affected but the expression of arv c was suppressed in arv-infected cells in the presence of various concentration of mg . taken together, the results suggested that proteasome inhibition decreased viral replication via suppression of protein translation. in this study, the level of ser -phosphorylated p was reduced significantly in the presence of mg in either bhk- cells pre-incubated min before infection or h.p.i. with mg in arv-infected bhk- cells (fig. a) . in comparison to without mg or dmso-treated groups, caspase activated in arvinfected bhk- cells treated with mg was reduced (fig. b) . addition of proteasome inhibitor in the medium also inhibited internucleosomal dna cleavage, demonstrating that arv-induced apoptosis could be suppressed by mg (fig. c ). fig. d shows that the transient expression of c was not affected in arv-infected bhk- cells in the presence of various concentrations of mg . this confirmed further that the mg could not affect host cell translation. it was also seen that the ele-vated level of ser -phosphorylated p ( fig. d ; upper panel) and apoptosis induction in bhk- cells (fig. d ; lower panel) was due to arv c expression. with the exception of mock-transfection with the plasmid pcdna . (−), both arv-infected cells without mg treatment and arv c-transfected cells with mg treatment showed internucleosomal dna cleavage ( fig. d ; lower panel). the s proteasome is a large multi-subunit complex that degrades selectively intracellular proteins. it is involved in a wide variety of mediated proteolytically intracellular process, such as apoptosis, cell cycle progression, transcriptional control, and metabolic regulation (hilt and wolf, ) . studies by many research groups have shown that various viruses have evolved sophisticated mechanisms to use or manipulate the host ubiquitinproteasome pathway for their own needs. retroviruses require the proteasome for budding from cells (schuber et al., ) , murine coronavirus for its transfer from endosome to cytoplasm during viral entry (yu and lai, ) , minute virus of virus (mvm) and reovirus for nuclear translocation (connolly et al., ; ros and kempf, ) , and human cytomegalovirus for inducing cell cycle progression (kalejta et al., ) . adenovirus requires active proteasomes to promote late gene expression (galinier et al., ) , coxsackievirus for viral rna transcription and protein synthesis (luo et al., ) , and tobacco mosaic virus for degradation movement protein (reichel and beachy, ) . to date, whether the ubiquitin-proteasome pathway-mediated arv replication and apoptosis induction is related to ubinquintin process or the proteasome degradation remains largely unknown. this study was therefore aimed at elucidating whether arv also uses the ubiquitinproteasome pathway for it's own benefit and other mechanisms for cell regulation. in this study, various aspects of the arv-proteasome interplay were characterized. a proteasome inhibitor mg was used to explore the effect of proteasome inhibition on arv replication and apoptosis induction by arv. it was observed that mg was fully active against arv when present at the beginning of the infection and a very limited effect was observed when present later after infection, suggesting that inhibition of ubiquitin-proteasome system did not affect virus entry and internalization and blocked arv replication at an early step in viral life cycle. infection with avian reovirus causes severe cpe and apoptosis in both avian and mammalian cells. during the past several years, it was demonstrated that arv c could induce apoptosis in both culture cells and chicken tissues lin et al., ) . studies by our laboratory have demonstrated further that arv-induced apoptosis by linking src kinase to p -mitochondrial pathway (lin et al., ; chulu et al., ) . a recent report demonstrated that p regulates bax expression and translocation into the mitochondria leading to cytochrome c release into the cytoplasm, activating the caspase pathway that finally leads to cell apoptosis (chulu et al., ) . to date, the levels of arv rna transcription and protein synthesis in infected cells in the presence of mg were not clear. in the present study, the arv rna transcription and protein synthesis were shown to be inhibited dramatically in the arv-infected cells after treatment with mg . the expression of ser -phosphorylated p and active caspase in arv-infected bhk- cells was reduced markedly following treatment with mg . this suggested that mg decreased arv infectivity and possessed the anti-apoptotic effect that may be due to reduced viral replication, especially down-regulation of c expression (fig. b-c) . how does the ubiquitin-proteasome pathway regulate arv replication? perhaps the ubiquitination of viral proteins is required for ubiquitin-proteasome mediated viral replication, such as ssrna-and dsrna-binding proteins ns and a which are essential for arv replication. elucidation of the ubiquitin-proteasome pathways involved in avian reovirus replication and induction of apoptosis will contribute important new information leading to better understanding of the mechanisms by which viruses in general cause cell death and diseases. further study is undergoing to address these questions. in conclusion, it was shown that proteasome inhibitor reduces arv replication through inhibition of viral rna transcription and protein synthesis, thus preventing arv-induced apoptosis. the results suggest that arv may have developed certain mechanisms, such as the ubiquitin-proteasome pathway, to inhibit apoptosis or viral replication by inactivating the p pathway. the avian reovirus genome segment s is a functionally tricistronic gene that express one structural and two nonstructural proteins in infected cells modification of late membrane permeability in avian reovirus-infected cells: viroporin activity of the s -encoded nonstructural p protein cloning and nucleotide sequencing of the s genome segment of avian reovirus s apoptosis induction by avian reovirus through p and mitochondrial pathways the ubiquitin-proteasome proteolytic pathway reovirus-induced apoptosis requires activation of transcription factor nf-kappab adenovirus protein involved in virus internalization recruits ubiquitin-protein ligase the ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction evidence that avian reovirus a protein is an inhibitor of the double-stranded rnadependent protein kinase proteasome: the world of regulatory proteolysis characterization of monoclonal antibodies against avian reovirus s c protein produced in insect cells and their application in detection of arv isolates monoclonal antibodies against different epitopes of nonstructural protein ns of avian reovirus s epitope mapping and functional analysis of a and ns proteins of avian reovirus human cytomegalovirus pp stimulates cell cycle progression by inducing the proteasome-dependent degradation of the retinoblastoma family of tumor suppressors tenosynovitis in chickens avian reovirus activates a novel proapoptotic signal by linking src to p avian reovirusinduced apoptosis related to tissue injury molecular evolution of avian reovirus: evidence for genetic diversity and reassortment of the s-class genome segments and multiple cocirculating lineages rapid characterization of avian reoviruses using polylogenetic analysis, reverse transcription-polymerase chain reaction and restriction enzyme fragment length polymorphism retardation of cell growth by avian reovirus p through the activation of p pathway proteasome inhibition reduces coxsackievirus b replication in murine cardiomyocytes protein architecture of avian reoviris s and identification of the cell attachment protein the ubiquitin-proteasome proteolytic pathway and proteasome inhibitors characterization of monoclonal antibodies against avian reovirus s protein a synthesized in escherichia coli degradation of tobacco mosaic virus movement protein by the s proteasome the ubiquitin-proteasome machinery is essential for nuclear translocation of incoming minute virus of mice proteasome inhibition interferes with gag polyprotein processing, release, and maturation of hiv- and hiv- avian reovirus c protein induces apoptosis in cultured cells protein coding assignment of avian reovirus s avian reoviruses proteins associated with neutralization of virus infectivity identification and characterization of rna-binding activities of avian reovirus non-structural protein ns synthesis in escherichia coli of arv core protein a and its dsrna-binding activity the ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry this work was supported by the national science council (nsc- - -b- - & nsc- - -b- - -my ), taiwan. key: cord- -tqw vx b authors: geerligs, h.j.; meinders, c.a.m.; snel, j.; duyves, w. title: the use of rt-pcr for determination of separate end-points for the strains ib h and ib d in titration of the combination vaccine poulvac ib(®) primer date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: tqw vx b poulvac ib(®) primer is a lyophilized vaccine containing two attenuated infectious bronchitis strains in one vial, ib h and ib d . for quantification of the viral content of the vaccine, dilution series of the final product are inoculated in embryonated chicken eggs. after the incubation period of seven days standard practice is for the embryos to be taken from each egg and examined visually for ib specific lesions; these readings are used to determine an end-point in viral titrations. the result is a titre value to which both strains contribute. however, it is not clear what the live virus titre is for strain ib h and for strain ib d . in order to determine end-points in the titration for each of the two strains, we collected the allantoic fluids from each egg after the incubation period and tested these for the presence of ib h and ib d by a strain specific reverse phase pcr. based on the data obtained by pcr we were able to determine an end-point for each of the two strains. for a given commercial batch of poulvac ib primer we determined titres of ( . ) eid( ) per vial for ib h and ( . ) eid( ) for ib d using pcr for end-point determination. these end-points matched well with the end-point determined for both strains cumulatively after visual examination, i.e. ( . ) eid( ) per vial. it is concluded that pcr is a suitable means to determine end-points in titrations of live viruses. infectious bronchitis virus (ibv), a coronavirus, is one of the foremost causes of economic losses within the poultry industry, affecting the performance of both meat-providing and egg-laying chickens (cavanagh, ; de wit et al., ) . the major reason for the high profile of ibv is due to the existence of a very large number of serotypes, although the virus is also highly contagious. in order to control the disease in poultry, both live and inactivated ib vaccines are used extensively. the nature of the protective immune response to ibv is poorly understood, but research has demonstrated that the surface spike protein, and particularly the amino-terminal s portion, is sufficient to induce good protective immunity. there is increasing evidence that only a few amino acid differences amongst s proteins are sufficient to have a detrimental impact on cross-protection (cavanagh, ) . as it is not always clear which serotype is responsible for any health problems in poultry, combinations of vaccines are widely used to generate broad protection * corresponding author. tel.: + ; fax: + . e-mail addresses: harm.geerligs@zoetis.com, harm.geerligs@pfizer.com (h.j. geerligs). (terregino et al., ) . another possibility is to use vaccines which contain a combination of different vaccine viral strains. zoetis is marketing a live combination vaccine for chickens named poulvac ® ib primer. this lyophilized vaccine contains two attenuated infectious bronchitis strains, ib h (macdonald and mcmartin, ) and ib d (kusters et al., ) . both vaccine strains have been attenuated by passage in embryonated chicken eggs, which is the usual method for attenuation of infectious bronchitis viruses and development of vaccines (macdonald and mcmartin, ; klieve and cummings, ; jackwood et al., ; liu et al., ) . in order to quantify the viral content of the vaccine, dilution series of the final product are inoculated in days embryonated chicken eggs. after the incubation period the eggs are opened and the embryos removed for examination of ib specific lesions. the product titre defined is the reciprocal value of the dilution causing embryonal lesions in % of the eggs. for the combination vaccine poulvac ® ib primer the result is a cumulative titre to which both ib strains contribute. in order to titrate both virus strains separately, strain specific neutralizing antisera have been tested. it has been shown that antiserum against strain ib d does not cross-react with strain ib h (kusters et al., ) and it was speculated that antiserum against ib h would not cross react with strain ib d . however, during titration experiments it was determined table pcr primers for detection of the strains ib h and ib d , with the respective pcr product size in base pairs. sequence - forward primer sequence - reverse primer length in base pairs ib d -atacaattatatcaaaccagc- -aatacagattgcttacaaccacc- ib h -aatactacttttacgttacac- -aatacagattgcttacaaccacc- that the antiserum against ib h strongly reacted with ib d . pcr has been used successfully to detect ib strains in various samples and it appeared to be possible to distinguish ib strains, including ib h and ib d , based on their reactivity with primers specific for the various strains (worthington et al., ) . here, we took advantages of the genomic level differences between the two ib strains and describe a pcr based approach to quantify the titres of each ib virus strain contained in the poulvac ib vaccine. lyophilized vials of a routine production batch of poulvac ® ib primer were used. during the manufacturing process ml of a mixture containing allantoic fluid with ib h , allantoic fluid with ib d and a freeze-dry stabilizer was filled per vial. the vials were lyophilized according to routine procedures and subsequently stored in the dark at ± • c. titrations of ib virus were performed according to standard procedures as described by doherty ( ) . specific pathogen free chicken (spf) eggs were obtained from valobiomedia, osterholz-scharmbeck, germany. three vials of each vaccine batch were tested per titration assay. the content of each vial of lyophilized product was reconstituted in ml water for injection. each sample was diluted further in nutrient broth (becton dickinson, breda, the netherlands) containing g/ml gentamicin sulphate (sigma-aldrich chemie bv, zwijndrecht, the netherlands), up to the beginning of the dilution series to be inoculated in eggs. in this series -fold dilution steps were made and per dilution days embryonated eggs were inoculated. after inoculation the eggs were incubated at • c and a relative humidity of %. after day incubation eggs with dead embryos were considered nonspecific deaths and discarded. after an incubation period of days the eggs were opened and the allantoic fluid harvested and stored at − • c. the embryos were removed and examined for the presence of specific lesions caused by the virus, i.e. dwarfing, curling and stunting of the embryo. dead embryos were considered positive for ibv. the titre, expressed as the number of eid /ml was calculated according to the method of spearmann-karber (finney, ) . allantoic fluids from the eggs were investigated for the presence of ib virus by reverse transcriptase pcr with strains specific reverse transcriptase pcr primers for ib h and ib d . the rna was isolated from each allantoic fluid samples using a high pure viral rna kit (roche, almere, the netherlands) according to the instructions of the manufacturer. for pcr analysis a pcr master mixture was prepared. for each sample to be analysed . l working solution forward primer ( m primer), . l working solution reverse primer ( m primer), . l taq-mix, . l × buffer, was mixed with . l pcr grade water. taq-mix and × buffer had been obtained from the super script one step reverse transcriptase pcr kit (invitrogen, bleiswijk, the netherlands). for one sample l of this mixture was added to a pcr tube containing . l of the rna sample. . l of pcr grade water was used instead in case of a negative control. the primers for the strains ib h and ib d and the size of the pcr products are indicated in table . the tubes were placed in the pcr machine (thermocycler, mj research ptc- , bio-rad, veenendaal, the netherlands) and the following steps were programmed: cdna synthesis min at • c, initial denaturation min at • c, cycles of s at • c, s at • c, s at • c. final extension was min at • c and reaction stop min at • c. the formation of pcr product was assessed by agarose gel electrophoresis. first the gel was prepared. tbe buffer was prepared by -fold dilution of a solution with . m tris, . m boric acid, . m edta in water. agarose was added to a concentration of % to the tbe buffer and ethydium bromide to a final concentration of . g per ml. the gel was allowed to solidify and used for electrophoresis of the pcr samples. the samples were allowed to go through the gel for h at v. a basepair ladder was used to determine the size of the bands in the agarose gel. gel electrophoresis was stopped when separation of the different bands in the base pair ladder was satisfactory. bands were made visible by ultraviolet illumination. in a first experiment, a dilution series of a batch of poulvac ® ib primer was prepared according to routine production qc procedures. the dilution series ranged from − to − . all dilutions were tested for presence of ib h and ib d by reverse transciptase pcr. none of the dilutions was positive for ib by pcr, see table . apparently, the quantities of virus in the dilutions series were below the detection level. the dilution series was inoculated in embryonated spf chicken eggs, eggs per dilution. after the routine incubation period samples of the allantoic fluids were tested also by pcr. it now appeared that after the egg passage dilutions up to − were positive by pcr for ib d and dilutions up to − were positive by pcr for ib h , see table . the embryos were also examined visually, and the results show that at a dilution of − all ( of ) embryos were affected by the virus. at a dilution of − one of embryos was affected. the only possible explanation for the presence of ib h and ib d in the allantoic fluids was that the viruses had been able to cause a productive infection in the eggs. it is concluded that in this test live virus titres were determined. in a second experiment a routine titration on poulvac ® ib primer was performed. this titration was performed in triplicate. after the incubation period the eggs were opened and the embryos were examined visually. of each triplicate a titre per vial was calculated (see bottom table under visual) and from the scores obtained, i.e. . , . and . log eid respectively, an average titre per vial was calculated, i.e. . log eid or . eid . analysis of allantoic fluids demonstrated for both strains there were clear endpoints in each triplicate so that a titre could be determined in a correct manner. the average titres determined for both strains were . eid per vial for ib h and . eid for ib d . if the sum of these values is determined the titre is . eid table titration of poulvac ib primer vaccine using strain specific pcr for detection of ib h or ib d . results from a dilution series before titration and allantoic fluid pools after titration, and results of visual examination after titration are shown. per vial, which is very near to the value of . eid obtained after visual examination. numerous publications are available in which viral titrations and possible issues have been described. for several viruses tissue culture methods can be used, in which a positive response can be observed by examination of monolayers of cells for the presence of a cytopathogenic effect (heldt et al., ) . if the virus does not form a cytopathogenic effect, the presence of live virus in the culture can be demonstrated by methods using colour agents (heldt et al., ) or specific antibodies (zielinska et al., ; yap and lam, ) . for avian viruses suitable substrates are needed and sometimes there are different possibilities (nicholas et al., ) . for titrations in embryonated eggs the presence of the virus in allantoic fluid can be tested using a specific property of the virus, for example its ability to cause haemagglutination (klimov et al., ) . the presence or absence of lesions on the embryos also can be used to determine the presence of live virus, such as has been demonstrated for ib viruses (de wit, ) . the use of pcr for screening samples from titrations for the presence of virus has not been described previously. initially pcr was used mainly for testing for the presence of viruses or other infectious agents in samples (tafuro et al., ) . currently, with the introduction of real time pcr it is possible to determine quantities of virus particles in samples (hiroshi kimura et al., ; isabel costafreda et al., ; spackman and suarez, ) . because of its specificity it is possible also to detect more than one virus strain in one test using pcr (van elden et al., ) . we used pcr for detection of two different ib virus strains in the allantoic fluids from all the individual eggs in a titration of a live ib combination vaccine. based on the results of the pcr we determined whether an allantoic fluid was positive for one or both of the viruses or not, and as such we were able to define a separate endpoint in the live virus titration for each of the two strains. it has to be clear that this is not the same as a quantitative pcr. in a quantitative pcr the number of genomic fragments is determined based on the number of cycles in the real time pcr required for a positive response. in such a pcr genomic fragments coming from live as well as 'dead' virus particles are determined. in the test described in this paper we determined whether an egg was positive for the presence of virus coming from a productive infection using pcr with a fixed number of cycles. only a live virus particle can cause a productive infection. if the egg would have been inoculated with a dead virus particle, it would not have given a positive response in the pcr. the titration method using pcr for testing the allantoic fluids is highly suitable to determine titres of different vaccine strains in one presentation. the pcr could be used also for end-point determinations of other ib strains, for example ib strains which do not have a very clear effect on embryos making it difficult to read egg titrations visually. furthermore, the use of pcr for end-point determination makes it possible also to perform titrations on a large scale using specialized equipment. it was demonstrated that pcr is a suitable means to determine end-points in titrations of live viruses. further research will be required to investigate repeatability and reproducibility of these methods for virus titrations. coronavirus avian infectious bronchitis virus detection of infectious bronchitis virus infectious bronchitis virus variants: a review of the history, current situation and control measures titration of avian infectious bronchitis virus in the tissues of experimentally infected chickens the spearman-kärber method a colorimetric assay for viral agents that produce cytopathic effects quantitative analysis of epstein-barr virus load by using a real-time pcr assay development, evaluation, and standardization of a real-time taqman reverse transcription-pcr assay for quantification of hepatitis a virus in clinical and shellfish samples attenuation, safety, and efficacy of in infectious bronchitis virus ga serotype vaccine immunity and cross-protection to nephritis produced by australian infectious bronchitis viruses used as vaccines influenza virus titration, antigenic characterization, and serological methods for antibody detection molecular epidemiology of infectious bronchitis virus in the netherlands s gene sequence heteronegeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives observations on the effects of the h and h vaccine strains of the infectious bronchitis virus in the domestic fowl a comparison of titration methods for live avian encephalomyelitis virus vaccines type a influenza detection and quantitation by real-time reverse transcriptase pcr rapid retrovirus titration using competitive polymerase chain reaction pathogenicity of a qx strain of infectious bronchitis virus in specific pathogen free and commercial boiler chickens and evaluation of protection induced by a vaccination program based on the ma and / serotypes simultaneous detection of influenza viruses a and b using real-time quantitative pcr a reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in western europe from infectivity titration of the fast-replicating and cytopathic hepatitis a virus strain hm a. by an in situ enzyme immunoassay development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis key: cord- -o hsr pm authors: an, dong-jun; kim, tae-young; song, dae-sub; kang, bo-kyu; park, bong-kyun title: an immunochromatography assay for rapid antemortem diagnosis of dogs suspected to have canine distemper date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: o hsr pm a new assay was developed for rapid and antemortem diagnosis of canine distemper (cd). this immunochromatography (ic)-based assay, which employs two monoclonal anti-cdv antibodies, was compared with nested pcr. when serial dilutions of purified cdv were tested, the cdv detection limits of the nested pcr and ic assays were × ( ) tcid( )/ml and × ( ) tcid( )/ml, respectively. nasal irrigation fluid, conjunctival swabs, and blood lymphocytes from dogs suspected to have cd were tested. preliminary ic experiments revealed that the optimal sample volume and reaction time were μl and min, respectively. relative to nested pcr, the sensitivity and specificity of the ic assay was maximal ( % and %, respectively) when conjunctival swabs were tested. this is significant because conjunctival swab specimens are easy to obtain in the early phase of cd infection. however, with blood lymphocytes and nasal samples, the ic assay was slightly less sensitive ( . % and . %, respectively) and specific ( . % and %, respectively) than nested pcr. since this novel ic assay does not require special instruments, it is a simple enough for dog owners to use. since early detection of cd would allow appropriate treatment and quarantine to be instituted quickly, such a test would help reduce the morbidity and mortality associated with cd help to prevent its spread to other animals. canine distemper virus (cdv) is a member of the genus morbillivirus of the paramyxoviridae and is related closely to the viruses responsible for measles, rinderpest, peste des petits ruminants, phocine distemper, dolphin distemper, porpoise distemper, and equine morbillivirus (fenner, ; haas and barrett, ; osterhaus et al., ) . canine distemper (cd) is a highly contagious disease that affects dogs of all ages. it is associated with high morbidity and mortality and occurs worldwide. dogs that have been infected naturally with cd show commonly systemic signs of the disease such as vomiting, diarrhoea and/or respiratory signs; sometimes they also exhibit generalized or localized myoclonus and the characteristic clin- * corresponding author. tel.: + ; fax: + . ical symptoms of cdv encephalomyelitis (frisk et al., ; moritz et al., ; okita et al., ) . in the recent outbreaks of cd in korea, the affected dogs could be classified clinically into two groups, namely, young dogs with cns signs associated with respiratory and/or gastrointestinal symptoms, and old dogs with cns signs alone. thus, the clinical signs of cd vary depending on the age and immune status of the host as well as the virulence of the virus strain and the environmental conditions (greene and appel, ) . acutely infected dogs shed the virus in all bodily secretions regardless of whether they are symptomatic or not. although the virus cannot be isolated from the bodily secretions of dogs with subacute distemper encephalomyelitis and persistent viral infection of the cns, they may still transmit the virus (appel, ) . to provide timely medical treatment that preserves the life of the infected dog (especially if neurological signs are present), it is important to have available an easy assay that can detect rapidly and accurately cdv. such an assay would also help to exclude other diagnoses and prevent the further transmission of the disease. at present, cd can be diagnosed by virus isolation, immunofluorescence assays (ifa), rt-pcr, and realtime pcr (frisk et al., ; hoyland et al., ; jozwik and frymus, ; shin et al., ) , all of which are relatively time-consuming and laborious techniques. the immunocomb antibody test kit (biogal galed labs, israel) for cd is also used frequently in animal hospitals to decide when to vaccinate a pet, as it reveals the anti-cdv igm and igg antibody levels. however, to use this test to determine whether a dog is infected with cdv, the animal has to be tested twice over several days. this delay in diagnosis is a serious limitation of this test. a new immunochromatography (ic)-based assay was developed for the antemortem diagnosis of cd. the sensitivity and specificity of this assay were compared with a nested pcr assay by testing conjunctival swabs, nasal irrigation fluid, and blood lymphocyte samples from dogs suspected to have cd. in total, specimens ( nasal irrigation fluids, conjunctival swab and blood lymphocyte samples) were obtained from dogs suspected of infection with cd. these samples were provided by nine private animal hospitals in seoul from may to april . all three samples (nasal, conjunctival and blood) were obtained from each of dogs at the time they presented at the animal hospital. of the remaining dogs, provided blood and either nasal irrigation fluids or conjunctival swab samples, and provided only blood. the conjunctival swabs and nasal irrigation fluids were each mixed with ml pbs (phosphate-buffered saline, ph . ) while blood lymphocytes were separated by using a leucosep tm tube (greiner, cat. no. , austria) . all of the samples were stored at − • c until use. the dogs suspected to have cd were maltese dogs (n = ), yorkshire terriers (n = ), shih tzu (n = ), korea jindo dogs (n = ), poodles (n = ), american cocker spaniels (n = ), pugs (n = ), and miniature schnauzers (n = ). most were young dogs who demonstrated mainly acute and systemic clinical signs. however, six dogs which were over years of age showed nervous symptoms. the choongang vaccine laboratory (daedeok valley, daejeon, korea) kindly provided us with the rockborn strain of cdv, canine coronavirus (ccv), canine parainfluenza viurs type (cpiv- ) and rabies virus. total rna was extracted from the nasal irrigation fluid, conjunctival swab and blood lymphocyte samples by using the microcolumn technique-based qiaamp viral rna mini kit (qiagen, cat. no. , usa) . the rna was then used in a reverse transcription reaction at • c for min and • c for min using a perkin-elmer gene amp pcr system . the cdna was amplified by using a one-step rt-pcr kit (qiagen, (table ) . amplification consisted of cycles of the same temperature profile used with the rt-pcr. the rt-pcr and nested pcr products were checked by electrophoresis on a . % agarose gel and were of the expected sizes ( and bp, respectively). hybridomas that produce mouse anti-cdv monoclonal antibodies (mabs) were generated as described by coyle et al. ( ) . briefly, the popliteal lymph node cells of balb/c mice (female, - weeks) that had been immunized in the footpad with purified cdv were fused with sp / myeloma cells. hybridomas that produced cdv-specific mabs in the screening test were selected and subcloned three times from single cells by limiting dilution. murine ascites fluid was produced in balb/c mice and igg was isolated by affinity chromatography using hitrap protein g hp column (ge healthcare, cat. no. - - , usa). western blotting was performed as described previously to confirm the specificity of the mabs (vihinen-ranta et al., ) . the cloned mabs were subtyped by using a mouse monobb id kit (zymed laboratory, cat. no. - , usa). the two selected mabs were designated as cdv-mab d and b . anti-cdv-mab d was dialyzed against mm carbonate buffer (ph . ) for h at • c. the ph of the . % (w/v) colloidal gold solution was adjusted to . with . m naoh and . mg/ml of anti-cdv-mab d was added. the mixture was gently mixed for min, blocked with % bovine serum albumin for min, and centrifuged at , × g for min. the gold colloid conjugate pellets were suspended in mm phosphate buffer (ph . ) containing . % casein and the optical density was adjusted to at nm with mm phosphate buffer (ph . ). the colloidal gold conjugate was stored at • c until use. the test strip was assembled with sample pad, conjugate pad, nitrocellulose membrane, and adsorption pad (cellulose paper). if a specimen containing cdv antigen is added to the sample pad, the gold conjugate migrates from the pad onto the cellulose membrane by capillary action. as the specimen and gold conjugate flow along the cellulose membrane, the cdv antigen in the specimen becomes sandwiched between the two antibodies, namely, the gold-conjugated anti-cdv-mab d and the anti-cdv-mab b . a signal is then detected on the nitrocellulose membrane (the test line). the control line is covered with mg/ml goat anti-mouse igg while the test line is covered with mg/ml cdv-mab b . the specimens were classified as true positive or true negative by the gold standard method, namely, the nested pcr, and the ic assay results were classified as false positive or false negative if they disagreed with the nested pcr results. the diagnostic sensitivity was calculated as true positive/(true positive + false negative) while diagnostic specificity was calculated as true negative/(true negative + false positive). the results of both calculations were usually expressed as percentages. several hybridoma cell lines that were found during screening to express an anti-cdv-mab were cloned by limiting dilution. two clones were then selected, namely, cdv-mab d and b . both produced igg isotype antibodies that were confirmed to be different clones by using the antigen and antibody complex test that the cdv antigen becomes sandwiched between the gold-conjugated anti-cdv-mab d and the anti-cdv-mab b on the capture line (data not shown). western blotting revealed that both purified monoclonal antibodies bound a kda cdv f (fusion) protein (fig. ) . a nested pcr assay served as the "gold standard" against which the ic assay was compared. the specificity of the nested pcr assay was first tested by using purified ccv, cpiv- , or rabies virus as the rna substrate. the nested pcr assay discriminated accurately cdv from all of these viruses (data not shown). the ic assay showed the same results (data not shown). the cdv detection limits of the ic and nested pcr assays were tested by using the rockborn cdv strain and diluting it serially -fold. the cdv detection limits of the nested pcr and ic assays were × tcid /ml and × tcid /ml, respectively. in total, nasal, conjunctival swab and serum samples obtained from dogs suspected to have cd were tested. preliminary assays using l, l, l, and l volumes of of the dog samples revealed that the best results were observed when l was used. when l was used, the optimal reaction time was on average min (fig. ) . these optimal conditions did not vary depending on whether the sample was a nasal, tear or blood sample. these parameters were then used when testing the remaining dog samples. of the samples, the ic assay yielded incorrect results with eight: six false negatives (three nasal irrigation and three blood samples) and two false positives (two blood samples) ( table ) . compared with the nested pcr assay, the overall sensitivity and specificity of the ic assay for all specimens was . % ( / ) and . % ( / ), respectively. the sensitivity and specificity of the test was highest when conjunctival swab samples were tested (both %). high sensitivity and specificity was also observed when nasal irrigation samples ( . % and %, respectively) and blood lymphocyte samples ( . % and . %, respectively) were tested (table ) . the antemortem diagnosis of canine distemper is based on the demonstration of viral antigens in scrapings and body fluids such as conjunctival and vaginal smears, tracheal washings and table clinical signs, age and breed of cd-positive specimens table analysis of the sensitivity and specificity of the ic assay relative to nested pcr results in detecting cdv in specimens from dogs suspected to have cd a ic assay result no. of samples that are positive or negative upon nested pcr total nasal irrigation fluid (n = ) conjunctival swab (n = ) blood lymphocytes (n = ) − total a the sensitivity and specificity of the ic assay relative to nested pcr results were, respectively, . % ( / ) and % ( / ) for nasal irrigation fluid samples, % ( / ) and % ( / ) for conjunctival swab samples, and . % ( / ) and . % ( / ) for blood lymphocyte samples. urine sediment (tipold et al., ) . various techniques have been used for this, including virus isolation, ifa, rt-pcr, and real-time pcr (frisk et al., ; hoyland et al., ; jozwik and frymus, ; shin et al., ) . however, the direct ifa gives false negative results in the subacute or chronic form of the disease (jozwik and frymus, ) , and the other methods tend to be labor-and time-consuming. the biogal's immuno-comb antibody test kit, which measures the anti-cdv igm or igg levels in the dog and is usually used to determine when to vaccinate, can be used as a subsidiary measure to deduce cd infection. however, with this method, the same dog has to be retested after several days to determine whether the antibody levels are increasing, which reflects an ongoing immune response and probable infection with cdv. this delay in diagnosis is a serious limitation of this method. a number of studies have examined which sample types are the most appropriate for use with pcr-based methods to detect cdv. one study using nested pcr analysis revealed that of blood, urine, saliva, and nasal swab samples from dogs suspected to have cd, . %, %, %, and . % tested positive, respectively (shin et al., ) . a similar study using nested pcr that detected cdv np rna revealed that of serum, whole blood, and cerebrospinal fluid (csf) samples, %, %, and % were positive, respectively (frisk et al., ; moritz et al., ) . in contrast, hospitalized dogs with neurological disturbances but lacking the typical findings of distemper were tested, rt-pcr analysis failed to detect cdv rna in any of the serum samples while only / ( %) whole blood and / ( %) csf samples were positive. however, / ( %) urine and all ( %) csf samples were positive (amude et al., ) . another study used rt-pcr to compare the various bodily secretions for the early detection of cdv in experimentally infected dogs . the conjunctival swab was found to be the most suitable specimen for early diagnosis of cd, as cdv amplicons were detected in the conjunctival swabs from all seven dogs at days - post-infection . compared to nested pcr in this study, the ic assay showed the highest sensitivity and specificity when conjunctival swabs were tested (both %). therefore, conjunctival swab specimens are the most suitable specimens for early antemortem diagnosis of cd, probably because there is persistent shedding of cdv in the eye, unlike in other bodily compartments. significantly, conjunctival swabs are easier to obtain than the other specimens. however, conjunctival swabs have to be collected in the early phase of infection . the eight samples that yielded disparate results with nested pcr and the ic test (two false positives and six false negatives) were inoculated into -day-old eggs to determine whether they were truly positive or negative. the six false negative samples, but neither of the false positive samples, induced pocks on the chorioallantoic membrane (cam) days after inoculation. these pocks were found by rt-pcr and nucleotide sequence analysis to contain cdv genes. thus, the overall sensitivity and specificity of the ic assay were . % and . % lower than those of the nested pcr, respectively. it is possible that vaccination may lead to a false positive if testing occurs within a few days after vaccination. indeed, one of the false positives results identified by the ic assay (which was then found to be a true negative) was a korea jindo dog that had been tested days postvaccination. notably, of the other dogs that were tested shortly after vaccination (two yorkshire terriers and one shih tzu were tested , , and days post-vaccination, respectively), all were true positives as determined by both the ic and nested rt-pcr assays. hence, it was not clear whether these animals were really infected or whether both assays have detected the vaccine strain ( table ) . the ic assay has the advantage over many other field diagnostic techniques used in clinical practice in that the test procedure is simple, rapid, and can be performed by owners as well as veterinarians. this is particularly relevant since animals suspected to have an infectious disease are generally not hospitalized immediately. due to these advantages, the ic assays detecting specific antibodies are available for other veterinary diseases, including cpv (oh et al., ). an ic test for simultaneous detection of specific antibodies against babesia caballi and babesia equi in the horse has also been developed by using recombinant b. caballi kda rhoptry protein and recombinant truncated b. equi merozoite antigen (huang et al., ) . however, the ic assays can be limited in terms of the amount of antigen they can detect, as large amounts of viral antigen are required to produce a clearly visible band. as a result, the interpretation of results may be affected by the subjectivity of the test operator. however, the ic assay developed for cdv is as good as nested rt-pcr when conjunctival swab samples are tested. in conclusion, even though the ic assay is slightly less sensitive and specific than nested pcr, it could be a useful aid for early diagnosis of cdv in infected dogs, especially when conjunctival swabs are used. this assay has the advantage over other techniques in that it can be completed within min without the need for special instruments. the ready availability of this assay to dog owners as well as veterinarians could help to reduce the morbidity and mortality of the disease as it would allow appropriate treatment to be instituted before full symptoms become evident. antemortem diagnosis of cdv infection by rt-pcr in distemper dogs with neurological deficits without the typical clinical presentation virus infections of carnivores a sample standardised protocol for the production of monoclonal antibodies against viral and bacterial antigens detection of canine distemper virus nucleoprotein rna by reverse transcription-pcr using serum, whole blood, and cerebrospinal fluid from dogs with distemper infectious diseases of the dog and cat rinderpest and other animal morbillivirus infections: comparative aspects and recent developments a comparison of in situ hybridisation, reverse transcriptase-polymerase chain reaction (rt-pcr) and in situ-rt-pcr for the detection of canine distemper virus rna in paget's disease immunochromatographic test for simultaneous serodiagnosis of babesia caballi and b. equi infections in horses comparison of the immunofluorescence assay with rt-pcr and nested pcr in the diagnosis of canine distemper comparison of tissue and fluid samples for the early detection of canine distemper virus in experimentally infected dogs the evaluation of diagnostic procedures for the detection of canine distemper virus infection one-step immunochromatography assay kit for detecting antibodies to canine parvovirus histopathological features of canine distemper recently observed in japan morbillivirus infections of aquatic mammals: newly identified members of the genus comparison of one-step rt-pcr and a nested pcr for the detection of canine distemper virus in clinical samples neurological manifestation of canine distemper virus infection detection of canine parvovirus antigens with antibodies to synthetic peptides this research was supported in part by the joint hbi corporation, anyang, kyunggi-do. key: cord- -ghbpga s authors: harcourt, jennifer l.; caidi, hayat; anderson, larry j.; haynes, lia m. title: evaluation of the calu- cell line as a model of in vitro respiratory syncytial virus infection() date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: ghbpga s respiratory syncytial virus (rsv) replication is primarily limited to the upper respiratory tract epithelium and primary, differentiated normal human bronchial epithelial cells (nhbe) have, therefore, been considered a good system for in vitro analysis of lung tissue response to respiratory virus infection and virus–host interactions. however, nhbe cells are expensive, difficult to culture, and vary with the source patient. an alternate approach is to use a continuous cell line that has features of bronchial epithelial cells such as calu- , an epithelial cell line derived from human lung adenocarcinoma, as an in vitro model of respiratory virus infection. the results show that calu- fully polarize when grown on permeable supports as liquid-covered cultures. polarized calu- are susceptible to rsv infection and release infectious virus primarily from the apical surface, consistent with studies in nhbe cells. the data demonstrate that polarized calu- may serve as a useful in vitro model to study host responses to rsv infection. respiratory syncytial virus (rsv) is the major cause of lower respiratory tract illness in infants and children worldwide, and infection can also result in serious disease in elderly and immunecompromised patients (falsey et al., ; hall et al., ; panitch, ; shay et al., ) . rsv enters the respiratory tract primarily through fomite or hand-to-nasal epithelium transmission following contact with infectious secretions (hall and douglas, ; hall et al., ) . infection is usually limited to the upper respiratory tract, and progression into the lower respiratory tract can result in serious complications of infection, including hospitalization for pneumonia and bronchiolitis. in humans, similar to other paramyxoviruses, rsv infection and replication primarily occurs in ciliated airway epithelial cells (wright et al., ) . as infection progresses, perivascular and peribronchiolar mononuclear cell infiltration is followed by necrosis of the airway epithelium (aherne et al., ; downham et al., ; ferris et al., ) . the mechanisms of cellular responses to rsv infection have been studied extensively in vitro in a variety of immortalized epithelial cell lines grown in monolayer cultures, including but not limited to vero, hep- , a , and ଝ the findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of cdc. * corresponding author at: national center for immunization and respiratory dis- madin-darby canine kidney (mdck) cells (kwilas et al., ; li et al., ; lupfer and pastey, ; roberts et al., ; stark et al., ; swedan et al., ; wright et al., ) . in contrast to observations made in rsv infected non-polarized epithelial cell lines, the in vivo response to rsv infection is directional. one ex vivo model, differentiated, polarized cell cultures of primary normal human bronchial epithelial cells (nhbe), has provided some insights into the mechanism of rsv infection and the cellular response to infection. this model system more closely mimics the airway epithelium structure than monolayer-cultured cells, and likely provides a better in vitro model of rsv infection and the associated cellular responses. in nhbe cells, rsv primarily infects ciliated lumenal columnar cells, and infectious virus is released primarily from the apical surface of nhbe (zhang et al., ) . these data from the polarized, differentiated nhbe model demonstrate the insights that can be gained using cell systems that more closely mimic the airway epithelium. however, nhbe cell systems are time-and labor-intense, costly to maintain, and require access to primary lung tissue samples. additionally they require substantial expertise in isolating and culturing epithelial cells from the lung tissue, and significant amount of time to first polarize and then differentiate at an air-liquid interface. given these limitations, an alternative to nhbe cells as an in vitro model of polarized airway epithelium cells was identified and evaluated. calu- cells originate from a human bronchus adenocarcinoma of a mixed phenotype, believed to be derived from submucosal gland serous cells (yoshikawa et al., ) . when grown on trans-well inserts, calu- polarize into apical and baso- fig. . calu- susceptibility to rsv infection. calu- cells were cultured as monolayers and infected with rsv strain a at moi , , or (a). at days and post-infection, the presence of rsv-f message rna was analyzed by one-step rt-pcr (a). tight junction formation and rsv g protein gene expression at days post-infection (moi = ) was evaluated by immunofluorescence assay (b). tight junction formation was evaluated by staining cells with anti-zona occludens - (zo- ) antibody (novus biologics) and -alexafluor conjugated anti-ms igg (invitrogen; red), rsv-g protein expression was evaluated by staining cells with -alexafluor conjugated anti-rsv-g protein monoclonal antibody - g (green), and nuclei were stained with dapi (blue). stained cells were visualized using a zeiss axioimager microscope at × magnification. lateral surfaces, but do not differentiate into multiple cell types and layers as observed with cultured nhbe (grainger et al., ) . polarized calu- are susceptible to infection by respiratory viruses, including influenza virus a, rhinovirus and severe acute respiratory syndrome -associated coronavirus (sars-cov) (grantham et al., ; saedisomeolia et al., ; yoshikawa et al., yoshikawa et al., , . the studies presented here evaluate polarized calu- as an in vitro model of rsv infection. to determine whether rsv infects and replicates in non-ciliated respiratory epithelial cells, monolayers of non-polarized calu- were infected with rsv strain a at a multiplicity of infection (moi) between and , and viral rna expression was determined. rsv-f viral mrna was detectable at both and days post-infection by rt-pcr, demonstrating that calu- cells can be infected by rsv (fig. a) . the expression of rsv surface proteins attachment, g, and fusion, f, was examined by immunofluorescence assay using anti-rsv-g ( - g) and anti-rsv-f ( - a) monoclonal antibodies. at seven days post-infection, there was little evidence of syncytia formation and rsv g (fig. b) and rsv f proteins were detected in a few small clusters of cells within the calu- monolayer (fig. b) . interestingly, tight junction formation, as indicated by immunos-taining for zona occludens (zo)- (fig. b) , was maintained during days of infection. rsv surface protein expression co-localized with tight junctions (fig. b) , consistent with the lack of any obvious cytopathology in differentiated, rsv-infected nhbe cells (zhang et al., ) . polarized calu- cultures may be generated by growing cells at an air-liquid interface (ali) or as liquid-covered cultures (lcc). calu- cells were evaluated in liquid-covered culture (lcc), in which they are primarily non-ciliated (grainger et al., ) . the polarization of calu- in lcc is dependent on several factors including growth medium, cell density and trans-well membrane pore size and composition. for these studies, calu- were seeded at a density of . × cells/cm onto polyester trans-well inserts ( . cm) with pores (corning) in eagles modified essential medium (emem, gibco) supplemented with % fetal bovine serum (emem- % fbs, complete medium), and polarization was demonstrated by trans-epithelial electrical resistance (teer) development and by a passive sodium fluorescein equilibration assay. following seeding into trans-well culture, the development of teer by calu- cultures was monitored using an epithelial voltohmmeter (world precision instruments). the resistance of calu- fig. . directional release of rsv from polarized calu- . apical and basolateral compartment media of trans-well cultured calu- was replaced every - days. resistance development was confirmed by a passive sodium fluorescein equilibration assay as previously described (a, (geys et al., (geys et al., , ), and is representative of independent experiments. resistance development of calu- infected with rsv-a (moi = ) at the apical surface (b), and apical (c) and basolateral (d) release of infectious virus were monitored for weeks post-infection. for resistance development, each value represents the median ± sem measurements from individual wells ( measurements/insert) and is presented as cm . the amount of infectious virus released from the apical (c) and basolateral (d) media of calu- infected at gradually increased after seeding into trans-wells, and peaked between and cm by days post-seeding (data not shown). calu- polarization was confirmed by measuring the passive diffusion of sodium fluorescein through the cell monolayer as previously described (geys et al., (geys et al., , . briefly, polarized cells were washed with non-fluorescent buffer, and sodium fluorescein ( mg/ml) was added to the apical compartment and non-fluorescent buffer to the basolateral compartment of cells. after an hour incubation, the amount of dye that diffused into the basolateral compartment was determined by spectrophotometric analysis of the basolateral sample compared to a sodium fluorescein standard curve. polarized calu- cultures do not allow equilibration of sodium fluorescein between apical and basolateral compartments. as the measurable resistance of polarized cells increased, the percent of sodium fluorescein that diffused into the basolateral compartment declined from % of the initial amount of dye added to the apical compartment when cells were non-polarized, to less than % when teer was ≥ cm ( fig. a) . calu- were considered to be completely polarized once the teer was ≥ cm and the amount of sodium fluorescein that diffused into the basolateral compartment was less than % of the initial amount in the apical compartment, the threshold of polarization (geys et al., (geys et al., , . polarized cells were infected at either the apical or basolateral surface with rsv strain a at an moi of , based on the concentration of cells initially seeded into the trans-well inserts. apical infections were performed by overlaying virus on the apical surface of polarized cells for h at • c. for basolateral surface infections, trans-well inserts were inverted and virus was applied directly to the basolateral surface and incubated in similar conditions. for both infections, inoculum was removed after incubation, wells that were infected at the basolateral surface were re-inverted into their original locations, and growth medium was added to all wells. at the indicated times post-infection, teer was measured, apical and basolateral media were collected, and virus was quantitated by standard plaque assay on vero cell monolayers as previously described (sullender, ) . there was little variability in the teer of calu- prior to apical infection (data not shown), but greater variability between individual trans-wells in the teer of rsv-a -infected and mock-infected calu- following infection (fig. b ). this decline in the stability of teer measurements was observed in both mock-and rsv-a -infected calu- , and occurred following infection at either the apical (fig. b ) or basolateral (data not shown) surface. the fluctuations in resistance observed following infection may in part be due to a disruption in the polarized state of cells caused by the infection process. despite resistance fluctuations following rsv infection, the overall teer of polarized calu- was not significantly decreased compared to mock-infected cells for at least weeks post-infection, suggesting that apical or basolateral rsv infection of polarized calu- does not significantly alter tight junction formation. these observations are consistent with rsv-infection in calu- monolayer cultures (fig. b) , and with rsv infection of polarized, differentiated nhbe (zhang et al., ) . in vitro and ex vivo studies have shown that rsv is released apically from infected, polarized epithelial cells (batonick et al., ; brock et al., ; roberts et al., ; wright et al., ; zhang et al., ) . virus release into the apical compartment of calu- infected at the apical surface was detectable as early as days post-infection (fig. c) . while the amount of virus release varied between time points and between individual samples at each time point, infectious virus was detectable for at least six weeks the apical surface was evaluated by plaque assay on vero cells from three individual samples, and is presented in pfu/ml for each sample. the lower limit of detection for the plaque assays is indicated with a dashed line. data is representative of independent experiments. fig. . anti-rsv-f antibody neutralizes rsv infection of polarized calu- . resistance development of calu- infected at the apical surface (a), and apical (b) and basolateral (c) release of infectious virus from apically-infected calu- (moi = ) were monitored for weeks post-infection. prior to infection, virus was incubated with anti-rsv-f ( - c) or anti-rsv-n ( - h) antibody at either g/ml or g/ml for h at • c. each teer value represents the median ± sem measurements from individual wells ( measurements/insert) and is presented as cm . the amount of infectious virus released from the apical (b) and basolateral (c) media of calu- infected at the apical surface was evaluated by plaque assay on vero cells from four individual samples, and is presented in pfu/ml for each sample. no virus was detectable in basolateral media (c) at day pi. *statistically different values between rsv-a -infected wells and wells which were infected with antibody-preadsorbed virus, as determined by two-tailed unpaired analysis, when p < . . post-infection (day pi, fig. c ). this persistent rsv infection of polarized calu- is consistent with findings in rsv-infected differentiated nhbe cultures, in which infection was detectable for at least days post-infection (zhang et al., ) . similar to apical infection, virus release after basolateral infection was detected as early as days post-infection, primarily from the apical sur-face, and was detectable for at least six weeks post-infection (data not shown). after either apical or basolateral infection, virus was detected in the basolateral media of cells only after day pi, corresponding with decreased teer in infected calu- (fig. d , and data not shown). the detection of virus in the basolateral media may be due to basolateral release of virus or to the mixing of apical and basolateral media in cultures due to decreased polarization. whether cells were infected at the apical or basolateral surface, greater than % of the virus release occurred into the apical compartment, demonstrating directionality in the response of calu- to rsv infection. in order to determine whether rsv entry into calu- was mediated by rsv-f surface protein, anti-rsv-f or anti-rsv-n monoclonal antibody (mab - c and mab - h, respectively) were incubated with virus for h at • c, prior to infecting polarized calu- cells. there was sample-to-sample variability in the amount of infectious virus released from infected cells at each time point examined. infection of calu- was inhibited by the neutralizing anti-rsv-f antibody (mab - c), a murine mab that recognizes the same epitope as palivizumab (anderson et al., ; johnson et al., ) , at g/ml but not g/ml while a control anti-rsv-n mab failed to inhibit infection at either or g/ml (fig. ) . the anti-rsv-f mab completely neutralized apical (fig. b ) and basolateral (fig. c ) release of rsv at g/ml at day (p < . ) and at day pi (p = . ). treatment with a control anti-rsv n mab did not significantly neutralize apical or basolateral rsv infection at the timepoints examined ( fig. b and c). consistent with protection from rsv infection, treatment with g/ml anti-rsv-f mab, but not of anti-rsv-n mab, protected against the accelerated decline in polarization observed in rsv-a infected cells (fig. a) . the teer of anti-f mab treated calu- was comparable to mock-infected controls as late as day pi. the data presented in this report demonstrate that calu- cells are susceptible to rsv infection when cultured as monolayers. infected cells expressed viral message and viral protein, but did not form obvious cytopathology, and infection did not appear to alter tight junction formation. the lack of obvious cytopathology and limited viral spread following rsv infection of calu- is in contrast to observations made in non-polarized epithelial cell lines used to study rsv infection, including hep- , where infection becomes widespread across the monolayer of cells. however, these findings are consistent with those made in cultured nhbe (zhang et al., ) and in human adenoidal epithelial cells (hae) and human adenoid organ culture (wright et al., ) , in which rsv spread is restricted and few cells appear to be infected with rsv. consistent with previous studies in polarized mdck (roberts et al., ) and in differentiated nhbe, polarized calu- released infectious virus primarily from the apical surface, and infection was persistent, detectable for at least weeks post-infection. persistent rsv infection of cells in culture has been reported for at least days post-infection of nhbe cells (zhang et al., ) . the persistent infection and release of infectious virus for at least weeks post-infection of calu- cells is consistent with observations of persistent infection in ex vivo, cultured nhbe, in animal models of rsv infection, and in some rsv-infected individuals (couch et al., ; dakhama et al., ; hegele et al., ; isaia et al., ; mejias et al., ; sikkel et al., ; zhang et al., ) . finally, consistent with previous reports in other cell lines, rsv infection in calu- could be inhibited by an antibody directed against the attachment protein of rsv. although calu- do not differentiate, the results indicate that polarized calu- respond to infection similar to polarized, differentiated nhbe cells, and more closely reflect in situ rsv infection than non-polarized epithelial cells. calu- are more readily available than nhbe cells, polarize into stable cultures more rapidly than nhbe cells, and demonstrate restricted, persistent infection and directional viral maturation similar to nhbe cultures, demonstrating that calu- are a useful in vitro model of polarized human lung epithelium-derived cells for studying cellular responses to rsv infection. pathological changes in virus infections of the lower respiratory tract in children neutralization of respiratory syncytial virus by individual and mixtures of f and g protein monoclonal antibodies human respiratory syncytial virus glycoproteins are not required for apical targeting and release from polarized epithelial cells apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells respiratory viral infections in immunocompetent and immunocompromised persons persistence of respiratory syncytial virus (rsv) infection and development of rsv-specific igg response in a guinea-pig model of acute bronchiolitis role of respiratory viruses in childhood mortality respiratory syncytial virus and influenza a infections in the hospitalized elderly sudden and unexpected deaths in infants: histology and virology in vitro study of the pulmonary translocation of nanoparticles: a preliminary study optimisation of culture conditions to develop an in vitro pulmonary permeability model culture of calu- cells at the air interface provides a representative model of the airway epithelial barrier palmitoylation of the influenza a virus m protein is not required for virus replication in vitro but contributes to virus virulence modes of transmission of respiratory syncytial virus possible transmission by fomites of respiratory syncytial virus immunity to and frequency of reinfection with respiratory syncytial virus persistence of respiratory syncytial virus genome and protein after acute bronchiolitis in guinea pigs persistence of viruses in the nasopharynx of apparently healthy children aged - years. results of investigations performed in - development of a humanized monoclonal antibody (medi- ) with potent in vitro and in vivo activity against respiratory syncytial virus respiratory syncytial virus grown in vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans anti-inflammatory effect of muc during respiratory syncytial virus infection of lung epithelial cells in vitro decreased replication of human respiratory syncytial virus treated with the proteasome inhibitor mg- respiratory syncytial virus persistence: evidence in the mouse model bronchiolitis in infants respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells lycopene enrichment of cultured airway epithelial cells decreases the inflammation induced by rhinovirus infection and lipopolysaccharide bronchiolitis-associated hospitalizations among us children respiratory syncytial virus persistence in chronic obstructive pulmonary disease respiratory syncytial virus infection enhances neutrophil and eosinophil adhesion to cultured respiratory epithelial cells roles of cd and intercellular adhesion molecule- antigenic analysis of chimeric and truncated g proteins of respiratory syncytial virus respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways a monoclonal antibody pool for routine immunohistochemical detection of human respiratory syncytial virus antigens in formalin-fixed, paraffin-embedded tissue growth of respiratory syncytial virus in primary epithelial cells from the human respiratory tract severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology the authors wish to thank dr. don latner for assistance with the immunofluorescence assays, and ms. elisabeth blanchard for assistance with cell maintenance. key: cord- -nx fg yn authors: mari, viviana; losurdo, michele; lucente, maria stella; lorusso, eleonora; elia, gabriella; martella, vito; patruno, giovanni; buonavoglia, domenico; decaro, nicola title: multiplex real-time rt-pcr assay for bovine viral diarrhea virus type , type and hobi-like pestivirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: nx fg yn hobi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (bvdv) and . as a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. the aim of the present study was to develop a multiplex real-time rt-pcr assay, based on the taqman technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging hobi-like strains. the assay was found to be sensitive, specific and repeatable, ensuring detection of as few as ( )– ( ) viral rna copies. no cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. analysis of field samples tested positive for bvdv- , bvdv- or hobi-like virus by a nested pcr protocol revealed that the developed taqman assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time rt-pcr assay previously developed for hobi-like pestivirus detection showed cross-reactivity with few high-titre bvdv- samples. the genus pestivirus belongs to the flaviviridae family and includes recognised species: bovine viral diarrhea virus and (bvdv- and bvdv- ), classical swine fever virus (csfv), and border disease virus (bdv) (simmonds et al., ) . pestiviruses have a positive single-stranded rna genome, approximately . kb in length, composed by a single open reading frame (orf), preceded and followed by untranslated regions ( and utr). the single orf encodes for a polyprotein that gives origin to smaller proteins by viral cleavage: n pro , c, e rns , e , e , p , ns /ns , ns a, ns b, ns a, ns b (simmonds et al., ) . among all the genomic regions, the utr, n pro and e are widely used for comparison and phylogenetic analysis (bauermann et al., ) . on the basis of the capacity to cause a cytopathic effect (cpe) in cell cultures, two bvdv biotypes are known, cytopathogenic (cp) and non-cytopathogenic (ncp), both involved in the pathogenesis of mucosal disease (md), a fatal outcome of bvdv infection in persistently infected (pi) calves (brownlie et al., ; bolin et al., ) . pestivirus infection leads to significant economic losses worldwide showing a wide range of clinical signs, including mild upper respiratory signs, a transient decrease in circulating white blood cells, and a low-grade, short-term fever. however, there are virulent strains that cause severe respiratory disease, gastroenteric disorders, hemorrhagic syndrome, and pneumonia (baker, ; brownlie, ; corapi et al., ) . reproductive disorders represent one of the most important consequences of the bvdv infection (houe et al., ) . infection of pregnant cows during the first trimester of gestation with ncp bvdv strains, leads to failure of fertilization, return to estrus, abortion, congenital malformations, stillbirths, or the birth of pi animals which may appear normal or sometimes smaller and with congenital malformations. they are constantly viremic and bvdv seronegative, representing the main source of bvdv infection in the herd through their body fluids, so that eradication programs rely on the detection and slaughtering of these animals (bauermann et al., ) . because of their rna nature, pestivirus genomes accumulate several mutations during replication that may lead to the emergence of new strains, lineages or species (kirkland et al., ; schirrmeier et al., ; vilcek et al., ) . recently, additional four pestivirus species have been proposed within the genus pestivirus: pestivirus of giraffe, pronghorn virus, bungowannah virus, and hobi-like pestivirus (bauermann et al., ) . the prototype strain (hobi d / ) of this emerging group of pestiviruses, also known as bvdv- or atypical pestiviruses (larska et al., ; liu et al., ) , was detected as contaminant of a batch of fetal bovine serum (fbs) imported from brazil (schirrmeier et al., ) . natural infections by hobi-like pestiviruses have been reported in south america (cortez et al., ; weber et al., ) , asia (kampa et al., ; haider et al., ; mishra et al., ) and italy (decaro et al., (decaro et al., , a (decaro et al., , c (decaro et al., , a (decaro et al., , b . the virus has been associated to respiratory disease (decaro et al., (decaro et al., , c (decaro et al., , a and abortions (decaro et al., a) . several molecular methods are available for the detection and characterisation of pestiviruses. most of them either do not detect hobi-like pestiviruses at all or detect them with low efficiency (schirrmeier et al., ; ståhl et al., ståhl et al., , . a recently developed real-time rt-pcr assay is able to detect hobi-like pestiviruses without providing any simultaneous detection of bvdv- and bvdv- (liu et al., ) . however, the oligonucleotides used in this assay cross-react with high-titre bvdv- samples (decaro et al., b (decaro et al., , b . on the other hand, the nested pcr approach established by sullivan and akkina ( ) misclassifies hobi-like viruses as bvdv- (decaro et al., b) . in order to detect all pestiviruses infecting cattle using a single tool, a novel real-time rt-pcr assay has been recently developed (losurdo et al., ) . in addition, a nested pcr (npcr) protocol has been set up for pestivirus typing (decaro et al., b) . this assay has been proven to be specific as no cross-reactions between bvdv- , bvdv- and hobi-like pestiviruses were observed. however, the test is time consuming and labor intensive and can be exposed to a high risk of crosscontamination due to the post-pcr manipulations. to overcome these limitations, we have developed a multiplex real-time rt-pcr assay for simultaneous detection of the different species of bovine pestiviruses, including the emerging hobi-like group, allowing a rapid, sensitive and specific diagnosis of pestivirus infection and characterisation of the viral species. the full-length genome of reference strains of bvdv- , bvdv- , hobi-like pestivirus, bdv and csfv were obtained from the genbank database (http://www.ncbi.nlm.nih.gov/genbank/index. html) and aligned using the bioedit software package (hall, ) . forward and reverse primers were designed using primer software, version . (http://frodo.wi.mit.edu/primer /) to amplify a -nt fragment of the utr conserved region of all aligned pestiviruses. three specific probes to detect bvdv- , bvdv- and hobi-like pestivirus were designed using beacon designer software, version . (premier biosoft international, palo alto, ca, usa) (fig. ) . primers and probes were synthesised by eurofins genomics and are reported in table . pestivirus strains bvdv- nadl (courtesy of dr ferrari, istituto zooprofilattico sperimentale di lombardia ed emilia romagna, brescia, italy), bvdv- / (decaro et al., ) , hobi-like strain / - italy (decaro et al., ) , bdv bd (buonavoglia et al., ) , and an rna extract of the csfv lapinised chinese vaccine (courtesy of istituto zooprofilattico sperimentale di lombardia ed emilia romagna, brescia, italy) were used to evaluate the specificity of the test. to rule out any cross-reactivity between bovine pestiviruses and other bovine viral pathogens, isolates of the following viruses were also tested: bovine coronavirus (decaro et al., ) , bovine rotaviruses (pratelli et al., ) , bovine respiratory syncytial virus (vaccine strain brsv/ , cattlemaster , zoetis italia srl), bovine parainfluenza virus (vaccine strain ts rlb , cattlemaster , zoetis italia srl), and bovine herpesvirus types (thiry et al., ) and (tempesta et al., ) . a total of pestivirus positive field samples were analysed. ninety-eight samples were recruited from a previous study and had been already characterised by the npcr assay (decaro et al., b) , whereas additional specimens were detected more recently during an epidemiological survey for hobi-like pestivirus (unpublished data). the analysed samples included tissue samples from aborted fetuses, respiratory specimens from calves with respiratory disease, fecal samples from calves with enteritis and edta-blood samples from pi animals. rna was extracted from all samples using qiaamp ® cador ® pathogen mini kit (qiagen s.p.a., milan, italy), according to manufacturer's instructions. rna standards for bvdv- , bvdv- and hobi-like pestivirus were obtained amplifying a fragment of the utr region of reference strains bvdv- nadl, bvdv- / and hobi-like pestivirus italy- / - , using the common forward primer (vilcek et al., ) and three different pestivirus specific reverse primers (bvd - r: -tctatgcacacataaatgtggta- , bvd - r: -actaccggtcactctgccaactctccta- , bvd - r: -tcggtacacacatacatgtgata- ), designed using primer software, version . . . the rt-pcr products were cloned into topo ® xl pcr cloning vector (invitrogen srl, milan, italy) and transcribed with ribomax tm large scale rna production system-t (promega italia, milan, italy) from the t promoter, according to the manufacturer's guidelines. after dnase treatment, the transcripts were purified using qiaamp ® rna easy kit (qiagen s.p.a., milan, italy) and quantified by spectrophotometric analysis. ten-fold dilutions of the rna transcripts, representing to copies rna l − of template, were carried out in a mixed fecal/nasal swab suspension from a calf that tested pcr negative for pestivirus rna (sullivan and akkina, ; decaro et al., b) . aliquots of each dilution were frozen at − • c and used only once. reverse transcription of l of duplicates of the standard dilutions and rna extracts was carried out using geneamp ® rna pcr kit (life technologies italia applera italia, monza, italy) in a l reaction volume containing pcr buffer × (kcl mm, tris-hcl mm, ph . ), mgcl mm, mm of each deoxynucleotide (datp, dctp, dgtp, dttp), rnase inhibitor u, mulv reverse transcriptase . u, random hexamers . u. reverse-transcription was carried out at • c for min, followed by a denaturation step at • c for min. the triplex real-time pcr targeting the utr gene of bvdv- , bvdv- and hobi-like pestivirus was performed on a cfx tm real-time system (bio-rad laboratories srl, milan, italy) in a -l reaction mixture containing . l of itaq tm universal probes supermix (bio-rad laboratories srl, milan, italy), nm of primers pesti-qf and pesti-qr, nm of probes bvd -pb and bvd -pb and nm of probe bvd -pb, and l of c-dna. the thermal protocol consisted of activation of itaq dna polymerase at • c for min, followed by cycles of denaturation at • c for s and annealing/extension at • c for min. the detection of the table primers and taqman probes used in the multiplex real-time rt-pcr assay for discrimination of bovine pestiviruses and other oligonucleotides used in the study. increasing fluorescent signal was carried out during the extension step of the reaction and the data was analysed with the appropriate sequence detector software (bio-rad cfx manager v. . , bio-rad laboratories srl). in order to verify the absence of rna losses during the extraction step and the presence of rt-pcr inhibitors in the rna templates, an internal control (ic), consisting of an rna synthetic transcript containing the m gene of canine coronavirus (ccov) type ii (decaro et al., ) , was added to the lysis buffer (avl buffer, qiagen s.p.a.) at a concentration of , rna copies ml − of buffer prior to nucleic acid extraction. the fixed amount of the ic added to each sample had been calculated to give a mean c t value in a genotypespecific real-time rt-pcr assay (decaro et al., ) of . with a s.d. of . as calculated by separate runs. samples in which the c t value for the ic was > . (average plus s.d.) were excluded from the analysis. specificity of the assay was evaluated by testing pestivirus reference strains and other bovine viruses including bovine respiratory syncytial virus, bovine coronavirus, bovine rotavirus, bovine herpesvirus and and bovine parainfluenza virus. serial ten-fold dilutions of the bvdv- , bvdv- and hobi-like pestivirus standards containing from to copies of rna transcripts and the correlate c t values were used to set the standard curves for respective absolute quantifications. bovine nasal and faecal swabs and edta-blood samples that had tested negative for pestivirus and distilled water were used as negative controls and blank, respectively. the sensitivity of the multiplex real-time rt-pcr assay was evaluated using -fold dilutions of edta-blood samples containing about , and copies of bvdv- , bvdv- and hobi-like pestivirus rna, respectively, made in a edta-blood sample from a calf tested negative for bvdv. the same sample dilutions were submitted to npcr (decaro et al., b) and to the hobi-like taqman assay (liu et al., ) for a comparison. intra-assay repeatability was evaluated testing times the same samples in one experiment, and the inter-assay repeatability was verified repeating the experiment times. clinical samples containing virus amounts spanning the whole sensitivity limits of the multiplex real-time rt-pcr assay were selected for repeatability evaluation. coefficients of variation (cvs) were calculated by dividing the standard deviation of each tested sample by its mean and multiplying that result by . the detection of pestivirus rna in clinical samples and rna transcript dilutions was carried out using a npcr protocol previously developed for the characterisation of bovine pestiviruses (decaro et al., b) . first-and second-step amplifications were carried out using superscript tm one-step rt-pcr for long templates (life technologies italia) and amplitaq gold (life technologies italia), as previously described (decaro et al., b) . oligonucleotides are reported in table . to compare the performance of the developed triplex assay with the only existing real-time rt-pcr assay claimed to detect specifically this group of viruses, all clinical samples were tested by means of the liu's assay (liu et al., ) . reverse transcription and realtime pcr were carried out as described for the triplex assay using oligonucleotides listed in table . no fluorescence signal was detected from either negative controls or distilled water and all of the other bovine pathogens, including the related pestiviruses bdv and csfv, were not detected by the developed multiplex real-time rt-pcr. the assay was proven to be species specific since bvdv- , bvdv- and hobi-like pestivirus were correctly detected by the specific probes and no cross-reaction between the three pestiviruses was observed. the standard curves generated for each pestivirus using ten-fold dilutions of standard rna covered a linear range of at least nine orders of magnitude (from / to copies of standard rna) and linearity was observed over the entire quantification range (slopes of − . , − . and − . for bvdv- , bvdv- and hobi-like pestivirus, respectively). coefficients of regression (r ) were . , . and . for bvdv- , bvdv- and hobi-like pestivirus, respectively. the sensitivity of the assay was set at rna copies for bvdv- and at rna copies for bvdv- and hobi-like pestivirus. nested pcr had the same sensitivity in the case of bvdv- and hobi-like pestivirus, but it was -log less sensitive than the taqman assay when bvdv- was processed. in addition, the specific taqman assay by liu et al. ( ) was able to detect as few as hobi-like pestivirus rna copies. the repeatability was evaluated by calculating the intra-and interassay cvs. bvdv- cvs ranged from . % (samples containing × copies rna l − ) to . % ( . × copies rna l − ); bvdv- cvs varied from . % ( × copies rna l − ) to . % ( × copies rna l − ); hobi-like pestivirus cvs were between . % ( × copies rna l − ) and . % ( × copies rna l − ). interassay cvs ranges were comprised between . % ( × copies rna l − ) and . % ( × copies rna l − ) for bvdv- , between . % × copies rna l − ) and . % ( × copies rna l − ) for bvdv- ; between . % ( × copies rna l − ) and . % ( × copies rna l − ) for hobilike pestivirus. the ic was detected in all the examined samples, with c t values below the threshold value of . , thus confirming the absence of rna losses during nucleic acid extraction or dna polymerase inhibition during real-time pcr. in order to rule out any interference between the speciesspecific taqman probes contained in the same mix, a pestivirus negative edta-blood sample was also spiked with low ( copies) and high ( copies) concentrations of standard rna of bvdv- , bvdv- and hobi-like pestivirus and the viral loads in the spiked samples were quantified using the developed assay. the rna titres of the pestivirus species were calculated correctly, showing that no interference occurred during detection and quantitation of the different viruses contained in the same sample (data not shown). by analysis of field samples tested positive by npcr (decaro et al., b) , there was a perfect agreement between gel-based and real-time rt-pcr assays. bvdv- and bvdv- were detected in and samples, respectively, whereas specimens that had been collected from two different cattle herds in southern italy tested positive for hobi-like pestivirus. the amount of rna detected in the samples covered a wide range of copies per microlitre of template, ranging from . × to . × (bvdv- ), from . × to . × (bvdv- ) and from . × to . × (hobi-like pestivirus). the liu assay (liu et al., ) was able to type correctly all hobi-like strains and did not recognise any of the bvdv- positive samples, but out of bvdv- strains were mistyped as hobi-like viruses. the bvdv- titres of the mistyped samples were above rna copies l − of template. several pcr-based methods have been developed for sensitive and rapid detection of bvdv in clinical samples (bhudevi and weinstock, ; young et al., ; la rocca and sandvik, ; yan et al., ; fan et al., ; zhang et al., ; losurdo et al., ) , but only few methods are currently available for unambiguous discrimination between bvdv- and bvdv- (sullivan and akkina, ; letellier and kerkhofs, ; baxi et al., ; leblanc et al., ) . the emergence of hobi-like pestivirus that causes clinical pictures overlapping those induced by bvdv- and bvdv- (bauermann et al., (bauermann et al., , (bauermann et al., , , posed several concerns as for the ability of existing diagnostic methods to efficiently detect this emerging group of pestiviruses (schirrmeier et al., ) . the panpestivirus rt-pcr developed by vilcek et al. ( ) , which is commonly used for bvdv molecular screening, fails to detect or detects with low efficiency hobi-like strains due to the presence of a mismatch at the end of primer that prevents the correct primer annealing. other conventional and real-time rt-pcr protocols are able to detect the novel pestivirus but do not provide any virus characterisation, which is helpful to assess virus epidemiology (elvander et al., ; letellier et al., ; gaede et al., ; hoffmann et al., ; losurdo et al., ) . a taq-man assay that was claimed to be specific for hobi-like pestivirus was recently developed (liu et al., ) , but this assay could not discriminate simultaneously bvdv- and bvdv- and showed partial cross-reaction with high-titre bvdv- samples (decaro et al., b (decaro et al., , b . this cross-reaction was confirmed by the present study since out bvdv- positive samples reacted as hobi-like strains when tested by the liu's assay. recently, a npcr assay was established for simultaneous discrimination of all pestiviruses infecting cattle, including hobi-like pestivirus (decaro et al., b) . this method was specific and reliable, but also cumbersome as it requires two separate steps, an rt-pcr followed by the nested amplification; in addition, it presents a certain risk of cross-contamination between positive and negative samples. to overcome the limitations of existing methods, we have developed a triplex real-time rt-pcr assay, based on the taq-man chemistry, which was able to differentiate efficiently bvdv- , bvdv- and hobi-like pestivirus. the assay was proven to be repeatable and linear over a range of at least orders of magnitude, from / to rna copies, thus ensuring an accurate measurement of pestivirus rna loads in clinical samples. in comparison with npcr, the real-time rt-pcr assay was equally or slightly more sensitive and less time-consuming. labeling the speciesspecific probes with different fluorophores enables a correct characterisation of the pestiviral strains contained in clinical samples. in addition, the -well format of the real-time pcr plates ensures a high throughput, with the chance to test simultaneously several samples, which is useful for large-scale epidemiological surveys. the developed assay is a closed system in which the tube is never opened post-amplification, and this reduces the possibility of cross-contamination of new samples with previously amplified products. a certain carryover may occur due to the separation between rt and fluorogenic pcr, but we preferred a two-step assay, since one-tube methods are less sensitive than a two-step rt-pcr procedure (nakamura et al., ; bustin, ) and the risk of rna degradation is increased if analyses are performed over a long period of time (postollec et al., ) . in order to further reduce the risk of contamination, we have strictly separated the different working steps and carried out pipetting in different laminar flow hoods. in addition, the two-step assay requires more work, thus reducing the laboratory capacity. another limitation of the study is that the analysed pestiviral strains were geographically homogeneous, so that theoretically less common subtypes or divergent viruses circulating in different countries could not be correctly characterised. however, sequence alignment of the oligonucleotide binding regions showed that they are conserved within the same viral species for bvdv- and bvdv- . as for hobi-like viruses, even the more divergent strains recently identified in asia (mishra et al., ; haider et al., ) displayed only few mismatches, which should be tolerated by the oligonucleotides, including the taqman probe (fig. ) . anyway, the assay needs to be validated with those divergent strains that seem to be widespread in the asian continent. the developed assay does not recognise bdv, which was recently detected in cattle (strong et al., ; braun et al., ) . however, at the moment the epidemiological situation does not require including bdv 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resembling mucosal disease associated with 'hobi'-like pestivirus in a field outbreak combination of reverse transcription real-time polymerase chain reaction and antigen capture enzymelinked immunosorbent assay for the detection of animals persistently infected with bovine viral diarrhea virus real-time rt-pcr detection of bovine viral diarrhoea virus in whole blood using an external rna reference comparison of conventional rt-pcr, reverse-transcription loop-mediated isothermal amplification, and sybr green i-based real-time rt-pcr in the rapid detection of bovine viral diarrhea virus nucleotide in contaminated commercial bovine sera batches this work was supported by grants from italian ministry of university, project pon / "micromap -sviluppo di una piattaforma tecnologica multiplex per diagnostica molecolare, portatile ed automatizzata, basata sulla logica strumentale del labon-chip, in grado di consentire applicazioni multiparametriche in campo infettivologico". key: cord- -k m i authors: fu, chao-yang; huang, he; wang, xiao-mei; liu, yong-gang; wang, zhi-guo; cui, shang-jin; gao, hong-lei; li, zan; li, jing-peng; kong, xian-gang title: preparation and evaluation of anti-sars coronavirus igy from yolks of immunized spf chickens date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k m i severe acute respiratory syndrome (sars) is a recently discovered viral disease, characterized by fever, cough, acute fibrinous pneumonia and high infectivity. specific pathogen-free (spf) chickens were immunized with inactivated sars coronavirus and their eggs were harvested at regular intervals. yolk immunoglobulin (igy) was extracted using the water dilution method, followed by further purification on a sephadex g- column. sds-polyacrylamide gel electrophoresis (sds-page), western blot and neutralization test results showed that the igy obtained was of a high purity and had a strong reactive activity with a neutralization titer of : . lyophilization and stability tests showed that lyophilized anti-sars coronavirus igy had promising physical properties, with no significant reduction in reactive activity and good thermal stability. all these data suggest that the anti-sars coronavirus igy could be a new useful biological product for specific antiviral therapy against sars. severe acute respiratory syndrome (sars) is a recently discovered viral disease, characterized by fever, cough, acute fibrinous pneumonia and high infectivity (chang, ) . outbreaks of sars during caused concern around the world as a serious health threat. comprehensive prevention and treatment measures were taken internationally, while at the same time research on the sars coronavirus and development of a vaccine, therapeutic drugs and biological products was commenced immediately. research has been successful and considerable progress has been made in all of these fields (chow et al., ) . analyses of nucleotide and amino acid sequences of the sars coronavirus demonstrated that it did not belong to any of the three known subgroups of coronaviruses and therefore was identified as a new kind of coronavirus (wang and ding, ) . recent progress has been made in production of biological products against sars. as high-titer human antiserum against sars coronavirus has been found to be capable of * corresponding author. tel.: + ; fax: + . e-mail address: xgkong@hvri.ac.cn (x.-g. kong). inhibiting the multiplication of the sars virus, the prospect of confirming passive immunity against sars for infected patients and for non-infected individuals at risk is feasible. passive immunization and short-term protection with high antibody titers has historically been an effective way to combat fulminating infectious disease. during the outbreak of sars in china in , some positive results were achieved by passive immunization when the sera of recovered sars patients were used. no adverse reactions were observed in the first vaccinated volunteers. in order to provide new and effective passive immunization against sars, basic studies on the sars coronavirus antibody should be carried out. these studies should include searching for better antibody sources, increasing output and improving techniques of production and purification. in this study, we have successfully immunized specific pathogen-free (spf) chickens, and then purified a high-titer anti-sars coronavirus yolk immunoglobulin (igy) with neutralizing activity against sars coronavirus. sars coronavirus antigen was prepared using sars virus strain bj and kindly provided by the chinese academy of military was inactivated by ␤-propiolactone and purified on a sucrose gradient centrifugation as described previously (yin and liu, ) . thirty -week-old spf leghorn hens were provided by the experimental animal center of the harbin veterinary research institute, the chinese academy of agricultural sciences, harbin, china. chickens were immunized by injecting . mg of sars coronavirus antigen into the pectoral muscle. the second and third booster injections of . mg antigen were administered and weeks later. blood was harvested before and after each immunization and eggs were harvested when they became available. the isolation of igy from individual eggs was performed using the protocol described by akita and nakai ( ) . briefly, the egg white was separated from the egg yolk and discarded. the yolk was mixed vigorously with sterilized water and left standing overnight at • c. the mixture was then centrifuged at × g at • c for min, and the supernatant was purified by chromatography the protein concentration in harvested eluate was measured by thin-layer gel scan. the recovery rate of crude protein before chromatography was found to be % while the purity was % after chromatography. the eluate was subjected to analysis by sdspolyacrylamide gel electrophoresis (sds-page) and western blot. the isolated igy had a high purity as confirmed by sds-page and purified igy had good biological activity as confirmed by western blot. the activity of igy in sera and yolks diluted at : in phosphate buffer saline (pbs) from immunized animals was assessed using an indirect elisa assay as described previously (huang et al., ) (fig. ) . after immunization of spf chickens with sars coronavirus antigen, there were no detectable antibodies against sars coronavirus in the yolks of eggs laid in the first weeks after the first immunization. the production of anti-sarsv antibody in yolks was found to commence weeks after the igy was produced in serum. an igy-neutralizing virus assay was performed in laminarflow safety cabinets. briefly, serially diluted samples of the igy were incubated with tcid of sars coronavirus at • c for h, then inoculated onto vero e cells and incubated at • c in a co incubator. uninfected vero e cells and sarspositive serum or spf chicken serum were used as controls for this experiment. the igy did not react with vero cells when tested by indirect fluorescent antibody technique. infectivity titers were calculated using a standard tcid assay. the highest antibody dilution that inhibited cytopathic effect in % of the vero e cells inoculated with this dilution was regarded as the % neutralization titer. the results of neutralization experiments (table ) indicated that yolk antibody till : dilution was effective in neutralizing the sars coronavirus, and were consistent with the elisa result when extracted yolk antibody was used. for the lyophilization procedure, yolk was isolated and mixed with phosphate buffer saline at a ratio of : (v/v), followed by centrifugation and degreasing. aliquots were loaded into -ml bottles to which less than . % (g/ml) merthiolate was added. each bottle was vacuumed and lyophilized according to standard methods. the samples were tested for antibody titers before and after lyophilization. igy after drying showed good physical properties including pale yellow color. the binding activity of igy in elisa did not change significantly after lyophilization (table ). stability testing of lyophilized igy demonstrated that igy binding did not decrease significantly until the temperature reached • c for min in hot water (table ) , indicating the igy had good heat stability. there was no change in igy activity under acid conditions from ph to after treatment at • c for h (table ) . furthermore, binding activity of lyophilized igy did not change after -month storage at either − , • c or room temperature (table ) . igy from spf chickens provides a rich source of antibody that is easy to obtain and is cost effective. the advantages of spf chicken igy over antibodies from other mammals include a high concentration and freedom from specific pathogens, making the use of igy a broader prospect for development (schade et al., ; tini et al., ) . the igy from spf chickens in our study maintained good biological and neutralization activity at environmental conditions. the recovery rate after purification by water dilution and the level of purity after further purification were promising, the binding of igy remained unchanged after lyophilization, also making it feasible for the product that had been prepared in this way to be used for passive immunization and short-term protection. since the first outbreak of sars transmission of the virus has been controlled and the potential for an epidemic has been alleviated by isolation of suspected patients in combination with drug therapy, based on clinical and epidemiological characteristics of sars. long-term control of sars, however, will require a combination of active and passive immunizations, drug therapy and other comprehensive measures. it is therefore of vital importance to be able to produce vaccines and passive immunization products for effective control of sars. the development of high-titer anti-sars coronavirus igy described in this study would appear to have potential as a new anti-sars biological product for passive immunization, as it effectively neutralized the sars coronavirus. in the present study, anti-sars igy was already prepared as a crude and purified product, while an indirect elisa test was used for quality control of igy production. to facilitate the process of mass production of purified igy, the techniques of degreasing and purification would be key points for further study. after evaluation using experimentally infected animal models, anti-sars igy produced in this study has the potential to be commercially manufactured. the efficency of capsules produced from crude igy or spray powder and intravenous solution manufactured from more highly refined igy will need to be determined as the next step in development of igy products. comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic e. coli strain clinical findings, treatment and prognosis in patients with severe acute respiratory syndrome (sars) molecular advances in severe acute respiratory syndromeassociated coronavirus (sars-cov) establishment of the detection method of anti sars yolk antibody and serum antibody polyclonal avian antibodies extracted from egg yolk as an alternative to the production of antibodies in mammals-a review generation and application of chicken egg-yolk antibodies animal virology, second ed we thank simon fenton and trevor bagust (faculty of veterinary science, university of melbourne) and cui xianlan (landcare research new zealand) for their critical reading and comments to assist the preparation of this manuscript. key: cord- - pu x rj authors: etemadi, mohammad reza; jalilian, farid azizi; othman, norlijah; lye, munn-sann; ansari, sara; yubbu, putri; sekawi, zamberi title: diversity of respiratory viruses detected among hospitalized children with acute lower respiratory tract infections at hospital serdang, malaysia date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: pu x rj background: the role of respiratory viruses as the major cause of acute lower respiratory tract infections (alrtis) in children is becoming increasingly evident due to the use of sensitive molecular detection methods. the aim of this study was to use conventional and molecular detection methods to assess the epidemiology of respiratory viral infections in children less than five years of age that were hospitalized with alrtis. methods: the cross-sectional study was designed to investigate the occurrence of respiratory viruses including respiratory syncytisl virus (rsv), human metapneumovirus (hmpv), influenza virus a and b (ifv-a and b), parainfluenzavirus , , and (piv , , and ), human rhinoviruses (hrv), human enterovirus (hev), human coronaviruses (hcov) e and oc , human bocavirus (hbov) and human adenovirus (hadv) in hospitalized children with alrtis, at hospital serdang, malaysia, from june to december , . the study was also designed in part to assess the performance of the conventional methods against molecular methods. results: viral pathogens were detected in ( . %) of the patients. single virus infections were detected in ( . %) patients; ( . %) were co-infected with different viruses including double-virus infections in ( . %) and triple-virus infections in ( . %) cases. approximately % of samples were found to be positive using conventional methods compared with % using molecular methods. a wide range of respiratory viruses were detected in the study. there was a high prevalence of rsv ( . %) infections, particularly group b viruses. other etiological agents including hadv, hmpv, ifv-a, piv – , hbov, hcov-oc and hev were detected in . , . , . , . , . , . and . percent of the samples, respectively. conclusion: our results demonstrated the increased sensitivity of molecular detection methods compared with conventional methods for the diagnosis of artis in hospitalized children. this is the first report of hmpv infections in malaysia. background: the role of respiratory viruses as the major cause of acute lower respiratory tract infections (alrtis) in children is becoming increasingly evident due to the use of sensitive molecular detection methods. the aim of this study was to use conventional and molecular detection methods to assess the epidemiology of respiratory viral infections in children less than five years of age that were hospitalized with alrtis. methods: the cross-sectional study was designed to investigate the occurrence of respiratory viruses including respiratory syncytisl virus (rsv), human metapneumovirus (hmpv), influenza virus a and b (ifv-a and b), parainfluenzavirus , , and (piv , , and ), human rhinoviruses (hrv), human enterovirus (hev), human coronaviruses (hcov) e and oc , human bocavirus (hbov) and human adenovirus (hadv) in hospitalized children with alrtis, at hospital serdang, malaysia, from june to december , . the study was also designed in part to assess the performance of the conventional methods against molecular methods. results: viral pathogens were detected in ( . %) of the patients. single virus infections were detected in ( . %) patients; ( . %) were co-infected with different viruses including double-virus infections in ( . %) and triple-virus infections in ( . %) cases. approximately % of samples were found to be positive using conventional methods compared with % using molecular methods. a wide range of respiratory viruses were detected in the study. there was a high prevalence of rsv ( . %) infections, particularly group b viruses. other etiological agents including hadv, hmpv, ifv-a, piv - , hbov, hcov-oc and hev were detected in . , . , . , . , . , . and . percent of the samples, respectively. conclusion: our results demonstrated the increased sensitivity of molecular detection methods compared with conventional methods for the diagnosis of artis in hospitalized children. this is the first report of hmpv infections in malaysia. most respiratory tract infections are caused by viruses or bacteria with viruses causing the highest proportion of infections. the major causes of acute respiratory infections (ari) in children are respiratory syncytial virus (rsv), parainfluenzavirus (piv), influenza virus (ifv), adenovirus (adv), and human rhinoviruses (hrv). human metapneumovirus (hmpv), human coronaviruses (hcov) hku , nl , e and hospitalized children is cost-effective. it is pivotal in directing active treatment early in the course of the illness following detection, reducing unnecessary antibiotic prescription, and limiting nosocomial transmission to high-risk patients (adcock et al., ; lanata et al., ; osiowy, ; woo et al., ) . in addition, assessment of the morbidity of specific etiological agents of alrtis identified using sensitive detection methods in hospitalized patients is important to determine agent-specific interventions such as vaccination against rsv (lanata et al., ) . extensive detection of infections will also expand our knowledge of the etiology of pneumonia and assist in determining which etiological agents should be considered for vaccine development (rudan et al., ) . recent developments in molecular diagnosis of respiratory viruses and the discovery of new viruses have renewed the interest in respiratory virus epidemiology. however, there is still a considerable deficiency in the diagnosis of viruses which cause alrti (ieven, ) . far too little attention has been paid to the epidemiology of respiratory viral infections in malaysia. therefore, in this study, the goal was to detect a panel of classical and newly discovered respiratory viruses including ifv, rsv, piv, adv, hmpv, hrv, hev, hcov, and hbov which cause alrti in children below years of age at hospital serdang. both conventional methods including direct immunofluorescence assay, cell culture and shell vial culture and molecular diagnostic techniques including multiplex pcr and sequencing were used. the survey was conducted at two -bed pediatric wards in hospital serdang, a government-funded multi-specialty hospital located in the district of sepang in the state of selangor, malaysia. the participants were children more than one-month-old and less than years of age who were admitted to the hospital between june , and december , with the diagnosis of alrti. patients with congenital or acquired immunosuppressive conditions, with conditions that posed a potential hazard in obtaining the nasopharyngeal samples (e.g. bleeding diathesis, severe respiratory compromise) as determined by the clinicians and children with incomplete data or inadequate samples were excluded from the study. all potential subjects (including careers of the patients) were briefed on the study before written informed consent was obtained by the pediatrician in-charge of the case. approval from the following authorities was obtained prior to the start of the study: the ministry of heath malaysia research and ethics committee (mrec) (nmrr- - - ), the medical research ethics committee, faculty of medicine and health sciences, upm (upm/fpsk/pads/t -mjketikaper/f (jmpp fep ( ) )) and the research ethics board of hospital serdang were obtained. blood samples for bacterial culture were collected by the nurses involved in the study and sent to the department of pathology, hospital serdang. nasopharyngeal aspirate (npa) was taken through both nostrils by inserting a disposable catheter (no. or no. ) connected to a mucus extractor. npas were transported in viral transport medium (vtm) to the laboratory and refrigerated at °c- °c until required. in order to avoid repeated freezing and thawing, all npas were processed upon receipt. the samples were vortexed vigorously for s and centrifuged at ×g for min. the supernatant was collected and set aside for virus isolation and genome extraction. the cell pellet was used in a direct immunofluorescence assay (dfa) after washing several times to remove mucus to avoid nonspecific fluorescence. most of the samples were detected with dfa at the same day of samples received followed by cell culture and genome extraction and cdna synthesis. the pcr reaction was performed when a bunch of samples were available. detection of the viruses was performed concurrently during the six months of the study period. the d ultra direct immunofluorescence assay respiratory virus screening & identification kit (diagnostic hybrids inc. (dhi), usa) which contains a blend of murine fluorescein isothiocyanate (fitc)conjugated monoclonal antibodies (mabs) was used as the first step to detect eight common viruses including rsv, hmpv, ifv type a and b, piv - , hadv. dfa negative samples were inoculated onto shell vial culture (svc). r-mix tm ready cells (dhi, usa), ready-to-use mixed cell monolayers comprising mink lung cells (mv lu) and human adenocarcinoma cells (a ), were used according to the manufacturer's recommendations. viral rna/dna was extracted from the filtered supernatant of npa using magmax viral rna isolation kit (applied biosystems, ambion, usa) according to the manufacturer's instructions. the concentration and purity of the extracts (a /a nm and a /a nm) were measured using a nanodrop (thermofisher scientific, usa). the first strand cdna synthesis was carried out on rna extracts in a final volume of μl by random hexamer primer using revertaid ™ h minus first strand cdna synthesis kit (fermentas, usa) following the manufacturer's instructions. the samples were incubated first for min at °c followed by min at °c. the reaction was terminated by heating at °c for min. three multiplex rt-pcr (mp/rt-pcr - ) and subsequently two hemi-nested multiplex pcr assays (hnmp/pcr - ) were carried out for the molecular detection of rna viruses (bellau-pujol et al., ) . mp/rt-pcr targeted influenza viruses a and b, rsv (types a and b), hmpv (a and b). mp/rt-pcr detected parainfluenza virus types , , and (a and b) (piv - ). the mp/rt-pcr contained primers for the detection of hrv, hev, hcov oc and e. an internal control consisting of glyceraldehyde- -phosphate dehydrogenase (gapdh) was included in mp/rt-pcr . hbov and hadv were detected in the samples by singleplex pcr and nested pcr respectively (allander et al., a; lu and erdman, ) . primer set hadvhexf /adhexr and nested primer set adhexf /adhexr were used to detect hadv hexon gene hyper-variable regions - (hvr − ). the pcr products were separated by electrophoresis in a . % agarose gel and visualized using ethidium bromide under uv light. the multiplex pcr was validated using pcr ® . -topo ® plasmid vector [topo ta cloning® kit (invitrogen, usa)]. hep- (atcc ccl- , usa), mrc- (atcc ccl- , usa), vero (atcc (ccl- , usa) and hela cells (atcc, usa) were purchased from american type culture collection (atcc) and cultured according to their guidelines. all rsv positive samples were cultured on vero and hep- cells. hadv positive samples were inoculated onto hela and hep- cells. mrc- and hela cells were used for samples which were positive for hrv. cell cultures with characteristic cpe for rsv and hadv were harvested and confirmatory testing was performed with dfa. the tube cultures showing cpe for hrv were harvested and confirmed by pcr. a second blind passage was performed after a week for cultures without characteristic cpe. data were analyzed using spss version . . all p-values were twotailed and p-values of < . were considered statistically significant. comparisons between the results obtained by molecular methods and conventional methods were evaluated by mcnemar's test and pairedsamples t-test. a total of children less than five years of age who fulfilled the inclusion criteria as outlined above and were hospitalized with alrtis during a -week period between june , and december , were enrolled in the study. dfa was the first conventional method of detection of common respiratory viruses used to identify eight viruses including ifv a & b, pivs - , rsv, hmpv and hadv. hrv, hev, hcovs, piv and hbov were excluded from immunological detection because of a lack of specific monoclonal antibodies. for each virus the pattern of immunofluorescent staining was specific and was used for confirmatory purposes. eighty eight ( . %) of the npa samples were dfa positive as follows: ( . %) for rsv, ( . %) for hmpv, ( . %) for hadvs, ( . %) for ifv a and ( . %) for pivs - . detection of rsv, hmpv, piv - , ifv-a & b, hadv in shell vial culture was attempted as the second stage of the conventional method on the remaining dfa negative npa samples. seven additional viruses were detected and a total of ( . %) samples were virus positive by dfa followed by shell-vial culture. therefore, this method was able to detect some viruses which were missed by dfa: rsv and one case each for hadv, hmpv and piv . for rsv, cpe was detected in of specimens representing a % recovery. twenty-four samples positive for hadvs were inoculated into hela and hep- cell lines. five of samples ( %) showed characteristic cpe. semi-confluent monolayers of hela and mrc- cell lines were used to isolate of the hrv positive samples detected using rt-pcr. the ability of the multiplex method to specifically detect multiple viruses in the same reaction tube was evaluated by testing a mixture of cloned plasmids of targeted viruses. analysis of the pcr products showed that each multiplex method simultaneously detected all three control viruses included in each reaction as well as the internal control with the expected band sizes. in the presence of all primer sets in the multiplex reaction, no mispriming was observed in the positive and negative control tubes. the specificity of the mp/rt-pcr products was confirmed by nucleotide sequence analysis. three multiplex rt-pcr (mp/rt-pcr - ) and subsequently hemi-nested multiplex pcr (hnmp/pcr - ) were carried out on nucleic acids extracted from clinical specimens. the specific products were clearly separated and identified on a . % seakem agarose gel, both for virus control and for clinical specimens. in total, samples ( . %) from the panel of were virus positive and ( . %) specimens were virus negative using this method. almost all viruses ( %) were detected in the first stage of the mp/rt-pcr - assay. in the second stage six ( / , %) additional viruses were detected by hnmp/pcr - . using normal and nested pcr, of the samples tested, were found to be positive for hbov and ( . %) for adenovirus. of these positive samples, single infections were documented in one hbov and three cases of hadv infection. in total, samples ( . %) were positive for respiratory viruses using the molecular method while ( . %) were negative. gapdh was successfully amplified from all of the npa samples tested by pcr indicating that there were no pcr inhibitors in the reactions. therefore, false negative results were excluded using this internal control. one hundred and twelve patients ( . %) were found to be infected with a single virus with the most frequently detected viruses being rsv ( %), hrv ( %), ifv-a ( . %) and hmpv ( . %)(table ). no single infection due to hcovs was observed. multiple respiratory viral infections were documented in ( %) samples; consisting of ( . %) double infections and ( . %) triple infections. the most frequently detected viruses in these patients were rsv ( , . %), followed by hadv ( , . %) and hrv ( , . %). dual infections of rsv with hadv and hrv were the most prevalent multiple viral infections found in the study ( / , % and / , %, respectively). culture of blood samples from patients revealed bacterial infections in only five ( %) cases including one patient with a single infection ( . %) with ἀ-hemolytic streptococcus viridans. four other samples were also virus positive and were considered to be nosocomial infections (blood cultures results were positive after h) as follows: rsv/burkholderia cepacia, hrv/ b. cepacia and hrv/ coagulase-negative staphylococcus. m. pneumonia infections were not identified in any of the patients. in total, specimens ( . %) from the panel of were virus positive by a combination of conventional and molecular methods, and seven ( . %) specimens were virus negative. the comparison between conventional and molecular methods is depicted in table . a greater number of samples ( . %) were found to be virus positive using molecular methods compared to conventional methods ( . %) (p < . , mcnamara's test). there were ( . %), ( . %) and ( . %) more positive samples compared with dfa, dfa plus svc and dfa plus svc plus conventional cell culture methods, respectively. on the other hand molecular assays were able to detect more viruses (p < . , paired-samples t-test). the monthly distribution of cases with respiratory tract viruses is shown in fig. . during the study period, a continuously persisting activity was seen for rsv, hrv, hadv, and hmpv. influenza a was detected from july to september, with a peak in august followed by a plateau from september onwards. an increased incidence of hrv and rsv cases was seen after the influenza. a peak from september onwards, peaking in october. for the viruses with low incidence, no distinct pattern was seen. recent developments in molecular diagnostics and the discovery of new viruses have created a renewed interest in the epidemiology of (van den hoogen et al., ) . regional determination of the epidemiology of specific viral infections will improve the treatment guidelines for doctors (irmen and kelleher, ) . the main goal of the current study was to detect a broad panel of respiratory viruses associated with hospitalized children with alrtis in malaysia. we report a high prevalence of respiratory viruses in hospitalized children (allander et al., ; jartti et al., ; jennings et al., ; richard et al., ) . this was achieved using highly sensitive nested pcr which was applied to a broad spectrum of viruses. npa samples from ( . %) patients were positive for single and/or multiple viruses. single virus infections were detected in approximately two-thirds of the samples compared with almost one-third of samples which were found to contain multiple viruses. the performance of conventional diagnostic methods and molecular methods was also evaluated using the samples in this study. as reported in other studies (coiras et al., (coiras et al., , freymuth et al., ; lasala et al., ; weinberg et al., ) we established that molecular methods were more sensitive than conventional methods for the detection of respiratory viruses in children hospitalized with alrtis. many respiratory viruses including rsv-a, and b, ifv-a, piv , piv , piv , hmpv, hrv-a and c, hev, hadv, hbov, and hcov-oc were detected in the patients in this study. rsv was the most prevalent virus detected in % of samples. previous publications have frequently identified rsv as the major viral pathogen associated with lrti in children (chan et al., ; grimwood et al., ; hall et al., ; richard et al., ; zamberi et al., ) . our results provide further supporting evidence that rsv infection is a frequent cause of hospitalization among children in tropical and developing countries (weber et al., ; who, ) . hrv was the second most prevalent virus and was detected in one-third of patients which is a similar infection rate to that reported in other studies (chung et al., ; kim and hodinka, ; lau et al., ; linsuwanon et al., ; miller et al., miller et al., , papadopoulos et al., ; peltola et al., ) . rsv and hrv have also been reported as the most common causes of lrtis in other studies (calvo et al., ; franz et al., ; gruteke et al., ; jennings et al., ; papadopoulos et al., ) . the finding of the current study is also consistent with study by nathan who found rv and hrv as the most detected virus associated with alrtis in a prospective study in malaysia (nathan et al., ) . co-infections with other viruses were found in approximately % ( / ) of hrv infections. the high prevalence of hrv in this study suggests that virus testing should be routinely aq hadv was the third ( . %) most common virus detected in this study. adenoviruses are responsible for . - % of all lrtis occurring in infants and children (chen et al., ; jennings et al., ; john et al., ; lee et al., ; rocholl et al., ) . our findings seem to be consistent with the detection rate of % among hospitalized children with alrtis in argentina (videla et al., ) . this high detection rate is especially important in developing countries with high prevalence of measles and malnutrition. hmpv was detected as the fourth most prevalent virus with an infection rate of %. this is consistent with a . - % detection rate among otherwise healthy children hospitalized with lrtis in several other studies (foulongne et al., a; lee et al., ; williams et al., ; wolf et al., ) . we demonstrated for the first time that hmpv could be an important cause of lrti in children in malaysia. the positivity rate is also comparable with a recent study of children hospitalized with alrti in subtropical brazil ( . %) (oliveira et al., ). the prevalence of hbov was between . % (bastien et al., ) to % (allander et al., b in these children with respiratory infections. in our study, hbov was detected for the first time in malaysia in of nasopharyngel aspirates giving a prevalence of . % (etemadi et al., ) . the detection rate was comparable to that ( . %) reported in france (foulongne et al., b) and sweden ( . %) (allander et al., b) but was lower than that reported ( . %) in singapore (tan et al., ) . sensitive multiplex pcr assays give useful information about the presence of multiple pathogens and their epidemiological and clinical effects (bellau-pujol et al., ) . in the current study, all of the multiple infections were diagnosed exclusively using pcr, which further supports the superiority of molecular methods over conventional methods (rovida et al., ; van de pol et al., ) . evaluation of the relative importance of each coexisting agent may play an important role in understanding the etiopathogenesis of these viruses (tsolia et al., ) . identification of all infectious agents is especially important in high-risk immonocompromised patients for appropriate antiviral therapy (leland and ginocchio, ) . in this study, multiple viral infections were found in % of patients and were usually combinations of rsv with hadv and/or hrv. the co-detection rate is similar to that found by calvo ( ) (calvo et al., ) continued presence of all of the surveyed viruses during the study period with no distinct seasonality. rsv and hadv was the most commonly detected co-infection. the high proportion of hadv co-infections ( . %) with other viruses in this study is comparable with ( %) hadv co-infection rates in a study by calvo ( ) (calvo et al., ) . the second most prevalent combination was rsv and hrv ( %). this combination has been reported as the most common coinfection in other studies (chung et al., ; papadopoulos et al., ; paranhos-baccalà et al., ; richard et al., ) . the high incidence of rsv and hrv co-infection can be explained by the substantial overlapping of the monthly distribution observed for these viruses during the study period (paranhos-baccalà et al., ) . quantification of the viruses in the samples may help to better understand the etiological role of each virus (rovida et al., ) . a study of the clinical features is required to clarify the disease severity in mixed infections (paranhos-baccalà et al., ) . a wide range of respiratory viruses were detected in this study facilitated by molecular diagnostic methods. molecular methods increased the detection rate by up to % compared with conventional methods. the high detection rate of hrv confirmed its association with severe lrti and hospitalization. our study also demonstrated that hmpv and hbov can be important causes of hospitalization of malaysian children. yearly variations in the incidence of respiratory viruses may influence their association with alrtis and therefore our six months study should be considered as a snapshot of viral alrtis in pediatric inpatients. effect of rapid viral diagnosis on the management of children hospitalized with lower respiratory tract infection cloning of a human parvovirus by molecular screening of respiratory tract samples cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third human 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we are grateful to all of the doctors and nurses of the pediatric department, hospital serdang for providing nasopharyngeal aspirates for the study. the authors declare that they have no competing interests. key: cord- -o glkhyv authors: houng, huo-shu h; norwood, david; ludwig, george v; sun, wellington; lin, minta; vaughn, david w title: development and evaluation of an efficient ′-noncoding region based sars coronavirus (sars-cov) rt-pcr assay for detection of sars-cov infections date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: o glkhyv the severe acute respiratory syndrome (sars) epidemic originating from china in was caused by a previously uncharacterized coronavirus that could be identified by specific rt-pcr amplification. efforts to control future sars outbreaks depend on the accurate and early identification of sars-cov infected patients. a real-time fluorogenic rt-pcr assay based on the ′-noncoding region ( ′-ncr) of sars-cov genome was developed as a quantitative sars diagnostic tool. the ideal amplification efficiency of a sensitive sars-cov rt-pcr assay should yield an e value (pcr product concentration increase per amplification cycle) equal to . . it was demonstrated that the ′-ncr sars-cov based rt-pcr reactions could be formulated to reach excellent e values of . , or % amplification efficacy. the sars-cov cdna preparations derived from viral rna extract and the cloned recombinant plasmid both exhibit the identical amplification characteristics, i.e. amplification efficacy using the same pcr formulation developed in this study. the viral genomic copy (or genomic equivalences, ge) per infectious unit (ge/pfu) of sars-cov used in this study was also established to be approximate – : . the assay’s detection sensitivity could reach . pfu or – ge per assay. it was preliminarily demonstrated that the assay could efficiently detect sars-cov from clinical specimens of sars probable and suspected patients identified in taiwan. the ′-ncr based sars-cov assay demonstrated % diagnostic specificity testing samples of patients with acute respiratory disease from a non-sars epidemic region. severe acute respiratory syndrome (sars) is a newly recognized infectious disease with significant health and economic impacts worldwide. sars was initially identified in guangdong province, china in november and subsequently in hong kong, singapore, vietnam, taiwan, countries in europe and in north america by march and april of (cdc, mmwr a; lee et al., ; cdc, mmwr b; who, ; riley et al., ) . the sars epidemic was caused by a previously uncharacterized coronavirus, sars-cov (peiris et al., a; drosten et al., ; ksiazek et al., ) . typical coronavirus particles were readily visible under electron microscope and the specific coronavirus sequences were detected by reverse transcription polymerase chain reaction (rt-pcr) from most sars-cov infected patients (peiris et al., a; drosten et al., ; lawrence, ) . sars-cov could be isolated and cultivated from sputum specimens and respiratory secretions of sars patients using vero cell cultures before the appearance of sars-specific antibodies in serum, average days following exposure (peiris et al., b; booth et al., ) . the incubation period for sars ranges from to days, though viremia can be documented approximately - days following exposure. the challenge of early detection of sars-cov infection is the low viremia of infected patients. low viremia yields low to modest specific signal/noise ratio making distinction of the early-infected victims from non-sars febrile patients difficult. initial efforts to control sars were directed toward identification of travel-associated cases that originated from china and other sars epidemic areas, such as hong kong (riley et al., ; lipsitch et al., ; twu et al., ) . despite enormous efforts to implement extensive control measures, there were still unrecognized cases of sars that led to nosocomial clusters and subsequent spread to other health-care facilities and community settings in different regions of the world (twu et al., ; seto et al., ) . several factors might contribute to difficulties to recognize cases of sars. early symptoms of sars are nonspecific and more commonly associated with other respiratory illnesses (updated interim us case definition for severe acute respiratory syndrome (sars), cdc, december ) . patients with sars who are immuno-compromised or who have chronic conditions, e.g. diabetes mellitus or chronic renal insufficiency may remain afebrile when acutely ill or have symptoms attributable to underlying disease, delaying sars diagnosis. the reappearance of sars in toronto, canada illustrates the possibility that future outbreaks of sars might occur during the flu season in different parts of the world. thus, it would not be adequate to rely solely on the preliminary clinical symptoms, such as fever and cough to detect and differentiate sars-cov infections from other common upper respiratory infections. there are various rt-pcr tests to detect sars-cov available using different sars-cov genomic sequences (drosten et al., ; peiris et al., b) . the formats of sars-cov rt-pcr assays range from conventional agarose gel based assays to fluorogenic real-time rt-pcr assays. real-time rt-pcr may be preferred format due to its simplicity and ability to generate quantitative data. however, most of the current real-time sars-cov rt-pcr assays have not been validated to document consistent detection sensitivity and specificity to detect low sars-cov titers during early stages of illness. an expected characteristic of sars-cov similar to other rna viruses, such as flaviviruses is the high rate of genetic mutations, which leads to evolution of new viral variants (leitmeyer et al., ; mangada and igarashi, ; sanchez and ruiz, ) . it is possible that the high mutation rate of sars-cov would render any given rt-pcr assay useless if the assay's target sequence is subject to mutational changes. thus, a negative test result may not rule out sars-cov infection especially for the patients showing early symptoms of sars. in this study, we report the development of a real-time rt-pcr assay based on the highly conserved -ncr of the genome as a quantitative sars diagnostic system (marra et al., ; ruan et al., ) . it was retrospectively demonstrated that the assay could be used as an efficient diagnostic to identify sars-cov positive samples derived from sars probable, or suspected patients during taiwan's sars outbreak. sars-cov rna and cdna preparation. the sars-cov strain used in this study originated from a deceased who physician in vietnam (us cdc isolate known as strain urbani; rota et al., ) and was obtained from the us army medical research institute of infectious diseases at fort detrick, maryland. twenty throat swabs of acute respiratory disease (ard) patients from fort jackson, south carolina were obtained from collections of the walter reed army institute of research, silver spring, maryland as negative controls for the sars-cov assay (mcneill et al., ) . the viral rna was extracted and transcribed into sars-cov cdna through reverse transcription (rt) reaction using a commercial rna extraction kit (qiagen inc.) and a rt kit (pe applied biosystems inc., foster city, california). rna was mixed with rt reaction mixture containing anti-sense sars-cov rt primer ( -cattattcactgtaccctcgatcg- , pmol/reaction) according to the manufacture's instruction. the rt mixture was incubated as followings, • c, min; • c, min; • c, min. the resultant cdna was stored at − • c until tested. fluorogenic probe/primers and pcr conditions. the fluorogenic probe (fam-tttcatcgaggccacgcggag-tamra) and its flanking primer pair (sense primer, -ggacttgaaagagccaccaca- & and anti-sense primer, -cattattcactgtaccctcgatcg- ) were designed based on the conserved sars-cov -ncr of canadian tor isolate (marra et al., ; genbank accession # ) and were purchased from pe applied biosystems inc. (foster city, california). pcr master mixture contained the following ingredients: x abi pcr buffer ii, mm mgcl , . mm dntps', nm hcv-u upper primer, nm hcv-l lower primer, nm hcv-p fluorogenic taqman probe labeled with -fam reporter dye and -tamra quencher dye, . unit taq dna polymerase (amplitaq gold, abi, foster city, california). two microliter of viral cdna derived from the rt reaction was mixed with l pcr master mixture. the pcr-cdna mixture was subjected to the following amplification conditions: • c, min heat activation of amplitaq gold enzyme; cycles of • c, s; • c, s. two commercially available fluorogenic thermocyclers were used to perform the real-time rt-pcr assay developed in this report, the rotor-gene real-time pcr thermocycler (westburg bv inc., leusden, the netherlands), and the mj opticon i dna engine (mj inc., massachusetts). construction and isolation of the recombinant plasmid containing the sars-cov -ncr. the sense and anti-sense primers of sars-cov were used to generate sars-cov cdna through cycles of pcr amplification. the sars-cov -ncr pcr amplicon was then cloned into the smai site of puc vector using ta-cloning protocol (mezei and storts, ) . the ligated vector-cdna mixture was transformed into e. coli jm host. one of the recombinant plasmid clones, phcv was identified as x-gal negative after incubating overnight at • c on selective lb agar plate containing ampicillin and adequate x-gal, iptg substrates. the phcv plasmid was extracted and purified by modified alkaline lysis and cscl-gradient centrifugation from ml of overnight lb broth culture containing mg/ml ampicillin. the assay was optimized by exploring combinations of assay parameters, such as mg + concentration, flanking primers/probe ratio and concentration, annealing temperature to achieve the highest possible detection efficiency of the sars-cov template. the optimal rt and pcr assay conditions obtained were described above, and were used throughout this study. various concentrations of viral rna were used to demonstrate the quantitative nature of the assay. fig. a shows typical sigmoid plots representing formation of the sars-cov specific pcr products from samples derived from vero cell culture containing five-fold of sars-cov using the fluorogenic sars-cov -ncr based rt-pcr system. sars-cov viral stock was serially diluted (dynamic range from , to . pfu/reaction) and used to generate viral cdna to demonstrate that the assay is a copy number dependent reaction. wells a -a contained serially diluted viral copy numbers, i.e. pfu/reaction, to yield sequential sigmoid curves arranged from high through low copy number (left to right). b -b wells were non-template controls that showed no noise, or background for this assay. (b) real-time amplification and detection of the cloned sars-cov -ncr, phcv using the fluorogenic sars-cov -ncr based rt-pcr system. five-fold serial dilutions of the cloned sars-cov plasmid, phcv (from , to ge/reaction) were used to demonstrate that the assay is a copy number dependent reaction. wells a -a containing serially diluted phcv yielded sequential sigmoid curves arranged from high through low copy number (left to right of the figure). b -b wells were non-template controls that showed no noise, or background for this assay. serial dilutions of viral rna by showing accumulation in fluorescence ( r) as amplification cycle number increases. threshold cycle (c t ) for each concentration of cdna is defined as the cycle number where the application software detects the increase of product fluorescence above the calculated background fluorescence (holland et al., ) . the c t values for the described viral cdna concentrations were automatically calculated by application software as the intercepts of the fluorescence signals and the base line of background fluorescence (set as the average of all tested samples from cycle to ). parallel sigmoid plots representing serial dilutions of sars-cov cdna template are shown and displayed in sequential order. higher concentrations of template cdna resulted in lower c t values, i.e. greater initial viral cdna template copy numbers required fewer amplification cycles or a smaller c t value to reach the detectable fluorescence level. these preliminary results demonstrated that the fluorogenic rt-pcr assay was capable of detecting sars-cov viral cdna in a dose-dependent fashion over at least seven serial five-fold dilutions ( . logs dilution). in addition, the assay was also capable of discriminating specific sars-cov viral stock cdna from background levels (non-template controls showed no fluorescence signal). thus far, the sars-cov cdna copy standards (presented in plaque-forming units, pfu) used in this study were obtained from laboratory derived sars-cov stock. live sars-cov cannot be safely used in most clinical laboratories and it is also difficult to maintain consistent viral rna standards for routine usage due to the labile nature of rna molecules. therefore, a recombinant plasmid containing the -noncoding sars-cov cdna was constructed to serve as a sars-cov genomic equivalence standard. the recombinant plasmid, phcv containing the sars-cov -noncoding region was constructed and isolated as a possible sars-cov genomic reference standard (see section ). fig. b demonstrated that the phcv plasmid can be used as a sars-cov genomic copy number standard for the assay developed in this study. the c t values of serially diluted plasmid concentration reflect a copy number dependent reaction. the slope of the phcv standard curve ( . ) is identical to the slope of sars-cov viral standard curve (see fig. ). this illustrates that the phcv dna and viral cdna can be amplified by the same method with the same amplification efficacy. fig. . comparison of quantitative sars-cov standard curves derived from direct viral cdna preparation and the cloned -ncr sars-cov plasmid dna, phcv . it was shown that both standard curves yield the same slope (two parallel lines taken from fig. a and b) . the ge/pfu ratio of : could be derived from the direct readout of intercepts difference at y-(ge/pfu). the ge/pfu ratio can also be calculated from the intercepts difference at x-(c t cycle) of . cycles between viral cdna (pfu) and cloned plasmid (ge) standard curves as followings: ge/pfu = e or . . = . the ideal amplification efficiency of any given double helical cdna substrate should yield a two-fold increase in pcr product during each amplification cycle. the e value, pcr product increment per amplification cycle of any quantitative real-time pcr assay can be estimated from the slope of standard curves consisting of c t values of serially diluted target cdna; either viral cdna preparations or the cloned plasmid, phcv may be used for this purpose (shown in fig. ) . the sars-cov -ncr pcr assay consistently yielded standard curves with the slope of . producing an e value of . (− /log(slope)) or amplification efficiency of e observed /e ideal × = %. it was demonstrated that the rt-pcr assay with % amplification efficiency could be used for consistent detect ion of the sars-cov viral rna extracted from samples containing as little as . pfu per reaction with an anticipated c t value of cycles (data not shown). fig. illustrates that the assay could be used to detect viral cdna preparations as well as the cloned sars-cov -ncr at the same amplification efficacy. a linear regression model was used to determine the number of infectious units (pfu) present in a given sars-cov preparation and the results from this calculation indicates that the ratio of defective to infectious particles could be deduced from the difference of standard curves of assays using viral cdna preparations as well as the cloned sars-cov -ncr. the ge/pfu ratio of : was derived from the direct readout of intercepts difference at y-(ge/pfu). the ge/pfu ratio was calculated from the intercepts difference at x-(c t cycle) of . cycles between viral cdna (pfu) and cloned plasmid (ge) standard curves as followings: ge/pfu = e or . . = . to demonstrate the utility of the sars-specific quantitative rt-pcr assay for diagnostic purposes, the assay was used for quantitative detection of sars-cov from nasopharyngeal aspirates obtained from patients that were diagnosed as probable or suspected sars cases using clinical criteria. the assay used l of extracted aspirate wash per reaction and the assay was set to run for cycles of amplification. cdc-pc was the sars positive control rna provided by the taiwanese cdc, and blank served as the negative control. the cloned phcv template (std - ) was used as the sars-cov ge copy control. * based on ge/pfu ration of : . * * below the cutoff threshold (sars negative). table indicates that the assay developed from this study could efficiently detect sars-cov from infected patients in taiwan. sars-cov rna provided by the center for diseases control (cdc) of the ministry of health, taiwan and the cloned phcv were used as positive controls. five out of samples tested were identified as sars-cov positive ( . % positive rate for sars identification). sars-cov rt-pcr positive samples indicated the presence of low concentrations of viral nucleic acid. unfortunately, no other laboratory testing results, such as the rise of sars-specific antibodies and sars-cov viral isolation were available to corroborate the rt-pcr data in this study. we further investigated and confirmed the specificity of sars-cov rt-pcr in this study. twenty clinical samples derived from acute respiratory disease (ard) patients caused by adenovirus in us were employed as negative controls for sars-cov assay (mcneill et al., ) none of these ard samples yield any positive fluorescence signal for sars-cov (specificity = %). the sars-cov genomic sequence differs significantly from other known coronaviruses, particularly in the -and -ncr (marra et al., ; ruan et al., ) . there are more than forty -ncr sars-cov sequences deposited in the genbank database, and a blast search demonstrated % homology among various sars-cov isolates obtained from different parts of the world. it was demonstrated that the -ncr based fluorogoenic rt-pcr developed in this study could be used as an efficient diagnostic assay for laboratory grown sars-cov detection and quantification. it was preliminarily illustrated that the assay has a wide dynamic range of detection (more than . logs) with excellent linearity (linear coefficient greater than . ) using cdna templates derived from serial dilutions of viral rna through reverse transcription (rt) reaction. the data presented in fig. showed the detection range of sars-cov from , through . pfu per assay. the c t value of cycles obtained for the . pfu/assay did not represent the ultimate end point of the assay. it was later demonstrated that the end point, or lower detection limit of the assay could reach as low as . pfu per assay with the c t value approximate at cycles (data not shown). the cloned -ncr sars-cov cdna phcv was also established in this study as a molecular copy standard, or genomic equivalent standard for the sars-cov assay. it was demonstrated in this study that the cloned cdna phcv could mimic the viral cdna derived from sars-cov rna preparations as a copy number standard for the assay. fig. showed the minimal detectable phcv copy number of two ge per assay with c t value greater than cycles. however, the population size of cloned sars-cov standard at two copies per reaction was too small to be represented as a "normal distribution" (gaussian distribution). thus, multiple repeats of the same assay using such a low substrate copy number, two copies per assay would yield the result of "hit or miss" phenomenon. in order to obtain more reproducible results of the assay, the cutoff, or end point of phcv for the assay was raised to - ge per assay with the c t value at cycles. based on the calculated sars-cov ge/pfu ratio from fig. , and the actual experimental data using serial dilutions of viral cdna preparations and cloned phcv , the lower detection limit of sars-cov assay could be conservatively defined as . pfu or - ge per assay. the traditional expression for viral titer, plaque-forming unit (pfu) per ml must be determined through a conventional viral plaquing assay. this culturing method identifies viable viruses that can generate cytopathic effects, or plaques, on the confluent lawn of susceptible host cells, such as vero cells (drosten et al., ) . the viral plaquing assay can only be used to score the total viable viruses that can infect the host cells. thus, it cannot be used to distinguish, or detect any viral contaminants that are capable of infecting the host cells used for the plaquing assay (porterfield, ; de madrid and porterfield, ) . in contrast, the srar-cov rt-pcr assay developed in this study is specific for the sars-cov detection. however, the sars-cov pcr assay developed from this study cannot be used to differentiate among living versus dead viral particles (freeman et al., ; bae et al., ; mackay et al., ) . any viral particle or genomic segment containing intact pcr target sequence, such as the -ncr sars-cov would be detected as one genomic equivalence (ge). the difference between pfu (viable viruses) and ge (total viral counts including defective and functional viruses) of sars-cov can be mathematically expressed as ge/pfu ratio (freeman et al., ; bae et al., ) . the ge/pfu ratio of approximate - : obtained in this study only represents the ge/pfu ratio of the vero cell derived sars-cov stock used in this study. the finding of high ge/pfu ratio from this study supported the hypothesis of sars-cov cultures and sars-cov infected clinical samples containing both infectious and defective (or subgenomic) viral rna, i.e. each sars-cov infectious unit is represented by multiple viral rna species. it is likely that the -noncoding based sars-cov rt-pcr assay could detect both infectious and defective viral rna from clinical samples (poon et al., ) . it is possible that viral stocks of different preparations might contain the same pfu per ml, but the total viral particles, or rna copy numbers would be different (bae et al., ; peccoud and jacob, ) . the pfu, infectious activity of any given viral stock is greatly affected by the percentage of viable virus. there are numerous factors that will determine, or affect the viability of each individual viral stock, such as culturing conditions (nutrients, ionic strength, incubation time and temperature), storage conditions (duration and temperature), and viral harvesting conditions. thus, it is difficult to obtain a universal reference viral stock containing a precisely standardized viral copy number with persistent viability data. it was demonstrated in this study that the cloned phcv plasmid could be used to replace viral cdna as a stable and rational sars-cov copy number standard for the sars-cov rt-pcr assay. the practical application of the sars-cov rt-pcr developed in this study is to accurately identify sars-cov in suspected sars patients. the key to the successful control of sars outbreaks is to identify sars-cov infected individuals in the early stages of infections. this will allow public health officers to apply adequate physical quarantine measures before the sars infected individuals become highly contagious. this requires the deployment of highly sensitive sars-cov diagnostics to detect low sars-cov titers. in this study, the assay was used to test a limited numbers of total rna extracts derived from sars suspected, or probable patients in taiwan. the tested results indicated that it is feasible to use the developed assay to identify the sars-cov infected patients with viremia loads ranging from . pfu through . pfu/ml. even though only a limited numbers of samples were tested in this study, the assay was confirmed to be specific, i.e. none of non-sars ard patients' samples tested positive using the sars-cov assay. even though we did not have specific information on the onset dates of disease for those clinical samples tested in this study. we demonstrated that the -noncoding based rt-pcr developed in this study could be used to detect both infectious and defective viral rna. thus, the assay should have fairly high diagnostic sensitivity of detecting sars-cov during the early stage of infections . it was previously reported that the mean time between onset of symptoms and sample collection ranged from . to . days for sars-cov detection directly from nasopharyngeal aspirate using real-time rt-pcr (peiris et al., a,b; tsang et al., ) . it was proposed to further verify and validate the assay developed in this study by using careful selection panel of reference samples consisting of wide dynamic range of naturally infected sars samples, i.e. from to pfu/ml as well as sars negative control samples. however, it is very difficult to obtain credible sars reference samples with known infectious titers since there is no known major outbreak of sars after the last multi-country outbreak in . the conserved -ncr feature of other rna viruses, such as dengue viruses had been reported and utilized to develop type-specific rt-pcr assays for dengue virus identification and quantification throughout the world (sudiro et al., (sudiro et al., , houng et al., houng et al., , . thus, it would be reasonable to predict that the -ncr based sars-cov specific rt-pcr assay can be used to detect different sras-cov originated from outbreaks of various geographic origins. it was demonstrated that the sars-cov -ncr based assay using canadian sars-cov tor sequence could be used to detect laboratory grown sars-cov urbani strain as well as various sars-cov infected samples in taiwan. based on the assay's excellent performance capacity (high detection sensitivity at . pfu/assay and robustness in detecting sars-cov of different origins and preparations including direct viral cdna as well as cloned recombinant plasmid) and superior specifications ( % specificity and % amplification efficacy) shown in this report, the -ncr based sars-cov rt-pcr should be able to serve as an efficient sars-cov diagnostics in the event of future outbreak. detection of yellow fever virus: a comparison of quantitative real-time pcr and plaque assay clinical features and short-term outcomes of patients with sars in the greater toronto area cdc update: outbreak of severe acute respiratory syndrome-worldwide severe acute respiratory syndrome-singapore a simple micro-culture method for the study of group b arboviruses identification of a novel coronavirus in patients with severe acute respiratory syndrome quantitative rt-pcr: pitfalls and potential detection of specific polymerase chain reaction product by utilizing the - exonuclease activity of thermus aquaticus dna polymerase quantitative detection of dengue virus using fluorogenic rt-pcr based on -noncoding sequence development of a fluorogenic rt-pcr system for quantitative identification of dengue virus serotypes - using conserved and serotype-specific -noncoding sequences the sars working group coronavirus confirmed as cause of sars a major outbreak of severe acute respiratory syndrome in hong kong dengue virus structural differences that correlate with pathogenesis transmission dynamics and control of severe acute respiratory syndrome real-time pcr in virology molecular and in vitro analysis of eight dengue type viruses isolated from patients exhibiting different disease severities the genome sequence of the sars-associated coronavirus epidemic spread of adenovirus type -associated acute respiratory disease between us army installations theoretical uncertainty of measurements using quantitative polymerase chain reaction coronavirus as a possible cause of severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study detection of sars coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-pcr assays early diagnosis of sars coronavirus infection by real-time rt-pcr a plaque technique for the titration of yellow fever virus and antisera transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection a single nucleotide change in the e protein gene of dengue virus mexican strain affects neurovirulence in mice effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) rapid diagnosis of dengue viremis by reverse transcriptase-polymerase chain reaction using -noncoding region universal primers microplate-reverse hybridization method to determine dengue virus serotype coronavirus-positive nasopharyngeal aspirate as predicator for severe acute respiratory syndrome mortality control measures for severe acute respiratory syndrome (sars) in taiwan world health organization. severe acute respiratory syndrome (sars): multi-country outbreak-update key: cord- -k uvqcmp authors: xia, hongyan; liu, lihong; nordengrahn, ann; kiss, istván; merza, malik; eriksson, ronnie; blomberg, jonas; belák, sándor title: a microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k uvqcmp this study describes a novel blocking microsphere-based immunoassay for highly sensitive and specific detection of antibodies against bovine viral diarrhoea virus (bvdv). the intra- and inter-assay variability are . % and less than %, respectively, and variability of bead conjugations is less than . %. the diagnostic performance of the assay was evaluated by testing a total of serum samples. based on a negative/positive cut-off value of . %, the assay has a sensitivity of . % and a specificity of . % relative to elisa. the new microsphere immunoassay provides an alternative to conventional elisa systems and can be used for high-throughput screening in the bvd control programmes. the genus pestivirus of the family flaviviridae consists of four approved species: bovine viral diarrhoea virus (bvdv- ), bovine viral diarrhoea virus (bvdv- ), classical swine fever virus (csfv), border disease virus (bdv); and a tentative species pestivirus of giraffe (thiel et al., ) . bvdv infections are usually very mild or inapparent clinically (baker, ) , but severe hemorrhagic syndrome is also observed in acute infection (perdrizet et al., ; corapi et al., ; ridpath et al., ) . diaplacental infection can lead to birth of persistently infected animals that serve as a reservoir for further spreading of the virus (sandvik, ) . bvd is considered as one of the major diseases with a worldwide economic impact in the cattle industry. for example, the cost of bvdv infection has been estimated about $ per cow in dairy herd in new zealand (reichel et al., ) , and may exceed $ per cow when infected with a high virulent strain or co-infected with other pathogens (pritchard et al., ; carman et al., ; houe, ) . sweden is one of the first countries to implement national bvdveradication programme since (lindberg and alenius, ) , and now the country is almost free of bvdv (ståhl et al., ; lindberg et al., ) . serological tests, including virus neutralization and elisa, have been used widely for detection of bvdv antibodies. the recent advance in microsphere-based flow cytometric technology has provided the possibility to develop multiplex diagnostic assays on a single platform. these assays can be designed more sensitive than conventional immunoassays due to the uses of small beads ( m) that leads to better reaction kinetics approaching liquid-phase conditions, and of chromophore phycoerythrin, an exceptionally bright reporter dye (krishhan et al., ) . the microspheres used in luminex xmap technology (luminex corp., austin, tx) are coded with unique combinations of fluorescent dyes, and can be immobilized with capture molecules, e.g. antibody. immunoassays can be developed in a similar way as elisa, but signals are detected and processed by luminex analyzer. by using xmap technology, a range of diagnostic assays has been developed in the recent years for the improved serological detection of viruses, e.g. respiratory syncytial virus that causes maladies (jones et al., ) , human immunodeficiency virus (faucher et al., ) , human papillomaviruses (dias et al., ) , equine arteritis virus (go et al., ) , and avian influenza virus (watson et al., ) . the objectives of this study were to develop a blocking microsphere-based immunoassay (bmia) for detection of antibodies against bvdv, and to compare the performance of the assay with a commercial elisa kit. a total of serum samples were evaluated in this study. these included clinical samples from sweden, where only bvdv- is present; and samples (including sera against bvdv- ) from svanova biotech ab, uppsala, sweden. bovine turbinate (tb) cells were maintained in f-dmem with % foetal calf serum, % l-glutamine, and % antibiotics. cells at % confluence were infected with oregon c v (kindly provided by prof. martin beer, institute of diagnostic virology, friedrich-loeffler-institut (fli), greifswald-insel riems, germany). at h post-infection, cultures were frozen at − • c. after thawing, . % nonidet p- (np- ) was added to the cell lysate and incubated at • c for h. following centrifugation at × g for min, the supernatant was taken as viral antigen. microplex microspheres were purchased from luminex corp (austin, tx). the coupling reaction was performed according to the manufacturer's instructions. briefly, million microspheres were resuspended in l of activation buffer ( mm monobasic sodium phosphate, ph . ). the microspheres were activated by l of mg/ml of n-hydroxysulfosuccinate (sulfo-nhs, pierce, rockford, il), followed by l of mg/ml -ethyl- - -dimethylaminopropyl carbodiimide (edc, pierce, rockford, il). after incubation at room temperature on an end-over-end rotator for min, the microspheres were washed twice with l of mm morpholineethanesulfonic acid (mes, ph . ) and resuspended in l of mm mes. two mabs wb and wb , which were described previously in blocking elisa (paton et al., ; kramps et al., ) , were added to the activated microspheres, respectively. after a -h incubation, the microspheres were washed twice by pbs-tbn (pbs, . % bsa, . % tween- , . % azide, ph . ) and resuspended in l of the buffer pbs-tbn and stored at • c in dark. the coupling reaction was confirmed by measuring fluorescent intensity on a luminex analyzer after incubation of microspheres with twofold serial dilutions ( . - g/ml) of r-phycoerythrin conjugated anti-mouse igg (sigma-aldrich co, st. louis, mo) for min. biotin labelling reaction was performed according to the manufacturer's instruction (pierce, rockford, il). briefly, g of wb or wb was incubated on ice with l of mm sulfo-nhs-lc-biotin for h. after removal of excess biotin using a desalting column, the biotinylated mabs were stored at − • c until use. the wb -conjugated microspheres ( microspheres/l), viral antigen, and diluted serum samples were mixed in equal volume ( l) and incubated for min on a plate shaker in dark. after addition of l of the biotinylated mab wb and incubation for another min, l of g/ml of r-phycoerythrin-conjugated streptavidin (prozyme, san leandro, ca) were added and followed by a further incubation for min. each sample was tested in duplicate if not indicated specifically. fluorescence intensity of each reaction was measure on the luminex analyzer, and median fluorescence intensity (mfi) was calculated based on the measurement of beads per sample. the results were expressed as inhibition percentage and calculated as the following: × (mfi negative control − mfi sample )/mfi negative control . the reproducibility of the assay was assessed by determining the level of both intra-assay and inter-assay variations. intra-assay variation within a plate was calculated as the mean percentage of coefficient of variation (cv%) for samples determined in triplicate. inter-assay variation was assessed by testing a panel of samples ( positive sera and negative sera) in three separate runs. elisa was performed with a p blocking elisa kit (svanova biotech ab, uppsala, sweden), according to the manufacturer's instruction. the kit had a sensitivity of . % and specificity of . % compared with virus neutralization test (svanova biotech ab, uppsala, sweden). receiver operating characteristics (roc) curve was generated to assess the diagnostic performance of the bmia. an roc curve is a plot of a test's true positive fractions (tpf) versus falsepositive fractions (fpf) for each possible cut-off value of the test and could achieve the best relationship between diagnostic sensitivity and specificity (detilleux et al., ; greiner and gardner, ) . sensitivity and specificity were calculated as the following formula: sensitivity = [true positives/(true positives + false negatives)] × ; specificity = true negatives/(true negatives + false positives) × . the analysis was performed by software medcalc (medcalc software, mariakerke, belgium). the coupling efficiency of each mab to microspheres was compared at different amount of mabs in coupling reaction with dilutions of a phycoerythrin-labelled anti-mouse igg (pe-igg). as shown in fig. , both wb and wb had a very low mfi value when g of mab was used in coupling reaction. at g/ml of pe-igg, g of wb gave an mfi value of , whereas wb gave an mfi value of . this indicated that, for the same amount of mab, wb had a higher coupling efficiency than wb . further increasing wb from g to g had less effect on the mfi values. therefore, wb ( g) was selected finally as the capture antibody for coupling to microspheres, and wb was used as detection antibody in this study. two key factors in the bmia were determined in a sandwich immunoassay: optimal serum dilution factor and the amount of detection antibody. a twofold dilution of serum sample gave the maximum difference in mfi values between positive and negative sera (fig. ) . titration of biotinylated detection antibody (wb bio) showed that the mfi values reached the plateau at about g/ml of wb -bio (fig. ) . to maximize the sensitivity of the assay, g/ml of wb were used in the assay, which corresponds to % of the maximum plateau mfi value. sensitivity and specificity of the bmia were compared with a commercial blocking elisa. a total of samples were tested in parallel by the two assays, and the results are presented in table . based on the nonparametric roc analysis of all samples, the cut-off value (percentage of inhibition) of the bmia was determined as . %. under this cut-off value, the bmia classified samples as positive (including bvdv- serum samples) and samples as negative (table ). comparing to blocking elisa, the bmia had a sensitivity of . % and a specificity of . %. fig. . selection of capture antibody according to its coupling efficiency. two mabs wb and wb at indicated amounts were coupled to million microspheres in a -l reaction volume, respectively. the median florescence intensity (mfi) was determined at various dilutions of anti-mouse igg-pe conjugate. the reproducibility of the bmia was evaluated by intra-and inter-assay variability, and variations in different preparations of conjugated microspheres. the mean cv% of samples tested in triplicate in one plate was . %. inter-assay variation was determined by testing samples in three separate runs. the mean cv% was below %. the reproducibility of the different preparations of conjugated microspheres was also investigated. the percentage of coefficient of variation (%cv) within three preparations of beads was less than . %. this study describes the development and evaluation of a microsphere-based immunoassay for rapid and sensitive detection of bovine viral diarrhoea virus antibodies. the diagnostic performance of the new bmia was compared to that of a commercial blocking elisa system, by testing a large panel of bovine sera. in general, the two assays worked consistently for detection of bvdv antibodies in samples. six samples were positive in bmia but negative in elisa. these samples had a percentage of inhibition value between % and % in blocking elisa. one reason for this discrepancy could be that these sera blocked binding of the viral antigen to the detection antibody in the bmia, leading to false-positive results. but more likely it was due to a slight higher sensitivity of the mia than elisa. only one sample was negative by the bmia but positive by elisa with a percentage of inhibition value of . % (just above the cut-off value of %). the observed discrepancies are in line with another comparison of elisa with a luminex assay for detection of human papillomavirus : six out of samples reacted differently in the two assays (faust et al., in press) . the bmia utilises two mabs against highly conserved ns protein of bvdv. as demonstrated, the assay is capable of detection antibodies against both bvdv- and bvdv- . it would be very interesting to detect antibodies, when available, against novel pestiviruses th/ khonkaen (liu et al., a) and sva/cont- (liu et al., b) , which have been proposed as a new species bvdv- (liu et al., c) . another direction would be to extend the assay for detection of antibodies against other viruses, e.g. bovine herpesvirus type , bovine respiratory syncytial virus, bovine coronavirus, bovine parainfluenza- virus, thus allowing a robust diagnosis of bovine respiratory diseases in a multiplex format. the power of a luminex assay lies in its multiplex capacity. khan et al. ( ) reported a multiplex microbead immunoassay for simultaneous detection of antibodies against six nonhuman-primate viruses. multiplexing can bring down the high cost of the bmia dramatically by reducing the number of elisa kit for each pathogen, labour input and hands-on time. this will, in turn increase the applicability of the assay. in summary, the bmia described above is an alternative to elisa for detection of antibodies against bvdv. the assay is reliable and sensitive, providing a powerful tool for the bvd surveillance programmes. in addition, this assay can be extended to a multiplex format allowing a robust, high-throughput diagnosis of bovine respiratory diseases. bovine viral diarrhea virus: a review severe acute bovine viral diarrhea in ontario severe thrombocytopenia in young calves experimentally infected with noncytopathic bovine viral diarrhea virus methods for estimating areas under receiver-operating characteristic curves: illustration with somatic-cell scores in 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bovine virus diarrhoea virus (bvdv) specific antibodies in cattle serum, plasma and bulk milk multiplexed microbead immunoassays by flow cytometry for molecular profiling: basic concepts and proteomics applications the control of bovine viral diarrhoea virus in europe: today and in the future principles for eradication of bovine viral diarrhoea virus (bvdv) infections in cattle populations virus recovery and full-length sequence analysis of the atypical bovine pestivirus th/ khonkaen maximum likelihood and bayesian analyses of a combined nucleotide sequence dataset for genetic characterization of a novel pestivirus sva/cont- phylogeny, classification and evolutionary insights into pestiviruses an elisa detecting antibody to conserved pestivirus epitopes bovine virus diarrheaclinical syndromes in dairy herds severe disease in a dairy herd associated with acute infection with bovine virus diarrhoea virus, leptospira harjo and coxiella burnetii does control of bovine viral diarrhoea infection make economic sense? multiple outbreaks of severe acute bvdv in north america occurring between and linked to the same bvdv strain selection and use of laboratory diagnostic assays in bvd control programmes molecular epidemiology of bovine viral diarrhoea during the final phase of the swedish bvd-eradication programme family flaviviridae a multiplexed immunoassay for detection of antibodies against avian influenza virus we thank ms. pia fällgren, the national veterinary institute (sva), uppsala, sweden for providing the serum samples. we are grateful to dr. siamak zohari (sva) for his invaluable comments. the work was supported by the award of excellence (excellensbidrag) provided to sb by the swedish university of agricultural sciences (slu). key: cord- -shrd s r authors: qin, zhao-ling; zhao, ping; cao, ming-mei; qi, zhong-tian title: sirnas targeting terminal sequences of the sars-associated coronavirus membrane gene inhibit m protein expression through degradation of m mrna date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: shrd s r sars-associated coronavirus (scov) m protein plays a key role in viral assembly and budding. recent studies revealed that m protein could interact with n protein in the golgi complex. in this study, we showed that scov m protein co-localized in the golgi apparatus with a golgi vector marker. to study m protein function, three candidate small interfering rnas (sirnas) corresponding to m gene sequences were designed, transcribed in vitro, and then tested for their ability to silence m protein expression. the plasmid, pegfp-m, encoding scov m protein as a fusion protein with egfp, was used for silencing and for reporter gene detection in hek t cells transfected with sirna constructs. the results showed that the mean green fluorescence intensity and m rna transcripts were significantly reduced, and that the expression of m glycoprotein was strongly inhibited in those cells co-transfected with m-specific sirnas. these findings demonstrated that the three m-specific sirnas were able to specifically and effectively inhibit m glycoprotein expression in cultured cells by blocking the accumulation of mrna, which provides an approach for studies on the functions of m protein and for the development of novel prophylactic or therapeutic agents for scov infection. severe acute respiratory syndrome-associated coronavirus (scov), a pathogenic agent of human coronaviruses, was identified in april . scov, a group four coronavirus, is enveloped, with a single-stranded positive-sense rna genome of about , nucleotides in length. based on the sequenced complete scov genomes, scov encodes at least five major structural proteins: spike (s), envelope (e), membrane (m), nucleocapsid (n) fouchier et al., ; rota et al., ) , and the newly identified orf a (shen et al., ) , which are common to all known coronaviruses. scov can cause severe acute respiratory distress syndrome with a diffuse alveolar damage (dad) at autopsy kuiken et al., ; nicholls et al., ) . at present, there is no vaccine or specific effective antiviral method available to treat this disease effectively. * corresponding author. tel.: + ; fax: + . e-mail address: qizt@smmu.edu.cn (z.-t. qi). rna interference (rnai) is a cellular process in which double-stranded rna (dsrna) molecules can silence targeted genes through sequence-specific cleavage of the corresponding rna transcript. transfection of small interfering rnas (sir-nas) into mammalian cells leads to the degradation of the target gene mrna or to the arrest of protein translation silence of the protein expression (fire et al., ; elbashir et al., ) . therefore, sirna-mediated rnai may provide a useful approach to study host cell-virus interactions and to serve as possible therapeutics in virus infection. sirnas have been employed as therapeutic molecules in human diseases including cancer, neurogenerative diseases and viral infectious diseases (shi, ; dy kxhoorn et al., ) . scov, as a rna virus, may also be an ideal target for the study of its biology and therapeutics by rnai method. to date, there are a number of published papers showing that sirna/shrna (short hairpin rna) target different regions or genes in the scov genome by various sirna/shrna generating strategies. rnaase iii specific sirna targeting the rna dependant rna polymerase (rdrp), or the s and n genes, could induce specific degradation of scov mrnas in human cells (zhu et al., ) . synthesized shrna targeting the n protein (tao et al., ) and rdrp (lu et al., ) , could inhibit the expression of target protein, and the latter significantly reducing the plaque forming ability of scov in vero-e cells. sirna targeting the e, m and n proteins , leader sequence, trs, -utr or the s gene qin et al., ; zhang et al., ) could effectively inhibit the expression of these targets. plasmid-derived sirna targeting to rdrp (meng et al., ; wang et al., ) , the leader sequence (b.j. t. li et al., ) , and to the non-structural protein (nsp ) could specifically inhibit the expression of target protein and also suppress the replication and propagation of scov in cultured vero e cell lines (ni et al., ) . the effect of sirnas/shrnas probably resulted in global reduction of subgenome synthesis and subsequent protein expression of scov. therefore, to screen more valid sirnas targets in scov genome will be important to provide more information for the development of better scov prophylaxis and therapy. among the five scov structural genes, m gene encodes a glycoprotein amino acids in length, which contains a short amino-terminal ectodomain (residues - ), three transmembrane helices (residues - , - and - ) and a -amino acid carboxy-terminal endodomain (voss et al., ; oostra et al., ) . m protein is the most abundant viral membrane glycoprotein and a key protein in viral assembly and budding through its interaction with n or s proteins (kuo and masters, ; he et al., ) . since scov m protein plays a key role in the viral life cycle, experiments were designed to test whether specific sirnas could inhibit the expression of m protein, which may hold promise for the development of scov gene-specific therapeutics. in this paper, the subcellular distribution of m protein was established in mammalian cells. sirnas transcribed in vitro were then introduced into the cells expressing the m protein. the results showed that the m protein is mainly located in the golgi apparatus, and that the specific sirnas corresponding to scov m gene specifically degraded m mrna, significantly inhibiting m protein expression. the base pair fragment of scov m gene was synthesized according to the published sequence (genbank accession no. ay ). the membrane protein coding region of this fragment spans from nt to nt in the full length scov genome. this fragment was used as the template to amplify the dna fragment encoding amino acid residues - of scov m protein by polymerase chain reaction (pcr). the forward primer was -gaattcgccaccatggcagacaacggtac- and the reverse primer was -ggatccatctgtactagcaaag-caatattgt- (the underlined sequences were ecori and bamhi sites, respectively). forward and reverse primers (final concentration . m) and the primestar tm hs dna polymerase (final concentration . u/l, takara) were used. the pcr conditions were denaturation ( • c, s), annealing ( • c, s) and extension ( • c, s) for cycles. then the ecori-bamhi fragment was inserted into the corresponding multi-cloning site of a eukaryotic expression vector pegfp-n (clonetech). the resulting plasmid was named pegfp-m, in which the enhanced green fluorescence protein (egfp) gene was located downstream of the m gene. identity of the m gene to the published sequence was confirmed by dna sequencing (bioasia co., shanghai). plasmid golgi/pdsred-n , which specifically locates to the golgi apparatus in mammalian host cells, was kindly provided by prof. yuwen cong (department of pathophysiology, beijing institute of radiation medicine). the sirnas corresponding to scov membrane gene were designed according to ambion's sirna guidelines. a scramble sirna sequence (wilson et al., ) and egfp sirna were used as negative and positive controls for silencing, respectively. all sequences of the sirnas were blast searched in the national center for biotechnology table sequences of template deoxynucleotides for sirnas used for target genes information's (ncbi), and were not found to have significant homology to genes other than the targets. the dna template (table ) used for sirna transcription was synthesized by bioasia co. the oligonucleotide-directed production of sirnas with t rna polymerase has been described previously (qin et al., ) . for each transcription reaction, m of each oligonucleotide template and t promoter primer were mixed and denatured by heating at • c for min. the following was then added to the mixture: × klenow reaction buffer, dntp mix (promega, usa), exo-klenow (takara, dalian) and nuclease-free water. the reaction was incubated at • c for min. the in vitro transcription was performed in l of transcription mix: l hybridization solution, l × t reaction buffer, l rntp mix, l t rna polymerase and l nuclease-free water. after incubation at • c for h, sense and antisense rnas generated in separate reactions were annealed by mixing both transcription reactions and incubating at • c overnight. the concentration of the generated dsrna was measured by the absorbance at nm in a biophotometer (eppendorf, germany). s nuclease and rnase-free dnase i (takara) were added to final concentrations of and u/g of sirna, respectively, for the digestion of ssrna and dsdna. sirnas were assessed by rna gel electrophoresis on % agarose. one volume of te-saturated (ph . ) phenol:chloroform:isoamyl alcohol ( : : ) was added to the in vitro transcribed sirna, and the sample then spun at , rpm in a microcentrifuge for min. the upper aqueous phase was collected in a fresh tube and mixed with one volume of chloroform:isoamyl alcohol ( : ) and centrifuged at , rpm for min. the upper, aqueous phase was then mixed with two volumes of ethanol and . volumes of mmol/l sodium acetate (ph . ), and the sample placed on ice for min, followed by centrifugation at , rpm for min. the pellet was washed with ml of % ethanol, and suspended in nuclease-free water for further use. the purity and integrity of sirnas were checked by agarose gel electrophoresis. human embryonic kidney (hek) t cells were grown at • c in dulbecco's modified eagle's medium (dmem) (sigma) containing % heat-inactivated fetal bovine serum (gibco brl, usa) supplemented with l-glutamine ( mm), streptomycin ( g/ml) and penicillin ( u/ml). twentyfour hours prior to transfection, the cells were seeded into -well plates at a density of . - × cells per well in fresh medium ( l/well) without antibiotics. for the transfection of adherent hek t cells, a total of . g of plasmid dna (pegfp-m, golgi/pdsred-n ) and/or nm of sirna mixed with lipofectamine tm (invitrogen, ca, usa) were used according to the manufacturer's instructions. the cells were incubated at • c for various times to optimize gene transcription and expression. the expression of m-egfp and golgi-red fusion proteins were observed directly under an inverted fluorescence microscope. expression of m-egfp (green) and golgi-red (red) in transfected cells was examined with a fluorescence microscope at and h after co-transfection. at h post-transfection, cells on glass cover slips were rinsed with phosophate-buffered saline (pbs), fixed with % paraformaldehyde for - min at • c, and then examined for m protein by confocal microcroscopy. then the cells were washed with pbs three times and stored in pbs at • c. images were viewed and collected by confocal fluorescence microscopy (leica microsystems heidelberg gmbh). expression of m-egfp in transfected cells was examined with a fluorescence microscope (olympus ck , japan) at , , and h after transfection. for flow cytometric analysis of m-egfp expression, the cells were harvested at h post-transfection and digested with . % trypsin, washed twice with pbs, and then resuspended in pbs to measure the fluorescence using a becton dickinson facscan flow cytometer with filters (emission, nm; excitation, nm). samples (about cells each) were counted and analyzed with cellquest software, using non-transfected hek t cells as control. the values were calculated as the percentage of the cell population that exceeded the fluorescence intensity of the control cells and the mean fluorescence intensity of this population. total rna from the test and control cells was extracted at h post-transfection using rnaex reagent (watson, shanghai) and digested with rnase-free dnase i (takara). one microgram of the rna was then reverse transcribed into cdna with oligo(dt) and the avian myeloblastosis virus (amv) reverse transcriptase xl (takara) according to manufacturer's recommendations. reactions without reverse transcriptase were performed in parallel and yielded no pcr products. the primers (forward primer, -ttggtgctgtgatcattcgt- ; reverse primer, -aaagcgttcgtgatgtagcc- ) for scov m gene were used for the semi-quantitative rt-pcr reaction as follows: denaturation ( • c, s), annealing ( • c, s) and extension ( • c, min) for cycles. glyceraldehyde- -phosphate dehydrogenase (gapdh) was used as an internal control. gapdh primers (forward primer, -tgggctacactgagcaccag- ; reverse primer, -aagtggtcgttgagggcaat- ) were synthesized based on the human gapdh mrna sequence (genbank accession no. bc ). the reaction conditions were as follows: denaturation ( • c, s), annealing ( • c, s) and extension ( • c, s) for cycles. then real-time quantitative pcr (lightcycler, roche) was performed as described (rajeevan et al., ) using sybr ® premix ex taq tm (takara). briefly, reactions were carried out in l volumes containing l of reverse transcription product. to quantitate scov m gene transcript levels, cdna from the hek t cells transfected with pegfp-m were diluted in a range from to . for each dilution, amplifications were performed using primers for m and gapdh genes so that the m mrna levels could be properly normalized. each cdna sample from the transfected hek t cells was run in parallel with the appropriately diluted sample. to perform analysis of relative expression levels of scov m gene using real-time pcr, the − ct method was used (livak and schmittgen, ) . the changes of scov m gene expression in sirna co-transfected cells, normalized to gapdh and relative to its expression in mock-transfected cells, were calculated for each sample. the primers for scov m and gapdh were the same as those described above. cells were harvested and lysed with % sds. equal amounts of total proteins were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and electrophoretically transferred to nitrocellulose membrane following the protocol suggested by the manufacturer (bio-rad, ca, usa). after blocking non-specific binding sites with % non-fat milk, the membrane was incubated with primary antibodies at overnight. the primary antibodies used were: anti-gfp mouse monoclonal antibody (santacruz, : dilution) and anti-gapdh mouse monoclonal antibody (kangchen, shanghai, : dilution). after washing, the blot was incubated with alkaline phosphatase-conjugated goat anti-mouse igg. immunoreactive bands were visualized with -bromo- chioro- -indolylphosphate/nitroblue tetrazolium (bcip/nbt) substrate (sino-american, shanghai). scov m gene was cloned into pegfp-n with a c-terminal egfp tag that could be used to monitor protein expression. at h post-transfection, the fluorescence was mainly in the cytoplasm of hek t cells, where it was condensed into discrete loci and spots (fig. ) , suggesting that m-egfp fusion protein was located in a particular cellular compartment. published data showed that m proteins of other coronaviruses were detectable in the golgi complex of mammalian cells. at h posttransfection, cells were analyzed by confocal microscopy, which showed that the m-egfp fusion protein was co-localized in the golgi apparatus with the golgi vector marker-golgi/pdsred-n (fig. ) . these results suggested that the golgi distribution of scov m protein was conformed, which was also consistent with the previous study (nal et al., ) . to silence the expression of scov m glycoprotein using rnai technology, specific sirna was made by in vitro tran- scription. the sense and antisense sirna templates were separately transcribed in vitro with t rna polymerase and annealed to form double-strand sirna as described in section . the overhanging leader sequence of the generated dsrna and dna template were digested with single-strand specific nuclease (s nuclease) and rnase-free dnase i, respectively. the double stranded sirnas of nt in length were obtained and found to be intact (fig. ) . semi-quantitative rt-pcr was performed to examine rna levels of scov m gene in sirna co-transfected cells. the data showed that egfp-sirna, m-sirna , m-sirna and m-sirna reduced the accumulation of m mrna (fig. a, upper panel, lanes , , and compared to lane ), while a control sirna with scrambled sequence had no effect on m mrna levels (fig. a , upper panel, lane compared to lane ) while gapdh mrna was not affected by the five sirnas (fig. a, bottom panel) . to more accurately quantify rna levels of the scov m gene, real-time quantitative pcr was performed using primers specific to the m gene and to gapdh. cdna from the hek t cells transfected with pegfp-m was diluted from to . for each diluted sample, amplifications were performed using primers for the m and gapdh genes so that the efficiencies of the target and reference genes were similar. each sample cdna from the transfected hek t cells was run in parallel with the diluted sample. the change in scov m gene expression in sirna co-transfected cells, normalized to gapdh and relative to m gene expression in mock-transfected cells, was calculated for each sample using − ct method. the results showed that scov m gene mrna levels were decreased about -, -and -fold in cells transfected with scov m-sirna , m-sirna and m-sirna , respectively, by h post-transfection. membrane gene transcript levels were decreased about -fold in cells transfected with egfp-sirna but not significantly changed in cells transfected with control sirna (fig. b) . to determine whether sirna could effectively silence scov m glycoprotein expression in cultured cells, pegfp-m and the various sirnas were co-transfected into hek t cells. the cells were examined microscopically at h post-transfection for green fluorescence. as shown in fig. , the fluorescence image was much stronger in the hek t cells transfected with control sirna compared to cells transfected with scov m-sirna , m-sirna and m-sirna ( fig. d-f, upper panel) . faint green fluorescence was observed in egfp-sirna transfected cells (upper panel b in fig. ) . the fluorescence intensity of hek t cells co-transfected with control sirna showed no significant difference from pegfp-m transfected cells (upper panels a and c in fig. ) . the bottom panels represent the corresponding image observed by light microscopy. specific silencing of the green fluorescence was confirmed by at least in three independent experiments. to further examine the silencing effect of scov m-sirnas, cells were collected and analyzed by fluorescence-activated cell sorting (facs) h after transfection. sorting was conducted (using cellquest software) to select cells that expressed egfp, using non-transfected hek t cells as a control. as shown in fig. , compared to the cells transfected with plasmid pegfp-m alone, the cells cotransfected with pegfp-m and control sirna gave no significant reduction of egfp expression, whereas the egfp-sirna gave an about . -and . -fold reduction in the percentage of fluorescent cell population and mean fluorescence intensity, respectively. percentage of fluo- protein expression by m-sirna was better than m-sirna or m-sirna . this was consistent with the data shown in figs. and . gapdh was not affected by any of the five sirnas tested (fig. , bottom panel) . these data indicate that sirnas silenced scov m glycoprotein expression by blocking the accumulation of m mrna. individuals with sars usually develop a high fever followed by severe clinical symptoms. as it is a newly emerging disease, a safe and effective vaccine is not yet available. the role of scov m protein in the viral life cycle, especially in viral assembly and budding, makes it an attractive target for anti-sars drug and vaccine research. rnai, induced by double-stranded rna molecules, can silence target gene expression. since its discovery in caenorhabditis elegans in , rnai has been found in many organisms. rnai could become a reasonable approach in experimental therapeutics for human viral pathogens both in acute and chronic infections (ketzinel-gilad et al., ) . a considerable body of work has demonstrated that rnai has great prospects in viral therapeutic applications due to its simplicity and specificity compared to many other anti-viral strategies. here, the recombinant m-egfp protein was expressed in hek t cells and spotty fluorescence was found in the transfected cells, which was condensed into discrete loci (fig. ) . using confocal microscopy, the results verified that the m-egfp fusion protein was predominantly located in golgi compartments (fig. ) , which is consistent with previous studies (nal et al., ) . however, the correct protein band could not be detected by western blotting analysis when the regular cell lysis buffer ( mm tris-cl, ph . , mm nacl, . % sodium azide, % triton x- , g/ml aprotinin and g/ml pmsf) was used (data not shown). it was speculated that this failure in detection may be due to its membrane-binding property (klumperman et al., ) . therefore, an alternative method was adopted using % sds to lyse the cells expressing scov m protein. three candidate m-specific sirnas were designed and transcribed in vitro to inhibit scov m protein. although sirnas can be acquired through several methods (donze and picard, ; miyagishi and taira, ; yang et al., ; svoboda et al., ; sui et al., ; lois et al., ; rubinson et al., ) , transcription in vitro by t polymerase is more convenient and reliable than other approaches. furthermore, the applications of these specifically designed sirnas, - nt in length, in animal models have recently made great progress, including the sirnas targeting hepatitis b virus (xuan et al., ) , japanese encephalitis virus (murakami et al., ) , human immunodeficiency virus (ping et al., ) . based on the extensive screening of the effective sirnas in vitro, some proper modifications were adopted to improve the efficacy and duration of sirnas for additional in vivo use. for example, endoribonuclease-prepared sirnas can efficiently inhibit hbv replication in a mouse model (xuan et al., ) , which might be a better therapeutic agent to fight against hbv. sirnas have also been pursued for the control of sars (de clercq, ) . some potent sirna inhibitors of scov in vitro were further evaluated for efficacy and safety in a rhesus macaque sars model using clinically viable delivery while comparing three dosing regimens. the results showed that specific sirna could mediate anti-sars effects either prophylactically or therapeutically, suggesting that a clinical investigation is warranted. this work underscores the prospects for sirna to enable a significant reduction in development time for new targeted therapeutic agents (b.j. t. li et al., ) . in this study, rt-pcr, real-time quantitative pcr and western blotting analyses showed that m-sirna , m-sirna , m-sirna could decrease the scov m gene transcript and translational levels compared to the sirna control (fig. ) . these data suggested that the effect of gene silencing induced by sirna should be sequence specific and entire open reading frame (orf) based. the sirnas transcribed in vitro can effectively down-regulate scov m rna and protein levels, corresponding to the reported mechanism that sirnas degrade target mrna. the resulting three scov m specific sirnas in this study were different from active sirna reported by other labs (he et al., ) , suggesting that these effective sir-nas could be further explored as a more efficacious therapeutic agents for scov infection. previous studies on the m protein revealed that it can interact with n protein (he et al., ) . in this work, when pegfp-m was co-transfected with full length scov n protein into hek t cells, the fluorescence of recombinant m-egfp was strongly enhanced (unpublished results). future work will aim to investigate the mechanism of how scov n protein can increase the expression of m protein using specific sirnas. inhibition of egfp expression by sirna in egfp-stably expressing huh- cells potential antivirals and antiviral strategies against sars coronavirus infections rna interference in mammalian cells using sirnas synthesized with t rna polymerase killing the message short rnas that silence gene expression duplexes of -nucleotide rnas mediate rna interference in cultured mammalian cells potent and specific genetic interference by double-stranded rna in caenorhabditis elegans aetiology: koch's postulates fulfilled for sars virus kinetics and synergistic effects of sirnas targeting structural and replicase genes of sars-associated coronavirus characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the sars coronavirus rna interference for antiviral therapy coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque sirna targeting the leader sequence of sars-cov inhibits virus replication analysis of relative gene expression data using real-time quantitative pcr and the (−delta delta c(t)) method germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors attenuation of sars coronavirus by a short hairpin rna expression plasmid targeting rna-dependent rna polymerase identification of effective sirna blocking the expression of sars viral envelope e and rdrp genes development and application of sirna expression vector inhibitory effect of rnai on japanese encephalitis virus replication in vitro and in vivo differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interference rna lung pathology of fatal severe acute respiratory syndrome glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins a and m coronavirus as a possible cause of severe acute respiratory syndrome modulating hiv- replication by rna interference directed against human transcription elongation factor spt silencing of sars-cov spike gene by small interfering rna in hek t cells validation of array-based gene expression profiles by real-time (kinetic) rt-pcr characterization of a novel coronavirus associated with severe acute respiratory syndrome a lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by rna interference the severe acute respiratory syndrome coronavirus a is a novel structural protein mammalian rnai for the masses inhibition of genes expression of sars coronavirus by synthetic small interfering rnas a dna vector-based rnai technology to suppress gene expression in mammalian cells rnai in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsrna potent and specific inhibition of sars-cov antigen expression by rna interference characterization of severe acute respiratory syndrome coronavirus membrane protein inhibition of severe acute respiratory syndrome virus replication by small interfering rnas in mammalian cells rna interference blocks gene expression and rna synthesis from hepatitis c replicons propagated in human liver cells inhibition of sras-cov replication by sirna esirnas inhibit hepatitis b virus replication in mice model more efficiently than synthesized sirnas short rna duplexes produced by hydrolysis with escherichia coli rnase iii mediate effective rna interference in mammalian cells rnase iii-prepared short interfering rnas induce degradation of sars-coronavirus mrnas in human cells silencing sars-cov spike protein expression in cultured cells by rna interference this work was financially supported by the special medical research project of cpla in the th five-year plan (no. z and js ). we thank dr. mark feitelson for critical reading of the manuscript prior to publication. key: cord- -jgq dz u authors: busson, l.; bartiaux, m.; brahim, s.; konopnicki, d.; dauby, n.; gérard, m.; de backer, p.; van vaerenbergh, k.; mahadeb, b.; de foor, m.; wautier, m.; vandenberg, o.; mols, p.; levy, j.; hallin, m. title: prospective evaluation of diagnostic tools for respiratory viruses in children and adults date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: jgq dz u aim: to compare the performances of molecular and non-molecular tests to diagnose respiratory viral infections and to evaluate the pros and contras of each technique. methods: two hundred ninety-nine respiratory samples were prospectively explored using multiplex molecular techniques (filmarray respiratory panel, clart pneumovir), immunological techniques (direct fluorescent assay, lateral flow chromatography) and cell cultures. findings: molecular techniques permitted the recovery of up to % more respiratory pathogens in comparison to non-molecular methods. filmarray detected at least % more pathogens than clart pneumovir which could be explained by the differences in their technical designs. the turnaround time under hours for the filmarray permitted delivery of results when patients were still in the emergency room. since the discovery of viruses in the twentieth century, considerable efforts have been made to improve the technics to detect and identify them. cell cultures were the first diagnostic tool to be used in the mid- s, and since then, new techniques have been developed to decrease the time to a result (immunofluorescence, lateral flow chromatography) or to boost the sensitivity (molecular techniques) (levine, ; ginocchio and harris, ) . many improvements have been made, and techniques combining speed and sensitivity are currently available, such as fully automated 'sample-in, result-out' multiplexed syndromic molecular tools (bluchan and ledeboer, ) . aside from being effective in terms of sensitivity and specificity, these latter diagnostic tools are able to recover non-cultivable viruses. as a consequence, questions regarding the usefulness of 'older' diagnostic methods regularly arise (leland and ginocchio, ; hodinka and kaiser, ) . meanwhile, important questions concerning these 'new' expensive rapid molecular techniques remain unanswered, such as their cost-effectiveness in terms of patient's management, or the clinical significance of detecting nucleic acids of micro-organisms that could be non-infectious at the time the sample is collected. the objective of this work was to compare the performances of antigen detection and cell cultures techniques routinely used since years for the diagnosis of respiratory viral infections in the setting of a tertiary care hospital to those of newer molecular techniques (clart pneumovir, genomica, coslada, spain and filmarray respiratory panel, biofire, biomérieux, marcy l'etoile, france). the study was initiated on the st of february (week ) and ended on the th of march (week ) in the saint-pierre university hospital, a tertiary general hospital with beds located in downtown brussels. this was during the peak of the - influenza season which was moderate in belgium and lasted from week to week . more than % of influenza a isolates collected in belgium were https://doi.org/ . /j.jviromet. . . received june ; received in revised form january ; accepted january a(h n )pdm . regarding influenza b, circulating strains were almost exclusively from the victoria lineage according to the belgian scientific institute of public health (belgian public health institute, ). the enrolment period was chosen in order to be sure to gather positive samples for influenza as it is intended to evaluate, in another article, the impact of the results on antiviral prescription. this choice obviously affects the prevalence of other viruses. adults and children attending the emergency room (er) and presenting with upper or lower respiratory symptoms were prospectively included if either intended to be maintained in the hospital or had any of the following conditions known to expose to a higher rate of complications of viral respiratory infections: chronic respiratory diseases such as cystic fibrosis or asthma, sickle-cell disease, asplenia, neuromuscular diseases, severe neurological affections, hereditary metabolic disorders including diabetes, congenital or acquired immunosuppression, heart defects, chronic nephropathies, chronic liver diseases and pregnancy. children under months of age with a fever without focus of infection were also included. upon inclusion, a respiratory sample was collected. nasopharyngeal aspirate (npa) samples were typically collected from children under years old, and nasopharyngeal swabs (nps) (flocked swab + utm ml, copan, brescia, italy) were collected from older children and adults. the samples were immediately sent to the microbiology laboratory for testing. prior to testing, npa were diluted with ml of viral transport medium composed of veal infusion broth (difco, becton dickinson, sparks, md, usa) supplemented with bovine albumin (sigma aldrich, st. louis, mo, usa). lateral flow chromatography (lfc) tests and the filmarray respiratory panel were used to test samples / , whereas direct fluorescent assays (dfa) and cell cultures were performed during working hours ( : am to : pm) from monday to saturday. the clart pneumovir test was performed once a week. lab results as well as clinical data from patients' chart were recorded and analyzed. antigen detection tests and cell cultures are routine tests performed for patients attending the emergency departments. filmarray and clart pneumovir were performed for the study. because only tests per day are reimbursed by the social welfare, the combination of lfc and dfa tests performed varied during the evaluation based on the most prevalent circulating viruses. from february st to the th , influenza (sofia influenza a + b, quidel, san diego, ca, usa), rsv (binaxnow rsv, alere, waltham, ma, usa) and human metapneumovirus (hmpv) dfa (argene, biomérieux, marcy l'etoile, france) tests were performed. fifty-nine samples were analyzed with this combination. from february th to march th , metapneumovirus detection was replaced by an adenovirus detection test (adenorespi k-set, coris bioconcept, gembloux, belgium). cell cultures were performed as follows: an aliquot of the sample was inoculated on confluent vero (african green monkey kidney), mrc (human lung) and llc-mk (rhesus monkey kidney) cell cultures (vircell, santa-fé, spain) in -well or -well tissue culture plates (greiner-bio one, frickenhausen, germany). cultures were incubated at °c in a % co atmosphere for weeks for the vero cultures plates and llc-mk cells and weeks for the mrc cells. the culture media were replaced weekly. cultures were examined every two to three days using an inverted microscope. hemadsorption was performed on the llc-mk cells at the end of the second week of incubation. the filmarray respiratory panel . is a closed 'sample-in, resultout' multiplex pcr system that integrates sample preparation, amplification, detection and analysis of results in approximately an hour. the panel detects the most common respiratory viruses: adenovirus, coronavirus ( e, hku , nl and oc ), human metapneumovirus, human rhinovirus/enterovirus (without distinction between the two), influenza a (with differentiations of h , h -pdm and h strains), influenza b, parainfluenza - and respiratory syncytial virus (rsv). the panel also detects bacteria; mycoplasma pneumoniae, chlamydophila pneumoniae and bordetella pertussis which were not evaluated in this study. tests were performed according to the manufacturer's instructions. the clart pneumovir test is a microarray technique that targets the same pathogens as the filmarray respiratory panel, with the exception of coronaviruses hku , nl and oc and the aforementioned bacteria. among enteroviruses, this technique only detects echoviruses, but it can differentiate them from rhinoviruses. the extraction of nucleic acids was carried out with the qiasymphony system sp (qiagen) using the qiasymphony virus/bacteria midi kit (input volume μl, output volume μl). the pneumovir assay was performed according to manufacturer's instructions, and detection and interpretation of the results were conducted by a carreader (genomica). as the molecular tests used were presumably more sensitive than the reference standard (viral culture), we constructed a "composite" reference standard to avoid bias in establishing the specificity of the evaluated tests. this "composite" reference standard was constructed as follows: when discrepant results between the molecular techniques were observed, a third molecular technique was performed (argene, biomérieux, marcy l'etoile, france). a sample was considered truly positive for a pathogen if at least molecular techniques were positive for this pathogen. for the targets included in the filmarray test but not in the clart pneumovir panel (coronaviruses hku , nl and oc ), only the specificity of the positive results were evaluated using the argene or laboratory developed tests. data were analyzed using two sided exact chi-square fisher's tests followed in case of statistical significance by chi-square trend tests. the statistical software used were medcalc v . . and ibm spss v . . a total of samples ( npa and nps) from patients were analyzed: samples were obtained from children, of whom were female and were male (mean age: years and months old; median: months old); and samples were obtained from adults, of whom were female and were male (mean age: years old; median: ). for children, % ( / ) of samples were positive for at least one pathogen. the pathogen detected, ranging from most to least, was rhino/enteroviruses ( ), influenza b ( ), influenza a ( ), coronaviruses ( ), adenovirus ( ), metapneumovirus ( ), rsv ( ) and parainfluenza ( ). for adults, % ( / ) of samples were positive, and the detected viruses were influenza a ( ), influenza b ( ), rhino/enteroviruses ( ), coronavirus ( ), adenovirus ( ), metapneumovirus ( ) and rsv ( ). no parainfluenza viruses were detected in the adult patients. all influenza a isolates were a(h n )pdm with the exception of one a(h n ) from one adult patient. influenza b strains were not subtyped. co-detection was more common in children than in adults ( . % vs . %; p < . ). table details the sensitivity and specificity observed for the different techniques, depending on the pathogens. table provides the rate of false negative results, partial agreement (meaning at least one but not all the expected pathogens were detected) and complete agreement (meaning all the expected pathogens were detected) of the non-molecular techniques and molecular techniques compared to the established standard. the filmarray test produced fewer false negative results and partial results compared to non-molecular techniques and the clart pneumovir test (p < . ). false negative results with molecular techniques were significantly more frequent in samples with codetections compared to those with only one pathogen: % vs % for the filmarray test (p = . ) and % vs % for the clart pneumovir test (p < . ). similar result was also observed for cell cultures when limiting the analysis to culturable viruses ( % vs %; p < . ). table reports the mean turnaround time (tat) of the techniques from the reception of the sample in the laboratory to the introduction of the results into the laboratory information system. the lfc and fil-marray techniques have the shortest tat, while cell cultures and the clart pneumovir test have the longest tat. the tat for negative cell cultures is about weeks as they are discarded after this delay. tables and provide the detection rates for different techniques for influenza a and b, depending on the duration of the cough or the age of the patients. there is an effect of cough duration for influenza a with the antigen detection test (p = . ) and cell cultures (p = . ), due to a decrease in diagnostic sensitivity with cough duration (p = . and p = . , respectively). the decrease is observed as soon as the cough duration exceeds days. an age effect was also observed for influenza a with the antigen detection test (p = . ) and cell cultures (p = . ), due to a decrease in sensitivity with age (p = . and p = . , respectively). the decrease is observed from the first age category with the antigen detection and from years for the cell cultures. no effect of cough duration or age was observed with the detection of influenza a for the filmarray and clart pneumovir tests or on the detection of influenza b with any method. as expected, molecular techniques were observed to be the most sensitive. however, the performances of the two evaluated techniques differed; lower sensitivity for the detection of several targets was observed for the clart pneumovir test, notably this technique only detects echoviruses among all enteroviruses. for adenoviruses, metapneumoviruses and rsv, the explanation probably relies more on technical considerations. indeed, false negative results with the filmarray and the clart pneumovir tests were not always for samples with a low viral load, as indicated by the cycle threshold (which correlates with the viral load) of the third molecular technique used to elucidate discrepant results between the first two techniques. as an example, the mean ct value and standard deviation of negative and positive samples for adenoviruses with clart pneumovir were . ( . ) and . ( . ) respectively (p = . ). the choice of primers and probes used to design tests can determine whether a certain strain of virus will be detected. although the filmarray test had a better sensitivity than the clart pneumovir test for the detection of adenoviruses, it may lack sensitivity in detecting certain strains, which could impact the management of immunocompromised patients (song et al., ) . a huge advantage of molecular techniques is that they allow the recovery of those pathogens that are difficult to culture, that will not grow in culture or for which no antigen detection tests are available. in the present study, the rate of complete detection of the pathogens present in the samples was far more important with molecular techniques compared to non-molecular ones. this has already been reported. (weinberg et al., ) viruses with poor detection using non-molecular techniques primarily include adenoviruses, metapneumoviruses and rhino/enteroviruses. there were also positive samples for coronaviruses (mahony, ) which, in our experience, do not commonly grow with routine cell lines, even if coronavirus nl was originally described in llc-mk (van der hoek et al., ) . the rate of co-detection was more important for children, which is a common finding and could be explained by notably increased, longerlasting viral shedding in children under months of age due to less developed mucosal immunity (sharma et al., ) . the co-detection of viruses in children is apparently not linked with a more severe outcome (comerlato scotta et al., ) , and the molecular detection of a virus in a respiratory sample is not always associated with symptoms. the type of detected virus and the age of the patient can be helpful to decide on a care plan (self et al., ) . as previously stated, false negative results with molecular techniques were significantly more frequent in samples with multiple pathogens compared to those with only one pathogen; this finding could be due to possible competition for the reagents when multiple targets are to be detected (bezerra et al., ) . the specificity of all molecular techniques was over %, except that for rhino/enteroviruses with the filmarray test ( . %). the differences observed between molecular techniques could also be due to primer and probe choices. overall, the clart pneumovir is less sensitive than the filmarray except for influenza b and its hands-on time and turnaround time are longer. only positive samples were analyzed for metapneumovirus using an antigen detection test, as it was substituted for adenovirus test during the evaluation. this test nonetheless appeared more sensitive than the cell culture tests, as no metapneumovirus was recovered from cell cultures. this finding was under the expected sensitivity, which is approximately % according to the literature (tang and crowe, ) . the apparent low sensitivity of non-molecular techniques observed for adenoviruses deserves comment. indeed, prolonged shedding after the primary infection is classically described for adenovirus. adenoviruses can also be detected in tonsillar tissue or isolated from a throat sample of up to % of healthy children (song et al., ; kalu et al., ) . these states of prolonged shedding or "latency" are more likely to be detected with molecular techniques. it is also noteworthy that % of adenoviruses detected in this evaluation were associated with one or more other pathogens, supporting the hypothesis that they could be bystanders in some cases. the sensitivity of cell cultures for rhino/enterovirus was also low and could be partially attributed to the fact that group c rhinoviruses do not grow on standard cell cultures (jacobs et al., ) . enteroviruses grow inconsistently on cell cultures, depending on their type, and no cell line enables the detection of all strains (stellrecht et al., ) . unfortunately, rhinoviruses and enteroviruses in this study were not typed. the presence of multiple viruses in a sample also influences cultures because the growth of the fitter or more abundant virus can mask the growth of others (george et al., ) . for example, on the false negative cultures for rhino/enteroviruses, were positive for another virus. likewise, for adenoviruses, for the false negative cell cultures, recovered another virus. molecular techniques supposedly do not suffer from this drawback, although as previously discussed, a higher rate of false negatives was observed when multiple pathogens were present in the sample. cell cultures, however, can enable the detection of unsuspected viruses. this detection was the case in this evaluation for one measles virus, which was not originally suspected, and herpes simplex viruses and cytomegaloviruses (cmv). in one case, cmv could explain the symptoms exhibited by a -month-old patient with a fever without focus of infection. the other cases were more likely to be recurrences or prolonged shedding. regarding the sensitivity of antigen detection tests for influenza, influenza a is more easily detected than influenza b, which has already been reported (busson et al., ) . the decreased sensitivity for influenza a with antigen detection and cell cultures, which depends on the age of patients, can be explained by the higher viral shedding in children, and also because for younger children, npa were preferred to nps. aspirates usually increase the detection rate for viruses over swabs (loens et al., ) . the decreased sensitivity of antigen detection and cell cultures when the duration of the cough increases is expected, because viral shedding is more important in the first days of the disease (aoki and boivin, ). the reason for the lack of similar findings for influenza b is unclear. the absence of sensitivity loss for molecular techniques, depending on the age of the patients and the duration of the cough, can be attributed to the high sensitivity of the techniques. usually, antigen detection tests have a faster turnaround time than cell cultures but a lower sensitivity except for viruses growing poorly, especially metapneumovirus and rsv, for which the sensitivity of antigen detection tests can be better than the one of cell cultures. the specificity of non-molecular tests was above %. false positive results in cell cultures can occur due to cross-contamination between wells on culture plates. antigen detection tests based on lateral flow chromatography are usually the easiest and fastest to perform. techniques based on immunofluorescence are slightly more time-consuming. currently, totally automated techniques with a short hands-on time, such as filmarray, can be realized in a time frame comparable to that of lfc tests. however, only one can be performed at one time, and the analyzer remains occupied for slightly more than an hour. the number of required instruments is based on the test volume at each facility and the desired tat. cell cultures remain the most time-consuming techniques, and results often arrive too late to impact patient management (ginocchio, ) . this was also the case for clart pneumovir in our setting, as it was only performed once a week due to the complexity of the corresponding analytical process. in belgium, non-molecular techniques for the diagnosis of respiratory viruses are reimbursed by the social welfare program, which is not the case for molecular techniques which are charged to the patients. during this evaluation, molecular techniques were free of charge for patients, but they cost approximately euros per patient. the necessity of molecular tests must be seriously considered before prescription. these tests should probably be reserved for the most severely ill patients, for whom rapid and comprehensive microbiological evaluation is most likely to impact patient management. as a comparison, antigen detection tests cost, including workforce, can range between . and euros depending on the test and cell cultures cost about euros. however, non-molecular tests are reimbursed by the social welfare in belgium permitting a broader prescription. the possibilities to implement molecular tests can vary between facilities and countries depending on local policies. using molecular rather than non-molecular techniques could impact patients' isolation strategies (richardson et al., ) , antibiotic/antiviral use or reduce of the length of stay (brendish et al., ; rogers et al., ) , but cost-benefit analyses are difficult to appraise because of many intertwined elements. molecular techniques have considerably increased our capacity to detect respiratory viruses in a timely manner. the main factors limiting a wide utilization of these techniques are cost and the difficulty to absorb the workload in large facilities with numerous samples to analyze per day. another drawback is that latent viruses or traces of genetic material may be detected. the interpretation of a positive result might be difficult, and it must be carefully correlated to the clinical history of the patient as the presence of a virus in the respiratory tract is a factor exposing to bacterial superinfection (vareille et al., ) . it is possible to quantify the viral load in a respiratory sample, but there are conflicting reports regarding correlations between viral load and outcome (granados et al., ; wishaupt et al., ) . technical improvements should be made to prevent variation in quantification caused by sample dilution with saline instilled during aspirates or by the variable quantity of sampled material with swabs. these improvements could render comparisons between studies more reliable and permit the establishment of a threshold, above which detected viruses are indeed involved in an ongoing infectious process. in addition, a positive antigen detection test usually correlates with a high viral load in a sample, and a positive cell culture can only be achieved with infective viral particles. whatever technique is used, fully understanding the benefits and limitations of each is crucial for interpretation. the choice of technique should depend, besides financial considerations, on the necessary sensitivity and speed based on symptoms, comorbidity and risk of a detrimental outcome for each patient. a tertiary care hospital should be able to offer diagnostic tools suitable for each individual case; if testing all patients with molecular techniques is not possible, the use of nonmolecular techniques is preferable over nothing at all. influenza virus shedding -excretion patterns and effects of antiviral treatment viral and atypical bacterial detection in acute respiratory infection in children under five years emerging technologies for the clinical microbiology laboratory routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial evaluation of rapid influenza diagnostic tests during the - epidemic: influences of subtype and viral load ap. duarte de souza et al. respiratory viral coinfection and disease severity in children: a systematic review and meta-analysis rapid and sensitive detection of respiratory virus infections for directed antiviral treatment using r-mix detection of respiratory viruses using non-molecular based methods reagents, stains, media and cell cultures: virology influenza and rhinovirus viral load and disease severity in upper respiratory tract infection is the era of viral culture over in the clinical microbiology laboratory? human rhinoviruses persistence of adenovirus nucleic acids in nasopharyngeal secretions: a diagnostic conundrum role of cell culture for virus detection in the age of technology the origins of virology optimal sampling sites and methods for detection of pathogens possibly causing community-acquired lower respiratory tract infections detection of respiratory viruses by molecular methods comparison of respiratory virus shedding by conventional and molecular testing methods in patients with haematological malignancy impact of a rapid respiratory panel test on patient outcomes respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia the developing human preterm neonatal immune system: a case for more research in this area performances of filmarray respiratory panel v . for detection of adenovirus in a large cohort of pediatric nasopharyngeal samples: one test may not fit all enteroviruses and parechoviruses respiratory syncytial virus and human metapneumovirus identification of a new human coronavirus the airway epithelium: soldier in the fight against respiratory viruses superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children pitfalls in interpretation of ct-values of rt-pcr in children with acute respiratory tract infection the author acknowledges the team of the virology none declared. this study was approved by the ethics committee of university hospital saint pierre (brussels). key: cord- -l elcfe authors: ganapathy, kannan; cargill, peter walker; jones, richard charles title: effects of cold storage on detection of avian infectious bronchitis virus in chicken carcasses and local antibodies in tracheal washes date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: l elcfe in order to test the survivability of infectious bronchitis virus (ibv) in dead chicken carcasses during h of cold storage, week-old specific-pathogen-free chickens were infected with virulent ibv massachusetts strain m , and were killed humanely days later. carcasses were stored in a cold room at °c. after , , , , or h of storage, necropsies were carried out. trachea, lung, kidney and rectum were collected for virus isolation by tracheal organ culture (toc) or embryonated chicken eggs (ece), and detection by nested reverse-transcriptase polymerase chain reaction (rt-pcr). ibv was detected by rt-pcr at all sampling times, except for and h of storage in kidney and h of storage in kidney and rectum. for ece, isolation was obtained at all sampling points, except at and h of storage in lungs. isolation by tracheal organ cultures was less successful, except from rectum. in addition to sampling for virus, tracheal washes were collected from each carcass to measure the ability to detect local antibodies after storage. levels of iga in tracheal washes remained high for up to h of storage, suggesting that accurate sampling for research purposes when required must be carried out within this time. infectious bronchitis caused by a coronavirus is an important disease in chickens, and it mainly affects respiratory and urogenital systems (cavanagh and naqi, ; dhinakar raj and jones, ) . diagnosis of infectious bronchitis virus (ibv) is confirmed by isolation of the virus using either chicken embryonated eggs (ece) or tracheal organ culture (toc) and detection by reverse-transcriptase polymerase chain reaction (rt-pcr) (cavanagh and naqi, ; gelb and jackwood, ) . tracheal swabs, oropharyngeal swabs and tissues such as trachea, lungs, kidney, oviduct and caecal tonsils are normally used for isolation (cavanagh and naqi, ; gelb and jackwood, ) . it is recommended that carcasses should be submitted to the laboratory as soon as possible but no reports are available to indicate an appropriate time limit, beyond which virus detection is impossible. this paper provides information on the probability of ibv recovery from target tissues in carcasses stored at • c for up to h post-killing. three different methods of demonstrating the presence of ibv, namely isolation by toc or ece, and detection by nested rt-pcr were used. the trachea is recognised as a main target organ for ibv infection, hence an important site for research into study local immune responses (dhinakar raj and jones, ; gillette, ; gomez and raggi, ) . in such investigation, tracheal washes are collected for detection of local antibodies (dhinakar raj and jones, ; hawkes et al., ) and this is normally done soon after killing. however, no details on the optimal time intervals between killing and collection of tracheal washes have been established. this experiment therefore provided the opportunity to measure the levels of iga and igg in tracheal washes of chicken carcasses stored at • c and sampled at the same intervals. white leghorn specific-pathogen-free chicken eggs (lohmann animal health, cuxhaven, germany) were incubated and hatched at our laboratory. chicks were housed in isolation rooms in an experimental house. food and water were provided ad libitum. the massachusetts strain m was used after numerous passages in ece. the titre was . log median egg infective dose per ml . prior to this, the virus had undergone passages in toc and passages in ece. chickens were inoculated when seven weeks old, with l of ibv by the oculo-nasal route. the birds were monitored for clinical signs and were humanely killed at days post-infection. carcasses were stored at • c. at , , , , and h of storage, four carcasses were randomly chosen for tracheal wash collection and virus detection. tracheal washes were collected as described by dhinakar raj and jones ( ) and stored at − • c until further use. they were assayed for ibv-specific iga and igg by indirect elisa (below). pieces of trachea, lung, kidney and rectum were aseptically collected for isolation or rt-pcr. a similar group of uninfected chickens kept in a separate isolation pen were used as a control. each trachea was scraped with a sterile surgical blade and the mucus and epithelium were vortexed in . ml of virus isolation medium [eagles serum-free mem with glutamine, streptomycin ( g/ml) and penicillin ( iu/ml)]. pieces of lung, kidney or rectum (after squeezing out faecal contents) were homogenised using a sterile pestle and mortar with sterile sand and . ml of the medium. subsequently, more medium was added to make a final : (w/v) dilution of the sample. prior to centrifugation, a sterile cotton swab was dipped into each of the tissue homogenates for rt-pcr detection of ibv. swabs were left to dry at room temperature then kept in a cupboard at room temperature until used. the tissue homogenates were centrifuged at × g for min and the supernatants were collected and stored at − • c until processed for virus isolation. three different methods were used to detect presence of ibv in the homogenised tissues. isolation of the virus was carried out in chicken tocs as described by cook et al. ( ) . three replicates of each homogenate underwent three passages and those tocs with complete ciliostasis at the third passage were subjected to rt-pcr, to confirm presence of the virus. virus isolation was carried as described by gelb and jackwood ( ) using specific-pathogen free chicken eggs of - days of incubation. this was done for up to three passages. after the third passage, eggs were incubated for days and chilled overnight. chorioallantoic fluid was collected aseptically and stored at − • c for until used. the embryos were examined for typical lesions of ibv. the chorioallantoic fluid from embryos with lesions consistent with ibv after up to three passages was considered positive for virus. those where embryo lesions were inconclusive after the third passage was subjected to rt-pcr to confirm the presence of ibv. the swabs were pooled according to tissues and sampling intervals. rna was extracted as described previously . briefly, each was dipped into ml of guanidine isothiocynate (solution d) containing -mercaptoethanol and being left at − • c for several hours. then, l of the sample was transferred to a new microcentrifuge tube and l of sodium acetate (ph . ) and l phenol chloroform were added. the mixture was vortex mixed and centrifuged for min. the top layer was transferred to l isopropanol, mixed and precipitated overnight at − • c. rna was precipitated using % ethanol and the resulting precipitate was suspended in a solution containing sterile tissue culture water, dithiothreitol and ribonuclease inhibitor. rt-pcr was conducted according to method of cavanagh et al. ( ) . ibv-specific iga and igg in tracheal washes were assayed using an indirect elisa (dhinakar raj and jones, ) except that the plates were coated with partially purified ibv m antigen. this was done by ultracentrifugation of previously clarified allantoic fluid at , × g for min and the resulting pellet was washed and resuspended in phosphate buffered saline (ph . ). the virus suspension was overlaid onto % sucrose and ultracentrifuged at , × g signs of sneezing, head-shaking and watery eyes were observed from day until day post-infection. at necropsy on days post-infection, no gross lesions were found. for trachea, virus was isolated at and h of storage only and in lungs between and h but from never more than two out of four chickens (table ) . at , and h of storage virus was isolated in the kidney, and was consistently detected in rectum. virus was consistently detected at all sampling points except at and h of storage in lungs. in total, rectum provided % recovery, followed by trachea and kidney ( %), and lungs ( %). ibv was detected at all sampling intervals in all four organs, except at , and h of storage for kidney and at h of storage for rectum (table ) . all control tissues remained negative for virus by isolation and rt-pcr. fig. shows levels of iga and igg in tw at different sampling intervals. levels of iga at , , and h of storage were similar and were significantly higher than and h of storage. however, no significant differences were found between the sampling intervals, except levels of iga at h of storage were significantly higher than h of storage. for igg, only trace amounts were detected. the main aim of this paper was to provide some information on the survivability of ibv in chicken carcasses during h of cold storage. this, in turn, should influence the ability to confirm a diagnosis on materials that is not fresh. the chickens were deliberately killed at days post-infection, as it is well recognised that it is more difficult to recover virus at this stage than at less than days post-infection (cavanagh and naqi, ; cook, ) . isolation was attempted in toc or ece, which have been reported to be equally sensitive for ibv recovery (cook et al., ; darbyshire et al., ; de wit, ) and also detected by nested rt-pcr cavanagh and naqi, ; elhafi et al., ) . in this study, it appears that irrespective of the ibv detection method, the virus was found in all tissues examined for up to h of storage. this suggests that in difficult circumstances, necropsy examination and tissue collection of carcasses kept at • c could be safely delayed up to h post-killing. interestingly, irrespective of the method of isolation, consistently higher numbers of recoveries were obtained from the rectum. it is known that clearance of ibv infection first occurs from the respiratory tissues followed by other tissues, with later clearance from kidney and digestive tract (lucio and fabricant, ). in addition, ambali and jones ( ) reported ibv isolation from rectal tissue and replication in the epithelium, suggesting that apart from excretion of the virus via this route, the tissue may play an important role in generating new virus when respiratory replication has declined. thus, the rectum could be an important but additional tissue for diagnosis of ibv. as for the kidneys, since we have used a non-nephrotropic virus, the frequency of virus detection was low and to be expected. the host, agent and environmental factors may have influenced the results of this experiment (dhinakar raj and jones, ) and a change in these factors, particularly the strain of virus used may have produced a different outcome. the nested rt-pcr employed in this study, is regularly used in our laboratory for detection of ibv in pooled swabs. for diagnosis of the disease, use of pooled swabs is a routine practice as it is convenient, and represents a flock rather than individual birds. in our study, we also used pooled swabs taken from homogenised tissues where rna was extracted for rt-pcr. this appears to be the first report where such an extraction has been performed. other workers have extracted rna from allantoic fluid (cavanagh and naqi, ; gelb and jackwood, ) , directly from tracheal or oropharyngeal swabs (capua et al., ; cavanagh et al., ; jackwood et al., ; elhafi et al., ) or directly from non-processed tissues (falcone et al., ) . in this study, positive pcr reactions were found on all occasions, except after , and h of storage for lungs and h of storage for rectum. this could have been due to low amount of viral genomes in the tissues or other non-specific inhibitory factors (kwon et al., ) , as positive isolations at these times were obtained by ece. generally, it appears that rt-pcr has detected positive samples at similar rate as ece, and was better than toc. this may demonstrate that direct swabs from homogenised tissues could be used for detection of ibv by nested rt-pcr, which may take no more than h. in researching into local immune responses induced by ibv, tracheal washes are collected frequently and assayed for iga and igg (hawkes et al., ; dhinakar raj and jones, ) . this study shows that levels of iga in the tracheal wash remain high for up to h of storage with slight decline at h of storage, and sharply drops thereafter. thus, where complex experiments on local immune responses are to be undertaken, and especially if multiple sampling needs to be done, it should be noted that for optimal detection of local immunoglobulins, tracheal washes should be collected within h of storage. early pathogenesis in chicks of infection with an enterotropic strain of infectious bronchitis virus co-circulation of four types of infectious bronchitis virus ( /b / , b and massachusetts) infectious bronchitis longitudinal field studies on infectious bronchitis and avian pneumovirus in broiler using type-specific pcr coronaviridae the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis virus comparative growth kinetic studies on avian infectious bronchitis virus in different systems detection of infectious bronchitis local antibody production in the oviduct and gut of hens infected with a variant strain of infectious bronchitis virus infectious bronchitis virus: immunopathogenesis of infection in chicken microwave or autoclave treatments destroy the infectivity of infectious bronchitis virus and avian pneumovirus but allow detection by reverse transcriptasepolymerase chain reaction rapid diagnosis of avian infectious bronchitis virus by the polymerase chain reaction inactivated rabies vaccine control and release: use of an elisa method a laboratory manual for the isolation and identification of avian pathogens local antibody response in avian infectious bronchitis: virus-neutralizing antibody in tracheobronchial secretions local immunity to avian infectious bronchitis in tracheal organ culture presence of viral antigens and antibody in the trachea of chickens infected with avian infectious bronchitis virus further development and use of a molecular serotype identification test for infectious bronchitis virus polymerase chain reaction a biotin-labelled dna probe for detection of infectious bronchitis virus in chickens tissue tropism of three cloacal isolates and massachusetts strain of infectious bronchitis virus key: cord- -u ebql a authors: lan, yungang; zhao, kui; he, wenqi; wang, gaili; lu, huijun; song, deguang; gao, feng title: inhibition of porcine hemagglutinating encephalomyelitis virus replication by short hairpin rnas targeting of the nucleocapsid gene in a porcine kidney cell line date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: u ebql a porcine hemagglutinating encephalomyelitis virus (phev), which causes porcine encephalomyelitis and is widespread among swine worldwide. rna interference (rnai) pathways have emerged as important regulators of virus–host cell interactions. in this study, two sirna expression plasmids (shn and shn ) were generated to target two different coding regions of the nucleocapsid protein (n) of phev. the shrnas were transiently transfected into a porcine kidney cell line, pk- , to determine whether these constructs inhibited phev production. our results revealed that both shrnas were highly capable of inhibiting viral rna genome replication, especially shn . next, stable transfection of shn was used to produce two sirna stably expressing pk- cell clones (shn - and shn - ), and these two lines were infected with phev. the analysis of cytopathic effects (cpe) demonstrated that shn - and shn - were capable of protecting cells against phev infection with high specificity and efficiency. furthermore, effective inhibition of viral replication persisted for up to h by a tcid( ) assay. these results indicated that rnai targeting of the n gene could facilitate studies of the specific function of viral genes associated with phev replication and may have potential therapeutic applications. consisting of - nucleotides (nt) (jana et al., ) . subsequent rnai studies have used synthetic small interfering rnas (sirnas) or short hairpin rnas (shrnas) extensively to study the interference of viral replication. thus far, the replication of various viruses, including many coronaviruses, has been effectively inhibited in vitro and in vivo (galan et al., ; lambeth et al., ; lan et al., ; wu et al., ; zhao et al., ; zhou et al., zhou et al., , zhuang et al., ) ; however, no reports have shown that the replication of phev in cell culture could be disrupted by shrnas targeting the n gene of phev. n is an extensively phosphorylated, highly basic, vital structural protein; its primary function is to form a helical ribonucleoprotein complex with viral rna (vrna) (wang et al., ) . n plays an important and necessary role as an enhancer of coronavirus replicon activity (almazan et al., ; chang and brian, ) . here, we constructed a single short hairpin rna (shrna) plasmid expression system that targeted the n gene and investigated whether shrnamediated rna interference could inhibit phev replication in pk- cells. the hev- n strain (genbank accession no.: ay ) was replicated in pk- cells (mengeling et al., ) . prior to being infected with phev, the cells were maintained in mem supplemented with % fetal bovine serum (fbs) and antibiotics ( g/ml streptomycin and u/ml penicillin) in a • c, % co incubator overnight. when % of the virus-infected cells showed cytopathic effects (cpe), the cultures were collected, purified by sucrose density gradient centrifugation, and stored at − • c until use. based on recent research (elbashir et al., ) and the experience of researchers from the ambion corporation (jacque et al., ) using genbank sequences (genbank accession no.: ay , nc ) for hev- n and vw , the conserved areas were selected, and ambion's online sirna target design tool was utilized to choose the two best target sequences for targeting n. blastn searches were conducted on all sequences to ensure gene specificity. the targeted oligonucleotides were inserted into the pgpu /gfp/neo plasmid vectors using the bbsi and bamhi restriction sites to produce shn and shn (sequences shown table ); the negative control shrna (shnc), which targeted gttctccgaacgt-gtcacgt sequences and did not match any gene, was purchased from shanghai genepharma co, ltd (shanghai, china). pk- cells were seeded in -well plates and incubated for h at • c in a % co atmosphere. when the cells were - % confluent, they were washed and overlaid with transfection complexes containing . g of shn , . g of shn , or . g of shnc, in l of mem medium mixed with lipofectamine tm (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. the transfection complexes were completely removed after incubating for h, and the cells were infected with tcid ( . ) of phev. non-transfected cells were used as a control. to study the inhibitory effects of rna interference on phev replication, the level of viral antigen produced in the pk- cells after shrna transfection and viral infection was examined h post-infection by an indirect immunofluorescence assay (ifa) using anti-phev serum. infected cells were visualized by ifa, as has been described previously (xu et al., ) . positive anti-phev serum and negative control serum were prepared, as described previously (yu et al., ) . cells were washed three times with pbs and incubated with cy -conjugated rabbit anti-pig igg (sigma, st. louis, mo, usa; : ) for h. after washing, the cells were photographed and examined with an olympus fv laser scanning confocal microscope. at h post-phev infection, the majority of the non-transfected cells and the cells transfected with shnc exhibited red fluorescence in the cytoplasm, indicating that these cells were virus-producing ( fig. a and c). by contrast, only a few cells in wells treated with shn and shn transfection displayed red fluorescence ( fig. d and e), indicating that most of these cells were effectively protected by shrnas and resisted viral infection. oligonucleotides for the quantitation of phev by taq-man real-time rt-pcr were designed using the corresponding genbank sequences (genbank accession no.: ay , nc ) for hev- n and vw ; the conserved areas were selected, and primers were designed for phev using primer express . . three primers were synthesized (jikang, shanghai, china) for quantification of the phev genome by real-time pcr: -agcgatgaggctattccgacta- (nfp primer), -ttgccagaattggctctactacg- (nrp primer), and -fttc-cgccaggcacggtactcccp- (taqman probe). the target region for real time rt-pcr spanned nucleotides - of the n protein gene of phev (genbank accession no.: ay ). total rna ( ng) was extracted and purified using the trizol ls reagent (invitrogen, carlsbad, ca, usa). pcr was used to amplify the n gene with the nfp and nrp primers under conditions of • c for min, followed by cycles of • c for s, • c for s, and • c for s, with a final extension at • c for min. the pcr product was gel-purified using an agarose gel dna extraction kit (axygen, union, ca, usa) and then cloned into the pgem- t vector (promega, madison, wi, usa). the resulting plasmid, pt-n, for which the correct sequence was confirmed by direct sequencing, was selected as a quantitative standard for the determination of the viral rna copy number. real-time pcr was performed with the abi prism ® sequence detection system using a quantitect tm probe pcr kit (qiagen, hilden, germany) under the conditions of • c for min, followed by cycles of denaturation at • c for s, annealing, and extension at • c for s. the quantitative standard curve for the determination of the phev genome copy number was created by real-time pcr of standard plasmid table list of shrna sequences in this study. sequences of shrna a shn (nt - ) sense: -caccggtactggtacagacacaacattcaagagatgttgtgtctgtaccagtaccttttttg- antisense: -gatccaaaaaaggtactggtacagacacaacatctcttgaatgttgtgtctgtaccagtacc- shn (nt - ) sense: -caccggctattccgactaggtttccttcaagagaggaaacctagtcggaatagccttttttg- antisense: -gatccaaaaaaggctattccgactaggtttcctctcttgaaggaaacctagtcggaatagcc- a the underlined sequences targeted the n gene, and the bold italic letters indicate the loop sequence. near the end of all shrna sense templates was a -nt poly(t) tract that is recognized as a termination signal by rna pol iii, which terminated shrna synthesis. the ends of the two oligonucleotides were non-complementary and formed the bbsi and bamhi restriction site overhangs that facilitated efficient directional cloning into the pgpu /gfp/neo plasmid vector. pt-n preparations at serial dilutions of , , , and copies/l. the specificity of the real-time pcr was confirmed by sequencing the product. to quantify the effect of shrna on viral replication at h postviral infection, the viral genome copy number was determined by real-time pcr, using the serially diluted plasmid pt-n as a standard. the r value of the standard curve was . , and the average amplification efficiency e was . . sequencing showed that the amplified fragment contained the expected portion of the phev genome, thereby demonstrating the specificity and reliability of the analysis. as shown in fig. , the copy number of the viral genome per nanogram of total rna calculated from the standard curve was . × copies/ng of viral genome in the total rna from the shnc-treated cells, which was similar to that observed in the mock transfected cells (non-transfected cells), whereas there were . × and . × copies/ng of the viral genome in total rna from cells treated with shn and shn , respectively. these values correspond to reductions of % and %, respectively. to substantiate further the inhibitory effect of shn and shn , the viral % tissue culture infectious dose (tcid ) was used to titrate phev at h post-infection. briefly, the cultures were collected, subjected to freeze-thaw cycles, serially diluted -fold from − to − , and added to pk- cells in -well plates. tcid was calculated using the reed and muench method (reed and muench, ) . the results (fig. ) showed that in control cells transfected with shnc, the phev tcid reached . at h postinfection, which was similar to that observed in non-transfected cells. in contrast, the titers at h post-infection were . and . for cells transfected with shn and shn , respectively, which corresponded to -and -fold reductions compared to shnc-transfected cells. our results demonstrated that shn and, especially, shn both blocked viral replication with very high efficiency, but the effects were transient (data not shown). to conform further the effect of using rna interference (rnai), stably transfected lines were created. for shn transfection, cells per well were plated in six-well plates and transfected with g of shrna duplex using lipofectamine according to the manufacturer's instructions. after h of incubation, the medium was replaced with mem containing mg/ml g . after maintenance for weeks in selection media, resistant cell clones, named shn - , shn - and shnc- , were selected, cultured, infected with tcid ( . ) of phev per well in -well plates, and incubated at • c for the various time periods indicated. to study phev-induced cpe (fig. ) at h post-infection, cells were examined by phase-contrast microscopy every h. the infected shnc- cells and non-transfected cells exhibited obvious morphological changes starting from the brims of the wells at h post-infection, such as loss of the monolayer as well as rounding and shrinking of the cells. analysis of cell morphology indicated that the negative control (shnc- ) had no inhibitory effect on phevinduced cpe compared with the mock control (non-transfected cells). in contrast, the trial group (shn - and shn - ) effectively blocked cpe in the cell cultures up to h (data not shown). in addition, infectious virus production was determined by a tcid assay. fig. shows that titers of phev reached . , . , and . tcid /ml at h, h and h post-infection, respectively, in shnc- , followed by a gradual decrease in titer from to h post-infection. in contrast, titers at h post-infection were . and . tcid /ml in shn - and shn - , corresponding to -and -fold reductions, respectively. titers at h postinfection were . and . tcid /ml in shn - and shn - , respectively, corresponding to -and -fold reductions. at h post-infection, the viral titers were still -and -fold lower in shn - and shn - , respectively, compared to that of shnc- . the data indicate that the shrna stably expressing cells experienced markedly inhibited infectious virus production, but the level of inhibition decreased as time progressed. to confirm the specificity of the rnai effect and the absence of interferon activity in stably transfected lines, shn - , shn - and shnc- were also challenged with tcid ( . ) of pseudorabies virus (prv) jl strain in a plaque counting assay, as has been described previously (ferrari et al., ) . no significant difference in plaque number and plaque size between mock control (non-transfected cells) and stably transfected lines was observed from to h after prv infection (shown in fig. ). phev is the only known cov that is neurotropic in pigs. recently, infection rates of phev have increased in some countries rho et al., ) ; thus, pigs without antibodies to phev, such as spf pigs, were threatened by a high risk of infection by phev (quiroga et al., ) . the study of phev replication and of the control or inhibition of this infection is of great significance because, currently, no effective preventative measures or cures exist for this disease. rnai provides effective antiviral defenses in plants and other organisms; so several studies have focused on harnessing rnai to inhibit viral infection (leonard and schaffer, ) . however, the effectiveness of sirna that has been transfected transiently into cells is restricted because of low transfection efficiency and the short-term persistence of silencing effects. recently, the fig. . viable viral production in two sirna stably expressing pk- cell clones (shn - , shn - ). tcid are the means of three repeat titrations at the time points indicated. *p < . vs. shnc stably expressing pk- cells (shnc- ). vector-based approach of shrna interference has been developed to achieve stable, long-term, and highly specific suppression of gene expression in mammalian cells siolas et al., ) . to explore the possibility of interrupting phev replication with sir-nas or shrnas, sirna expression plasmids targeting the n gene in phev have been synthesized. n plays an important role in viral packaging. in the process of viral packaging, n acts as a bridge between the m protein and the viral rna. the interaction of n and mrna may affect on viral rna transcription and replication. in addition, n is highly conserved among coronaviruses, is expressed during viral infection and has a strong immunogenicity. these features make the n gene coding region a good rnai target site. our results have shown that fig. . the stably transfected lines (shn - , shn - , shnc- ) failed to alter significantly plaque number or size (not shown) from to h after tcid of prv incubation. at , , and h after virus infection, respectively, the medium was removed, cells were fixed and stained, and the number of plaques was counted. all data were collected in triplicate wells and in three independent experiments. the replication of phev in cell culture can be disrupted by shrnas targeting the n gene of phev. it is clear from this study that the dna vector-based shrna approach, that is, the use of rnai expression plasmids directed against the phev n gene, could effectively block expression of the viral target gene and inhibit viral replication. in addition, sirna therapy can be directed at the different levels of viral function, such as transcription or translation. based on the present data and the advantages of sirna technology, we believe that sirna is a potential therapeutic agent against chronic phev infection. the nucleoprotein is required for efficient coronavirus genome replication cis requirement for n-specific protein sequence in bovine coronavirus defective interfering rna replication analysis of gene function in somatic mammalian cells using small interfering rnas a comparative study of pseudorabies virus (prv) strains with defects in thymidine kinase and glycoprotein genes host cell proteins interacting with the end of tgev coronavirus genome influence virus replication vomiting and wasting disease associated with hemagglutinating encephalomyelitis viruses infection in piglets in jilin a hemagglutinating virus producing encephalomyelitis in baby pigs modulation of hiv- replication by rna interference rna interference: potential therapeutic targets targeting marek's disease virus by rna interference delivered from a herpesvirus vaccine in vitro inhibition of porcine hemagglutinating encephalomyelitis virus replication with sirnas targeting the spike glycoprotein and replicase polyprotein genes antiviral rnai therapy: emerging approaches for hitting a moving target in vitro inhibition of csfv replication by retroviral vector-mediated rna interference characteristics of a coronavirus (strain n) of pigs virus infections of porcines hemagglutinating encephalomyelitis coronavirus infection in pigs a simple method of estimating fifty percent end points detection and genetic analysis of porcine hemagglutinating encephalomyelitis virus in south korea synthetic shrnas as potent rnai triggers interactions of sars coronavirus nucleocapsid protein with the host cell proteasome subunit p inhibition of sars-cov replication by sirna in vitro inhibition of classical swine fever virus replication by sirnas targeting npro and ns b genes dna-mediated protection against classical swine fever virus small interfering rna inhibits sars-cov nucleocapsid gene expression in cultured cells and mouse muscles inhibition of porcine transmissible gastroenteritis virus (tgev) replication in mini-pigs by shrna effective inhibition of porcine transmissible gastroenteritis virus replication in st cells by shrnas targeting rna-dependent rna polymerase gene procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection order -he-s-ns . -ns . -e-m-n- (genbank accession no.: nc ). phev was first isolated in in canada from suckling piglets with encephalomyelitis and has since been isolated from swine worldwide. it was first isolated in primary cultures of pig kidney (pk- ) cells through visible cytopathic effects (cpe) and infectivity assays (greig et al., ; mengeling et al., ) . when piglets younger than weeks are infected with phev, their mortality rates range from to % (pensaert, ) .rna interference (rnai) is an accurate and potent gene silencing method that uses double-stranded rna (dsrnas) molecules this study was supported by the national natural science foundation of china (nos. , , , ) key: cord- -aul ifso authors: yuan, wen; wang, jing; xu, fengjiao; huang, bihong; lian, yuexiao; rao, dan; yin, xueqin; wu, miaoli; zhu, yujun; zhang, yu; huang, ren; guo, pengju title: development of a duplex real-time rt-pcr for the simultaneous detection and differentiation of theiler’s murine encephalomyelitis virus and rat theilovirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: aul ifso theiler’s murine encephalomyelitis virus (tmev) and rat theilovirus (rtv), the member of the genus cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. cardioviruses infection may cause serious complications in biomedical research. to improve the efficiency of routine screening for cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (rt-pcr) assay was developed for simultaneous detection and differentiation of tmev and rtv. the duplex assay was specific for reference strains of tmev and rtv, and no cross-reaction was found with seven other rodent viruses. the limits of detection of both tmev and rtv were × ( ) copies rna/reaction. reproducibility was estimated using standard dilutions, with coefficients of variation < . %. clinical samples were evaluated by both duplex real-time rt-pcr and conventional rt-pcr. for clinical samples， samples were positive for tmev and samples were positive for rtv using duplex real-time rt-pcr approach, whereas only samples were positive for tmev and samples were positive for rtv when conventional rt-pcr was applied. mixed infections were found in samples when analyzed by conventional rt-pcr whereas samples were found to be mixed infection when duplex real-time rt-pcr was applied. this duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses. theiler's murine encephalomyelitis virus (tmev) is a positivesense, single-stranded rna virus classified as a member of the genus cardiovirus in the family picornaviridae, along with encephalomyocarditis virus (emcv) and mengovirus (ozden et al., ; pevear et al., ) . tmev is a common cause of asymptomatic enteric infections in mice housed in non-barrier animal colonies (brownstein et al., ) . in addition, tmev spreads to the central nervous system (cns) to cause poliomyelitis (flaccid paralysis) and more rarely, encephalitis (thompson et al., ) . tmev is divided into two subgroups based on neuro-virulence in mice after intra-cerebral inoculation. the first subgroup consists of highly neuro-virulent strains represented by gdvii and fa that cause acute and fatal encephalitis. the second subgroup comprises less virulent strains, such as bean and da, that produce a persistent infection in the cns of mice that results in mononuclear cell inflammation and demyelination (lehrich et al., ; lipton, ) . this demyelinating disease has immune parameters and histopathology similar to those of chronic progressive multiple sclerosis (ms) and thus is extensively studied as an infectious model for ms. tmev infection is one of the most common viral infections detected in contemporary laboratory mouse colonies. recent reports document that laboratory rats can also be naturally and experimentally infected with the tmev virus, the newborn rats inoculated intracerebrally with the tmev virus showed clinical signs similar to those in mice, characterized by flaccid paralysis of the hind limbs, ruffled hair coat, tremor and weight loss (rodrigues et al., ) . rat theilovirus (rtv), also referred to as theiler-like virus of rats (ohsawa et al., ) , or rat encephalomyelitis virus, is a newly characterized rat cardiovirus that has been shown to be related, but genetically distinct from, other viruses in the tmev group. the first report of a tmev-like pathogen infecting rats occurred in http://dx.doi.org/ . /j.jviromet. . . - /© elsevier b.v. all rights reserved. when a small group of sprague-dawley (sd) rats were identified with central nervous system deficits and histopathologic lesions that resembled those of mice infected with tmev (mcconnell et al., ) . the original isolate, known as mhg, was reported to cause paralysis in suckling rats and mice following intracranial inoculation. it was further recognized that subclinically infected rats developed serum antibodies that cross-reacted with tmev antigens (hemelt et al., ) . the seroprevalence data indicate rtv is one of the most prevalent viral pathogens infecting research rat colonies (drake et al., ; dyson, ; livingston and riley, ; ohsawa et al., ; rodrigues et al., ) . since cardioviruses infection poses a greater epizootic threat for colony-bred mice and rats used in biomedical research, rapid diagnosis is essential to prevent transmission of infection throughout a research animal facility. viral diagnosis is based commonly on antigen or antibody detection. serologic methods such as multiplexed fluorescence immuneassay (mfia), enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence assay (ifa) has typically been used to diagnose tmev and rtv infections in rodents (drake et al., ; khan et al., ; laborde et al., ; lipton et al., ; pritchett-corning et al., ). however, serologic assays cannot detect tmev and rtv infections directly in immunodeficient strains of rodents that do not generate a humoral immune response, and the time required for host seroconversion in immunocompetent rodents may prevent rapid definitive diagnosis by serologic testing during an epizootic. serologic assays also do not give information if an agent is still present in an animal and if it is still infective. in addition, serologic assays to detect antibodies to rtv in rats have historically used mouse tmev strains as antigens, exploiting the antigenic cross-reactivity of these viruses with rtv (easterbrook et al., ; pritchett-corning et al., ) . therefore, the two groups of viruses may not be distinguishable. for the detection of contamination in biological materials, the mouse/rat antibody production (m/rap) test is still applied occasionally, although polymerase chain reaction (pcr) tests have been developed (bootz et al., ) . this test requires the use of animals, is time consuming and expensive. pcr has the advantages of high sensitivity and high specificity, which enables it to detect infections in experimental mice and biological materials and even environmental contamination. different pcr methods, either conventional or real-time pcr based assay, have been developed and have proven a useful adjunct diagnostic method for detection of tmev and rtv (bootz et al., ; drake et al., ; trottier et al., ) . more recently, multiplex real-time pcr has been reported to be as robust as simplex real-time pcr in detecting a broad range of different rodent pathogens but its application for the detection of cardioviruses has not yet been reported (pang et al., ) . the aim of this study is to develop a rapid, sensitive and specific duplex real-time rt-pcr assay for the simultaneous detection of tmev and rtv, and provide a useful tool for the routine health monitoring of these two viruses in laboratory rodents and for the screening of contaminated biological materials. theiler's mouse encephalomyelitis virus bean strain (tmev), mouse hepatitis virus a strain (mhv), reovirus type dearing strain (reo- ), rat coronavirus strain (rcv), sendai virus sendai/ strain (sendai), pneumonia virus of mice number strain (pvm) were obtained from american type culture collection (atcc). rat theilovirus y strain (rtv) and murine norovirus guangzhou/k / /chn strain (mnv) were obtained from intramural stocks, and rna for lymphocytic choriomeningitis virus armstrong strain (lcmv) were kindly provided by dr. zheng ming he, laboratory animal institute, national institutes for food and drug control. tmev, rtv, reo- , sendai, and pvm were propagated in bhk- cells (atcc ccl- ), mhv and rcv was propagated in nctc (atcc ccl . ), mnv was propagated in raw . cells (atcc tib- ). all viral stocks were stored at − • c until use. clinical samples were collected from four different sources: ( ) stool and cecum samples were collected from mice and rats in guangdong province of china during the period - for routine health monitoring services, these laboratory animals had been reared under barrier colonies; ( ) sprague-dawley (sd) rats and km mice were obtained from a non-barrier laboratory rodent colony, all animals were euthanized under co anesthesia, and the cecum specimens were collected; ( ) wild rats (rattus norvegicus) were live-trapped around the non-barrier laboratory rodent colony mentioned above, and the ceca were collected from the rats according to the same sampling procedures; ( ) different cultured cell line samples were obtained from intramural stocks. in addition, twenty cecum content and spleen samples collected from eight specific pathogen free (spf) mouse strains (balb/c, c bl/ , dba, fvb, , icr, km and nih) and two rat strains (sd and wistar) were used to evaluate specificity of the duplex real-time rt-pcr assay, these animals were reared under barrier colonies and were confirmed as serology negative for tmev and rtv by a commercial elisa kit (xpressbio, maryland, usa). subsequently, a %(w/v) suspension of all stool, cecum and spleen specimens was prepared with phosphate-buffered saline (pbs; . m, ph . ), clarified by centrifugation at g for min at • c, and stored at − • c until used. the experimental protocols were approved by the institutional animal care and use committee at guangdong laboratory animals monitoring institute. the rnas of the reference viruses and clinical samples were extracted using qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. rna concentrations were measured using a uv-vis spectrophotometer nanodrop c (thermo scientific, usa) and adjusted to about ng/l in nuclease-free h o to normalize the different extractions. all rna samples were stored in − • c until use. the whole genomic nucleotide sequences of tmev and rtv were obtained from genbank database. sequence alignments were performed using the clustal w method from the mega . program (dnastar inc., madison, wi). specific primer and probe combinations were created using primer express software (applied biosystems inc., foster city, ca, usa) for individual detection of tmev and rtv. specific primers and probe for tmev were developed from the a protein region of the viral genome (genbank accession no. x ) and labeled with fam as a fluorescent detector. primer/probe combinations for rtv were developed using sequence from the leader peptide (genbank accession no. eu ) region of the viral genome and labeled with joe as the fluorescent dye. the primers and probes used in the present study are listed in fig. and table . the duplex real-time rt-pcr was performed in a one-step format, using the one-step primescript tm rt-pcr kit (takara biotechnology co., dalian, china). a reaction mixture was prepared containing (per vial) l of sample rna, . m of each primer and . m of each probe (table ) , . l of × one-step rt-pcr buffer, . u takara ex taq tm hs, . l primescript tm rt enzyme mix ii, and the remaining volume was adjusted to l with rnase-free water. the passive reference dye rox was included in the reaction mixture. the cycling parameters for rt-pcr included min at • c for reverse transcription, s at • c for taq hs activation, followed by cycles for amplification with • c for s and • c for s. fluorescence signals were collected at the end of each amplification step. the reaction was carried out using abi equipment and software (applied biosystems inc., foster city, ca, usa). a simplex real-time rt-pcr assay was performed in the same manner as the duplex assay, except that only one set of primers/probe was included. for sensitivity determination, rna transcripts of the selected region of tmev and rtv were synthesized in vitro. the amplification of each target sequence was performed by conventional rt-pcr on template rnas from tmev (strain bean ) and rtv (strain y ) using the primers indicated in table (tmev-f /tmev-r for tmev, and rtv-f /rtv-r for rtv). the dnas obtained were gel-purified, quantified by spectrophotometry and stored at − • c until use. each dna was inserted into a pgem-t vector (promega, madison, wi, usa) using a t-a cloning strategy according to the manufacturer's instructions, and transferred into competent escherichia coli bacteria for propagation. plasmid dna was purified from insert-containing colonies, using tianprep mini plasmid kit (tiangen, beijing, china). virus-derived dna inserts were revealed by conventional pcr using specific primers (as above), the identity and integrity of the positive plasmids had been verified by sequencing. the homogeneity of the plasmid preparations was assessed by agarose gel electrophoresis. the dna concentration of each plasmid preparation was determined by spectrophotometry using a nano-drop c (thermo scientific, usa). in vitro transcription was performed using the ribomax tm large scale rna production systems (promega, madison, wi, usa) with t rna polymerase over linearized plasmid dna, following the manufacturer's instructions. transcribed rna was treated with rnase-free dnase and purified with the qiaamp viral rna mini kit (qiagen, hilden, germany). rna transcripts were stored at − • c until use. additionally, rna concentration was determined by spectrophotometry. rna copy number was determined by the following formula: amount (rna copies/l) = (x(g/l)rna × . × )/(nt transcript length × ). after the transcription, purification and rna quality evaluation, -fold series dilutions containing × to × rna copies were prepared using healthy mice total rna extracts ( ng/l). the specificity of the duplex real-time rt-pcr assay was determined by evaluation of rna extracted from positive cultures of the following rodent viral pathogens: tmev, rtv, mhv, reo- , rcv, sendai, pvm, mnv and lcmv, and from twenty cecum content and spleen samples of ten spf rodent strains. ten-fold serial dilutions of the rna standard of tmev and rtv ranged from . × to . × copies/ng of total rna were tested to determine the detection limits of the duplex real-time rt-pcr assay. the serially diluted rna standard was also used to establish a standard curve for tmev and rtv by plotting the threshold cycle (ct) and the viral copy logarithm. based upon the duplex real-time pcr standard curve, the correlation coefficient (r ) was obtained. these assays were performed in parallel and in triplicate on the same dilutions. the standard rna ranged from . × to . × copies/ng of total rna of tmev and rtv were used to evaluate the interassay and intra-assay reproducibility of duplex real-time rt-pcr. ct values were measured in multiple replicates. the threshold cycle of each concentration was obtained and calculated. for comparative purposes, conventional rt-pcr were developed for detection tmev and rtv in clinical samples. two sets of primers used for conventional rt-pcr were designed and described in table . the conventional rt-pcr was performed for the separate detection of tmev and rtv as previously described (bootz et al., ) with little modification. briefly, the first strands of cdna were synthesized with primescript tm st strand cdna synthesis kit (takara biotechnology co., dalian, china) according to the manufacturer's instructions, the pcr reaction mixture in l reaction volume comprised of l × premix taq tm (takara biotechnology co., dalian, china), . l of forward and reverse primers ( m), l of cdna, the remaining volume was adjusted with rnase-free water. the reactions were carried out using a program that included initial denaturation at • c for min, followed by cycles of denaturation at • c for s, • c for s, • c for s, and a final extension at • c for min. pcr products were analyzed by electrophoresis in % agarose gels, stained with gelred (biotium, hayward, ca, usa) and visualized by ultraviolet light. serial dilutions of each rna standards ranged from × to × copies/ng of total rna were tested with the duplex assay, the fluorescent signals were detected with × to × copies/ng of total rna; however, no specific signals were detected with × copies/ng of total rna and the negative control for the duplex assay (fig. ) . therefore, the detection limit of the duplex real-time rt-pcr assay was approximately × copies of rna per reaction for both tmev and rtv. standard curves were constructed from the ct value; the duplex assay was able to detect dilutions from left to right on the amplification plot (labeled - , red amplification curves represent tmev, and blue amplification curves represent rtv) correspond to × , × , × , × , × , × , × , and × copies/ng of total rna, respectively. the threshold cycles < are considered positive results. the × copies/ng of total rna dilution and negative control amplification plot (labeled ) did not reach the threshold value, and therefore is considered to be negative result. the detection sensitivity was × copies of rna per reaction for both tmev and rtv. each rna standard over a linear span of × to × copies/ng of total rna (fig. a, b) . the dynamic range of the assay encompassed eight orders of magnitude, with a strong linear relationship between the ct values and the log of the input number of copies (tmev, r = . ; rtv, r = . ). the slopes of each standard curve were − . for tmev and − . for rtv. the standard curves generated from the duplex assay were similar to those generated from the single assay (fig. c, d) . no obvious interference or inhibition was observed in duplex real-time rt-pcr when compared with simplex assay the specificity of the duplex real-time rt-pcr assay was validated using genomes rna of tmev, rtv, seven other rodent viruses and samples of ten spf rodent strains. in the fam channel, all samples tested negative except for tmev, while in the joe channel, all samples tested negative except for rtv (fig. ) . the results suggest that the designed primer pairs and probes were highly specific and selective for their target viruses and exhibited no cross-reactivity with other viruses. to determine whether viral genomic rna influences amplification efficiency, we tested the duplex assay in the presence of large amounts of another viral rna. in the presence of × copies/ng of rtv genomic rna, the tmev standard curves were similar to those generated from the standard dilutions alone (fig. a) . similarly, in the presence of × copies/ng of tmev genomic rna, the rtv standard curves were similar (fig. b) . these results indicate that the genomic rna does not inhibit pcr amplification each other. to evaluate intra-and inter-assay variability, a tenfold serial dilution of the mixed rna standards from × to × rna copies/ng of total rna was used in the duplex assay. ct values were measured in multiple replicates, and the mean of each ct value and standard deviation (sd) and coefficients of variation (cv) were calculated. intra-assay variability was determined using five replicates per batch, and inter-assay variability was examined by running the same standards with three replicates on four consecutive days ( table ). the coefficients of variation were less than % in the intraand inter-assays, indicating that the assay was highly reproducible. a total of clinical samples obtained from four different sources were simultaneously tested using the duplex real-time rt-pcr method and conventional rt-pcr. as shown in table , from duplex real-time rt-pcr analysis, ( . %) and ( . %) of the total clinical specimens were positive for tmev and rtv, respectively, and the viral loads of positive tmev and rtv were . × to . × copies/ng of total rna and . × to . × copies/ng of total rna, respectively (fig. ) . ( . %) and ( . %) samples were positive for tmev and rtv by conventional rt-pcr, respectively. all positive samples of tmev and rtv detected by conventional rt-pcr were positive by the duplex fig. . specificity of duplex real-time rt-pcr. the specific fluorescent signals were detected from rna of tmev and rtv. no cross-reactions were detected from mhv, reo- , rcv, sendai, pvm, mnv, lcmv, and the cecum content and spleen samples collected from ten spf mouse and rat strains. real-time rt-pcr. moreover, there are more samples positive for tmev and more samples positive for rtv by duplex realtime rt-pcr when compared with the result from conventional rt-pcr. these results indicated that duplex real-time rt-pcr was more sensitive than conventional rt-pcr. in addition, samples co-infected with tmev and rtv were all from rodent samples collected from non-barrier colony and wild colony. no co-infections of the two viruses were found in the barrier colony. different cultured cell line samples were also negative for tmev and rtv. cardioviruses infection is ubiquitous and worldwide in its distribution in colony-bred and feral mice and rats. the greater prevalence of cardioviruses in rodent had made tmev and rtv a more serious concern to biomedical researchers. since cardioviruses are easily spread by the fecal-oral route, and viruses are characterized by resistance to inactivation, periodic reintroduction, and relatively long shedding periods. elimination of these viruses from research facilities requires improved detection. the diagnosis of tmev or rtv in clinical as well as symptomless infections depends upon positive serology, m/rap, or rt-pcr amplification of viral rna. this study describes the development of a new duplex real-time rt-pcr assay for the detection of tmev and rtv using differently labeled taqman probes for each virus. the primer/probe combinations were highly specific, and no cross-reaction was detected by using seven other viral pathogens of rodent. meanwhile, the cecum content and spleen samples collected from ten spf mouse and rat strains were also negative by the duplex realtime rt-pcr assay. this clearly demonstrates that the tmev and rtv primer/probe combinations are specific to their specified virus. cell-adapted virus culture supernatant, recombinant plasmids and in vitro-transcribed rna can be used for determination of the sensitivity of real-time pcr (trottier et al., ) . in this study, the in vitro-transcribed rna containing the partial gene of the tmev and rtv virus were used to determine the sensitivity of duplex real-time rt-pcr. by using different concentrations of primers, probes, and other reagents, reaction temperatures and times, the conditions of the duplex real-time rt-pcr were optimized (data not shown). the duplex assay was sensitive enough to detect and quantify a very low copy number without mutual interference; the limits of detection of both tmev and rtv were × copies rna/reaction. the sensitivity of this duplex assay is equivalent to that of the simplex assay. nevertheless, duplex pcr takes the same amount of time and effort but produces more data. the system was also specific and sensitive even in the presence of large amounts of another viral rna. the low mean cv of the duplex real-time rt-pcr indicated that this assay was able to generate reproducible results. furthermore, clinical samples collected from four different sources were simultaneously evaluated using the duplex realtime rt-pcr and conventional rt-pcr. the results showed that the duplex real-time rt-pcr assay was more sensitive and effective than conventional rt-pcr in the examination of clinical samples, where samples and samples were negative by conventional rt-pcr, respectively, but they were positive by the duplex real-time rt-pcr. the tmev and rtv positive rate of clinical samples obtained from barrier colony-raised mice or rats was . % ( / ) and . % ( / ), respectively, which is much higher than the rate of . - . % reported in north america and europe (pritchett-corning et al., ), indicating that cardioviruses had a high prevalence in china in the past years. the positive rate of non-barrier colony and wild colony was higher than that of barrier colony. in additional, mixed infection of tmev and rtv was detected in non-barrier raised laboratory rodent and wild rats. as wild mice and rats are the natural host of cardioviruses (lipton et al., ; ohsawa et al., ; rodrigues et al., ) , it is necessary to prevent introduction of non-barrier colony raised or feral mice and rats, or contaminated bedding from an outside source, into a clean (barrier conditions) laboratory rodent colony. thus, strict adherence to standard biological containment measures and barrier conditions is advisable. in conclusion, duplex real-time rt-pcr assay developed in this study can be used to specifically and simultaneously detect tmev and rtv. it is a convenient, rapid, and sensitive diagnostic tool for routine health monitoring of viral infection in laboratory mice and rats, and for the screening of contaminated biological materials. comparison of the sensitivity of in vivo antibody production tests with in vitro pcr-based methods to detect infectious contamination of biological materials duration and patterns of transmission of theiler's mouse encephalomyelitis virus infection differential susceptibility of sd and cd rats to a novel rat theilovirus vivo tropisms and kinetics of rat theilovirus infection in immunocompetent and immunodeficient rats management of an outbreak of rat theilovirus a survey of rodent-borne pathogens carried by wild-caught norway rats: a potential threat to laboratory rodent colonies comparison of mhg virus with mouse encephalomyelitis viruses simultaneous serodetection of highly prevalent mouse infectious pathogens in a single reaction by multiplex analysis evaluation of an indirect enzyme-linked immunosorbent assay for routine screening of theiler's murine encephalomyelitis virus antibodies in mice colonies demyelinative myelopathy in mice induced by the da virus serological evidence that mus musculus is the natural host of theiler's murine encephalomyelitis virus theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination diagnostic testing of mouse and rat colonies for infectious agents isolation and characterization of a neurotropic agent (mhg virus) from adult rats: proceedings of the society for experimental biology and medicine genetic analysis of a theiler-like virus isolated from rats theiler's virus genome is closely related to that of encephalomyocarditis virus, the prototype cardiovirus analysis of the complete nucleotide sequence of the picornavirus theiler's murine encephalomyelitis virus indicates that it is closely related to cardioviruses contemporary prevalence of infectious agents in laboratory mice and rats theiler's murine encephalomyelitis virus in nonbarrier rat colonies a spontaneous epizootic of mouse encephalomyelitis enhanced detection of theiler's virus rna copy equivalents in the mouse central nervous system by real-time rt-pcr key: cord- -fvq hezk authors: hornyák, Ákos; bálint, Ádám; farsang, attila; balka, gyula; hakhverdyan, mikhayil; rasmussen, thomas bruun; blomberg, jonas; belák, sándor title: detection of subgenomic mrna of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (p-sg-qpcr) date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: fvq hezk feline infectious peritonitis is one of the most severe devastating diseases of the felidae. upon the appearance of clinical signs, a cure for the infected animal is impossible. therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (fcov) and the manifestation of feline infectious peritonitis is of paramount importance. in the present work, a novel real-time rt-pcr method is described which is able to detect fcov and to determine simultaneously the quantity of the viral rna. the new assay combines the m gene subgenomic messenger rna (sg-mrna) detection and the quantitation of the genome copies of fcov. in order to detect the broadest spectrum of potential fcov variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (priproet) principle. this technology was chosen since priproet is very robust to tolerate the nucleotide substitutions in the target area. therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. the detection specificity of the new assay was proven by positive amplification from a set of nine different fcov strains and negative from the tested non-coronaviral targets. examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. the sensitivity of the p-sg-qpcr method was high, since as few as genome copies of fcov were detected. the quantitative sg-mrna detection method revealed more than – , times increase of the m gene sg-mrna in organ materials of feline infectious peritonitis cases, compared to those of the enteric fcov variants present in the faeces of normal, healthy cats. these results indicate the applicability of the new p-sg-qpcr test as a powerful novel tool for the better detection and quantitation of fcov and for the improved diagnosis of feline infectious peritonitis, this important disease of the felidae, causing serious losses in the cat populations at a global scale. feline infectious peritonitis is one of the most severe devastating diseases of the felidae. upon the appearance of clinical signs, a cure for the infected animal is impossible. therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (fcov) and the manifestation of feline infectious peritonitis is of paramount importance. in the present work, a novel real-time rt-pcr method is described which is able to detect fcov and to determine simultaneously the quantity of the viral rna. the new assay combines the m gene subgenomic messenger rna (sg-mrna) detection and the quantitation of the genome copies of fcov. in order to detect the broadest spectrum of potential fcov variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (priproet) principle. this technology was chosen since priproet is very robust to tolerate the nucleotide substitutions in the target area. therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. the detection specificity of the new assay was proven by positive amplification from a set of nine different fcov strains and negative from the tested non-coronaviral targets. examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. the sensitivity of the p-sg-qpcr method was high, since as few as genome copies of fcov were detected. the quantitative sg-mrna detection method revealed more than - , times increase of the m gene sg-mrna in organ materials of feline infectious peritonitis cases, compared to those of the enteric fcov variants present in the faeces of normal, healthy cats. these results indicate the applicability of the new p-sg-qpcr test as a powerful novel tool for the better detection and quantitation of fcov and for the improved diagnosis of feline infectious peritonitis, this important disease of the felidae, causing serious losses in the cat populations at a global scale. published by elsevier b.v. feline infectious peritonitis is currently the leading cause of infectious death among young domestic and wild felidae. the disease has a worldwide distribution, especially in cats originated from purebred catteries (pedersen, ; foley et al., ) . the causative agent of feline infectious peritonitis, feline coronavirus (fcov), belongs to the coronaviridae family within the order nidovirales (almeida and tyrell, ; siddell et al., ) . fcov, together with human, porcine, and canine coronaviruses is a member of alpha coronavirus genus (de groot et al., ) . regarding pathogenicity, fcov comprises two biotypes: the feline infectious peritonitis viruses (fipv) and the feline enteric coronaviruses (efcov). the existence of at least two serotypes of fcov (types i and ii) has been shown based on in vitro neutralisation assays using monoclonal antibodies. each serotype includes viruses of both the fipv and efcov biotypes (hohdatsu et al., a,b) . type i and ii viruses differ mainly in their in vitro growth characteristics, as the members of the serotype i can rarely if ever be propagated in cell-cultures. in the field, the prevalence of fcov type i appear to be higher, as approximately - % of feline infectious peritonitis cases are due to infection with type i viruses (hohdatsu et al., ; herrewegh et al., ; addie et al., ; kummrow et al., ) . biotype efcov is not a significant cause of morbidity in cats. it may produce mild enteritis, but most experimental or natural infections remain subclinical. it has been observed that the incidence of feline infectious peritonitis in a cat population is strongly correlated with the efcov → fipv mutation rate (vennema et al., ) . fipv causes a fulminant and fatal disease of cats with manifestations of anorexia, chronic fever, and malaise. in addition, ocular and neurological disorders can occasionally occur. there is a vital need to develop reliable and robust laboratory diagnostic method for the detection and identification of fipv biotype in all doubtful cases, considering the profoundly different biology and clinical consequences caused by the two biotypes of fcov. initially, the determination of antibody titre by indirect immune fluorescence method (burleson et al., ) served for diagnosis followed by the detection of viral rna from blood (kennedy et al., ) . the reliability of these diagnostic approaches were questioned due to the inconsequent correlation of the positive results and the disease outcome. simons et al. ( ) published a more sophisticated method, targeting subgenomic mrna (sg-mrna) production of the virus, detected by conventional rt-pcr. this method was supposed to detect only the intensively replicating viral biotype, since viral sg-mrnas are served to express the viral structural protein genes. however, the suitability of this test to detect fipv was questioned by can-sahna et al. ( ) on the basis of the high ratio of positive results obtained from the samples of clinically healthy cats. these observations indicate that an assay capable of fcov sg-mrna quantitation in the different organs and body fluids would be of very high importance, considering the fact that low-level virus replication occurs even in the case of efcov infection (herrewegh et al., ) , and hopefully the exact determination of the viral replication rate by measuring the viral sg-mrna level would be decisive for the establishment of a correct diagnosis (de groot-mijnes et al., ) . considering this important diagnostic need, a real-time sg-mrna detection and measuring system, termed p-sg-qpcr was developed, as a novel tool identification and quantitation of fcov in order to obtain an improved system for the reliable diagnosis of feline infectious peritonitis. the novel assay is based on the fret principle (förster resonance energy transfer; förster, ) and uses the primer-probe energy transfer (priproet) technology. this robust real-time pcr technology was previously successfully applied to detect a wide variety of viruses comprising vesicular disease viruses, hepatitis e virus, classical swine virus porcine reproductive and respiratory syndrome virus and porcine circovirus (rasmussen et al., ; hakhverdyan et al., ; bálint et al., ) . compared to the most wide-spread real-time pcr system, the taqman method, priproet is providing a higher flexibility in the detection of varying target nucleic acids. priproet is able to overcome multiple mutations located in the targeted region of the viral genome and to amplify a wide range of nucleic acids despite of the variations in the targeted regions (rasmussen et al., ) . this robust feature made the priproet technique suitable for the detection of the fcov, a highly variable rna virus. in this study, the robustness and flexibility of priproet was combined, providing a wide range detection of the virus variants, with the capability to quantitate subgenomic mrna, allowing the quantitative assessment of virus replication, thus the estimation of the development of feline infectious peritonitis. the quantitative realtime pcr (p-sg-qpcr) enabled us to detect and differentiate a wide range of closely related coronaviruses such as fcov, canine coronavirus (ccov) and transmissible gastroenteritis virus (tgev) and to estimate the tendency of virus replication in various organs, by a single method. thus, the p-sg-qpcr assay was found as a powerful novel tool for the improved diagnosis of feline infectious peritonitis. a collection of coronaviruses of human, feline, canine, porcine, bovine, murine and avian origin, summarised in table , was included in the specificity, sensitivity and reproducibility tests of the p-sg-qpcr assay. faecal samples from a total of healthy domesticated cats were collected in sweden in . small intestine, mesenteric lymph node, spleen, liver, lungs and kidney samples of nine diarrhoeic cats, previously diagnosed as fcov infected were also included in the tests. furthermore, a total of organ samples, seven body fluids, and four faecal samples obtained from swedish and five hungarian cats perished with feline infectious peritonitis. a collection of separated leukocyte samples obtained from clinically possible feline infectious peritonitis cases, collected in hungary between and , and was also subjected to molecular examination. diagnoses were achieved on the results of pathological, histopathological examinations and positive findings of immunohistology and gel based conventional rt-pcr for feline infectious peritonitis (kiss et al., ) . the genomic rna from all the above-mentioned samples was tested by the sybr green rt-qpcr described by escutenaire et al. ( ) , while the sg-mrna levels were examined by the p-sg-qpcr assay. approximately g pieces of the faecal samples and organs were homogenised in sterile ceramic mortars using sterile quartz sand. they were diluted in ml sterile phosphate-buffered saline (pbs), and then were centrifuged at × g for min to remove cell debris and bacterial contamination. the supernatant was collected and used for virus isolation and pcr. after homogenisation, samples were stored at − • c. feline leukocytes were purified from whole blood on histopaque- (sigma-aldrich, saint louis, mo, usa) according to the manufacturer's recommendations. the cells were resuspended in pbs to the original volume of the whole blood they originated from. one part of the sybr green positive specimens, including feline faecal samples collected from healthy cats and organ samples of five recently perished cats diagnosed with feline infectious peritonitis in sweden were subjected to standard virus isolation procedure in -well tissue culture plates (greiner bio-one gmbh, frickenhausen, germany), incubated for h at • c in a % co atmosphere. following two blind-passages, cells were scraped off in l supernatant, and examined by the genomic and subgenomic pcr assays described above to detect virus replication. strain fipv df- was grown in felis catus whole foetus cells (fcwf- ) in minimal essential medium (emem) (sigma-aldrich) containing mm l-glutamine, . g/l sodium bicarbonate, . mm non-essential amino acids, and . mm sodium pyruvate, supplemented with % foetal calf serum (fcs). three-day-old cell cultures were split into : ratio; the remaining cells were adjusted to a concentration of × cells/ml and plated into -well plates (greiner), incubated overnight at • c in a % co atmosphere. on the next day cells were infected with serial ten-fold dilutions of fipv df- , in four wells in parallel, incubated for h at • c allowing the virus to adsorb to the cell receptors and then the inoculates were replaced with emem containing % fcs. virus titre was determined on the basis of cytopathic effect (cpe) observed on the third day post inoculation by reed-muench method (reed and muench, ) . the titration was repeated three times under the same conditions. viral rna was isolated from cat and dog faecal samples, homogenised organ samples of perished cats and feline leukocytes with qiaamp viral mini kit (qiagen, hilden, germany) according to the manufacturer's recommendations. l of the % (w/v) suspension of organ and faeces homogenates, as well as the leukocyte suspension were diluted with l pbs before rna extraction. after extraction, the volume of eluted rna was complemented to l. the concentration of the extracted samples' total rna-s was between and ng/l. the highly conserved membrane (m) gene regions of feline, canine and one porcine coronaviruses were aligned with clustal x . program in order to design the reverse primer and probe capable of detecting both serotypes of fipv. the reverse primer was modified with a donor fluorophore ( carboxyfluorescein, fam) at the end. the subgenomic forward primer was designed to bind to the leader sequence of the coronaviral sequences available in the genbank. the probe was designed to bind upstream of the reverse primer without any nucleotide gap, and was labelled with a reporter fluorophore (texas red, txr) on its end, allowing the real-time detection of the bps long pcr product. gc content, stability, primer-dimer and hairpin formation were analysed by the oligo analyzer . (http://www.idtdna.com). primers and probe were synthesised and hplc purified by thermo electron gmbh (http://www.thermohybaid.de). sequences of the primers and probe are summarised in table . for performing the p-sg-qpcr assay the titanium tm one-step rt-pcr kit (clontech laboratories, palo alto, usa) was applied. the amplification reaction parameters (primer and magnesium concentration, annealing temperature and period, number of cycles, melting temperatures of the reference viruses) were optimised by titration of the different variables in order to achieve low cycle threshold (c t ) values and high fluorescence signal. after optimisation, the reaction mix was as follows: × one-step buffer, . mm dntps, nm forward primer, nm reverse primer labelled with fam, nm probe labelled with txr, u of recombinant rnase inhibitor, . rxn × titanium tm taq rt enzyme mix and l template rna in a total reaction volume of l. the themostabilizing reagent, the gc-melt tm , and the oligo (dt) primer were not included in the reaction mix to gain more intensive fluorescence signal. all reactions were run in the corbett research rotor-gene real time amplification system (rg- , corbett research, mortlake, nsw, australia), the thermal profile initiated with reverse transcription at • c for h, followed by initial denaturation at • c for min, and cycles of [ • c for s, • c for s, • c for s]. fluorescence signal data were collected for s after the primer annealing, where the wavelength of the source was - nm and the detector was nm. after cycling, melting point analysis was used to confirm the specificity of amplification at - • c with s holds at each elevation step of • c. the nine reference feline coronavirus strains, the three ccov strains; / , cb/ , / and porcine tge purdue strain were involved in the specificity tests. the melting point analysis reveals grouping of the viruses into four different genotypes in this very conserved region of the m gene. the four colours denote the four melting points of the genotypes. a positions refer to the genome of - fipv strain (ay ). b according to escutenaire et al. ( ) . positions refer to the genome of sars tor (ay ). the sensitivity of the system was determined by using known amounts of recombinant rna prepared from fipv df- as follows: conventional rt-pcr using qiagen one-step rt-pcr kit (qiagen) was carried out with the unlabelled version of the priproet primers. a specific t promoter sequence was added to the end of the forward primer. the pcr product was gel purified using the qiaquick gel extraction kit (qiagen, hilden, germany). the purified dna samples were in vitro transcribed using megascript ® t kit (ambion, austin, tx, usa). the rna concentration was determined with nanodrop nd (nanodrop, wilmington, de, usa). the standard curve was generated from a serial ten-fold dilution ( - copy numbers) of the recombinant rna in rnase free water (ambion, huntingdon, uk). furthermore, in order to assess the detection limit of subgenomic mrnas, sensitivity was evaluated from a serial ten-fold dilution from to of fipv df- grown in fcwf cell culture. inoculation of the cells was followed immediately by rna extraction of the different virus dilutions. virus titre based on cpe in fcwf was compared to the characteristic parameters of the assay: threshold cycle (c t ), fluorescents signal, and melting point. the specificity of the assay targeting the m gene subgenomic mrna was verified by gel electrophoresis, sequencing the amplicons and by melting point analysis. subsequently, the capability of the pcr assay to detect various fcov strains was tested. nine reference feline coronavirus strains representing both serotypes and biotypes were subjected to the specificity tests. on the other hand, non-feline coronaviruses were tested to exclude the crossreactivity of the assay, including human, canine, porcine, avian, bovine and murine coronavirus strains, representing all the three genus of coronavirinae (table ) . the efficiency of the assay was calculated using the equation of e = (− /m) − , where "e" is the amplification efficiency and "m" is the slope of the standard curve. all tests for sensitivity and specificity were performed in triplicate in order to assess the intra-assay variability. inter-assay variability was calculated in two different ways: ( ) comparison of the obtained c t values of the standard samples included in each experiment; ( ) examination the c t values of the same set of rna samples in three distinct runs. no signs of virus induced cpe were found in the case of the efcov positive faecal samples and feline infectious peritonitis positive organ samples after the third passage. neither the sybr green rt-qpcr nor the p-sg-qpcr assay could display fluorescent signals and characteristic melting points during the examination of cell culture supernatants for the detection of their genomic and subgenomic rnas. the mean titre of fipv df- strain reached . tcid / l (/ml) based on the typical cpe, such as cell fusion and rounding of cells at the edges of the cythopathic foci. the sensitivity of p-sg-qpcr was determined by generation of standard curves from a serial ten-fold dilution of both the recombinant rna and fipv df- grown in fcwf cell culture. the detection of the different dilutions of the recombinant rna was linear in the range of . × up to . × copies per reaction (fig. ) . this result indicated a sensitivity of - viral genome copies per pcr assay. the detection limit of the p-sg-qpcr test for subgenomic mrna detection from the fcwf cell cultured df- virus strain was tcid / l. the specificity of amplification was confirmed by gel electrophoresis of the p-sg-qpcr amplicons, followed by sequencing (data not shown). the p-sg-qpcr was able to detect all nine reference strains of fcov as well as tgev and ccov. the melting point analysis revealed four different genetic variants in this very conserved region of the m gene with the following average temperature values: ( ) . • c (ucd , ucd , ucd , black, - , df- ); ( ) . • c (ucd ); ( ) . • c (nor , - , ccov cb/ and / ); ( ) . (tgev purdue) (table ; fig. ). the efficiency of the assay was defined from the standard curve that displayed a linear inversely proportional relationship between the logarithmic amount of copy concentration and the c t of the original samples. reaction efficiency was found to be with an r value of . (fig. ) . the intra assay variability was assessed using different amounts of rnas of the nine feline cov strains. these samples were used in three replicates in the same sg-qpcr run and the intra-assay variability was obtained from two statistical table . parameters: standard deviation was . and the standard error of the mean (sem) was . . the inter assay variability was determined by the same rna set testing it three times and the standard deviation was obtained as . , the standard error of the mean (sem) was . (data not shown). the feline faecal samples from sweden were analysed for the presence of fipv-specific nucleic acid by both sybr-green qpcr and by the new p-sg-qpcr method, in parallel. both assays revealed the presence of fcov in several samples, but to a different extent. the sybr-green method detected positives ( . %), while the p-sg-qpcr assay revealed only ( . %). the c t values of the priproet positive samples varied between . and . (mean: . ; sd: . ) reflected markedly different sg-mrna copy number in the intestines of the cats: × - × copies/ l % (w/v) faeces suspension. all the nine diarrhoeic fcov shedder cats were sybr-green positive, but only five of them proved to be p-sg-qpcr. the c t value of the fcov positive samples varied between . and . (mean: . ; sd: . ), which is equivalent with approximate × - × copies/ l % (w/v) faeces suspension (table ) . table the ct values and the melting points of the nine reference coronavirus strains obtained with the p-sg-mrna qpcr. the representatives of the four genotypes can be seen in the alignment with violet, yellow, green and blue backgrounds. type c t pea k none of spleen, liver, lungs and kidney samples of the nine fcov positive cats showed positive results when tested with the two realtime pcr methods. the feline organ samples representing cats succumbed to feline infectious peritonitis in sweden and hungary were analysed by sybr-green qpcr, while only organ samples by the p-sg-qpcr. a total of ( . %) of samples analysed by both methods were positive by sybr-green qpcr and ( . %) by the p-sg-qpcr. the p-sg-qpcr c t values ranged between . and . (mean: . ; sd: . ), which is equivalent with × - × copies/ l % (w/v) tissue suspension. the detailed analysis of the feline infectious peritonitis positive cat samples by sybr-green qpcr method revealed that the following organs harbour fcov most frequently: lungs / ( %), liver / ( %), kidney / ( . %), mesenteric lymph node / ( %), spleen / ( %) and gut / ( . %). based on the results obtained from the newly developed p-sg-qpcr, the list of organs most frequently harbouring sg-mrna in quantitative order is the following: mesenteric lymph node / ( %), spleen / ( . %), lung / ( . %), liver / ( %), bronchiolar lymph node / ( . %), gut / ( . %), kidney / ( . %). analysis of body fluids and excreta collected from the carcasses gave the following results: heart blood / ( %), ascitic fluid / ( %) and faeces / ( %). interestingly, the three tonsils involved in the examinations consequently remained fcov negative with both two qpcr methods; / ( %). the p-sg-qpcr c t values in the gut, the primary replication site of fcov reflected significantly lower viral replication: the values ranged between . and . (mean: . ; sd: . ), which is equivalent with × - × copies/ l % (w/v) faeces suspension ( table ) . the melting point analysis revealed only . • c difference regarding the organs of the same carcass, but resulted in four different groups regarding the different animals: . • c average temperature (one cat of eight, . %), . • c ( / , %), . • c ( / , %) and . • c ( / , . %). the obtained p-sg-qpcr c t values revealed higher subgenomic m gene mrna transcription levels in the different organs compared to those of the faecal samples. this phenomenon can be explained by the lower cell density of the faecal samples, or the lower replication rate of the virus in the intestinal epithelial cells. furthermore, the c t values of the sg-mrna positive gut samples demonstrated a much smaller deviation compared with those of the organ samples. the leukocyte specimens of clinically sick cats displaying fever, lymphocytopenia, hypergammaglobulinaemia, in a few cases enlargement of the abdomen were subjected initially to the sybr-green qpcr method revealing positive cases ( . %). in contrast to the previous findings, the p-sg-qpcr led to the same result. the obtained c t values ( . , . , . and . respectively) correspond to approximately × - × copies/ l leukocyte suspension (table ) . one sample showed c t value of . . as the c t can be excluded from this series due to its abnormal high value, the mean value of . corresponds to copies/ l leukocyte suspension of sg-mrna in the leukocytes of sick cats suffering with feline infectious peritonitis (confirmed earlier by pathological, histopathological, immunohistological and rt-pcr examinations). the melting points of the five amplicons were . • c, . • c, . • c, . • c and . • c. the follow-up investigations revealed that all five cats perished with fip in days after the p-sg-qpcr examinations (data not shown). the diagnosis of feline infectious peritonitis, this major viral disease of the felidae is extremely difficult, due to several factors in the complicated infection biology of fcov. first, no exact genetic marker(s) has been identified yet in the viral genomes that would allow the differentiation of the two fcov biotypes: fipv and efcov. second, during the viral replication genomic alterations are generated continuously by this virus, leading to the rapid appearance of a variety of viruses in the body of the host. since reliable means for the molecular determination of the differences between the avirulent and virulent variants of fcov are not available, the diagnosis of feline infectious peritonitis should address other biological characteristics of the virus ways today. in the present work, two peculiar features of the infection biology of feline coronaviruses were utilised. on the one hand, the enhanced capability of the fipv biotype to replicate intensively in various organs and tissues of the host animal was exploited (stoddart and scott, ; kipar et al., ; rottier et al., ) . on the other hand, the ability of nidovirales to initiate sg-mrna synthesis during replication phase was addressed. in the past few years, by the use of sg-mrna pcr techniques (gillim-ross et al., ; simmons et al., ) , replicating covs have been detected successfully. for this reason, to facilitate correct feline infectious peritonitis diagnosis and prognosis, the present study aimed at developing a p-based real-time pcr assay to detect the virus and to determine the level of virus replication, in a single diagnostic platform. the flexibility of the p-sg-qpcr technique is indicated by the fact that cov strains even with three mismatches in their genomes at the primer and probe binding sites could simultaneously be detected by this novel method (table ) , without decrease of the fluorescent signal. therefore, the p-sg-qpcr method may be regarded as a real-time amplification system with unique capability for the identification of all previously published fcov m gene sg-mrnas. table alignment of feline, canine and porcine cov strains; viruses even with substitutions could be demonstrated by the sg-mrna detecting qpcr system. sense, unlabeled primer region with grey, m probe (labelled with txr) region with violet, m antisense primer (labelled with -fam) region with green background are depicted. ucd- sequence data on the m gene has not been available in the genbank yet. the representatives of the three other genotypes can be seen in the alignment with yellow, green and blue backgrounds (see table ). cov strains belonging to all three genus of coronavirinae did not give specific fluorescent signals, excluding the cross reactivity of the test. the sensitivity of the assay was determined in two different ways, and the detection limit proved to be as low as copies in each case. the efficiency of the p-sg-qpcr test was calculated by the establishment of two separate calibration curves: one from serial -fold dilution of the recombinant rna, while the other from the serial -fold dilution of fipv df- virus grown on fcwf cell line. both standard curves displayed a linear detection of sg-mrna in the range of - range with a correlation coefficient (r ) of . and efficiency of %, and r of . and efficiency of %, respectively. difference between the two efficiency values may be attributed to the superior purity of the recombinant rna standard. these results revealed a significant difference between rate of positive results on feline faecal samples using the genomic sybr green and the p-sg-qpcr assays. efcov can be detected in a high proportion of intestinal samples of cats by pcr (herrewegh et al., ; foley et al., ) . in contrast, the limited number of psg-qpcr positive results may indicate a relatively low percentage of animals with extensive virus replication level in the intestinal tract. since the p-sg-qpcr assay is demonstrating intensive virus replication, it is tempting to speculate that this test is capable of identifying the persistent virus-shedder cats, which contribute to the maintenance cov infection in the catteries, and their ratio can reach even % (addie et al., ) , and this group includes the cats with the risk of the fatal fipv variant emerging. however, the majority of cats shed efcov intermittently for weeks or months, in table frequency of genomic fcov positive samples by sybr green method and frequency of fcov sg-mrna positive samples by priproet method completed by sg-mrna copy number. the copy number data refer to l of % (w/v) tissue and faeces suspension or l of leukocyte suspension. sybrgreen: priproet sg-mrna load (copies/ l sample) faecal samples from healthy cats / ( . %) / ( . %) × - × faecal samples from feline enteric coronavirus positive diarrhoeic cats / ( %) / ( . %) × - × faecal samples from cats succumbed with feline infectious peritonitis / ( %) / ( %) × - × organ samples from feline enteric coronavirus positive diarrhoeic cats / ( %) / ( %) organ samples from cats succumbed with feline infectious peritonitis / ( . %) / ( . %) × - × leukocytes (heart blood) from cats succumbed with feline infectious peritonitis / ( %) / ( %) × - × leukocytes from possible feline infectious peritonitiscases / ( . %) / ( . %) × - × some cases at very low level (addie and jarrett, ; lutz et al., ) . hence, to avoid false negative diagnosis, negative results of the p-sg-qpcr method should be interpreted with caution, and in these cases this assay is a method of choice complementing the widely used genomic real-time qpcr assays in the determination of fcov status. another explanation for the surprisingly low copy number of the fcov sg-mrna in the gut may be explained with the balanced immune response of the host animals that keeps fcov replication at low level, and as a consequence, the subgenomic rna levels may reach much lower rates then their genomic counterparts. whichever explanation is true, evaluation of quantitative pcr results has to take into consideration the inhibitory compounds of the faecal samples (dye et al., ) . the different melting point values of the faecal samples indicating variable number of nucleotide substitutions at the probe-binding site (rasmussen et al., ; hakhverdyan et al., ) revealed at least four different fcov virus genetic variants in the targeted region of the m gene. comparing these melting points with those of the reference viruses it can be concluded that the most frequent virus types in the swedish collection may be the ucd like, the - -like fcov, the nor and / ccov strains. although recombination cannot be excluded in some of this fcovs the majority of the viruses detected must correspond with these strains, because the m gene is the most conserved structural protein gene. the spleen, liver, lungs and kidney samples of the nine fcov positive cats were all negative with the applied two different real-time pcr methods, reflecting an fcov negative status of these organs. analysis of the organ samples obtained from feline infectious peritonitis cases revealed a high copy number of fcov, which is not surprising, since several earlier publications reported enhanced virus replication in the cats diseased with feline infectious peritonitis. the negative results in the case of a few samples are supposed to be a consequence of the different distribution of fipv in various organs. the most frequently affected organs are the liver, the lungs, the kidneys and mesenteric lymph nodes with - % positive result rate. this is a reflection of their dense vascularisation, rendering them particularly susceptible to the virus that is transported by the infected monocytes and macrophages. a slight difference was observed in the same organs regarding their sg-mrna contents, where the order of frequency was: mesenteric lymph nodes, spleen, lungs and liver ranging from % to % positive result. concluded by their enhanced sg-mrna content these are the main susceptible organs, which the fcov possessing its altered genome can first colonise and then intensively multiply. the high rate sg-mrna positive results observed in the body fluids demonstrate the value of the clinical and pathological samples allowing readily diagnosing feline infectious peritonitis. the p-sg-qpcr c t values revealed an average of times more coronaviral sg-mrna in the feline infectious peritonitis positive samples than in the faecal samples of the clinically healthy fcov young shedder animals, in some organs (liver, spleen) even - , times more, proving the very high virus multiplication rate in the diseased cats. interestingly, the basic organ of fcov replication the gut, displays virus depletion in all examined feline infectious peritonitis cases, the maximal virus replication occurred at a remarkably low level, never even exceeding copy numbers. these data suggest that the mutated fcov variants are prone to abandon this organ after acquiring successful access to other organs via monocytes and macrophages. the interesting phenomenon that the same melting point detected in the different organs within the body may be explained by the absence of concomitant cov infection. compiling these findings, it can be hypothesised that once an fcov variant had colonised the enteric system of a cat, the virus could either be eliminated, or kept under control by the specific immune response of the host animal, or the continuous/ intermittent high level virus replication in the worst case lead to the generation of the highly virulent fatal fipv variant by mutation. the different melting points of the fipv amplicons obtained from different carcasses suggest the same phenomenon that was observed in the case of the fcov faecal sample collection, i.e. at least four different fipv variants existed among cats that have suffered from and succumbed to fip. the swedish fipv cases revealed a melting point between nor , / , - and the tgev purdue strains, and may represent a still non-characterised fipv variant regarding the targeted stretch of the m gene. on the contrary, the five hungarian fipvs can be classified into four different groups based on their melting point values: (i) resembling to the ucd ( %); (ii) two comparable to the nor , / and - ( %); (iii) similar to the swedish strains ( %); and (iv) having a melting point similar to the tgev ( %). this difference is in accordance with the previously described existence of different genetic patterns of fcov isolates in geographically diverse areas. the p-sg-qpcr analysis of the leukocytes revealed a relatively low prevalence of feline infectious peritonitis among the uncertain cases, underlines the limited capability of the exclusive use of clinical and conventional laboratory examinations for the establishment of a correct diagnosis. the consequently high fcov copy numbers of the leukocyte samples demonstrate (with one exception) the feasibility of this diagnostic tool to obtain a correct prognosis from the blood samples of live animals. in contrast to the faecal and organ samples, the same melting point was obtained from the leukocytes, which raised several questions that can be answered only by the examination of a sufficient amount of blood samples. taken together, these results indicate the applicability of the new p-sg-qpcr test as a novel assay for the better detection and quantitation of fcov replication in various organs, and/or in the separated monocyte-macrophage fractions of the diseased cats. in combination with genomic quantitative pcr methods, the assay also provides a practical tool for the identification of persistently infected strong shedder individuals in a closed cat populations e.g., in catteries. furthermore, this method facilitates the early diagnosis of fip, allowing a prompt treatment of the infected cats. considering these practical strengths, this assay provides a practical novel tool for the better understanding of the infection biology as well as for improved control of feline infectious peritonitis, this mysterious viral disease of the felidae. use of a reverse-transcriptase polymerase chain reaction for monitoring the shedding of feline coronavirus by healthy cats persistence and transmission of natural type i feline coronavirus infection the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture virology: a laboratory manual development of primer-probe energy transfer real-time pcr for the detection and quantification of porcine circovirus type the detection of feline coronaviruses in blood samples from cats by mrna rt-pcr revision of the family coronaviridae. taxonomic proposal of the coronavirus study group to the ictv executive committee natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease evaluation of real-time rt-pcr for the quantification of fcov shedding in the faeces of domestic cats sybr green real-time reverse transcription-polymerase chain reaction assay for the generic detection of coronaviruses patterns of feline coronavirus infection and faecal shedding from cats in multiple-cat environments zwischenmolekulare energiewanderung und fluoreszenz discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-pcr development of a real-time pcr assay based on primer-probe energy transfer for the detection of swine vesicular disease virus the molecular genetics of feline coronaviruses: comparative sequence analysis of the orf a/ b transcription unit of different biotypes persistence and evolution of feline coronavirus in a closed cat-breeding colony feline coronavirus type ii strains - and - originate from a double recombination between feline coronavirus type i and canine coronavirus characterization of monoclonal antibodies against feline infectious peritonitis virus type ii and antigenic relationship between feline, porcine, and canine coronaviruses a study on the mechanism of antibody-dependent enhancement of feline infectious peritonitis virus infection in feline macrophages by monoclonal antibodies the prevalence of types i and ii feline coronavirus infections in cats morphologic features and development of granulomatous vasculitis in feline infectious peritonitis correlation of genomic detection of feline coronavirus with various diagnostic assays for feline infectious peritonitis prevalence and genetic pattern of feline coronaviruses in urban cat populations feline coronavirus serotypes and : seroprevalence and association with disease in switzerland fcov shedding pattern of privately owned cats under field conditions experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd , fipv-ucd , and fipv-ucd virologic and immunologic aspects of feline infectious peritonitis virus infection development of a novel quantitative real-time rt-pcr assay for the simultaneous detection of all serotypes of foot-and-mouth disease virus quantitative multiplex assay for simultaneous detection and identification of indiana and new jersey serotypes of vesicular stomatitis virus a simple method of estimating fifty percent endpoints acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein a mrna pcr for the diagnosis of feline infectious peritonitis isolation and identification of feline peritoneal macrophages for in vitro studies of coronavirus-macrophage interactions feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses the work was financially supported by the formas grants , by the award of excellence (excellensbidrag) provided to s.b. by the swedish university of agricultural sciences (slu), by the jános bolyai fellowship of the hungarian academy of sciences and by the tÁmop- . . .b- / "development of a complex educational assistance/support system for talented students and prospective researchers at the szent istván university" project. grateful thanks are due to swedish colleagues stina ekman, bodil ström-holst, peter thorén and to the cat owners from all over sweden for providing us a valuable collection of clinical samples for detailed investigations. cat organ and leukocyte samples were kindly provided in hungary by colleagues béla lakatos and zoltán demeter. moreover, the authors thank colleagues canio buonavoglia, dave cavanagh, lia van der hoek, ali kheyar, istván kiss, matthias niedrig, and peter rottier for supplying several coronavirus reference strains and isolates. key: cord- - o jo uk authors: chen, hao-tai; zhang, jie; sun, de-hui; chu, yue-feng; cai, xue-peng; liu, xiang-tao; luo, xue-nong; liu, qing; liu, yong-sheng title: rapid detection of porcine circovirus type by loop-mediated isothermal amplification date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: o jo uk a method of loop-mediated isothermal amplification (lamp) was employed to develop a rapid and simple detection system for porcine circovirus type (pcv ). the amplification could be finished in min under isothermal condition at °c by employing a set of four primers targeting the cap gene of pcv . the lamp assay showed higher sensitivity than the conventional pcr, with a detection limit of five copies per tube of purified pcv genomic dna. no cross-reactivity was observed from the samples of other related viruses including porcine circovirus type (pcv ), porcine parvovirus (ppv), porcine pseudorabies virus (prv) and porcine reproductive and respiratory syndrome virus (prrsv). the detection rate of pcv lamp for clinical samples was . % and appeared greater than that of the pcr method. the lamp assay reported can provide a rapid yet simple test of pcv suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction. porcine circovirus (pcv) is a small, non-enveloped, spherical single-stranded dna virus, and can be classified as a member of the genus circovirus of the family circoviridae . two distinct genotypes of pcv, designated pcv type (pcv ) and pcv type (pcv ) have been identified. pcv shares an approximate % nucleotide sequence homology with pcv . pcv was identified as a contaminant of porcine kidney cell cultures and considered nonpathogenic for swine . however, pcv is now generally accepted as the major infectious agent involved in postweaning multisystemic wasting syndrome (pmws) (bolin et al., ) . clinical signs of pcv infection in pigs include progres-sive weight loss, paleness, dyspnoea and, occasionally, diarrhoea and icterus. histopathological findings include histocytic infiltration and lymphocyte depletion of lymphoid tissues, interstitial pneumonia and, less frequently, hepatitis and nephritis (kim and chae, b; segalés and domingo, ) . two major open reading frames (orfs) have been recognized for the genome of pcv ; orf , called the rep gene, which encodes a protein of . kda involved in virus replication (mankertz et al., ) , and orf , named the cap gene, which encodes the major immunogenic capsid protein of . kda (cheung, ; nawagitgul et al., ) . the capsid protein has the type-specific epitopes (mahe et al., ) , which suggests that orf contributes to the development of pmws and thus has potential for protective immunization in a vaccine (liu et al., ) , and for type-specific diagnosis (blanchard et al., ) . epidemiologic data suggest that the virulence of pcv is strongly related to the presence of the capsid protein (cho et al., ) . accumulated evidence indicates that pcv replicates in the lymph nodes, lung, liver, spleen, heart and kidney of infected pigs. this results in impairment of the immune system through degradation of lymphoid tissues (kennedy et al., ) and through changes in the proportions of lymphocyte subsets present in peripheral blood (darwich et al., ) . the presence of pcv in tissues of pigs with pmws had been proven by virus isolation, polymerase chain reaction (pcr), in situ hybridization and immunohistochemistry kim and chae, a) . real-time pcr is a sensitive assay for the detection of pcv (brunborg et al., ; chung et al., ; olvera et al., ) . although specialized equipment such as a thermal cycler is needed, pcr-based detection methods are commonly accepted because of their high sensitivity and specificity. loop-mediated isothermal amplification (lamp) is a novel amplification method which was developed originally by notomi et al. ( ) . the most significant advantages of lamp are the ability to amplify specific dna sequences under isothermal conditions between and • c and a visible result within - min. the method has been applied successfully to the detection of human influenza a virus, severe acute respiratory syndrome coronavirus and newcastle disease virus (hong et al., ; pham et al., ; poon et al., ) . however, the use of lamp for detecting pcv has not been reported to date. in this study, we evaluated the potential of lamp for the development of a simple and rapid detection system for pcv . the pcv -bj vaccine strain was used to develop a lamp method (beijing bio-pharmaceuticals corporation). monolayers of pk- cells (atcc ccl- ) grown in -cm culture flasks were infected with an inoculum of pcv and cytopathic effects (cpe) were monitored daily. upon observation of between and % cpe, the supernatant from the infected culture was collected, centrifuged at × g for min and stored in aliquots at − • c until use. field isolates of pcv , porcine parvovirus (ppv), pseudorabies virus (prv), and porcine reproductive and respiratory syndrome virus (prrsv) were identified by conventional pcr (or rt-pcr) and sequencing. a total of clinical samples that were diagnosed as pcv positive are shown in table , including peripheral blood and tissues of lymph nodes, lung, liver, kidney, heart and spleen. these clinical samples were taken from pcv -antibody-positive pigs tested by elisa. among them, samples were identified as positive by pcr and sequencing and the other eight samples were identified by virus isolation. dna was extracted from blood, lymph nodes, lung, liver, kidney, heart and spleen samples taken from pcv -infected and healthy pigs, using a dneasy tissue kit (qiagen) according to the manufacturer's instructions. after extraction, dna was eluted in l of elution buffer and stored at − • c. rna was extracted directly from prrsv samples by using trizol reagent (invitrogen). complementary dna (cdna) was synthesized using l of the eluted rna with oligo(dt) primers and the reverse transcriptase kit (takara corp., japan) according to the manufacturer's instructions. the highly conserved sequences in the capsid protein-coding region of pcv were selected as the target for lamp and pcr. a set of four primers for the cap gene was designed for lamp by alignment of six pcv genomic sequences (accession nos. ay , ay , dq , dq , ef and ef ). primers f, b, fip and bip for lamp are shown in table , and the f and b primers were also used in pcr. ; lanes - , different pcv copy numbers subjected to pcr ( , , , , and copies/tube, respectively); lanes - , different pcv copy numbers subjected to lamp assay ( , , , , and copies/tube, respectively). pcr products showed a specific amplification for the cap gene of pcv -bj with a detection limit of copies, whereas detection limit for lamp was five copies per reaction. the lamp reaction was carried out in a conventional water bath by mixing . m each of fip and bip primer, . m each of f and b primer, . mm each deoxynucleoside triphosphate, u of bst dna polymerase (new england biolabs) using the manufacturer's supplied × buffer (containing mm of mgso , . m betaine) and l of extracted template dna or cdna in a . ml eppendorf tube. the amplification reaction was performed at • c for min and then terminated by heating at • c for min. lamp products were analyzed by . % agarose gel electrophoresis. pcr was carried out in a l reaction volume containing . mm of each deoxynucleoside triphosphate, and l of × buffer, u of taq polymerase (nippon gene), m each of primers f and b, and . l of extracted dna or cdna. the amplification regime was min at • c, followed by cycles of • c for min, • c for s and • c for min, with a final elongation for min at • c. the pcr was carried out in the gene amp pcr system (applied biosystems). pcr products were subjected to electrophoresis on a . % agarose gel. the detection limit of lamp was tested and compared with pcr by using the same templates at identical concentrations. serial dilutions of , , , , and copies of dna from pcv -bj strain were used in this assay. in addition, clinical samples were analyzed with the lamp reaction and the sensitivity of detection was compared between lamp and pcr. to assess the specificity of lamp, potential cross-reactions with dna of pcv , ppv, prv and cdna of prrsv were examined. pcv -bj strain genomic dna was used as the positive control and dna extracted from healthy swine tissues was used as the negative control. pcr products were sequenced with an automated abi model a stretch dna sequencer. dnastar software was applied to align the sequences and blast searching of genbank was used to assess homology with the known capsid protein gene sequences of pcv . a successful lamp reaction with pcv -specific primers at • c for min produced many bands of different sizes upon agarose electrophoresis, since the lamp products consisted of several inverted-repeat structures. the amplification by lamp showed a ladder-like pattern, whereas the pcr product was a specific dna band. the result indicated that the detection limit of the pcv lamp was five copies per reaction whereas that of pcr was copies (fig. ) . the detection sensitivity of lamp was therefore fivefold better than for conventional pcr. in order to evaluate the optimal tissues for viral detection and to compare the sensitivity of pcv detection by lamp and pcr, dnas from tissue samples of blood, heart, lung, liver, kidney, lymph nodes and spleen from pcv -infected pigs were extracted and subjected to lamp and pcr. there was a % positive detection rate for both pcr and lamp on extracts of lung, kidney and heart tissue. however, lamp showed higher sensitivity than pcr for the detection of pvc dna in blood, lymph nodes, liver and spleen tissue samples (table ) . overall, the detection rate of pcv lamp for clinical tissue samples was . % and appeared better than that for the pcr method. dna extracted from tissues of healthy animals, pigs infected with pcv , ppv and prv, and cdna from prrsv were used as templates for pcv lamp. agarose gel electrophoresis analysis indicated that the pcv lamp reaction did not detect pcv , ppv, prv, or prrsv, and gave a negative reaction with tissues of healthy swine. only with the pcv -bj dna did the pcv primer set give a positive reaction (fig. ) . pmws was first observed in piglets of a high-health herd in canada in (harding and clark, ) , and appeared to be an emerging disease that affected swine herds in many countries of north america, europe and asia choi et al., ) . pcv (which differs markedly from pcv ) was commonly found in pigs with pmws . several researchers reported that ppv, prv and prrsv could also reproduce symptoms typical of pmws rodriguez et al., ; rovira et al., ) . thus, the development of a simple and rapid diagnostic tool that could detect pcv and differentiate it from pcv , ppv, prv and prrsv in the same samples would be of significance for epidemiological surveillance and prediction of the severity of pmws outbreaks in swine herds. lamp is a new diagnostic method which is quite simple, requiring only a conventional water bath or heat block for incubation under isothermal conditions. another useful feature of lamp is that its products can be observed directly, by naked eye, because a white precipitate of magnesium pyrophosphate forms in the reaction tube (mori et al., ) . adding sybr green i to lamp reactions can increase the ease and sensitivity of detection by the naked eye (iwamoto et al., ) . some samples from blood, lymph nodes, liver and spleen that were positive by lamp were not detected as positive by pcr. the greater sensitivity of lamp (as compared to pcr) for detecting pcv detection accords with the sensitivity reported for lamp methods used to detect newcastle disease virus, japanese encephalitis virus, mumps virus and west nile virus (okafuji et al., ; parida et al., ; parida et al., ; pham et al., ) . the lack of cross-reaction observed with pcv , ppv, prv and prrsv suggest that the pcv lamp system possesses reliable specificity in addition to high sensitivity. the presence of pcv in blood and many tissues following natural infection (darwich et al., ; kennedy et al., ) was confirmed by the pcv lamp method using, in the present study, clinical samples of blood, lymph nodes, lung, liver, kidney, heart, and spleen. the optimal tissues for pcv lamp are probably the blood, lymph nodes, lung, kidney and heart because these gave a % detection rate in the lamp assay. lamp is a simple and timesaving procedure, allowing results to be obtained within h, whereas the pcr method typically requires - h. compared with pcr, the lamp method appears to be a fast and sensitive tool for the clinical diagnosis of pcv infection. nonetheless, the reliability of this assay should be further evaluated by large-scale investigation. in conclusion, a pcv lamp assay was developed 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loop-mediated isothermal amplification of dna rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification comparison of porcine circovirus type load in serum quantified by a real time pcr in postweaning multisystemic wasting syndrome and porcine dermatitis and nephropathy syndrome naturally affected pigs real-time reverse transcription loop-mediated isothermal amplification for rapid detection of west nile virus development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of japanese encephalitis virus loop-mediated isothermal amplification for rapid detection of newcastle disease virus detection of human influenza a viruses by loopmediated isothermal amplification aujeszky's disease virus infection concurrent with postweaning multisystemic wasting syndrome in pigs experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus postweaning multisystemic wasting syndrome (pmws) in pigs: a review this work was supported in part by grants from the national key technologies r&d program of china (no. bad a ). this study was also supported by the national high-tech r&d program (no. aa a - - ) and the national natural science foundation of china (no. and no. ). key: cord- -gb vgs authors: mekata, tohru; kono, tomoya; savan, ram; sakai, masahiro; kasornchandra, jiraporn; yoshida, terutoyo; itami, toshiaki title: detection of yellow head virus in shrimp by loop-mediated isothermal amplification (lamp) date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: gb vgs reverse transcription loop-mediated isothermal amplification (rt-lamp) assay was developed for detecting the structural glycoprotein gene of yellow head virus (yhv). the rt-lamp assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, sensitivity and rapidity under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. the whole procedure is very simple and rapid, and reaction time and temperatures were optimized for min at °c, respectively. detection of gene amplification could be accomplished by agarose gel electrophoresis. the standardized rt-lamp procedure was used to detect yhv in the heart and gill from infected shrimp. thus, the rt-lamp assay is extremely rapid, cost-effective, sensitive and specific and has potential usefulness for rapid diagnosis for yhv detection in shrimp. yellow head virus (yhv) is an enveloped, rod-shaped particle (approximately nm × nm) with prominent surface projections (approximately nm) and an inner helical nucleocapsid (chantanachookin et al., ; wang and chang, ; loh et al., ) . based on the virion morphology and the presence of a single-stranded rna genome (wongteerasupaya et al., ) , yhv was previously reported as a rhabdovirus . however, it was subsequently demonstrated that the yhv genome is positive-sense rna (tang and lightner, ) . sequence identity, genome organization and gene expression have indicated that gill associated virus (gav) and yhv are related to coronaviruses, toroviruses and arteriviruses and are classified in new taxa (family roniviridae, genus okavirus) within the order nidovirales (cowley et al., (cowley et al., , sittidilokratna et al., ; cowley and walker, ) . * corresponding author. tel.: + ; fax: + . e-mail address: itamit@cc.miyazaki-u.ac.jp (t. itami). the development of a loop-mediated isothermal amplification (lamp) assay for detection of white spot disease virus (wsdv) dna was described by kono et al. ( ) . the lamp assay is a novel approach to nucleic acid amplification that amplifies dna with high specificity, selectivity and rapidity under isothermal conditions. therefore, a thermal cycler is not needed. the lamp assay originally described by notomi et al. ( ) is based on the principle of the reaction performed by a dna polymerase with strand displacement activity and a set of two specially designed inner primers and two outer primers. lamp is highly specific for the target sequence because of the recognition of the target sequence by six independent sequences in the initial stage and by four independent sequences in the later stages of the lamp reaction. the amplification efficiency of the lamp method is extremely high because of its no time loss for thermal change, based on its isothermal reaction. therefore, the lamp assay has the advantage in specificity, selectivity and rapidity over other nucleic acid amplification methods (mori et al., ) . the lamp assay is also useful for rna template detection upon the use of reverse transcriptase (rtase) together with dna polymerase. in this paper, the rt-lamp assay for detection of yhv rna in shrimp is described. black tiger shrimp (penaeus monodon) with or without yhv clinical signs were collected from shrimp farms in songkhla, thailand. shrimp samples were kept on ice for rna extraction. rna extraction from the gills was carried out using an rna extraction kit (high pure rna tissue kit (roche diagnostics, germany) according to the manufacturer's instructions. briefly, gill tissues ( - mg) were homogenized with the lysis buffer. rna was then eluted from spin columns in a final volume of l of elution buffer and stored at − • c until use. yhv-specific rt-lamp primers were designed according to published sequence of yhv structural glycoprotein gene (gen-bank accession number: af ; jitrapakdee et al., ) using primer explorer version (https://primerexplorer.jp/ lamp . . /index.html). a set of four primers composed of two outer and two inner primers was designed. the two outer primers are known as the forward outer primer (f ) and the backward outer primer (b ), which helps in strand displacement. the inner primers are known as the forward inner primer (fip) and the backward inner primer (bip). each primer has two distinct sequences corresponding to the sense and anti-sense sequences of the target, one for priming in the first stage and the other for self-priming in later stages. fip contains f c region (complementary to f ), a tttt spacer and the f region. bip contains the b c region (complementary to b ), a tttt spacer and the b region. fip and bip were hplc-purified. the location of the primers within the rna fragment is shown in fig. . the rt-lamp was carried out in a total volume of l reaction mixture with a loopamp rna amplification kit (eiken chemical co. ltd., japan) according to the manufacturer's instructions. briefly, l of target rna was mixed with l ( pmol) of each yhv-fip and yhv-bip, . l ( pmol) of yhv-f and yhv-b , . l of × reaction mix, . l of distilled water and . l enzyme mix containing bst dna polymerase and amv reverse transcriptase. after incubation at • c for , , or min, the reaction was terminated by heating at • c for min. the reaction temperature ( , and • c) was also optimized. the rt-lamp products were electrophoresed in a % agarose gel to determine the optimal condition. ten-fold serial dilutions ( − to − diluted) of rna extracted from yhv-infected shrimp was used as a template for rt-lamp according to determined conditions. after the reaction, rt-lamp products were electrophoresed on a % agarose gel and visualized using a gel document system (ultra-violet products, japan). nested rt-pcr was carried out using a commercial kit, iq (farming intelligene technology corporation, taiwan) for detecting yhv and gav. the first-step rt-pcr was carried out in l reaction volume containing . l of rt-pcr premix (reaction buffer, dntps and yhv/gav-specific primers), . l of iqzyme dna polymerase ( u/l), . l of rt enzyme mix, . l of rna extracted from yhv. ten-fold serial dilutions ( − to − ) of template rna were used to determine the sensitivity of the detection. the amplification regime was min at • c, min at • c followed by cycles of • c for s, • c for s and • c for s, then final elongation for s at • c and s at • c. after rt-pcr reaction was completed, . l of nested pcr premix and . l of iqzyme dna polymerase were added. two-step pcr reaction profile was followed by cycles of • c for s, • c for s and • c for s, then final elongation for s at • c and s at • c. two-step pcr products were electrophoresed in a % agarose gel to visualize the specific products. to determine specificity of rt-lamp method, rt-lamp was carried out with the different sources of rna template, i.e. rnas or dnas of wsdv-infected shrimp, taura syn-drome virus (tsv)-infected shrimp or healthy shrimp using commercial rna extraction kit, high pure rna tissue kit. instead of using the commercial kit for extraction of rna from shrimp, . m naoh (wang et al., ) was used. rna was extracted from gill tissues of shrimp samples showing yellow head disease clinical signs. twenty-five micrograms of sample was homogenized in l . m naoh on ice. then, l homogenization was diluted with l tris-hcl buffer. the same weight of shrimp gill sample was used for the extraction of rna using high pure rna tissue kit following the manufacturer's instruction. a series of -fold dilutions ( − to − diluted) of extracted rna were used as template for rt-lamp. rt-lamp was performed using determined condition. rt-lamp products were electrophoresed and analyzed in a % agarose gel. the rt-lamp was carried out using rna as template in order to determine the optimal temperature and reaction time. rt-lamp products were detected at both and • c. however, the product at • c showed a clearer reaction bands and this temperature was used as an optimal temperature. no amplification of template was found in the reaction time of and min. for the reaction time of and min at • c, lamp products were detected. however, for the complete amplification, the reaction time of min was selected as an optimal reaction time. the results are shown in fig. . lane , molecular size marker (/x /hinc ii digest); lanes - , rt-lamp and nested rt-pcr carried out using concentrations of rna ( − , − , − , − , − , − and − ), respectively. all products were electrophoresed on a % agarose gel and stained with ethidium bromide. in order to determine the sensitivity of detection limit, rt-lamp and nested rt-pcr were carried out using various concentrations ( − to − dilution) of rna extracted from yhv-infected shrimp as template. rt-lamp detected at a concentration of − dilution as a template, while the nested rt-pcr detected − diluted. the sensitivity of detection limit by rt-lamp is times lower than that of nested rt-pcr (fig. ) . the cross-reaction with other shrimp disease viruses, i.e. wsdv, tsv and healthy shrimp rna was also carried out to determine the specificity of rt-lamp method. positive result for rt-lamp was found none of them (fig. ) . this indicates that this rt-lamp method is a high specificity for yhv. the template rna extracted by the commercial rna extraction kit (high pure rna tissue kit; roche diagnostics) provided higher sensitivity for rt-lamp ( − dilution) than that by quick method ( − dilution) using . m naoh (fig. ) . in this study, the rt-lamp diagnostic protocol was carried out for the detection of yhv in shrimp. two sets of primer (outer and inner) used were able to amplify a bp sequence of structural glycoprotein gene. the optimal condition of rt-lamp reaction for the detection of yhv-rna was shown as • c and min. however, it was also found that the reaction could be terminated within min. this suggests rt-lamp is a more rapid method for the detection of shrimp virus, compared to the rt-pcr method which takes - min for rt reaction and at least - h for conventional pcr method. in addition, it was shown that rt-lamp method used for yhv detection specifically reacts only to yhv-infected shrimp. no cross-reaction with wsdv-infected shrimp was found. fig. . rt-lamp products with different source of rna/dna template, i.e. from heart of yhv-infected shrimp (positive), tsv-infected shrimp (negative), wsdv-infected shrimp (negative) and healthy shrimp (negative). rt-lamp products: lane , molecular size marker (/x /hinc ii digest); lanes and , yhv-infected shrimps; lanes and , tsv-infected shrimp; lanes and , wsdv-infected shrimp; lane , healthy shrimp. all the products were electrophoresed on a % agarose gel and stained with ethidium bromide. fig. . rt-lamp products with various concentrations of template ( − , − , − , − , − and − ) extracted using commercial kit and quick method. (a) commercial rna extraction kit: lane , molecular size marker (/x /hinc ii digest); lanes - , − , − , − , − , − and − , respectively. (b) . m naoh method: lane , molecular size marker (/x /hinc ii digest); lanes - , − , − , − and − . rt-lamp was carried out using series of concentrations of rna. all products were electrophoresed on % agarose gels and stained with ethidium bromide. the sensitivity of rt-lamp was found to be times lower than that of nested rt-pcr using an iq kit for the detection of yhv/gav. however, nested rt-pcr detection needs at least - h, comparing rt-lamp within one hour. as an efficient diagnostic method, the rapidness for detection should be also considered. the sensitivity of rt-lamp also depends on the quality of rna template, as the results showed quick method for rna extraction using . m naoh. this rapid technique resulted in lower sensitivity for the detection of virus, compared to using a commercial kit, although this method may take only min for rna extraction. therefore, for the confirmatory diagnosis shrimp showing the typical signs of yellow head disease that are heavily infected with the virus can be diagnosed with this method in short period of time for rna preparation. this study proposed the first rt-lamp protocol as an alternative method for the rapid detection of yhv with high sensitivity and specificity. this protocol is useful for the detection of low concentration of yhv from several tissues of cultured shrimp during early stages of infection. additionally, this method could be used as both screening and confirmatory diagnosis for suspected shrimp, even though the virus titer is relatively low. this technique is recommended as an applied protocol for health management program and disease surveillance of shrimp in hatcheries as well as in grow-out pond, in order to prevent the disease outbreak. histology and ultrastructure reveal a new granulosis-like virus in penaeus monodon affected by yellow-head disease yellow head virus from thailand and gill-associated virus from australia are closely related but distinct prawn viruses gillassociated virus of penaeus monodon prawns: an invertebrate nidovirus with orf a and orf b genes related to arteri-and coronaviruses the complete sequence of gill-associated virus of penaeus monodon prawns indicates a gene organisation unique among nidoviruses identification and analysis of gp and gp structural glycoproteins of yellow head nidovirus of penaeus monodon shrimp detection of white spot syndrome virus in shrimp by loop-mediated isothermal amplification viral pathogens of the penaeid shrimp detection of loopmediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation yellow-head virus: a rhabdovirus-like pathogen of penaeid shrimp loop-mediated isothermal amplification of dna the complete orf b-gene sequence indicates yellow head virus is an invertebrate nidovirus a yellow head virus probe: application to in situ hybridization and determination of its nucleotide sequence a simple method of preparing plant samples for pcr yellow head virus infection in the giant tiger prawn penaeus monodon cultured in taiwan yellow-head virus of penaeus monodon is an rna virus this study was supported partly by a grant-in-aid for science research from the ministry of education, science, culture, sports, science and technology of japan. key: cord- -t svobg authors: bruijnesteijn van coppenraet, l.e.s.; swanink, c.m.a.; van zwet, a.a.; nijhuis, r.h.t.; schirm, j.; wallinga, j.a.; ruijs, g.j.h.m. title: comparison of two commercial molecular assays for simultaneous detection of respiratory viruses in clinical samples using two automatic electrophoresis detection systems date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: t svobg two molecular assays were compared with real-time rt-pcr and viral culture for simultaneous detection of common viruses from respiratory samples: a multiplex ligation-dependant probe amplification (mlpa) and a dual priming oligonucleotide system (dpo). in addition, the positive detections of mlpa and dpo were identified using two different automatic electrophoresis systems. a panel of culture-positive and negative samples was tested by the molecular assays for the presence of influenza a and b virus, respiratory syncytial virus, human metapneumovirus, rhinovirus, coronaviruses, parainfluenza viruses and adenovirus. one hundred and twenty-nine ( %) samples were positive as detected by at least one method. sixty-nine ( %) samples were positive by cell culture (excluding human metapneumovirus and coronaviruses), ( %) by rt-pcr, ( %) by mlpa and ( %) by dpo. the mlpa yielded results in one attempt for all samples included while ( . %) samples had to be repeated by the dpo assay due to inconclusive results. the mlpa assay performed well in combination with either electrophoresis system, while the performance of the dpo assay was influenced by the electrophoresis systems. both molecular assays are comparable with real-time rt-pcr, more sensitive than viral culture and can detect dual infections easily. results can be obtained within day. pneumonia is a serious illness with significant morbidity and mortality rates. a wide variety of bacteria and viruses is held responsible for causing pneumonias, in children as well as in adults, although in about - % of patients the etiology is not established (johnstone et al., ; michelow et al., ) . since signs and symptoms at presentation rarely point to a specific pathogen, it is usual to start empirical therapy aimed at bacterial pathogens. considering the frequency of viral etiology, antibacterial therapy is often employed inadequately and unnecessarily. in comparison with conventional detection techniques, multiplex real-time pcr has been shown to be more sensitive and specific, yielding results within h (bonzel et al., ; templeton et al., ) and enabling direct detection of viruses that are difficult to culture (falsey et al., ; fouchier et al., ; mahony, ) . consequently, the results of nucleic acid amplification tests may contribute to timely treatment decisions. in this study, two commercial molecular assays, both designed for simultaneous detection of the most common viruses from a variety of respiratory samples, were compared with real-time rt-pcr and viral culture: a multiplex ligation-dependant probe amplification (mlpa) and a dual priming oligonucleotide system (dpo). the mlpa technique employs two probes which ligate in the presence of target-specific complementary sequences. the probes consist of a target-specific sequence, common primer sequences and a stretch of nucleotides that allow specific detection based on length differences (reijans et al., ) . the dpo technique employs targetspecific primers of double the normal oligo length which contain a polydeoxy-inosine linker to gain specificity and sensitivity for the multiplex detection (drews et al., ) . in addition, positive detections by the mlpa and dpo assays were identified by two different automatic capillary electrophoresis detection systems. prior to the validation using patient samples, a pilot study was carried out using dilutions of virus strains to confirm all specific detections included in the molecular assays (except human corona hku virus). the viral targets included in the molecular assays are listed in table . the validation panel consisted of viral cell culture-positive and culture-negative respiratory samples. eighty-seven samples with positive culture detections for one of the viral targets (or positive rt-pcr results for coronavirus or hmpv) were submitted and culture-negative samples were matched based on sample type and age of the patients (range week to years old, median age months). the samples were collected from several different years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) to ensure some strain variability. this panel of respiratory samples, consisting of bronchioalveolar lavages (n = ), sputa (n = ) nasopharyngeal lavages (n = ), nasopharyngeal (n = ) and throat (n = ) swabs, was stored at − • c prior to nucleic acid extraction. the samples were submitted to the molecular assays without knowledge of the cell culture results that had been obtained prior to storage at − • c. respiratory samples were cultured at • c using four different cell lines on coverslips: hel (human embryonic lung fibroblast), vero (african green monkey), hep- (larynxcarcinoma) and llc-mk (rhesus monkey kidney) using minimum essential medium (mem) with mm l-glutamine and earle's bss adjusted to . g/l sodium bicarbonate, % fetal bovine serum and addition of penicillin ( , u/l), streptomycin ( , g/l) and amphotericin b ( . mg/l). all cultures were examined daily for cytopathic effect (cpe). in addition, all cultures were examined on days and using immunofluorescent (if) antibodies against common respiratory pathogens including rsv, adenovirus, influenza virus a and b, and parainfluenza virus , and (d dfa respiratory virus screening/id kit, itk diagnostics bv, uithoorn, the netherlands). dilution series of cultured rsv and influenza virus b were submitted to mlpa, dpo and real-time rt-pcr and a tcid experiment in hep- cells for rsv and llc-mk cells for influenza virus to determine the % infective dose for cell culture. each dilution was prepared in maintenance culture medium and l of these dilutions was used in all culture wells as well as in nucleic acid extraction. eight dilution series per virus were used. positivity was checked using if and cpe after and days. extraction of total nucleic acids was performed with the nuclisens easymag (biomérieux, boxtel, the netherlands). the protocol used was specific a with "on board" lysis and l of silica mix. a mix of l sample with l saline ( . %) was used in the extraction. the mlpa protocol required an internal control which was added after lysis to the extraction mixture. nucleic acid extracts were eluted in l elution buffer and stored at - • c before molecular testing. the mlpa (respifinder dc twostep kit, pathofinder, maastricht, the netherlands) was conducted according to the manufacturers' protocol using an abi thermocycler (applied biosystems, foster city, ca, usa). in short: l nucleic acid extract was added to l pre-amplification mix. second, a hybridisation step was carried out with l of diluted pre-amplification product and l fresh hybridisation reaction mix. ligation and final pcr were carried out simultaneously in a third reaction mix consisting of l mix and l hybridisation product. pcr products were stored at − • c until electrophoresis. the total mlpa protocol including extraction was performed in approximately h. the dpo assay (seeplex rv detection kit, seegene, rockville md, usa) was conducted according to the manufacturers' protocol using an abi thermocycler. in short: a reverse transcription step was carried out with l rt reaction mix after an initial denaturation step with l reaction mix and l nucleic acid extract. the following pcr was carried out in two reaction mixes consisting of either l of rv mix a or b and l cdna. after pcr, the product was stored at − • c until electrophoresis. the total dpo protocol including extraction was completed in approximately h. all samples included in the validation panel were submitted to two-step real-time rt-pcr. rt-pcr was carried out in sim- table . all reactions were analysed using two microcapillary electrophoresis systems: the agilent (agilent technologies, santa clara, ca, usa) and the experion (bio-rad laboratories, hercules ca, usa), as prescribed by the respective manufacturers. input of both systems was l amplicon. the results were examined by two different technicians to exclude interpretation differences. the identifications obtained in the agilent were used in the calculations. defined as true positives were samples that yielded positive viral detections by more than one method (culture, dpo, mlpa or real-time rt-pcr). samples that remained negative in all methods were considered true negative. positive results obtained by only one method were considered unconfirmed or false-positive. all calculations were performed using the microsoft excel version . the tcid cut-off of the rsv culture was determined at dilution . in both if and cpe, the tcid of influenza virus type b was . in if and . in cpe. the highest dilution that yielded a specific reaction of rsv in both the mlpa and the dpo was . the highest dilution that yielded a specific reaction of influenza virus in the mlpa assay was and in the dpo assay . the results of the simplex real-time rt-pcr assays were identical to those of the mlpa. the actual input in the molecular assays is -fold lower than in culture because of the extraction protocol. these results show that the analytical sensitivity for the rsv detection is slightly greater for culture compared to the molecular methods. the greatest sensitivity for influenza virus was obtained by the mlpa and rt-pcr and the lowest by the dpo assay. the specific detections included in the two molecular assays were validated using nucleic acid extracts from cultured virus strains. all virus strains were identified successfully and specificity was confirmed. of the total collection of samples that were used in the validation panel, five samples were excluded. one sample was inhibited repeatedly in the mlpa and of four samples an insufficient volume was left to perform all tests. a total of samples were included in the comparison: ( %) samples were positive for one or two viral targets, including unconfirmed (false) detections. sixty-nine ( %) samples were cell culture-positive, ( %) samples were rt-pcr positive, ( %) samples yielded positive mlpa results and ( %) samples were found positive by the dpo. in table , the performance per method and detection rate are summarised. in table , the virus specific detections have been summarised per method. both tables show the number of detections which exceeds the amount of positive samples due to mixed infections. the mlpa results obtained with both detection systems were identical. all virus detections, except for the inhibited samples, could be identified in a single attempt (with no interpretation differences between technicians). one sample was inhibited repeatedly in the mlpa and therefore excluded. for the dpo, the agilent performed better than the experion: the agilent showed a clearer difference between background signals and specific signals. the agilent enabled clear interpretation for (of ) samples in one attempt while the experion yielded clear interpretation for only (of ) samples. of the repeated electrophoresis results for six samples ( . %: one with the agilent, five with the experion), the two technicians disagreed on the interpretation and therefore the complete dpo protocol was repeated. for six other samples the experion run hampered and for these only the electrophoresis run was repeated. additionally, six ( . %) of the samples were inhibited using the dpo and three ( . %) using the mlpa. the calculated amplicon sizes differed slightly between the agilent and the experion electrophoresis systems. the amplicon sizes were calculated based on the markers included with the kit reagents. because of the greater performance of the agilent, all virus detections of the mlpa and dpo, as mentioned in the results, were based on the combination with the agilent electrophoresis. all samples were tested by the simplex real-time rt-pcrs to confirm the results from the comparison. of positive viral detections that were found only by one method (those unconfirmed or false detections are listed in brackets in table ), a specific rt-pcr was repeated. seven of detections could be confirmed. these included six mlpa detections (three rhinovirus, two coronavirus and one adenovirus detection) and one dpo detection (a rhinovirus detection). all seven had high threshold cycle (ct) values: ranging between ct . and ct . . of mixed infections found initially, were detected by more than one method. mlpa detected dual infections of which were confirmed, rt-pcr detected dual infections, viral cell culture detected one dual infection and dpo detected dual infections of which were confirmed (table ) . of the virus detections involved in mixed infections, the distribution of viruses is shown in table . the performance of two commercial molecular detection assays, the mlpa and the dpo, was compared using a panel of respiratory samples. both molecular assays yielded comparable sensitivities to that of rt-pcr and significantly greater sensitivities in respiratory specimens than viral culture (excluding hmpv and coronaviruses for which cell culture was not undertaken), which is in accordance with earlier publications (kim et al., ; reijans et al., ; yoo et al., ) . the mlpa assay appeared to have a greater sensitivity for all viral targets compared to that of the dpo and similar sensitivity compared to that of real-time rt-pcr. the overall specificity of the dpo assay appeared greater compared to that of mlpa. how-ever, the sensitivity of the mlpa decreases the specificity due to a number of false-positives. it might well be that these so-called false-positive detections were in fact accurate detections as shown by the seven additional detections that were confirmed by rt-pcr during discrepancy testing. the low viral load in these samples was confirmed by the high ct values that were obtained with the repeated extraction and rt-pcrs. comparison of the analytical sensitivity with the sensitivity obtained in the respiratory samples for influenza virus type b showed similar differences between the mlpa and the dpo assays. the differences in sensitivity for rsv were less obvious, but the greater detection rate of the molecular assays can be explained by the greater potential to detect co-infections. these two viruses were chosen for the tcid sensitivity comparison because of the difference in sensitivity observed in the validation panel. the sensitivity of the dpo assay might be enhanced by lowering the amount of internal control (ic), because the strength of the ic signal compared to the specific target signals might be due to competitive amplification. the type-specific viral detections per molecular assay (table ) could all be differentiated except for parainfluenza virus types - , which are detected as a cluster by the mlpa and coronavirus oc /hku and e/nl which are not further differentiated by the dpo assay. in the sample panel used here coronavirus hku was not present because all oc /hku dpo detections were identified as oc by the mlpa. at least one parainfluenza was detected by the mlpa and identified as such by rt-pcr. in two cases, virus was isolated by culture (influenza b virus and parainfluenza ) but were not detected by the molecular assays. these viruses were found in patients with dual infections, parainfluenza combined with enterovirus in a neonate and influenza b virus and herpes simplex virus in bronchoalveolar fluid in a -year-old patient, and were only found by specific if tests on cover slips. the amount of virus in the original clinical sample was probably very low and duration of storage or handling might have influenced the quality of the sample, although a laboratory contamination might be a plausible explanation as well. the molecular assays are comparable in several aspects: the multiplex format, target identification based on length differences of the amplicons and the most common respiratory viruses are included. however, the dpo assay is faster and easier to perform than the mlpa, which consists of more hands-on steps. on the other hand, the mlpa has several advantages over the dpo. first, it con- table positive detections per target organism. between square brackets [+] are additional possible positives (unconfirmed or false-positive). in the last column "detected as mixed infection", all positive detections were considered and therefore the percentages were calculated versus "all positives". a total of samples were included in the comparison. tains an internal control added prior to extraction and therefore controls the extraction as well as inhibition, while that of the dpo is added after the extraction. second, the difference between the performances of the electrophoresis systems was only apparent for the dpo assay for which samples had to be repeated in the experion run (inhibited samples not taken into account). overall, peaks were sharper and seemed more pronounced by the experion compared to those obtained by the agilent. however, this resulted in false-positive and/or false target identifications due to background signals. as the mlpa showed virtually no background, this problem was not encountered by the mlpa and the results of the two electrophoresis systems were identical for the mlpa assay. both assays are unable to produce quantitative results whereas real-time rt-pcr can estimate semi-quantitative results per sample. however, in both assays the relative peak-height provides information on the viral load compared to that of the internal control and between multiple virus detections. another drawback of both assays is the fact that sample cups have to be opened after the formation of cdna. therefore the risk of cross-contamination exists and a sufficient number of control samples should be included. the molecular assays revealed a high frequency of double infections (table ). specifically coronaviruses ( %), rsv ( %) and rhinovirus ( %) were frequently detected as a mixed infection. by viral culture only one dual infection was identified. this is probably due to overgrowth of the faster growing virus which is identified as the infecting agent. following a positive identification, the cultures are usually not examined further and dual infections could thus be missed easily. the viruses that are involved commonly in dual infections, such as coronavirus, cannot be detected easily in culture. several studies have pointed out that a more severe clinical course is associated with dual infection (caracciolo et al., ; johnstone et al., ; paranhos-baccalà et al., ) . therefore, the importance of a sensitive test that is able to identify dual infections is emphasized. asymptomatic infection with these respiratory tract viruses is described both as uncommon (falsey et al., ; kumar et al., ) as well as common (peltola et al., ; zalm et al., ) . the importance of low viral load detections, therefore, is often unclear and has to be correlated with the clinical diagnosis. in conclusion: the mlpa assay has a greater sensitivity, is easier to interpret and has a greater success rate than the dpo (in particular when the dpo was combined with the experion). the dpo needs less "hands-on" time than the mlpa. however, both assays are more rapid and sensitive than viral culture and are good alternatives for real-time rt-pcr. frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase 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reverse transcriptase-pcr assay using a dual priming oligonucleotide system respiratory pathogens in children with and without respiratory symptoms rt-pcr protocols and previously unpublished oligo sequences were kindly provided by dr. a. beerens of the laboratory for infectious diseases groningen, the netherlands. key: cord- -uzfsv df authors: bellau-pujol, s.; vabret, a.; legrand, l.; dina, j.; gouarin, s.; petitjean-lecherbonnier, j.; pozzetto, b.; ginevra, c.; freymuth, f. title: development of three multiplex rt-pcr assays for the detection of respiratory rna viruses date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: uzfsv df three multiplex hemi-nested rt-pcr assays were developed to detect simultaneously rna respiratory viruses: influenza viruses a, b and c, human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), parainfluenza virus types – (piv- , - , - and - ), human coronavirus oc and e (hcov) and rhinovirus (hrv). an internal amplification control was included in one of the rt-pcr assays. the rt-pcr multiplex and the hemi-nested multiplex detected and . tcid of rsv a, respectively, and . and . tcid of influenza virus a/h n , respectively. two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. almost all samples ( / ) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. this method also detected an additional viruses and co-infections. the overall sensitivity ( %), rapidity and enhanced efficiency of these multiplex hemi-nested rt-pcr assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses. human respiratory tract infections are caused by numerous viruses, including influenza viruses a, b and c, parainfluenza viruses - (piv- , - , - and - ), human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), human coronaviruses oc and e (hcov), human rhinoviruses (hrv), adenoviruses and some human enteroviruses (hev). the direct diagnosis of such viral respiratory infections is based on the use of conventional methods such as isolation by cell culture and antigenic detection (gardner and mcquillin, ) . even though these methods are effective and often with the aim of providing a tool capable of detecting an increasingly complete panel of viruses (aguilar et al., ; coiras et al., coiras et al., , echevarria et al., ; fan et al., ; grondahl et al., ; osiowy, ; templeton et al., ) . here, we describe the development and evaluation of three multiplex rt-pcr methods for the detection of rna viruses involved in respiratory diseases. this retrospective study tested nasal aspirates from children hospitalised in paediatric units of the university hospital of caen and flers hospital between october and march . each nasal aspirate was collected in ml of viral transport medium ) and a ml aliquot was frozen at − • c. two groups of specimens, positive and negative, were assessed. the first group included specimens positive for a respiratory virus, selected by chronological order. ninety-one contained viruses detected by conventional methods: direct fluorescence assay and viral isolation technique. the number of samples has been limited, according to the representativeness of the most frequent viruses in respiratory diseases. this group comprised samples positive for hrsv, for influenza a virus, for influenza b virus, for hrv, for piv- , for piv- , piv- and for hcov oc . twenty samples positive for hmpv detected by a specific pcr (freymuth et al., a, b) were included in this group. no influenza c virus, hcov e, or piv- were identified during the period of study. the negative group included randomly selected clinical specimens not found to contain any viruses according to conventional methods. the following reference strains were used as positive controls to determine the sensitivity and specificity of our methods: hrv- , atcc vr- ; influenza c virus: c/paris/ / ; influenza a virus: a/h n /panama/ / ; influenza b virus: b/victoria/ ; hrsv a (atcc vr- ); hmpv canada/s ; hcov oc (atcc vr- ); hcov e (atcc vr- ); piv- sendaï .e ; piv- • : lyon/ / ; piv- : lyon/ / . immonofluorescence assay was used to detect viruses as previously described (freymuth et al., ) , using fluorescein-conjugated monoclonal antibodies directed against influenza viruses a and b, hrsv, piv- , - , - and adenovirus (ad) (imagen©; dako diagnostics). slides were examined under a microscope using a hemi-quantitative method. for viral isolation, embryonic lung fibroblasts (mrc ) were cultured in -cm flasks. they were then inoculated with . ml of each sample, incubated at - • c and observed for cytopathology during weeks. ad, hrsv and some hrv strains are likely to replicate in this cellular type. when samples were negative in immunofluorescence assay, we also attempted to isolate them using huh cells (nakabayashi et al., ) that had been grown in -well tissue culture plates, as described previously (vabret et al., ) . after days of incubation, cultures were examined for cytopathogenic effects and the cells were scraped and tested by immunofluorescence assay. when immunofluorescence assay was negative but the culture was positive, pcr specific for hrv (savolainen et al., ) , hcov e and hcov oc (vabret et al., ) were carried out using culture supernatants. rna was extracted from l of each sample, using a commercial reagent (qiaamp viral rna mini kit ® , qiagen). whenever possible, the extracts were tested immediately after extraction. if this was not possible, they were divided into aliquots and kept frozen at − • c. each aliquot was used only once to avoid the loss of viral genomic material during repetitive freezing and thawing. three multiplex rt-pcr methods, targeting respiratory viruses, were developed ( fig. ) . each multiplex method detected four viruses: influenza viruses a, b, hmpv (a and b) and hrsv (a and b) for multiplex ; piv- , - , - and - (a and b) for multiplex ; hrv, influenza c virus, hcov oc and e for multiplex . an internal control was included in multiplex to check the extraction step and the presence of inhibitors of the rt-pcr assay. this control consisted of glyceraldehyde- -phosphate dehydrogenase (gapdh) gene, which is normally transcribed in nasal mucosis cells. this gene was amplified with specific primers (table ) . primers targeted specifically the haemagglutinin neuraminidase genes of piv- , - (echevarria et al., ) and - (karron et al., ) , the phosphoprotein gene of vip- a and - b (aguilar et al., ) , the nucleocapsid gene of hrsv sub-groups a and b (cane and pringle, ; freymuth et al., ) , the matrix protein genes of influenza viruses a and b (donofrio et al., ) , the matrix protein gene of hmpv (this study), the haemagglutinin-esterase gene of influenza c virus (zhang and evans, ) , the m gene of oc and e (vabret et al., ) and the vp /vp and hypervariable region in the -non-coding region of hrv (savolainen et al., ) . the sequences of the primers, as well as their annealing temperatures and amplicon sizes are given in table . positive controls were included in each multiplex rt-pcr. these consisted of four rnas extracted from virus- infected cells and mixed together. for example, the multiplex positive control was a mixture of influenza a and b rna, hrsv rna and hmpv rna. as a negative control, h o was used instead of nucleic acid. each multiplex rt-pcr was a single-step combined rt-pcr amplification, performed using the one-step rt-pcr kit from qiagen. the reaction mixture contained l of × rt-pcr buffer ( . mm mgcl ), . mm datp, dgtp, dctp and dttp, . m of each of the primers ( primers in the multiplex for the negative group) and l of enzyme mix. a . l aliquot of rna extract was added to give a final volume of l. the cycling conditions for the three rt-pcrs were: an initial cycle at • c for min and • c for min; followed by cycles at • c for s, • c ( • c for multiplex ) for s and • c for min; and a final incubation at • c for min. the q-solution provided in the kit was used for multiplexes and ( l/reaction). our preliminary assays showed that q-solution was not necessary in multiplex . multiplex rt-pcr products were visualised after electrophoresis on an ethidium bromide-stained % agarose gel. the products of multiplex rt-pcrs and were subjected to hemi-nested multiplex pcr. the principle is to amplify part of one or several dna fragments resulting from rt-pcr. for each virus, an internal primer was designed and used together with the corresponding anti-sense primer used for rt-pcr (table ) . for hemi-nested multiplex pcr , the reaction mixture contained: % buffer (applied biosystems, roche ® ), . mm dntps, . m each "hemi-nested" primer (mia , mib , hmpv and vrsi), . m each of the following primers: p cane, mia , mib and hmpv , . u of amplitaq ® dna polymerase (applied biosystems, roche ® ) and l water q.s.p. we added . l of each multiplex rt-pcr product to this mixture. for hemi-nested multiplex pcr , the reaction mixture contained: % buffer (applied biosystems, roche ® ), . mm dntps, m each heminested primer (pis i, para i, para i, pi i), . m each following primers: pis −, pip −, para - , pip +, . u of amplitaq ® dna polymerase (applied biosystems, roche ® ) and l water q.s.p. we added . l of each multiplex rt-pcr product to this mixture. the cycling conditions for hemi-nested pcrs and were: • c for min; followed by cycles of • c for s, • c for s, • c for s; and a final incubation at • c for min. hemi-nested pcr products were visualised after electrophoresis on an ethidium bromide-stained % agarose gel. when a multiplex rt-pcr product was thought to be a virus (due to its size), a specific hemi-nested pcr was carried out to confirm the virus identity. the hemi-nested pcr mixtures were similar for all four viruses (hcov oc and e, hrv and influenza c virus): % buffer (applied biosystems, roche ® ), . mm dntps, m of each primer (table ) , . u of amplitaq ® dna polymerase (applied biosystems, roche ® ) and l water q.s.p. we added . l of each multiplex rt-pcr product to this mixture. cycling conditions were: • c for min; followed by cycles of • c for s, • c for s, • c for s; and a final incubation at • c for min. hemi-nested pcr products were visualized after electrophoresis on an ethidium-bromide stained % agarose gel. the primers used for rt-pcr have been described and individually evaluated in the original publications (table ) . the primers used for hemi-nested pcr were designed using blast (http://www.ncbi.nlm.nih.gov/) and the proligo site. these primers were designed to optimise amplification (g + c content, melting temperature and length) and to be usable in identical amplification conditions, to amplify fragments of sizes sufficiently different to allow them to be distinguished on a gel and to avoid the formation of primer-dimers whenever possible (elnifro et al., ) . a second fragment is frequently visible in the hemi-nested pcr assays. it corresponds to an additive amplification of the rt-pcr fragment, because of the persistence of rt-pcr primers in the rt-pcr products. the presence of several pairs of primers in a pcr increases the probabilities of mispairing and obtaining nonspecific amplification products, in particular the formation of primer-dimers. the oligo software can theoretically detect such interactions. in practice, this is one of the major difficulties encountered when designing multiplex pcrs. the use of q-solution, supplied in the one-step rt-pcr qiagen©kit, reduced this phenomenon. q-solution was included in multiplex rt-pcr strategies and ; preliminary studies showed that it was not necessary in multiplex rt-pcr . this solution reduces the number of non-specific reactions, but in some cases it can affect the hybridisation of primers and thus reduce amplification efficacy. a range of q-solution concentrations were used to determine the concentration that reduced nonspecific reactions maximally without affecting sensitivity: a concentration of l/reaction mix was found to be optimal. the analytical sensitivity of the method was assessed first by testing successive dilutions of various viral strains (influenza virus a, hrsv a, piv- , hrv and hcov oc ) with multiplex rt-pcrs , and and classical rt-pcr specific for each virus tested (as described in the original publications). for influenza a virus and hrsv, the signal was lost at the same dilution in multiplex rt-pcr and monospecific rt-pcr, showing that the sensitivity of these two methods was identical. similar results were obtained with hrv. however, the multiplex method was found to be more sensitive than the mono-specific method for the detection of piv- and hcov oc ( table ). the analytical sensitivity of the method was also assessed by quantifying two prototype strains (influenza virus a/h n and hrsv a) by tcid and rt-pcr. the rt-pcr multiplex and the heminested multiplex detected and . tcid of rsv a, respectively, and . and . tcid of influenza virus a/h n , respectively. the ability of the multiplex methods to detect several viruses in the same extract was assessed by testing combinations of four viral strains prepared from culture supernatants. each multiplex assay simultaneously detected all four viruses: four distinct bands of the expected sizes (table ) were visible on the electrophoresis gels (fig. ) . however, we noted that hcov e and hrv amplicons were too similar in size to be distinguished accurately (respectively, and bp). a hemi-nested pcr is therefore indispensable to distinguish between these two viruses. the analytical specificity of the method was checked by including the following in each multiplex rt-pcr: an appropriate positive control, a control associating a strain of chlamydia pneumoniae, a strain of mycoplasma pneumoniae and a strain of adenovirus (ad ). no non-specific amplification products were observed (data not shown). the multiplex methods were assessed on samples ( positive and negative nasal aspirates) collected from children hospitalised in the caen university hospital or flers hospital between october and march . the positive samples included viruses detected by the classical immunofluorescence assay and cell culture methods, and hmpv strains detected by an rt-pcr. most ( / ) of the viruses detected by conventional methods were also detected by the multiplex method (table ) . all the samples positive for hrsv ( ), influenza a virus ( ), influenza b virus ( ), piv- ( ), piv- ( ), piv- ( ) and hcov oc ( ) were detected by the multiplex method. in comparison with the conventional methods, the negative predictive value and sensitivity of the multiplex method for the detection of these viruses were % (with a confidence interval of . to with a % risk for influenza a virus, . to for influenza b virus; . to for hrsv, . to for piv- , . to for piv- , . to for piv- and . to for hcov oc ). the multiplex method detected hrv in of the samples positive for this virus. the two others, in which hrv was detected by culture in mrc cells, were not confirmed; moreover, one hmpv was detected in one of these two samples and one piv- in the other. all the samples positive for hmpv was confirmed by the multiplex method, giving a sensitivity of % compared to the specific rt-pcr that detected the virus in the previous study. nearly all of the viruses ( / ) were detected during the first stage of the multiplex reaction, i.e., before the heminested step. in the other nine cases, the virus was only detected at the hemi-nested step: hrsv and piv- . six of the seven negative results for hrsv and the two piv- corresponded to samples that were positive according to the culture method but negative according to immunofluorescence assay (table ) , showing that the viral load in the samples was probably low. the viruses that were only detected during the second step of the multiplex pcr were all confirmed by mono-specific hemi-nested pcr. in addition to the viruses detected by the conventional methods and the hmpv detected by rt-pcr, the multiplex method detected further viruses, consisting of co-infections ( / or . % of aspirates): co-infections associating two viruses and one co-infection associating three viruses (hmpv, hrsv and hrv) ( table ). among these viruses, could potentially be detected by the usual cell culture and/or immunofluorescence assay: hrv, hrsv and hcov oc . fifteen of the ( . %) co-infections involved an hrv. in the negative group, the multiplex assays identified viruses in of the samples, i.e., in % of them. fortytwo of these viruses could theoretically be detected by the conventional methods: hrv, hrsv, piv- , piv- , influenza a virus, influenza b virus. the others were: hmpv and piv- (table ) . furthermore, seven co-infections were detected by the multiplex method, which is equivalent to % of the extracts tested ( / ) and % of the viruses detected ( / ). all these co-infections involved an hrv. two of them involved three viruses: piv- , hrv and hmpv in one case, and hrsv, hrv and hmpv in the other (table ) . a wide range of viruses can cause respiratory infections and currently - % of these aetiologies remain unidentified in hospitalised children (freymuth et al., ) . this may be due to the lack of sensitivity of some of the detection methods and to the fact that some respiratory viruses are not systematically sought (e.g., piv- , influenza c virus and hcov). the aim of this study was to develop rapid, sensitive and specific molecular methods for the detection of a large panel of respiratory rna viruses that are more powerful than co-infections hrv + piv- hrv + piv- hrv + piv- + hmpv hrv + hrsv + hmpv hrv + influenza a virus hrv + hrsv total no. of positive samples the classical immunofluorescence assay and culture methods. during preliminary trials, we attempted to adapt the multiplex method to the detection of adenovirus, which cause frequent respiratory tract infections (freymuth, ) . but this affected the detection of other viruses, and we considered that it was preferable to search for adenovirus in a multiplex pcr assay including other dna respiratory pathogens. in this study, three multiplex methods for the detection of respiratory viruses were developed and tested on nasal aspirates from hospitalised children. all the viruses initially detected by the conventional methods were confirmed by the multiplex method, with the exception of two hrv identified after culture in mrc cells. it was not possible to check this result by repeating the cell culture (insufficient sample volume). the absence of inhibitors in the rt-pcr step was confirmed by the internal control. given the large genotypic diversity of hrv, it is probable that the primers used here were not adapted to some genotypes, even though they have been shown to amplify over serotypes (savolainen et al., ) . the overall sensitivity of the multiplex method (rt-pcr and hemi-nested pcr) was % compared to conventional methods, but the non-nested multiplex rt-pcr had a sensitivity of only %. in fact, seven hrsv and two piv- were not detected by the first step of the multiplex method; this was undoubtedly due to a low viral load. it is also very likely that the freezing and thawing steps altered the samples. the heminested multiplex pcr gave a sensitivity of % for these two viruses. in an evaluation of the commercially available multiplex rt-pcr (hexaplex©, prodesse), hindiyeh et al. found that hrsv were more difficult to detect (sensitivity of %) than influenza a virus ( . %), influenza b virus ( %) and piv - ( %) (hindiyeh et al., ) . for the other viruses (influenza a, b and c viruses, piv- , - , hrv and oc ) the first step of our method (rt-pcr) alone gave a sensitivity equivalent to that obtained with the conventional tools. a hemi-nested multiplex pcr was not developed due because of the persistence of non-specific amplification products. several multiplex methods for the simultaneous detection of several respiratory viruses have been published. grondahl et al. described a multiplex rt-pcr-hybridisation method targeting nine microorganisms (hev, influenza viruses a and b, hrsv, piv- , - , ad, m. pneumoniae and c. pneumoniae). they found that its sensitivity was low, particularly for hrsv: of the hrsv detected by eia were negative (grondahl et al., ) . puppe et al. assessed this method in and confirmed that the sensitivity never exceeded % and was particularly low for piv- ( %) (puppe et al., ) . the multiplex rt-pcr-hybridisation technique, hexaplex was described in by fan et al. for the detection of seven respiratory viruses: hrsv a and b, influenza viruses a and b, piv- , - and - (fan et al., ) . four studies have found that the hexaplex©method had a good sensitivity ( - % depending on the study and the virus) and was more efficient than conventional methods (fan et al., ; hindiyeh et al., ; kehl et al., ; liolios et al., ) . two multiplex nested rt-pcr methods were developed by coiras et al. for the detection of respiratory viruses. the first one was able to detect six viruses (influenza viruses a, b and c, hrsv-a, -b and adenovirus) more efficiently than conventional methods, and the method detected additional viruses, of which were also detected by individual rt-pcrs. the sensitivity and specificity of the methods were, respectively, and % (coiras et al., ) . in , the authors described a second multiplex rt-pcr, which detected eight other respiratory viruses: piv- , - , - and - , hcov oc and e, hrv and hev. all samples found to be positive by immunofluorescence assay and/or cell culture ( / ) were confirmed by multiplex , which also detected additional viruses (coiras et al., ) . finally, in , templeton et al. described two realtime multiplex rt-pcr methods for the detection of seven respiratory viruses: influenza viruses a and b, hrsv, piv- , - , - and - (templeton et al., ) , and syrmis et al. an rt-pcr-hybridisation method for the detection of seven viruses: influenza viruses a and b, ad, piv- , - , - and hrsv (syrmis et al., ) .as well as being highly sensitive, our multiplex methods can identify a large number of viruses that are not detected by the conventional methods: viruses among the aspirates in the positive group and viruses among the aspirates in the negative group, which is equivalent to . and . % of samples, respectively. according to statistical rules, the selection of test samples does not allow the calculation of the positive predictive value or of the specificity of the multiplex method. furthermore, as believed by other authors , the specificity of the method is difficult to interpret given that the reference method itself has limits. there are several possible explanations why these samples were negative according to conventional methods (a). the multiplex method can have given false positive result. substantial precautions have been taken to prevent contaminations of reaction tubes with previously amplified products or target rna or dna from other specimens, and the existence of false positives due to contaminants was ruled out by the absence of unexpected bands in the negative controls (b). the immunofluorescence assay to detect antigens is associated with sensitivity problems when the viral load is low (casiano-colon et al., ) , whereas molecular methods are theoretically more sensitive (c). using the cell culture method it is difficult to detect several viruses in a given sample, as the development of one virus can mask or inhibit that of another (d). conventional methods only detect replicative viruses, which gives them a good diagnostic value. the hypothesis that the viruses detected uniquely by the multiplex method are non-replicative is probably true in some cases (e). the most consistent argument is that cell culture methods are not adapted to all viruses, particularly to hrv, hmpv, piv, hcov and influenza virus c. in this study, hrv that were detected uniquely by the multiplex method ( in the negative group and in the positive group) are likely to be serotypes that are difficult or impossible to culture. however, only mrc and huh cells were used for viral isolation technique, and the traditional cell lines, such as mdck, hela r or nci-h cells, were not used. this could explain the greater quantity of viruses that were found using the multiplex rt-pcr assay in relation to the cell cultures. the detection of hrv is considerably improved by the use of molecular biology techniques (savolainen et al., ; coiras et al., ; gilbert et al., ) . a retrospective study carried out in caen between and revealed hrv infections in hospitalised children, % of which were identified by cell culture and % of which were identified only by rt-pcr (guittet et al., ) . nevertheless, it is important to point out that the detection of hrv in nasal aspirates may be associated with interpretation problems, given that this virus can persist for weeks or longer after the acute phase (jartti et al., ) . the multiplex rt-pcr was able to detect all the hmpv previously identified. furthermore, a high proportion of hmpv was found in the negative group: / , i.e., % of samples and nine others in the positive group. hmpv is responsible for - % of viral respiratory tract infections in hospitalised children worldwide (van den hoogen et al., ; boivin et al., ; freymuth et al., a, b) . until now hmpv was not targeted by any multiplex protocol. its clinical impact and its prevalence fully justify its detection in routine diagnosis, alongside hrsv. three type piv were detected by this method: one in the positive group and two in the negative group. it is very difficult to culture this serotype in vitro and immunofluorescence assay has only been possible for a short while, since the production of a first monoclonal anti-piv- antibody. for these reasons piv- is practically never sought in virology laboratories (echevarria et al., ) . but it has been shown that it can cause bronchiolitis or pneumonia in young children and immunodepressed subjects and its prevalence appears to be higher than originally thought (aguilar et al., ; lindquist et al., ) . three hcov oc were detected by the multiplex pcr, two of which were positive with the cell culture method. the hcov oc , which are generally considered to cause colds, have been described to cause lower respiratory tract infections (pneumonia and bronchiolitis) in infants and the elderly . given that few virology laboratories seek hcov, it is probable that their pathogenic role is underestimated . the influenza c virus is generally considered to cause non-severe influenza. however, its pathogenic potential is not well known and its frequency is undoubtedly highly underestimated, as shown by the presence of anti-influenza c antibodies in a large proportion of the french population (manuguerra et al., ) . gapdh, transcribed by nasal mucous cells and used as an internal control in the multiplex rt-pcr, has the advantage of being always present in the sample. this internal control was used in multiplex only, as it caused disturbances in amplifications results of the other rt-pcr multiplex assays and in the hemi-nested pcr multiplex . theoretically, since those rt-pcr multiplex assays used the same amplification kit and the same extraction protocol, the amplification of gapdh would be similar in the three rt-pcr assays. a gapdh amplification product was observed for all nasal aspirates tested, indicating that no enzyme inhibitors were present. however, it has never been validated for use as an internal control in respiratory samples. the techniques described by coiras et al. include an internal control supplied with the promega©kit; once again no amplification inhibitors were detected during clinical evaluations (coiras et al., (coiras et al., , . syrmis et al. used an endogenous human retrovirus (erv- ) as an internal control and found that only out of samples tested were amplification negative (syrmis et al., ) . dingle et al. created a stable internal control based on a modified rna fragment of hepatitis delta (dingle et al., ) . among the respiratory samples tested, only two cases of inhibition were detected. it is possible that the dilution of samples in the transport medium overcomes the effect of inhibitors (syrmis et al., ) . the multiplex methods described in this study detected numerous co-infections in the positive ( %) and negative ( %) groups. the rt-pcr and described by coiras et al., respectively, detected . and % of co-infections among the positive samples (coiras et al., (coiras et al., , . the method described by templeton et al. detected . % of co-infections (templeton et al., ) . of the four studies that have evaluated the hexaplex technique, only that by kehl et al. ( ) reported cases of co-infection: % among the positive samples, none of which were confirmed by cell culture. the studies by osiowy ( ) , puppe et al. ( ) and syrmis et al. ( ) did not describe any cases of co-infection. in our % of co-infections in the positive group and % of coinfections in the negative group involved hrv. the rate of viral co-infection among hrv infections appears to be very variable: from to % (guittet et al., ) . three cases of triple infection were observed: one in the positive group and two in the negative group (cf. tables and ). two cases associating hrsv, vip and hrv were previously described by gilbert et al. ( ) who used a protocol involving three distinct rt-pcrs, specific for each virus. the studies that have revealed co-infections have tended to find that they are not a factor associated with severity. brouard et al. 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the detection of seven common respiratory viruses in respiratory samples rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses , , , and simultaneous infection with respiratory syncytial virus and other respiratory pathogens human coronavirus infections: importance and diagnosis comparaison of three non-nested rt-pcr for the detection of influenza a viruses direct diagnosis of human respiratory coronaviruses e and oc by the polymerase chain reaction an outbreak of coronavirus oc respiratory infection in normandy, france concurrent outbreaks of rhinovirus and respiratory syncytial virus in an intensive care nursery: epidemiology and associated risk factors clinical impact and diagnosis of human metapneumovirus infection detection and identification of human influenza viruses by the polymerase chain reaction key: cord- -oihcay w authors: choudhary, manohar l.; anand, siddharth p.; heydari, mostafa; rane, grishma; potdar, varsha a.; chadha, mandeep s.; mishra, akhilesh c. title: development of a multiplex one step rt-pcr that detects eighteen respiratory viruses in clinical specimens and comparison with real time rt-pcr date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: oihcay w rapid and accurate diagnosis of viral respiratory infections is crucial for patient management. multiplex reverse transcriptase polymerase chain reaction (mrt-pcr) is used increasingly to diagnose respiratory infections and has shown to be more sensitive than viral culture and antigen detection. objective of the present study was to develop a one-step mrt-pcr that could detect respiratory viruses in three sets. the method was compared with real time rt-pcr (rrt-pcr) for its sensitivity and specificity. clinical specimens from pediatric patients with respiratory symptoms were used in the study. ( . %) samples were detected positive by mrt-pcr. of these ( . %) exhibited presence of a single pathogen and ( . %) had multiple pathogens. rrt-pcr detected ( . %) positive samples, where ( . %) exhibited one virus and ( . %) showed co-infections. concordance between mrt-pcr and rrt-pcr was . % and kappa correlation . . sensitivity and specificity of mrt-pcr were . % and . % while that of rrt-pcr was . % and . % respectively. rhinovirus ( . %) was the most frequently detected virus followed by respiratory syncytial virus b ( . %), h n pdm ( . %), parainfluenza virus- ( . %) and metapneumovirus ( . %). in conclusion, mrt-pcr is a rapid, cost effective, specific and highly sensitive method for detection of respiratory viruses. respiratory viral disease is a serious public health problem globally and has social and economic impact. respiratory viruses cause disease that ranges from mild respiratory illness to fatal pneumonia and contribute significantly to morbidity and mortality worldwide (williams et al., ) . clinical presentations of respiratory viral infections are very similar to each other and etiological diagnosis based on clinical parameters is difficult. rapid diagnosis is essential for prompt patient management and public health action. diagnosis of viral respiratory infections has been based on the use of conventional methods such as isolation by cell culture and antigen detection. although these methods are effective and often complementary, they have certain limitations. cell culture is considered to be the "gold standard" for virus detection, but it is too laborious and time consuming. antigen detection is insufficiently sensitive and/or specific. sensitivity and specificity of respiratory virus detection have improved considerably with the advent of nucleic acid amplification tests (nats; bellau-pujol et al., ; fox, ; freymuth et al., ; liolios et al., ; vabret et al., ; weinberg et al., ) . to enable a rapid response to a potential outbreak, it is desirable to have fast, accurate and comprehensive diagnostic methods that are capable of detecting simultaneously and subtyping viruses. however, it is very expensive to use virus specific pcr assays. several groups have developed multiplex rt-pcrs to identify respiratory viruses in clinical samples (bellau-pujol et al., ; bharaj et al., ; brittain-long et al., ; coiras et al., ; gunson et al., ; jansen et al., ; kim et al., ; lee et al., ; mahony et al., ; pabbaraju et al., ; wang et al., ) . the preference of one test over the other depends on its specificity, sensitivity and turnaround time as well as cost in resource limited settings. the purpose of this study was to develop a one step mrt-pcr that could detect respiratory viruses including influenza a viruses, h n pdm , seasonal h n , h n , influenza b viruses, respiratory syncytial virus (rsv) a and b, human metapneumovirus (hmpv), parainfluenza viruses (piv) , , , , rhinovirus, enterovirus, corona viruses oc , e, nl and hku in three sets in human clinical samples and to compare it with rrt-pcr. primers used in this assay were designed using dual priming oligonucleotide (dpo) technology which allows specific detection of viruses without any non specific amplification (chun et al., ) . conserved regions of target genes were chosen to design the forward and reverse primers. for mrt-pcr, different viral genes with their pcr product sizes are given in table . primer sequences can be provided on request. the above mrt-pcr primers were obtained from sigma (bangalore, india). multiplex rt pcrs were carried out in tubes (sets) (set : influenza a, subtype seasonal h , h and pandemic h ( ), influenza b; set : rsv-a, rsv-b, hmpv, piv- , - , - ; and set : piv- , corona virus oc /hku , corona virus e/nl , rhinovirus/enterovirus). the mrt-pcr did not differentiate between corona virus oc and hku ; e and nl ; and between rhinovirus and enterovirus. monoplex one step real time rt pcrs (rrt-pcr) for influenza a, subtype seasonal h , h , pandemic h ( ), influenza b and internal control rnasep were carried out as per protocol provided by centers for disease control and prevention, usa (cdc) (cdc protocol, ). for rsv-a and b, piv- , - , - , corona virus oc , e, nl and rhinovirus, the rrt-pcr assay was performed as described (gunson et al., ) , for hmpv (maertzdorf et al., ) and for piv- as described on the following website www .appliedbiosystems.com/cms/groups/mcb/cms .pdf. for rrt-pcr, all influenza, rsv-a, rhinovirus, piv- , and coronavirus nl taqman probes were labelled with fam (fluorescein amidite); rsv-b, piv- , - , - , coronavirus oc and e were labelled with vic. real time pcr primers and taqman probes were obtained from applied biosystems, usa. tissue culture grown viruses used as positive controls for influenza b, seasonal influenza a h n , h n , h n pdm , rsv-a and b, hmpv, piv- , - , - and enterovirus. clinical samples positive for piv- , rhinovirus, human corona virus oc /hku and e/nl were confirmed by sequencing and used to check primers cross reactivity. nasal, nasopharyngeal or throat swab specimens of ili (influenza like illness)/sari (severe acute respiratory illness) patients collected from july to august and referred to national institute of virology by different hospitals in pune for diagnosis of h n pdm were used in this study. the study was approved by the institutional ethics committee. a total of clinical specimens of pediatric patients were tested by conventional mrt-pcr and monoplex rrt-pcr. one hundred and fourteen influenza positive samples ( seasonal h n , seasonal h n , h n pdm and influenza b) were used for validation of the multiplex assay set . rna was extracted using the magmax automated rna extractor as per manufacturer's instructions. fifty microlitres of clinical sample was used for rna extraction and finally eluted in l of elution buffer. amplification for conventional mrt-pcr was carried out on geneamp pcr system using invitrogen superscript iii one step rt-pcr kit. master mix for mrt-pcr comprised of l × buffer, l enzyme mix, m of forward and reverse primer each and l of rna template to make a l reaction. negative and positive controls were run with each experiment. thermal cycling conditions were • c for min for reverse transcription, initial denaturation at • c for min, cycles of three steps − s at • c, s at • c, s at • c, and final extension at • c for min. l of pcr products were mixed with gel loading dye and run on a % agarose gel containing ethidium bromide and visualized on a uv transilluminator. real time rt-pcr assay was performed on applied biosystems' abi machine using invitrogen superscript iii one step qrt-pcr kit. a typical l pcr reaction comprised of m of each forward and reverse primer, m of taqman probe, . l × buffer, . l superscript iii enzyme and l rna template. thermal cycling conditions for rrt-pcr were • c for min for reverse transcription, initial denaturation at • c for min, cycles of s at • c, s at • c. the specificity of the multiplex pcr assay was evaluated by cross reaction tests with known viral isolates and different panels of sequence confirmed known clinical respiratory samples as reference material. the assay set was also tested on influenza a h n , h n , h n , and newcastle disease virus procured from national institute of virology's virus repository and who (world health organization) influenza panel for qa/qc. to analyze sensitivity, known positive samples were subjected to monoplex pcr. the forward primer was tagged with t promoter and by gel electrophoresis in a % agarose gel, bands of expected size were observed. these bands were extracted from agarose gel using qiagen gel extraction kit (qiagen, germany). purified pcr product was quantified using a nanoplex nanophotometer (implant, germany). in vitro transcription was carried out using ribomax t in vitro transcription system from promega as per manufacturer's instructions (promega, usa). further, rna was purified using qiagen's rna extraction kit and estimated using the nanoplex nanophotometer. rna copy number was calculated and fold serial dilutions of the in vitro rna were performed. copy number referred to the number of copies of the target gene used to ascertain the limit of detection of the assay. the concentrations tested for each target in set were to copies and in sets and were copies to copy of template rna per reaction. the limit of detection (lod) was defined as the lowest concentration at which each target could be detected consistently. pcr products were detected by gel electrophoresis. positive controls were prepared by pooling in vitro rna of each set. initial pcr was set up using monoplex primers for the relevant target gene with the same mastermix composition and thermal cycling conditions as described for the mrt-pcr. pcr product was purified using qiagen's pcr purification kit according to the manufacturer's instructions. the purified product was used to set up cycle sequencing reaction with each sequencing primer as follows: mastermix: × sequencing buffer - l, cycle sequencing reaction mix rr - l, primer - l, ddw - l and template - l. thermal cycling conditions: • c for min; cycles of • c − s, • c for s and • c for min. the above cycle sequencing product was purified using qiagen's dyeex cycle sequencing purification kit as per manufacturer's instructions. applied biosystems xl capillary sequencer was used for sequencing and results were analyzed using sequencing analysis . software. the sequences were confirmed using ncbi blast. statistical analysis was carried out using pasw statistics software. the agreement between two diagnostic tests was calculated by means of concordance and kappa statistics. the sensitivity and specificity of one test with reference to the other and vice versa were also computed. specificity of the mrt-pcr assay was evaluated by cross reaction tests against known respiratory virus isolates/positive samples and a who qa/qc panel for influenza viruses showed no cross reactivity amongst different viruses. a product of the expected size was obtained for each viral target in the presence of all the primers of the respective multiplex pcr and the specific products could be distinguished on agarose gel electrophoresis based on their size. for confirmation of the observed bands, sequencing of the amplified pcr products was carried out. for validation of set of the multiplex assay, respiratory clinical specimens previously positive for influenza viruses by rrt-pcr were used and results were % concordant. co-infection was observed in one sample where both seasonal influenza a/h n and a/h n detected, this was confirmed by sequencing of the hemagglutinin (ha) and neuraminidase (na) genes. set of the mrt-pcr detected specifically only h n pdm , seasonal h n , h n and all other influenza-a viruses were detected as a universal influenza-a product of bp. in the who influenza panel, samples exhibited bands corresponding to influenza-a only. these samples were confirmed as influenza a h n by sequencing the matrix gene. analytical sensitivity of the conventional multiplex assay was determined by testing fold serial dilutions of the quantified rna transcripts of each target. set detected rna copies per reaction for each target. set detected rsv-a at copies per reaction; hmpv, piv- , - and - at copies per reaction and rsv-b at rna copies per reaction. set detected enterovirus at copy per reaction; piv , rhinovirus and corona oc /hku , e/nl at rna copies per reaction in the mrt-pcr. the experiments were repeated three times using all three sets to obtain consistent limits of detection for each virus in the mrt-pcr table ). supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . / j.jviromet. . . . overall, concordance between both the methods was . % and kappa correlation was . . sensitivity and specificity of mrt-pcr were . % and . % respectively considering rrt-pcr as the "gold standard". conversely sensitivity and specificity of rrt-pcr were . % and . % respectively considering mrt-pcr as the "gold standard" (table ) . for seasonal h n , h n pdm , influenza b, rsv-a and b and piv- , % concordance were observed using both the methods. the sensitivity and specificity for rhinovirus using mrt-pcr were . and . % and rrt-pcr were . % and . % respectively. thirty seven out of samples positive for rhinovirus/enterovirus in mrt-pcr but negative by rrt-pcr were selected randomly and sequenced for the -utr region. sequencing result confirmed samples as rhinovirus ( . %) and as enterovirus ( . %). for hmpv, sensitivity and specificity were % and . % for mrt-pcr while the sensitivity and specificity were . % and % respectively for rrt-pcr. the samples positive in the mrt-pcr, but negative by rrt-pcr was confirmed to be positive for hmpv by sequencing. sensitivity and specificity by mrt-pcr were % and . % for piv- and those for rrt-pcr were . % and % respectively. among piv- positive clinical samples, approximately % samples showed amplification bands corresponding to piv- and piv- which were tested further using monoplex primers. they were negative for piv- and piv- indicating that the bands were nonspecific. for piv- , the sensitivity and specificity were % and % for rrt-pcr respectively. four out of the samples negative by rrt-pcr were sequenced and found to be piv- . randomly selected corona virus oc /hku positive samples were sequenced and confirmed as corona virus oc . rhinovirus [ / ( . %) ] was the respiratory virus detected most frequently followed by rsv-b in samples ( . %), h n pdm in / ( . %), piv- in samples ( . %) and hmpv in samples ( . %). corona virus e was detected in samples by rrt-pcr. as shown in supplementary table , rhinovirus was detected most frequently in co-infection followed by rsv. all coinfections were confirmed by performing monoplex pcrs with their respective primers. major respiratory viral pathogens recorded globally are rsv, influenza virus, metapneumovirus, parainfluenza virus, rhinovirus, enterovirus, adenovirus and bocavirus. a number of studies have attempted for development and evaluation of multiplex pcrs for detection of various respiratory viruses (auburn et al., ; bellau-pujol et al., ; boonsuk et al., ; brittain-long et al., ; coiras et al., ; jansen et al., ; kim et al., ; mahony et al., ; suwannakarn et al., ; wang et al., ) . this is the first study using single step mrt-pcr for the detection of respiratory viruses from clinical specimens. for the conventional mrt-pcr, conserved regions of genes were selected (table ) . each set in the assay produced distinctively sized pcr products which could be visualized and easily differentiated by agarose gel electrophoresis. the only limitation of this multiplex pcr is lack of an internal control to check quality of the samples. the mrt-pcr is sensitive as determined by testing fold serial dilutions of quantified rna transcripts of each target. multiplex pcr set detected rna copies/reaction for each target which is comparable with multiplex pcr developed by kim et al. ( ) . an influenza typing kit available commercially, the seeplex a/b typing kit has a sensitivity and specificity of . % and % in detecting h n pdm when compared to the cdc real time rt-pcr (choi et al., ) while sensitivity and specificity of our mrt-pcr was %, showing excellent concordance with cdc's real time rt-pcr. multiplex rt-pcr detected ( . %) samples positive for one or more of the respiratory viruses listed above and real time pcr detected ( . %) samples as positive (supplementary table ). this low detection rate by rrt-pcr was mainly due to the low sensitivity of detecting rhinovirus and metapneumovirus. previously, detection rates between % and % have been reported (brittain-long et al., ; broor et al., ; coiras et al., ; kim et al., ; mahony et al., ; maitreyi et al., ; yeolekar et al., ) . co-infections have been described previously in approximately - % of infected patients in different studies (brittain-long et al., ; coiras et al., , kim et al., mahony et al., ; wang et al., ) . one of the biggest advantages of mrt-pcr is its ability to detect co-infections which are often undetected in viral culture with immunofluorescence detection. several cases of co-infection were detected using both the pcr methods. multiplex rt-pcr detected ( . %) samples with co-infection including samples in which viruses were detected whereas rrt-pcr detected ( . %) samples with co-infection. one case of co-infection with influenza a/h n and a/h n viruses was also detected. co-infection with different influenza viruses occurs naturally and plays an important role in evolution, epidemiology and pathogenicity of the virus. concordance between both the methods was % for seasonal h n , h n , h n pdm , influenza b, piv- , rsv-a and b. lower detection rates by rrt-pcr were due to the lower sensitivity of this system for rhinovirus and hmpv. this may be due to sequence variation in primer probe binding regions in indian subtypes. frequency of piv- , piv- , piv- and coronavirus was low; a larger number of samples are required to better assess their clinical sensitivity and specificity. the multiplex rt-pcr scores over the monoplex rrt-pcr in a resource limited settings. the cost of mrt-pcr for the viruses discussed here is approximately us$ whereas for the real time pcrs it is us$ . moreover, the real time pcr machines are very expensive when compared to a conventional thermal cycler. the one step multiplex rt-pcr assay developed in the present study provides a simple, rapid and cost effective method for the detection of different respiratory viruses from human clinical specimens. the mrt-pcr is as sensitive and as specific as rrt-pcr based assay. this mrt-pcr assay can be used for respiratory virus surveillance as well as diagnosis. detection of nine respiratory rna viruses using three multiplex rt pcr assays incorporating a novel rna internal control transcript development of three multiplex rt pcr assays for the detection of respiratory rna viruses 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real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay rapid detection of respiratory viruses by centrifugation enhanced cultures from children with acute lower respiratory tract infections comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections typing (a/b) and subtyping (h /h /h ) of influenza-a viruses by multiplex real-time rt pcr assays comparison of three non-nested rt pcr for the detection of influenza-a viruses molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-pcr assays superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children estimates of worldwide distribution of child deaths from acute respiratory infections respiratory viruses in acute respiratory tract infections in western india the authors would like to thank indian council of medical research (icmr) for funding the study. technical team of influenza group and atul walimbe for statistical analysis is acknowledged. key: cord- -tm hn hz authors: mockett, a.p.adrian title: envelope proteins of avian infectious bronchitis virus: purification and biological properties date: - - journal: j virol methods doi: . / - ( ) - sha: doc_id: cord_uid: tm hn hz immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (ibv). the purified proteins were inoculated into rabbits to produce antisera. the rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. ibv-infected chickens produced antibodies to both the spike and membrane proteins. both these antibodies were at their highest concentration about – days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. neutralizing antibodies were at their highest concentration days after inoculation. a second inoculation of virus at days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. the neutralizing, anti-spike and anti-membrane antibodies all reached highest concentrations – days after this inoculation. the advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed. avian infectious bronchitis virus (ibv) is a coronavirus whose principal site of replication is the ciliated epithelial cells of the respiratory tract mucosa of chickens. viral replication occurs in the cytoplasm of the cell and virions are formed by budding from the endoplasmic reticulum. there are three viral structural proteins: spike (s; peplomers), membrane (m) and nucleocapsid (n). s and m proteins are both glycosylated and parts of them are exposed at the surface of the virion. the spike protein consists of two glycopolypeptides, s and $ , which have molecular weights of kda and kda, respectively (cavanagh, ) . the membrane protein is present as a number of distinct species which have molecular weights ranging from kda to kda; the molecular weight differences are associated with the various degrees of glycosylation. n protein ( kda) is associated with the viral rna. the ibv spike protein, associated with the outer projections, plays an important part in the infection of cells. chicken antisera to this protein and spike-specific monoclonal antibodies (mockett et al.. ) can neutralize the infectivity of the virus. a similar function has been found for the spike protein of murine coronavirus mhv- (collins et al., ; fleming et al., ) and the porcine coronavirus tgev (garwes et al., ) . the spike protein of ibv also contains strain-specific determinants (mockett et al., ) . the membrane protein appears to be a more highly conserved antigen and it is possible that only a small amount (approx. kda) is exposed at the viral surface (boursnell et al., ) . the nucleocapsid protcin interacts with the viral rna to form a helical nucleocapsid. the objectives of this work were to produce immunoadsorbents using monoclonal antibodies which have been prepared previously (mockett et al., ) and to purify the virus-coded proteins of the viral envelope in a single step in relatively large amounts. this allowed hyperimmune rabbit antisera to the proteins to be produced and tested for neutralizing antibodies. in addition the sequential humoral antibody response of chickens after ibv infection has been studied using the purified viral proteins and whole virus in elisas and compared to the results using the neutralization test. the massachusetts m strain of ibv was grown in the allantoic cavities of i -day-old embryonated chicken eggs and purified on isopycnic sucrose gradients as described by cavanagh ( ) . purified virus was pelleted in a x ml rotor at , x g for h at °c and resuspended in phosphate-buffered saline (pbs). an equal volume of pbs containing % (wt./vol.) np was added, mixed using a dounce homogeniser and incubated for h at °c. the material was centrifuged for min in an eppendorf microcentrifuge and the resulting supernatant, containing soluble viral components, was used for the affinity chromatography purification. monoclonal antibodies (designated a and c! ) to the spike and membrane proteins respectively of ibv strain m were prepared (mockett et al., ) . the gammaglobulin fraction of ascitic fluids containing either anti-spike or anti-membrane monoclonal antibodies was isolated by salt precipitation using a final concentration of % (wt./vol.) na so . for the spike immunoadsorbent . mg of gammaglobulin was coupled to . mg of cnbr-sepharose b (pharmacia) according to the manufacturers' instructions and for the membranc immunoadsorbent . mg was coupled to the same amount of gel. unreactive groups on the gel were blocked using m ethanolamine, pit . , and any non-covalently bound proteins wcre removed by repeated washings with . m nahco~ buffer, ph . , containing . m naci and . m acetic acid buffer, ph . , containing . m naci. the immunoadsorbent was stored in pbs containing . % nan~ at °c until used. it was washed twice with m nh scn in pbs containing . % octylglucoside, four times with pbs and twice with pbs containing % np before use. all wash volumes were ml. the solubilised virus preparation was mixed with the immunoadsorbent for h at °c using a rotary stirrer. the gel was poured into a chromatography column and washed with pbs containing . % np ( ml) and pbs containing . % octylglucoside ( ml). m nh,scn in pbs containing . % octylglucoside was added and fractions of ml collected. the absorbance at nm of each of the fractions was read using a sp pyeunicam spectrophotometer. the fractions in the absorbance peak were dialysed against pbs. a sample of each fraction was then subjected to electrophoresis in a polyacrylamide gel. those fractions containing detectable viral protein were pooled and constituted the purified protein prepar~,tion. ten per cent polyacrylamide slab gels containing sds were used (laemmli, ) and after electrophoresis samples were stained first with coomassie brilliant blue r- and then silver (morrisey, ) . new zealand white rabbits were used. samples of purified proteins were mixed with an equal volume of freund's complete adjuvant and inoculated intramuscularly into the rabbits. a similar inoculation was given wk later. after wk the same antigen in incomplete freund's adjuvant was inoculated subcutaneously. five months later blood was collected from the ear vein and the resulting serum stored at - °c until used. the houghton poultry research station line of rhode island red chickens was used. ibv (m ) was inoculated intratracheally ( ciliostatic dose fifty (cd ); darbyshire et al., ) into chickens and sequential blood samples were taken from the wing vein (see fig. for times after inoculation). sera from the blood samples were stored at - °c until used. a serum pool for each time of sampling was made by mixing an equal volume of serum from each of the eight samples. three different antigens were used for the ei,isas: spike protein, membrane protein and ib virus. the purified spike and membrane proteins were used at a dilution of : in carbonate buffer whilst the purified ib virus was used at a : dilution. antigens were adsorbed for i h at °c. in the second step chicken sera were serially diluted in . m nacl containing . ~ c np (saline/np ) from an initial . iog dilution. any specifically bound antibody was detected using a rabbit anti-chicken igg serum ( : ) and a goat anti-rabbit igg alkaline phosphatase conjugate ( : , ) (sigma), both diluted in saline/np . the substrate was p-nitrophenyl phosphate ( mg/ml)in diethanolamine buffer and the reaction stopped using m naoh. each step was for min at °c, the reaction volumes pl and the plate was washed three times with pbs containing . c~ tween between each step. titres were calculated graphically (mockett and darbyshire, ) using an absorbance value of . as the cut off. the chicken antisera were tested for neutralizing antibody to ibv as described by darbyshire et al. ( ) . rabbit sera were precipitated with °~ (wt./vol.) nazso and the resulting gammaglobulin tested because rabbit sera have high concentrations of non-specific inhibitors of ib virus replication. the viral proteins purified by affinity chromatography are shown in fig. . the spike protein, which is composed of two polypeptides, was the only protein detected in the two fractions shown using the sensitive silver staining procedure. similarly the membrane protein was not contaminated with other proteins, although this protein did not stain as well as the spike. there were other stained bands present, but these are artifacts sometimes observed, even in the absence of protein, with this staining procedure. the purification was highly reproducible. the purified viral proteins were inoculated into rabbits and the gammaglobulin fraction of the antisera tested for neutralizing activity. only the anti-spike gammaglobulin neutralized the virus. however the membrane protein was a good immunogen because the rabbit anti-membrane sera tested in the elisa had high activity against the whole virus: in fact the titres were higher than those of the rabbit anti-spike sera (see table ). the results of testing sera from ibv-infected chickens for antibody to spike and membrane proteins showed that both anti-spike and anti-membrane antibodies were produced early after infection (see fig. ). peak titres were between and days after infection. the antibody response to the whole virus had a similar profile. however, the the second inoculation of virus induced an anamnestic antibody response. the elisa detected a similar increase in antibody titres using spike, membrane and whole virus -the peak was at - days after infection. the neutralizing antibody response was also similar and the peak titres were at days after infection which contrasted to the slow rise to the peak titres after the primary inoculation. this paper describes the application of affinity chromatography using monoclonal antibodies for the purification of the two viral structural proteins present at the surface of the ib virion -spike and membrane. a previous report has described procedures for the purification of these viral proteins and also nucleocapsid protein, the only other major structural protein (cavanagh, ) . ibv was solubilised in np detergent and centrifuged in a sucrose gradient containing this detergent in order to purify the nucleocapsid protein. the addition of m naci to the sucrose solutions was required for the purification of the spike and membrane proteins, as they co-migrated in gradients containing low salt concentrations. however, the nucleocapsid protein could not be purified in gradients containing high salt concentrations. the yield of material from these gradients was relatively low, due to the limited number of fractions which contained purified viral components. in other studies purified spike material contained some nucleocapsid protein and the membrane preparation contained other proteins which were thought to be of cellular origin. there are a number of advantages in using affinity chromatography. by making use of the specificity of the antibody pure material can be isolated, even from a crude mixture of proteins. the method is very quick and easy and the immunoadsorbent can be used several times. thus, relatively large amounts of purificd material can be obtained. the availability of spike and membrane proteins in a highly purified form will allow more biochemical, structural and immunological studies to be done. the conditions used to solubilise the virus did not dissociate the two spike polypeptides, therefore, both s i and $ were detected in thc material eluted from the anti-spike immunoadsorbent. the results of experiments using the rabbit antisera to the viral proteins confirmed the biological importance of the spike protein as only antibodies to this protein neutralized the infectivity of the virus. thc chicken, about days after an ibv infection, has antibodies to both the spike and membrane proteins in its serum but only very low concentrations of neutralizing antibodies. thc profile of neutralizing antibodies shown in this paper agrees with previous published findings (holmes, ; mockett and darbyshire, ; hawkes et al., ) . the results show that anti-spike antibodies produced early after infection are non-neutralizing, as assessed by our in vitro technique. this raises the question as to the function of these antibodies in thc chicken. previous evidence has shown only the spike protein to be capable ofeliciting neutralizing antibodies. there is a possibility that the anti-spike antibodies could be neutralizing in vivo and the function of the anti-membrane antibodies could be similar. the possible role of these antibodies in protection remains to be resolved. purified viral proteins can be used to determine which is responsible for protecting thc chicken from infection. protection tcsts such as thc ciliostasis (darbyshire, ) and the mixed infcction (escherichia coil and ibv) (smith et al., ) tcsts are available. it is only by using methods such as affinity chromatography that stflficientl~, large amounts of pure viral proteins can be made available to enable such tests to bc done. . \.'iru~ rc~ i . pov, cll virolog, , ~. darbyshire artwrlght, lg":'bl vet ph. i). i hcsis, i. tnl,ct~,it', oi ]'he author wishes to thank ms. ,i.k.a. ('ook for her help with the neutralilatton tests, ms. debra southee for her excellent technical assistance and i)r. i'.i).k. br()wn for useful discussions. key: cord- -ccdj authors: vabret, astrid; mouthon, franck; mourez, thomas; gouarin, stephanie; petitjean, joëlle; freymuth, françois title: direct diagnosis of human respiratory coronaviruses e and oc by the polymerase chain reaction date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: ccdj an rt-pcr-hybridization was developed that amplified genetic material from the m protein gene of hcov- e and hcov-oc . the analytic sensitivity of these original primers were compared with primers defined in the n gene and described previously. the results show that . tcid( ) of hcov- e and . tcid( ) of hcov-oc can be detected by this molecular method using the original method. detection of hcov- e and hcov-oc in clinical specimens is possible using this method: respiratory specimens ( sputum and nasal aspirates) were tested with this rt-pcr-hybridization and human coronavirus are detected ( %). the method could provide a useful tool for demonstrating the role of human coronavirus in infections of the respiratory tract. human coronaviruses were described initially in patients infected with rhinitis. they belong to a group of viruses that concern human and different animal species. they are implicated in diseases involving the respiratory tract, the digestive system, and the central nervous system (vabret et al., ) . apart from rhinitis, human coronavirus is associated with more severe pul-monary infection (myint, ) . as for other respiratory viruses (influenza viruses, rhinoviruses), they are associated with bronchitic hyperactivity even in non-atopic patients (trigg et al., ; freymuth et al., ) . epidemiological inquiries have led to the conclusion that these viruses circulate widely in seasonal oubreaks. coronaviruses are enveloped viruses, pleiomorphic with a long ( kb) rna molecule. the human strains are divided into two distinct antigenic groups which are both represented by a prototype virus, hcov- e and hcov-oc . few detection methods of coronavirus are available at present. consequently, these viruses are sought rarely in diagnostic laboratories, and the associated clinical symptoms associated are not defined. some molecular detection methods were described recently for screening for human coronavirus: pcr amplification, simple or nested, with primers defined in the n protein gene stewart et al., ) . in this paper, two methods are described for the routine detection of two groups of human coronavirus. they consist of a pcr, where primers are defined in the m protein gene, followed by molecular hybridization using nonradioisotopic probes. the alignment of the nucleotidic sequence of the m genes of the e and oc shows a homology of %, insufficient to define a common system of detection (sequences extracted from genbank™, software gcg™). those detection systems have been developed on both the prototype strains hcov- e and hcov-oc , and are compared to the other method using primers defined in the n protein gene as published stewart et al., ; sizun et al., ) . the two cell line-adapted strains of prototypes human coronavirus ( e and oc ) were obtained from atcc, rockville, md. hcov- e was propagated by inoculation into a human embryonic lung diploid fibroblast cell strain (mrc ) at - days old and incubated for h at c in eagle's basal medium (mbe, gibcobrl) supplemented with . % sodium bicarbonate, % fetal bovine serum and antibiotics (penicillin u/ml, and gentamycin mcg/ml). the cytopathic effect produced by hcov- e in mrc is not characteristic and appears as an extensive lysis. the identification of coronavirus is carried out by using an indirect immunofluorescent test with a monoclonal antibody (mouse igg mab, - h. , obtained from talbot, p.j. canada). strain hcov- e was amplified by several passages in cell cultures so as to obtain a viral suspension with a titer of × tcid /ml. laboratory stocks of hcov- e were kept at − °c, and used for further experiments. hcov-oc was propagated by inoculation into a -day old human rectal tumor cell strain (hrt ) and incubated for or h at °c in a humidified atmosphere containing % co in rpmi medium (gibcobrl) supplemented with % fetal bovine serum and antibiotics. the isolation of hcov-oc required the presence of trypsin (porcine pancreatic trysin, sigma t , . mg/ml) to cleave the hemagglutinin-esterase protein. the identification of hcov-oc was carried out by an indirect immunofluorescent test using a monoclonal antibody (mouse igg mab - oc. , obtained from talbot, p.j canada.). after a few passages, a viral suspension with a titer of × tcid /ml was obtained. the laboratory stocks of this viral suspension was kept at − °c and used for all experiments. infectious virus titers of samples used for evaluation of diagnostic techniques were measured using the immunofluorescent method described previously. susceptible cells (mrc or hrt ) were inoculated with logarithmic dilutions of cell culture supernatant in a -well plate. after or days of incubation, the number of infected wells were determinated by the immunofluorescence test. infectious titers were calculated by the karber method. each one of the logarithmic dilutions was stored for min in order to extract rna. for the control of specificity, strains of human respiratory syncytial virus, sub-group a and b, human adenovirus type , influenza virus a (h n ) and b, herpes simplex virus, cytomegalovirus strain ad , rhinovirus type , parainfluenza virus type and were isolated in cell culture. for rt-pcr, ml of each one of the tenfold dilutions of the viral suspension were mixed with ml of rnazol™ b (bioprobe, france), and rna was extracted by a guanidium isothiocyanate procedure as recommended by the manufacturer. rna was precipitated from the extract with cold isopropanol and purified by washing with % cold ethanol. the extracted rna was resuspended in ml of distilled water treated with diethylpyrocarbonate (depc) and ml of rnasin ® ribonuclease inhibitor (promega, madison, wi). the rt-pcr used our original primers and probes defined in the m gene of hcov- e and hcov-oc , and the other primers and probes defined previously in the n gene of these two viruses (stewart et al., ; myint et al., ; jouvenne et al., ) (tables and ) . rt-pcr was carried out in ml of a reaction mixture containing ml of extracted rna, ml of mm dntps, ml of cdna primer at mm, ml of × mgcl mm (geneamp ® perkin elmer), u ( . ml) of rnasine (promega, madison, wi), u ( ml) of avian myeloblastosis virus reverse transcriptase (promega), . u ( . ml) of taq polymerase (perkin-elmer cetus), and ml of sterile water. the final mixture was overlaid with mineral oil, and the rt-pcr was carried out in an omnigene thermocycler (hybaid): first min at °c, then min at °c, then cycles: denaturation, °c, s; annealing, at a variable temperature (tables and ) corresponding to the primers of reaction, s; extension, °c, s; final extension °c, min. each rt-pcr test included water controls that were treated identically to the virus samples throughout. pcr amplification products were detected by agarose gel electrophoresis and by a dna enzyme immunoassay (gen-eti-k deia, sorin). this test is based on the hybridization of amplified dna with a single stranded dna, %-biotinylated probe, coated on the wall of a microtiter plate with a streptavidin-biotin bond. the hybrid of the probe and dna was detected by using an anti-ds-dna monoclonal antibody and by the addition of an enzyme tracer (antimouse igg conjugated to horseradish peroxidase). the optimal concentration of the probe required for the test was . ng/ml for all the probes used. the assay was carried out as recommended by the manufacturer, and an index value was defined as od sample value/od cut-off value. a positive rt-pcr-eia was defined by a dna fragment visualized at the right position on agarose gel associated with a positive hybridization (index value \ ). since many studies show that dna enzyme immunoassay (deia) is superior to gel electrophoresis for detection of pcr amplicons, and as it has been shown by experience gained with different probes used in this system that absorbance values greater than the cut-off indicate that specific hybridization has taken place, we consider as positive a negative gel electrophoresis associated with a hybridization index \ (freymuth et al., ; garcia et al., ; levy et al., ; cantaloube et al., ) . fig. shows that dna enzyme immunoassay (gen-eti-k deia, sorin) increases the sensitivity of the detection of the hcov rna. after extraction of tenfold dilutions of a hcov-oc viral suspension, the results of rt-pcr using primers defined in m gene (mf , mf ) show that the viral suspension and first dilution ( − ) are positive by gel detection and that the hybridization index is greater than . for the − dilution, no band was detected on the agarose gel when the hybridization index was positive and equal to four. the positivity of this detection was confirmed by the vizualization of a band at the right position ( pb) resulting from the heminested rt-pcr using primers mf , mf , and mf . the analytic sensitivity of the rt-pcr-eia molecular method was determined by considering that the highest positive dilution represents the limit of detection. since the infectious titer of the viral suspension is known, it is possible to deduce the correlation between the limit of molecular detection and the infectious titer (tcid ). from october to february , respiratory specimens (sputum) were taken from adult patients who suffered from an acute illness of the lower respiratory tract. a total of nasal aspirates were collected from children who suffered from an acute attack of asthma. all these respiratory specimens (sputum and nasal aspirates) were resuspended in ml of viral transport medium and frozen at − c. nucleic acids were extracted by rnazol b™ (bioprobe, france) and the rt-pcr using primers defined in gene m and described previously were carried out from these frozen samples. positive and negative control were included and treated in the same way as the virus sample. pcr amplification assays were carried out on the extracted rna of the two prototype strains using the different primers defined in n and m genes (tables and ) generate unique fragments having the expected molecular level and visible on agarose gel under ultraviolet light. for hcov- e, bands were located at and pb in n gene for assays using primers defined by stewart et al. ( ) and by myint et al. ( ) , and at pb in m gene for assays using our original primers. for hcov-oc , bands are located at and pb in n gene for assays using primers defined by stewart et al. ( ) and by myint et al. ( ) and at pb in m gene for assays using original primers. these pcr amplification products hybridize specifically with the corresponding probes in the hybridization test with an index value greater than . to assess the sensitivity of the detection of hcov by rt-pcr hybridization, the infectious titers (tcid ) of tissue culture-grown viruses in mrc cells for hcov- e and in hrt for hcov-oc were determined. two dilution series were made from each viral suspension. nucleic acid was extracted from each dilution for cdna synthesis and pcr. thus, the end point of detection of infectious virus could be directly compared with the end point of viral detection by rt-pcr-hybridization. the results are summarized in table . for hcov- e, rt-pcr-hybridization detected and . tcid using the primers defined in n gene by myint et al. ( ) and stewart et al. ( ) respectively, and . tcid with our primer set defined in the m gene. for hcov-oc , rt-pcr-hybridization with the primers defined in n gene by myint et al. ( ) and stewart et al. ( ) are not sensitive. they did not detect less than tcid while our prime set defined in m gene detected . tcid . thus, comparing the end points, rt-pcr-hybridization in m gene is -times more sensitive for hcov- e and times more sensitive for hcov-oc than the viral isolation technique. from october to february , a total of respiratory specimens were collected from adult patients suffering from an acute lower respiratory tract illness and from children with an acute attack of asthma. all the specimens were analysed for hcov- e and oc by rt-pcrhybridization using primers and probes defined in the m gene. as shown in table , hcov were detected in six of sputum ( %) from adult patients (three hcov- e and three hcov-oc ), and six of nasal aspirates ( %) of children (two hcov- e and four hcov-oc ) the diagnosis of a coronavirus respiratory infection is difficult. in the first place, beside prototype strains, very few wild strains grow in culture. the reference detection technique is electron mi- value of the limit of molecular detection for each system of detection of human coronaviruses e and oc by rt-pcr using different primers and probes defined in n and m genes primers/probes references hcv value of the limit of detection gene myint et al., tcid /ml e n stewart et al., n . tcid /ml m - . tcid /ml n oc myint et al., tcid /ml stewart et al., n \ tcid /ml m - . tcid /ml croscopy. electron microscopy is not a sensitive technique, and it requires an experienced technician. in respiratory samples, images of coronavirus are very hard to differentiate from other cellular structures. search of intracellular viral antigen by direct immunofluorescence on respiratory cells is disappointing, and many diagnostic laboratories do not undertake this test. in our laboratory, only one marketed antibody (piv- , argène france) was used between and for the systemic search of coronavirus e in nasal aspirates sampled from hospitalised children, and bronchoalveolar liquids (bal) from hospitalised adults. among the tests done, only six samples (five bronchoalveolar liquid and one nasal aspirate) were found positive for hcov- e (data not shown). the sensitivity of this antibody has not been defined in viral diagnosis and it is possible that the epitopes recognized are only slightly or not expressed by the cells infected by the wild-type hcov- e. furthermore, the specificity of that monoclonal antibody is not defined. in order to validate the results of immunofluorescence and to devise a diagnostic method for respiratory samples, we have developed a detection system by rt-pcr-hybridization, sensitive and specific. the use of nested pcr, similar to that published in the literature has been discarded because of the high comtamination risk that it represents when many samples are tested. each amplified pcr product is submitted to a molecular hybridization that uses a specific probe recognising hcov- e or hcov-oc , thus on one hand allowing a control of its specificity, and on the other hand an increase of its sensitivity. the test is simple and does not require the use of radioactive materials. in the literature, the primers allowing the amplification of human coronavirus were chosen mainly in the gene of the n nucleocapside protein. two reasons justify this choice: this protein is a priori well conserved and the correspondent rnam is present in large amounts in the infected cell (van der most and spaan, ) . we decided to compare two of these primers with an original system defined in the m protein gene. of all the protein components of the virion, the m protein is the most abundant. it is a transmembranous protein with a n-terminal hydrophilic ectodomain and three hydrophobic regions containing three transmembrane helixes (rottier, ) . the nucleotidic sequence of its gene is a priori conserved. the results obtained show that in the prototype strains and in the detection, these primers permit table detection of hcov- e and hcov-oc by rt-pcr-hybridization using original primers defined in the m gene in nasal aspirates from children suffered from an acute attack of asthma and respiratory specimens of adults with acute illness of low respiratory tract sputum nasal aspirates detection of hcv- e detection of hcv-oc number of co-infection e and oc total of positive ( %) ( %) specimens a very sensitive detection assay on a scale of . tcid for hcov- e and . tcid for hcov-oc . these methods, therefore, allow the detection of less than one infectious particle. the phenomenon can be explained by the synthesis in cell culture of many defective particles. these particles contain identifiable genetic material, but do not have the capacity to infect other cells. one inconvenient aspect of this method is that it requires a different detection system for the two types of coronavirus. the low percentage of homology of the nucleotidic sequence between the n and m protein genes does not permit a common detection system. only one recent publication by stephensen et al. ( ) suggests a detection of the polymerase gene (orf b) and the development of a coronavirus consensus pcr. this approach is interesting as it can be used to detect a new coronavirus, or at least a variant of the prototype strains, although the sensitivity of this method is still to be defined. the use of classical diagnostic methods provides diagnosis in only % of the samples received when there is a suspicion of viral respiratory infection (freymuth et al., ) . the development of molecular methods to detect viruses that are not identified by these classical detection protocols (especially coronavirus and rhinovirus) seems useful (ieven and goossens, ) . the results obtained by using the rt-pcr-hybridization in the m protein gene on different respiratory samples can validate the use of this technique. these are concordant with the recent results of nokso-koivisto et al. ( ) . these researchers were looking for e and oc coronavirus in many samples ( nasal aspirates and medium ear sample) that came from a prospective cohort of -month old children, followed over a period of months and sampled during each infectious episode. the method used was a rt-pcr duplex hcov- e and hcov-oc , followed by a molecular hybridization on microplates. the primers are localised in the gene of the n nucleocapside protein and are different from those used in this study. the results show that the coronavirus was found in . % of the samples ( in total, hcov-oc and hcov- e), of which half were obtained from children presenting other infectious diseases (nokso-koivisto et al., ) : bronchitis, pneumonia, quinsy, laryngitis, conjunctivitis, and exanthema. in order to compare the sensitivity of the different molecular methods described for the detection of coronaviruses e and oc , it is advisable to apply in parallel many respiratory specimens since the percentage of positive samples found in the studies is low, around %. it is also necessary to correlate those results with the clinical signs presented by the patients, and to study controls without respiratory clinical signs. in conclusion, even if the isolation by culture and the search for intracellular viral antigens remain the 'gold standard' of detection techniques of respiratory classical viruses (respiratory syncytial virus, and influenza virus), the use of molecular methods for viruses, such as coronavirus, seems justified in a diagnostic approach in order to determine the clinical implications and the importance of those viruses in respiratory pathology. the molecular technique of detection defined in this study has numerous advantages: it is simple to carry out, its analytical sensitivity on the prototype strains is high and the localisation of the primers in the m protein gene is useful. evaluation of four pcr sustems amplifying different genomic regions for molecular diagnosis of gb virus c infections les virus responsables d'infections respiratoires en pédiatrie. bilan de aspirations nasales réalisées chez l'enfant sur une période de six ans detection of respiratory syncytial virus by reverse transcription-pcr and hybridization with a dna enzyme immunoassay detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae in exacerberations of asthma in children evaluation of enzyme immunoassay for hepatitis b virus dna based on anti-double-stranded dna relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory sequence analysis of the membrane protein gene of human coronavirus e evaluation and comparison of pcr and hybridization methods for rapid detection of cytomegalovirus in clinical samples human coronavirus: a brief review evaluation of nested polymerase chain methods for the detection of human coronaviruses e and oc respiratory coronavirus infections in children younger than two years of age the coronavirus membrane glycoprotein comparison of immunofluorescence with monoclonal antibodies and rt-pcr for the detection of human coronaviruses e and oc in cell culture phylogenetic analysis of a highly conserved region of the polymerase gene from coronaviruses and development of a consensus polymerase chain reaction assay detection of coronaviruses by the polymerase chain reaction bronchial inflammation and the common cold: a comparison of atopic and non-atopic individuals infections à coronavirus humains, importance et diagnostic coronavirus. replication. transcription, and rna recombinaison we are very grateful to p.j. talbot (canada) who supplied us with monoclonal antibodies and agreed to read this report. key: cord- - p q x authors: gueudin, m; vabret, a; petitjean, j; gouarin, s; brouard, j; freymuth, f title: quantitation of respiratory syncytial virus rna in nasal aspirates of children by real-time rt-pcr assay date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: p q x a method was developed for the quantitation of respiratory syncytial virus (rsv) based on real-time rt-pcr using a lightcycler instrument. a control real-time rt-pcr was undertaken on gapdh mrna (a human housekeeping gene) was carried out to standardise the non-homogeneous respiratory samples. the real-time rt-pcr method was one log more sensitive for the detection of rsv according to the endpoint dilution technique than the culture method or a conventional qualitative rt-pcr-hybridization-eia. no cross-reactivity was observed with any of the viruses that could be found in the respiratory tract. rsv and gapdh were quantified in nasal aspirates from children hospitalised for acute respiratory tract disease: ( . %) were positive according to the immunofluorescence assay (ifa), ( . %) were culture-positive and ( %) were positive according to our real-time rt-pcr method. the sensitivity, specificity, positive and negative predictive values of the real-time rt-pcr were , , , %, respectively. the samples found to be positive for rsv were classified according to the severity of the disease. the mean number of rsv rna copies was higher in the severe disease group than in the non-severe group . × ( ) vs . × ( ) (p= . ). however, the mean ratio of rsv rna copies to gapdh mrna copies was . in the severe group, and . in non-severe group (p=ns). human respiratory syncytial virus (rsv) is the most common etiological agent of serious respiratory tract disease in infants and young children worldwide (chanock and parrott, ) . its clinical manifestations vary from mild disease of the upper respiratory tract to severe bronchiolitis or pneumonia. it is the most frequently isolated agent from hospitalised infants (freymuth et al., ) . outbreaks of rsv infection occur in the winter, when the two major groups of rsv (a and b) are both circulating (freymuth et al., ) . some authors have shown that a strains result in more severe infections than b strains (hall et al., ) . several groups have used quantitative culture methods to evaluate the relationship between disease severity and the amount of rsv in the nose (buckingham et al., ) . the aim of our study was to quantify rsv genomic rna by use of real-time pcr using a light-cycler instrument (wittwer et al., ) . as for other paramyxoviridae , rsv particles are mostly cell-associated in infected tissues. as nasal aspirates are non-homogeneous, a method is suggested for the standardisation of samples based on the quantitation of the mrna of a housekeeping gene, gapdh (which is probably expressed at a constant rate). this study describes two real-time pcr techniques designed to quantify rsv genomic rna and gapdh mrna. these methods were then used to assess the rsv loads in respiratory specimens from children admitted to hospital with acute respiratory tract disease. seventy-five nasal aspirates were collected in ml of viral transport media from children admitted to hospital with acute respiratory tract disease during the winter Á/ . two hundred microlitre fractions were used to inoculate the mrc- and a cell lines. ifa using an fitc-conjugated monoclonal antibody (imagen tm respiratory syncytial virus, dako, france) was used to detect the rsv antigen in nasal aspirates or in culture, as described previously (freymuth et al., ). an aliquot of each nasal aspirate was stored at (/ c in transport media. samples from children hospitalised with a rsv infection were quantified by real-time rt-pcr. these children were divided into non-severe and severe disease groups on the basis of clinical data. the severe disease group included children who required oxygen therapy for more that h and children who were being ventilated mechanically for respiratory failure. all the other children (n / ) had non-severe disease. a -bp dna fragment derived from the n gene of the rsv strain a was generated by conventional rt-pcr with primers ppn and ppn (table ). the pcr product was cloned into pcr † . -topo from the topo ta cloning kit (invitrogen, cergy pontoise, france) according to the manufacturer's instructions. the fragment was cloned downstream of the t rna polymerase promoter. another plasmid was generated using a dna fragment derived from the human gapdh gene with the gapdh and gapdh primers (as used for real-time pcr for gapdh). the clones were designated pn and pgapdh, respectively. after the transcription of these plasmids, we verified the orientation of the insert by enzymatic restriction: pn was digested with xba i and pgapdh with bgl i. the plasmids were then purified with the qiagen plasmid maxi kit (qiagen gmbh, germany). to obtain transcripts of equal length, we cut mg of each plasmid with spe i. we transcribed ml of each plasmid with t rna polymerase from the riboprobe † in vitro transcription system (promega, madison, usa) according to the manufacturer's instructions. to remove all dna, a dnase treatment was performed. the dna-free tm (ambion inc., austin usa) system was used; this removes dna without the need for phenol/chloroform extraction or heating. the rna concentration was estimated by spectrophotometry and the average of four measurements was used. aliquots were stored at (/ c. once an aliquot had been thawed it was never refrozen. for each quantification, two standard aliquots were subjected to serial fold dilutions in depc (diethylpyrocarbonate)-treated water. after dnase treatment, we ensured that the rna standard did not contain any dna by carrying out pcr without the reverse transcription step. our results showed that no amplification occurred. rna was extracted from . ml of the stored nasal aspirates by use of the high pure rna isolation kit (roche molecular diagnostics, france) according to the manufacturer's instructions. the sample and ml of lysis/binding buffer were placed in a filter tube. the tube was centrifuged at )/g for s. one hundred microlitres of restored dnase was added and the mixture incubated at room temperature for min. the sample was then washed three times and finally eluted with ml of double distilled water. the template rna was never frozen before use. the standards ( ml) were diluted serially and the extract from the unknown samples ( ml) were simultaneously subjected to reverse transcription using the omniscript tm reverse transcriptase kit (qiagen gmbh, germany). samples were incubated at c for h and then at c for min. rsv was reverse transcribed using n primer (table ) , which targeted rsv genomic rna in the gene n. gapdh was reverse transcribed using the gapdh primer, which targeted mrna corresponding to the human gapdh gene. as the table oligonucleotide primers and probes used to detect human gapdh mrna and genomic rna from the n gene of rsv efficiency of the reverse transcription can vary (ferre, ; van milaan et al., ) , we used plasmidtranscribed rna to standardise the assay. a lightcycler instrument (roche molecular biochemicals, france) was used to amplify and to quantify the amplification product after each cycle. a hybridisation probe system was used for detection. fluorescence was measured after the annealing step and was based on the fluorescence resonance energy transfer (fret). primers n and n generated a -bp product ( table ). the product was detected with the hybridisation probes sn (fluorescein) and sn (red ). the amplification reaction was undertaken in a ml mixture with the lightcycler (lc)-dna master hybridisation probes (roche molecular biochemicals, france). we carried out hot-start pcr; ml of lc-mix was incubated with . ml of the antitaq † antibody (clontech, france) for min at room temperature. we then added . mm mgcl , . mm each primer, . mm sn and . mm sn . finally, ml of cdna was added to ml of pcr mixture in each capillary tube and amplified as follows: c for min for one cycle, followed by denaturation at c s, annealing at c for s and extension at c for s for cycles. primers gapdh and gapdh generated a -bp fragment ( table ). the hybridisation probes s gapdh (fluorescein) and s gapdh (red ) were used for fret. the pcr mixture was amplified with lc-faststart dna master hybridisation probes (roche molecular biochemicals, france). the reaction mixture contained ml of lc, . mm mgcl , . mm each primers, . mm s gapdh and . mm s gapdh. finally, ml of cdna was added to ml of this mixture and amplified as follows: c for min for one cycle, followed by denaturation at c for s, annealing at c for s and primer extension at c for s for cycles. to assess the detection limit of the quantitative rt-pcr we also isolated rsv in culture and used a qualitative rt-pcr method described previously (cane and pringle, ; freymuth et al., ) . briefly nasal aspirates were cultured for days and then rsvpositive cultures were identified by immunostaining with an anti-rsv antibody (ref. - , argene, france). for the qualitative rt-pcr different primers and a different probe were used for the n gene to those employed in the quantitative rt-pcr. the amplified products were detected by a hybridisation assay on microplates using the gen-eti-k deia † kit (sorin biomedica, france). many viruses can be found in nasal aspirates from children admitted to hospital with acute respiratory tract disease. rna and dna extracted from cell cultures infected with different respiratory viruses were screened by the real-time rt-pcr for rsv to assess the specificity of this assay. no cross-reactions were detected with the following virus strains: parainfluenza virus types , and , influenza virus a/h n and a/ h n , influenza virus b and c, echovirus , coronavirus oc and e, rhinovirus , and adenovirus . the sensitivity of the real-time pcr was determined by the use of serial dilutions of rsv transcripts ( Á/ transcripts/ml). each dilution was being quantified four times in duplicate. the detection rate was % for the Á/ transcripts/ml dilutions and % for the transcripts/ml dilution. the same experiment was carried out for the rt-pcr for gapdh: the detection rates were , and % for , and transcripts/ml, respectively. to assess the intra-assay reproducibility of the realtime rt-pcr assays for rsv and for gapdh, an extract from an rsv-positive sample was submitted to consecutive assays and measurements in the same run. in the rt-pcr assay for rsv, the quantitative mean value of viral rna was . log with a standard deviation of . log and a coefficient of variation of . %. in the rt-pcr assay for gapdh, the quantitative mean value of gapdh mrna was . log with a standard deviation of . log and a coefficient of variation of . %. to estimate the inter-assay variability of the real-time rt-pcr assay for rsv, we studied two batches of rna; one with a low number of viral copies, and the other with a high number of copies. the aliquots were stored at (/ c. in four consecutive experiments, an aliquot of each extract was quantified twice. a new standard curve, a new rt and a new real-time pcr were performed each time. the coefficient of variation was % when the rt-pcr assay for rsv was performed with the low copy number extract and % with the high copy number (table ) . as the analytical sensitivity of the real-time rt-pcr assay for rsv has previously been estimated using rsv plasmids, we wanted to evaluate it on viral strains. we used serial -fold dilutions ( ( Á/ ( ) of five rsv strains (rsv a long, a wt no. , a wt no. , b and b wt no. ) to infect mrc- cells. rna extracted from these strains was detected by the qualitative rt-pcr assay for rsv described previously and quantified by the real-time rt-pcr assay for rsv on a lightcycler instrument. for all rsv strains the least sensitive method was the qualitative rt-pcr assay ( table ). the culture method detected three of the five rsv strains at one more dilution than did rt-pcr-hybridisation-eia. finally, the real-time rt-pcr assay for rsv appeared to be the most sensitive assay, detecting one more dilution than the culture method for three of the five viral strains. rsv infection was detected in clinical specimens by direct ifa and by a viral isolation technique. rsv and gapdh rnas were quantitated by the two real-time rt-pcr assays. of the nasal aspirates, ( . %) were ifa-positive, ( . %) were culture-positive and ( %) were positive according to the real-time rt-pcr assay for rsv. of the samples found to be positive by the real-time rt-pcr assay for rsv, six could not be quantitated as the number of rsv rna copies contained was below the detection limit of the technique; two of these six samples were also culturepositive. twenty-six nasal aspirates were positive according to all techniques: ifa, culture and real-time rt-pcr assay for rsv; eight were positive according to both the culture method and real-time pcr, and five were positive according to ifa and real-time rt-pcr. to estimate the sensitivity, specificity, positive and negative predictive values of the real-time rt-pcr assay for rsv a gold standard was required for rsv detection. theoretically, the gold standard is the isolation in cell culture. to overcome the problem associated with the relative insensitivity of the culture method with clinical specimens (particularly due to bacterial contamination) we considered samples to be positive if they were found to be positive by at least two of the three technique. thus ( %) true-positives were obtained. a specimen that was found to be positive by only one technique was considered to be a false-positive. there were eight false positive results: two cases with ifa, three cases with the culture method and three cases with real-time rt-pcr assay for rsv. the latter three cases were probably true-positives because the real-time rt-pcr assay was the most sensitive method tested. the sensitivity, specificity, positive and negative predictive values of the three techniques were calculated (table ) . on clinical specimens, the real-time rt-pcr was the most sensitive ( %), followed by the culture method ( %) and ifa ( %). the number of rsv rna copies was calculated and of gapdh mrna copies in the specimens where the viral load could be calculated (fig. ) . the number of rsv rna copies ranged from . to . log with a mean of . log and an sd of . log. the analysis of the gapdh mrna copies confirmed that the number of cells in nasal aspirates differed from one sample to another, ranging from . to . log with a mean of . log and an sd of . log (fig. a) . as rsv particles are cell-associated in cultures and in clinical samples and as the number of copies of the housekeeping gene (gapdh) is proportional to the number of cells in the sample, these results can be expressed as the ratio of the number of copies of rsv rna to the number of copies of gapdh mrna. this allows the results obtained from the different nasal aspirates to be compared with each other, and ensures that the number of cells in each nasal sample (and thus the quality of the sampling) is not an interfering factor. most of the specimens had comparable and low ratios of below (mean ratio: . ), and had a ratio indicating a rather high rsv rna level. next, the rsv rna was quantitated according to the severity of the disease (fig. ) . the rsv copy number ranged from . )/ to . )/ with a mean of . )/ copies and a sd of . )/ copies ( fig. a) . the mean number of rsv rna copies was higher in the severe disease group than in the non-severe disease group ( . )/ vs . )/ ), and the difference between the two groups was close to the significant threshold (p / . with the krustal Á/wallis test). the ratio of rsv rna copies to gapdh mrna copies ranged from . to . with a mean of . in the patients with severe disease, and from . to . , with a mean of . in the patients with non-severe disease (fig. b) . the difference between the two groups was not significant according to the test of krustal Á/wallis test. the sensitivity of the real-time rt-pcr assay for rsv on clinical samples was %, which is significantly higher than the sensitivity of conventional techniques for the detection of rsv in nasal aspirates. this method gave the same results as ifa and the culture method for ( . %) of the nasal aspirates tested. eight aspirates were found to be positive by just one of the three methods. the two samples found to be positive only by the ifa method are likely to be false-positives. three samples were positive only in one of the two types of cell culture (mrc or a- ). the presence of some inhibitors of the rt or pcr steps can be ruled out as gapdh mrna was amplified in these samples. however, we cannot exclude the possibility that the cell cultures that were inoculated in microplates were cross contaminated. three nasal aspirates were only found to be positive by real-time rt-pcr (even after checking on another extract). as this technique is the most sensitive of the three tested, and as these three samples had a very low viral load, it is likely that they are actually true-positives, and that conventional techniques were not sensitive enough to detect such low levels of virus in these cases. several groups have developed rt-pcr techniques to detect rsv rna in respiratory samples from children with lower respiratory tract infections (cubie et al., ; freymuth et al., freymuth et al., , henkel et al., ; paton et al., ) .these methods usually detect about Á/ % more rsv-positive samples than conventional methods for rsv detection. in this paper, we have shown that quantitative real-time rt-pcr is more sensitive than qualitative rt-pcr in infected cell cultures, and than isolation of respiratory samples in cell culture, identifying % more cases as being rsvpositive. however, real-time rt-pcr can also be used as a qualitative method. in this application it has many advantages over the conventional qualitative rt-pcr. real-time rt-pcr is faster and cheaper taking just min on the lightcycler apparatus, and including the detection of the amplification product. it is also a closed system, which prevents cross contamination. we attempted to estimate the detection limit of the real-time rt-pcr. the analytical sensitivity of the real-time rt-pcr was rsv rna copies/ml in vitro. however, this value is not absolute as it was obtained from a standard that was quantified by measuring absorbency at nm which is not very accurate. however, as rsv rna in respiratory samples was always quantitated using the same standard, the results are comparable and reliable. the amounts of rsv rnas in quantifiable positive nasal aspirates seemed to be roughly related to the severity of the acute respiratory tract disease. the difference was close to the threshold for significance, but this result should be interpreted with caution because of the low number of samples included in the study. an association between high rsv titres and the severity of bronchiolitis has been reported previously in a one study using other virological techniques. in a study on infants with rsv infection, buckingham et al. ( ) reported that nasal aspirates from infants with severe infection contained more rsv than those from infants with nonsevere disease: mean . / . vs . / . log pfu/ ml, p / . . conversely, hall et al. ( ) showed that peak titres in nasal washings could not be correlated with the severity of the disease, but that infants with bronchiolitis did appear to have a higher mean titre than those with pneumonia: . vs . log tcid /ml; p b/ . . based on the rsv/gapdh ratio, it was found that there is no statistically significant difference between the children with severe or non-severe disease. in other words, if the quantitative results are standardised according to the number of cells in the sample, thus accounting for the quality of the sampling, the severity of the disease does not appear to be linked to the rsv load. the mean rsv/gapdh ratio tended to be higher in cases of severe bronchiolitis, which makes it necessary to undertake further investigations to confirm whether there is indeed a link between the severity of rsv infection and the quantity of virus in nasal aspirates. nevertheless, factors other than intensive viral replication are likely to play a role in the severity of the bronchiolitis. these factors can include inflammatory responses and the genetic susceptibility of the patients. moreover, viral concentrations in tracheal secretions decrease within h of endotracheal intubation in some infants with severe rsv infection (malley et al., ) . in conclusion, real-time rt-pcr assay for rsv is a new tool for the detection of rsv in respiratory samples. the quantitation of rsv rna showed that nasal aspirates from children with severe bronchiolitis contain more rsv copies than those from children with non-severe disease, but the difference is no longer observed when the viral load is related to the number of cells contained in the sample. nevertheless, the realtime rt-pcr assay for rsv can be used as a qualitative test and, in this case, its rapidity and low cost may make it useful for diagnostic purposes. nasal quantity of respiratory syncytial virus correlates with disease severity in hospitalised infants respiratory syncytial virus heterogeneity during an epidemic: analysis by limited nucleotide sequencing (sh gene) and restriction mapping (n gene) acute respiratory disease in infancy and childhood: present understanding and prospects for prevention detection of respiratory syncytial virus in acute bronchiolitis in infants quantitative or semi-quantitative pcr: reality versus myth les virus responsables d'infections respiratoires en pédiatrie. bilan de aspirations nasales réalisées chez l'enfant en une période de six ans prevalence of respiratory syntical virus subgroups a and b in france, to detection of respiratory syncytial virus, parainfluezavirus , adenovirus and rhinovirus sequences in respiratory tract of infants by polymerase chain reaction and hybridisation quantitative shedding patterns of respiratory syncytial virus in infants occurrence of groups a and b of respiratory syncytial virus over years: associated epidemiologic and clinical characteristics in hospitalised and ambulatory children improved detection of respiratory syncytial virus in nasal aspirates by seminested rt-pcr reduction of respiratory syncytial virus (rsv) in tracheal aspirates in intubated infants by use of humanized monoclonal antibody to rsv f protein rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification the lightcycler: a microvolume multisample fluorimeter with rapid temperature control we would like to thank ultan power for critical review of the manuscript. key: cord- -nrluar e authors: park, eun-mee; park, sun-whan; lee, ye-ji; lee, won-ja; choi, wooyoung title: production of ebola virus-like particles in drosophila melanogaster schneider cells date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: nrluar e in this study, we generated recombinant virus-like particles (vlps) against family filoviridae, genus ebolavirus, species zaire ebolavirus, strain makona (ebov) in drosophila melanogaster schneider (s ) cells using the ebov makona. s cells were cotransfected with four viral plasmids encoding ebov makona proteins and protein expression was analyzed by immunoblotting. we confirmed that ebov makona proteins were successfully expressed in s cells. additionally, we further examined the formation of intracellular and extracellular vlps by electron microscopy. evlps were produced by sucrose gradient ultracentrifugation of s cells transfected with ebov makona genes, and production of vlps was confirmed by immunoblot analysis. collectively, our findings showed that the s cell system could be a promising tool for efficient production of evlps. family filoviridae, genus ebolavirus (ebov) causes severe viral hemorrhagic fever in humans and other primates (feldmann and goisbert, ) . previous outbreaks of ebov in central africa are associated with serious public health problems. indeed, since its identification in during an outbreak in zaire (world health organization, ) , there have been over outbreaks of ebov in africa (del and gaurmer, ) . cases were reported between and and patients died. , of , patients died in ebov outbreak that occurred in (cdc, ) . - % of patients infected with ebov die. the ebola virus variant makona was the causative agent of the recent outbreak of ebola during to in west africa, which was the largest outbreak to date. the recently isolated ebov makona variant, from a clinical case of ebov disease (evd) in makona, has been shown to have a varying case fatality rate (cfr) of - % at later stages of the outbreak (who ebola response team, ) . therefore, in this study, we applied s cells for production of species zaire ebolavirus, strain makona (ebov makona) vlps using the makona outbreak strain. ebov belongs to the filoviridae family and contains single-stranded, negative-sense rna genome of kb. the particles resemble long, stretched filaments and consist of three compartments, including the nucleocapsid (np), matrix space, and envelope. additionally, the particles measure nm in diameter and range from to nm in length. the genome of ebov contains seven genes, which encode gp, np, vp , vp , vp , vp , and l proteins (sanchez et al., ; falasca et al., ) . virus-like particles (vlps) mimic the structure of virions and have nonreplicating, noninfective properties because the particles lack the infectious genome and are only composed of structural or capsid proteins (kushnir et al., ) . vlp-based vaccines are safe and high immunogenic. moreover, vlps stimulate innate and humoral immunity and activate antigen presentation in antigen-presenting cells (pushko et al., ) . the development of vaccine candidates derived from vlps may be a promising approach for efficient antibody production. indeed, several vlp vaccine candidates for a variety of viruses have been developed. vaccines based on vlps for hepatitis b virus (hbv), papillomavirus, and influenza virus a have been evaluated in clinical trials in human or have been approved for clinical use (shirbaghaee and bolhassani, ; pushko et al., ) . vlps are produced in several systems, including bacteria, mammalian cells, yeast, plants, and insect cells. insect system is a promising system for high yield and rapid production of vlps (vicente et al., ) . moreover, insect systems can induce post-translational modifications similar to mammalian cells, facilitating the assembly of vlps (fang et al., ; liu et al., ) . vlp-based vaccines, including hepatitis c virus (hcv), papillomavirus, enterovirus type a (ev ), influenza virus a, and sars-coronavirus (cov), have been developed in insect systems (baumert et al., ; acosta-rivero et al., ; lechmann et al., ; senger et al., ; lopez-macias et al., ; ho et al., ) results in a high yield of hiv- vlps and proper cleavage of envelope precursor protein (yang et al., ) . in a previous study, vp / double-layered rotavirus-like particles containing wa capsid protein were produced in s cells transformed with a bicistronic expression system consisting of encephalomyocarditis virus (emcv)-derived internal ribosomal entry site (ires) element (lee et al., ) . glycoprotein (gp), nucleoprotein (np), minor matrix protein (vp) , and vp originating from ebov makona was artificially synthesized. the amplified inserts were cloned into the bglii and xbai sites of the plasmid pmt/v /his (invitrogen, carlsbad, ca, usa) and were confirmed by sequencing. np, gp, and vp of ebola virus have been shown to facilitate assembly and release of vp evlps (kallstrom et al., ) . therefore, to assess ebov protein expression, drosophila s cells were transfected with ebov plasmids using transfection reagent (tranit- ; mirus, usa); at h before harvesting, cells were treated with μm cuso to induce protein expression. the cells and culture supernatants were harvested, and ebov protein expression was confirmed by immunoblot analysis. the cells were lysed in ripa buffer (invitrogen) for min on ice, and lysates were collected after centrifugation at , rpm for min at °c. the culture supernatants were collected and concentrated using a kda centrifugal filter with ultracel- membrane (millipore, germany). to visualize the released particles, the concentrated supernatants were centrifugated through % sucrose and then negatively stained. the cell lysates and concentrated supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis after heating at °c for min, and the proteins were electrotransferred to polyvinylidene difluoride membranes. the membranes were blocked in tbs/tween solution containing % skim milk for h at room temperature. the membranes were then incubated with primary anti-v (invitrogen), anti-np (alpha diagnostics, usa), anti-vp (cosmo genetech, south korea), and anti-gp antibodies for h at room temperature, washed three times with tbs/tween solution, and incubated with secondary anti-mouse-ap, anti-rabbit-ap, or anti-human-ap antibodies for h at room temperature. finally, membranes were washed three times, and proteins were detected using a bcip/nbt substrate kit (invitrogen). as shown in fig. a , we confirmed that ebola viral proteins were efficiently expressed in ebov makona-transfected s cells. the formation of vlps similar to ebola virus particles was visualized in both ebov makonatransfected s cells and culture supernatant by transmission electron microscopy (tem; fig. b ). as negative control for vlps formation, we used cells which were transfected with gp, np, and vp without vp . as shown in fig. b , filamentous particles of approximately nm in diameter and nm in length were observed in culture supernatant of ebov makona-transfected s cells. next, the harvested cells were disrupted by repeated freezing and thawing three times, and the debris was removed after centrifugation at , ×g for min. cell lysates collected by centrifugation were overlaid on top of a continuous sucrose gradient ( %, %, %, %, and %) and ultracentrifuged at , rpm for h at °c using a sw ti rotor (beckman, usa). the procedure for sucrose gradient ultracentrifugation is illustrated in fig. a . to analyze the production of evlps, fractions were collected from top to bottom, and the expression of ebov proteins was confirmed by immunoblotting. as shown in fig. b , abundant amounts of ebov proteins were detected in fractions and . from this result, we found that evlps were mainly concentrated in fractions and . we then aimed to recover the evlps. to this end, fractions and were overlaid on top of a % sucrose cushion and ultracentrifuged at , rpm for h at °c using a sw ti rotor (beckman). the pelleted evlps were resuspended in pbs and subjected to immunoblot analysis to confirm the expression of evlps. as shown in fig. c , viral proteins were detected in the vlp pellets; analysis of the formation of evlps by tem is ongoing. collectively, these findings showed that evlps were effectively produced in an s cell system and that the s cell system may be a promising strategy for effective production of evlps. vlps have been produced in several expression systems and have been shown to provide protective effects against ebov infection in animal model. vaccination with t cell derived-ebola vlps protects nonhuman primates (nhps) or rodents from ebov infection (warfield et al., , a swenson et al., ) . antibodies against ebov are produced in serum of cynomolgus macaques vaccinated with t cell-derived evlps. high active specific antibodies against ebov are produced in serum of cynomolgus macaques vaccinated with t cellderived evlps. additionally, tumor necrosis factor (tnf)-α production in evlp-vaccinated individuals is strongly increased in cd + t cells (warfield et al., a,b) . immunization with kun-vlps generated by transfection with kun replicon rna containing ebov gp protects makona pigs and nhps from challenge with ebov infection (reynard et al., ; pyankov et al., ) . moreover, in previous works, evlps have been produced in insect systems, resulting in activation of the immune response. vlps produced in sf cells using a baculovirus system stimulated cytokine secretion in dendritic cells, and immunization of mice with the vlps increased igg a antibody production through induction of the th -biased immune response (ye et al., ) . in a previous report, high yields of hiv- vlps were produced in hiv- envelope protein-overexpressing s cells, resulting in proper cleavage of envelope precursor protein (yang et al., ) . therefore, we suggested that s cells may be a promising system for efficient production of evlps. in this study, we used s cells for the production of vlps and examined evlps production following overexpression of ebov proteins. we showed that viral proteins of ebov makona were efficiently expressed in s cells and confirmed the formation of evlps by tem. we purified evlps by sucrose gradient ultracentrifugation. collectively, our findings suggested that the evlps produced by the s cell system in this study may be useful for the development of efficient vlp-based vaccines against ebov. to the best of our knowledge, this is the first successful demonstration of vlp production from the new outbreak strain in the s expression system. further in vivo studies on the vlps of ebov are necessary to characterize immune responses to this ebov strain and to study the protective effects of vlp-based vaccines against ebov infection. we have no conflicts of interest to declare. in vitro assembly into virus-like particles is an intrinsic quality of pichia pastoris derived hcv core hepatitis c virus structural proteins assemble into virus like particles in insect cells outbreaks chronology: ebola virus disease ebola: implications and perspectives molecular mechanisms of ebola virus pathogenesis: focus on cell death differences in the post-translational modifications of human papillomavirus type b major capsid protein expressed from a baculovirus system ebola haemorrhagic fever assembly of human severe acute respiratory syndrome coronavirus-like particles biophysical characterization and conformational stability of ebola and marburg virus-like particles analysis of ebola virus and vlp release using an immunocapture assay virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development vaccine hepatitis c virus-like particle induce virus-specific humoral and cellular immune response synthesis of double-layered rotavirus-like particles using internal ribosome entry site vector system in stably-transformed drosophila melanogaster use of baculovirus expression system for generation of virus-like particles: success and challenges safety and immunogenicity of a virus-like particle pandemic placebo-controlled trial of adults in mexico development of virus-like particle technology from small highly symmetric to large complex virus-like particle structure a kunjin replicon virus-like particle vaccine provides protection against ebola virus infection in nonhuman primates kunjin virus replicon-based vaccines expression ebola virus glycoprotein gp protect the guinea pig against lethal ebola virus infection sequence analysis of the ebola virus genome: organization, genetic elements, and comparison with the genome of marburg viruses enhanced papillomavirus-like particle production in insect cells different applications of virus-like particles in biology and medicine: vaccination and delivery systems vaccine to confer to nonhuman primates complete protection against multistrain ebola and marburg virus infection large-scale production and purification of vlp-based vaccines advances in virus-like particle vaccine for filoviruses induction of humoral and cd + t cell responses are required for protection against lethal ebola virus infection filovirus-like particles produced in insect cells: immunogenicity and protection in rodents ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenges ebola haemorrhagic fever in zaire hiv- virus like particles produced by stably transfected drosophila s cells: a desirable vaccine component ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutrializing antibodies this study was supported by korea centers for disease . key: cord- - cmduez authors: callison, scott a.; hilt, deborah a.; boynton, tye o.; sample, brenda f.; robison, robert; swayne, david e.; jackwood, mark w. title: development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: cmduez it is important to rapidly differentiate infectious bronchitis virus (ibv) from disease agents like highly pathogenic avian influenza virus and exotic newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. in this study, we report the development and testing of a real-time rt-pcr assay using a taqman(®)-labeled probe for early and rapid detection of ibv. the assay amplifies a -bp product in the ′-utr of the ibv genome and has a limit of detection and quantification of template copies per reaction. all strains of ibv tested as well as two turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. evaluation of the assay was completed with tracheal swab samples. a total of samples collected from ibv antibody negative birds were negative for ibv by the real-time rt-pcr assay. we tested tracheal swabs submitted to two different diagnostic laboratories and found . % of the tracheal swabs positive for ibv by real-time rt-pcr, whereas only . % of the samples were positive by virus isolation, which is the reference standard test. we also collected a total of tracheal swabs at six different time points from birds experimentally infected with different dosages of ibv and found that, independent of the dose given, the viral load in the trachea plateau at days post-inoculation. in addition, an inverse relationship between the dose of virus given and the viral load at days post-inoculation was observed. finally, we tested total tracheal swab samples, from a flock of commercial broilers spray vaccinated for ibv in the field. the percentage of birds infected with the ibv vaccine at , , and days post-vaccination was %, %, and %, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. this observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. the real-time rt-pcr test described herein can be used to rapidly distinguish ibv from other respiratory pathogens, which is important for control of this highly infectious virus. the test was extremely sensitive and specific, and can be used to quantitate viral genomic rna in clinical samples. infectious bronchitis (ib) is a highly infectious disease of the upper-respiratory tract in chickens (cavanagh and naqi, ) . it can also affect the kidneys and reproductive tract. it is of economic importance to the poultry industry due to the high morbidity and production losses associated with the disease. the etiologic agent of ib is infectious bronchitis virus (ibv), an enveloped, positive-sense, single-stranded rna virus that belongs to the coronaviridae family. the virus contains four structural proteins; nucleocapsid surrounding the viral rna, an integral membrane glycoprotein, a small envelope protein, and a spike glycoprotein located on the surface of the viral envelope, which contains epitopes that induce virus-neutralizing and serotype-specific antibodies. although control of ibv is primarily through the use of live attenuated vaccines, the disease is difficult to control because different serotypes of the virus do not cross-protect. therefore, it is imperative to quickly and accurately detect the presence of the virus within an infected poultry flock so that subsequent flocks can be properly vaccinated. it is also important to rapidly differentiate ibv infections from other upper-respiratory diseases like avian influenza, newcastle disease, infectious laryngotracheitis, and avian mycoplasmosis so that appropriate measures against those diseases can be taken in a timely manner. current diagnostic assays for ibv include virus isolation in embryonating eggs, tracheal organ culture, or cell culture immunoassays, and molecular assays that detect the viral rna (gelb and jackwood, ) . virus isolation is considered the reference standard; however, it is expensive and time consuming because several passages may be required to detect the virus. immunoassays use ibv-specific monoclonal antibodies to detect the virus in direct or indirect fluorescent antibody and enzymelinked immunosorbent assay (elisa) formats. although faster and simpler than virus isolation, immunoassays tend to lack specificity and sensitivity and none detect all strains or types of ibv karaca et al., ; naqi et al., ) . molecular assays for the detection of ibv are commonly used because they provide highly specific and sensitive results in a timely manner. molecular assays use the reverse transcriptase-polymerase chain reaction (rt-pcr) to detect viral rna directly from a clinical sample or from virus isolated in a laboratory host system. when rt-pcr is used to amplify the spike glycoprotein of ibv, it can be coupled with restriction fragment length polymorphism (rflp) or nucleic acid sequencing to identify the type of the virus (cavanagh et al., ; jackwood et al., ; keeler et al., ; kingham et al., ; kwon et al., ) . the constant threat of globally important diseases like avian influenza and newcastle disease, which must be differentiated from ib, in diagnostic investigations, makes it extremely important to rapidly identify the causative agent of any upperrespiratory disease or changes in egg shell quality and egg production in chickens. in this study, we developed a taqman ®based real-time rt-pcr assay for rapid detection of ibv viral rna directly from clinical samples. we examined the sensitivity and specificity of the test and evaluated it using known negative, known positive, and clinical samples from commercial chickens. virus isolation was performed in specific pathogen-free (spf) - -day-old embryonating chicken eggs as previ- ously described (gelb and jackwood, ) . other viral and mycoplasma strains used in this study are listed in table . extraction of rna from clinical samples was performed with the high pure rna isolation kit (roche, indianapolis, in) or the rneasy kit (qiagen, valencia, ca). extractions from allantoic fluid and tracheal swabs taken from experimentally infected birds as well as rna extracted for sensitivity and specificity analysis and for test evaluation was conducted with the magmax total rna isolation kit (ambion, austin, tx) following the manufacturer's suggestions. a sequence alignment ( fig. ) of the -untranslated region (utr) of six strains of ibv (genbank accession nos. ay , m , ay , ay , ay , ay ) was used to identify conserved sequences within this region to design primers and a probe for the real-time rt-pcr assay. a forward primer ibv gu ( -gct ttt gag cct agc gtt- ) located at nucleotide positions - of the ibv m strain genome sequence (genbank accession no. ay ); a reverse primer ibv gl ( -gcc atg ttg tca ctg tct att g- ) located at nucleotide positions - of the ibv m strain genome sequence, and a taqman ® dual-labeled probe ibv g probe ( -fam-cac cac cag aac ctg tca cct c-bhq - ) located at nucleotide positions - of the ibv m strain genome sequence were designed to amplify and detect a -bp fragment of the -utr gene. the primers were synthesized by integrated dna technologies (coralville, ia), and the probe was synthesized by biosearch technologies (novato, ca). primers and probe were utilized in a l reaction containing . l of quantitect probe rt-pcr × mix (qiagen, valencia, ca), . l of rt enzyme (qiagen), primers to a final concentration of . mol, probe to a final concentration of . mol, . l of water, and l of rna template. the reaction was conducted in a smartcycler (cepheid, sunnyvale, ca) at • c for min; • c for min with optics off; cycles of • c for s followed by • c for s with optics on. for each reaction, the cycle threshold (c t ) number was determined corresponding to the pcr cycle number at which the fluorescence of the reaction exceeded u of fluorescence, which is the default value for the smartcycler. runoff rna transcripts corresponding to the first nucleotides of the mass ibv genome were generated to use as standards in the assay. transcripts were generated from a plasmid containing the first ∼ bp of the mass genome of ibv downstream of a t promoter, which was created using the -primer a-beau u ( -tacactagccttgcgc-taga- ) and -primer a-beau l ( -gcacgcca-aagtcccatag- ). briefly, rna was extracted from the mass strain of ibv using the high pure rna isolation kit (roche diagnostics corporation). the purified rna was resuspended in diethylpyrocarbonate (depc) treated water and used in the rt-pcr reaction as previously described (jackwood et al., ; lee and jackwood, ) . the amplified product was cloned into the pcr-xl-topo vector (invitrogen inc., carlsbad, ca) per the manufacturer's directions and sequenced to verify its accuracy. the plasmid was linearized at base pair relative to the start of the genome with xmn i (new england biolabs, beverly, ma). the linearized dna was gel purified and used as template with a ribomax t in vitro transcription system (promega, madison, wi) per the manufacturer's recommendations. the length of the runoff rna transcripts was verified by agarose gel analysis, and the concentration was determined using a biophotometer (eppendorf, hamburg, germany). ten-fold serial dilutions of the runoff rna transcripts containing × to × copies of template per microliter were made and l of each dilution was used as template in the assay. the limit of detection/quantification and reproducibility of the assay were determined by six independent runs. the limit of detection was defined as the lowest rna concentration yielding a c t value where the fluorescence of the reaction exceeded u of fluorescence. the limit of quantification was defined as the lowest rna concentration on the standard curve that maintained linearity. the average number of ibv genome copies for each group of experimental samples was estimated using the equation derived from the standard curve. to test the specificity of the assay, rna or dna from different pathogens known to infect the avian upper-respiratory tract (table ) was used in the test. we further tested the specificity of the assay using negative samples obtained from commercial layer chickens housed from day of age in positive pressure horsfal isolation units at poultry diagnostic and research center (pdrc, athens, ga). the birds were monitored for serum antibody titers to ibv using the proflock ® ibv elisa kit (synbiotics). a total of serum samples were collected at weeks ( samples) and weeks ( samples) of age. sterile polyester tipped applicators (mdci ltd., west sussex, u.k.) were used to take tracheal swabs from each bird at and weeks of age ( total samples). each swab was placed into a . ml microcentrifuge tube containing ml of sterile × pbs (ph . ). the tubes were mixed using a bench-top vortex, and stored at − • c until needed for rna extraction. the sensitivity of the assay was determined using clinical tracheal swab samples submitted to delaware (agriculture research laboratory, university of delaware, georgetown de , usa) and maryland (maryland diagnostic laboratory, salisbury, md , usa) laboratories. clinical samples consisting of tracheal swabs from commercial chickens experiencing upper-respiratory disease, submitted as routine diagnostic cases to the delaware (georgetown, de) and maryland (salisbury, md) state diagnostic laboratories were tested by the real-time rt-pcr assay, as well as, by virus isolation at those laboratories (gelb and jackwood, ) . the sensitivity was calculated with the formula: se = tp/(tp + fn), where; se = sensitivity, tp = true positives, which were samples positive by virus isolation (gelb and jackwood, ) and by the real-time rt-pcr assay, and fn = false negatives, which were samples positive by virus isolation and negative by the real-time rt-pcr assay. the real-time ibv rt-pcr test was evaluated with known positive samples obtained from ibv inoculated specific pathogen-free (spf) chickens divided into four groups of chickens each and housed in four positive pressure horsfal isolation units located in four separate filtered air positive pressure rooms at pdrc (athens, ga). at day of age, blood and tracheal swab samples were taken from two birds in each group. the next day, each bird was inoculated intranasally with l of the arkansas dpi strain of ibv with the following titers; group no virus, group was given . × embryo infectious dose (eid )/ml, group was given . × eid /ml, and group was given . × eid /ml. the birds were examined twice daily for clinical signs and tracheal swabs were taken from five birds in each group on , , , , , and days post-inoculation (d.p.i.). tracheal swabs were placed in l of × pbs (ph . ) and stored at − • c until used for rna extraction. in addition, a total of tracheal swab samples ( birds swabbed at , , and days post-vaccination) from a commercial broiler flock that was spray vaccinated with a combination mass/ark commercial vaccine at days of age were also tested. after collection, each tracheal swab was placed into . ml microcentrifuge tubes containing ml of sterile × pbs (ph . ). the tubes were mixed using a bench-top vortex and stored at − • c until used for rna extraction. the primers and taqman ® probe targeted a highly conserved region of the -utr (fig. ) and amplified a -bp product (data not shown). the probe was designed to anneal to the same strand as the ibv gl primer. this strategy allowed for the fewest guanine residues within the probe sequence and placed the -end of the probe only bp away from the -end of the ibv gl primer. the detection and quantification limits were determined using c t values obtained for each reaction containing from to copies of the standard rna. the values were plotted against the log of the number of template copies and a linear equation (y = − . x + . ) with a r value = . was generated (fig. ) . the assay maintained linearity for at least six orders of magnitude. using the slope from the linear equation, the overall efficiency of the assay was estimated to be . %. fig. . the assay standard curve was generated by plotting the c t values vs. log of -fold serial dilutions ( - ) of standard rna corresponding to the -utr of the ibv genome. an overall reaction efficiency of . % was estimated using the standard curve slope as indicated by the formula. the assay was negative below template copies. therefore, the limit of detection and quantification were both determined to be template copies. the standard deviation of the mean c t values obtained for each reaction containing from to copies of the standard rna calculated from six independent runs, ranged from . to . cycles. the assay amplified rna from all ibv strains and the two tcov strains listed in table , whereas no amplification signal was observed from any of the other pathogens tested. a search of the genbank database using the blastn analysis program (http://www.ncbi.nlm.nih.gov/blast/) indicated that additional strains of ibv would likely also be detected by the assay (saibbk, dq ; h , ay ; partridge/gd/s / , ay ; peafowl/gd/kq / , ay ; cal , ay ; k , ay ; cu , ay ; cu , ay ; cu , ay ; cu-t , ay ; cu , ay ; cu , ay ; ko , ay ; ma , ay ; lx , ay ; bj, ay ). three hundred and forty commercial layers not exposed to ibv, were negative for ibv antibodies ( serum samples) by commercial elisa (synbiotics) at weeks of age when the experiment was terminated. maternal antibodies were detected in of serum samples taken from those birds at weeks of age. a total of tracheal swabs ( swabs at weeks of age and swabs at weeks of age) taken from those birds were negative for ibv in the real-time rt-pcr assay. a total of out of ( . %) clinical samples submitted to the delaware and maryland diagnostic laboratories were positive by the ibv real-time rt-pcr assay, whereas only out of ( . %) samples were positive by vi. the two tests were the same for out of ( . %) samples. only one sample was ibv real-time rt-pcr negative and vi positive, while samples were ibv real-time rt-pcr positive and vi negative. using virus isolation as the reference standard, the calculated sensitivity for the assay was . . known positive tracheal swabs taken from three groups of chickens given different doses of the arkansas dpi strain of ibv and one group of negative control birds are presented in fig. . the mean copy number for each group was calculated using a standard curve (fig. ) . at d.p.i., viral load in the trachea correlated with the dose given to each group with a fold difference of viral rna measured between groups and , and a -fold difference between groups and . at d.p.i., the virus load in the trachea plateaued at approximately × rna copies regardless of dose. at d.p.i., birds that received the lowest dose of virus (group ), had the highest copy number of viral genomes (approximately , copies), whereas birds in group , which received the highest dose of virus, had the lowest number of viral rna copies (approximately copies). three of the samples from the negative control birds in group , fig. . the viral load in each sample was quantified using the standard curve (fig. ) and the average viral genome copy number per group was calculated. group ( a) received no virus, group ( a) received a l dose of virus with a titer of . × eid /ml, group ( a) received a l dose of virus with a titer of . × eid /ml, and group ( a) received a l dose of virus with a titer of . × eid /ml. error bars indicate ± s.d., and d.p.i. = days post-inoculation. had recorded c t values of . , . , and . , at , , and d.p.i., respectively. two of those samples ( . and . ) were below our calculated limit of detection and thus considered negative. the other sample was retested and also found to be negative (data not shown). tracheal swabs taken from an ibv spray vaccinated commercial broiler chicken flock at days post-vaccination, had out of samples test positive, whereas at days postvaccination, out of samples were positive and at days post-vaccination, out of samples were positive. in this report we present the development and evaluation of a real-time taqman ® -based rt-pcr assay for the detection and quantification of ibv genomic rna directly from tracheal swabs. the target region for the assay was the highly conserved -utr of the ibv genome, which resulted in amplification of all ibv strains tested. it also amplified the -utr of two strains of tcov. this result was not unexpected because tcov is closely related to ibv and likely arose from a recombination event within the spike glycoprotein gene (guy, ; jackwood et al., ) . all other tcov genes including the -utr appear to be similar to ibv (guy, ) . with the exception of the closely related tcov, the assay was specific to ibv and did not detect any of the other pathogens tested. like all coronaviruses, ibv generates a -co-terminal nested set of viral mrnas when it replicates in the cell. generally, only the full-length viral genome is packaged in the virus particle. by targeting the highly conserved extreme -end of the genome, but not the leader sequence (nucleotides to ), which is found on all viral subgenomic mrnas, the assay only utilizes full-length viral rna as template, which should make it more sensitive than other rt-pcr methods that target the spike gene, and more accurate as a quantification tool for viral load in biological samples. the limit of detection/quantification and reproducibility of the assay was evaluated by generating a standard curve with rna run-off transcripts. the standard curve was generated from six independent runs on samples containing from to copies of rna and the limit of detection and quantification were both determined to be template copies. using the standard curve equation, the efficiency of the assay was calculated to be . %. in addition, the assay appears to be highly reproducible based on standard deviations of the c t values, which ranged from . to . cycles. we found that the real-time rt-pcr assay was extremely sensitive in a diagnostic laboratory setting. of the clinical samples submitted to the delaware and maryland diagnostic laboratories, % were positive for ibv by the real-time assay whereas only % of those samples were positive by virus isolation. the window of time that ibv can be detected following infection with the real-time rt-pcr test described herein is days post-infection, which likely represents a significant improvement over virus isolation and explains the higher rate of detection in the tracheal swab samples. for virus isolation, it is generally recommended that tissues such as cecal tonsil and kidney or cloacal swabs be taken in addition to tracheal swabs because the virus is known to persist there (gelb and jackwood, ) . not only does that practice significantly increases the work load it also can yield misleading results because vaccine viruses can also persist in those tissues and be shed in the feces (cavanagh and naqi, ) . it is important to evaluate any new diagnostic test with a number of known negative and positive samples. known negative samples, obtained from commercial layer chicks maintained in isolation units and monitored for antibodies to ibv by elisa, all tested negative for ibv in the real-time rt-pcr assay. since we wanted to insure the birds remained free of ibv, we choose commercial layer chicks with maternal antibodies to the virus as an added insurance against infection. for known positive samples, we used spf chicks with no maternal antibodies and infected them with three different dosages of the arkansas dpi strain of ibv. an additional negative control group was also maintained during that experiment. three samples from the negative control group had c t values of . , . , and . . after retesting, all of the samples were below our calculated limit of detection and considered negative. cross-contamination of samples especially during the rna extraction process is a real concern with any rt-pcr reaction, but it is recognized that precautions should be taken to limit contamination at every step of the procedure. the viral load in the trachea of birds given different dosages of ibv showed that regardless of the initial dose of virus, a similar maximal amount of viral rna was detected at d.p.i. more studies will be necessary to determine the course of virus replication in vivo, but it appears that the tracheal epithelium can support a finite amount of virus and that virus replication continues until that limit is reached. for the dosages used in our study, the maximum level of viral load was reached within days post-infection. it is interesting to note and quite appropriate that the requirements for efficacy of ibv vaccines outlined in section . title of the code of federal regulations (http://www.access.gpo.gov/nara/cfr/waisidx / cfr . html) requires that detection of challenge virus be conducted d.p.i. we also observed that the level of viral rna in the trachea of birds, which received a high dose of virus, declined more quickly than levels in birds receiving a low dose of the virus. loss of ciliated columnar epithelium and presumably the associated virus replicating in those cells, is a common lesion with ibv infection. birds receiving a high dose of ibv can develop clinical signs and lesions within h, whereas natural spreading virus generally requires h or more before clinical signs and lesions occur (cavanagh and naqi, ) . presumably, high dosages of the virus would accelerate the loss of the epithelial cells in the upper-respiratory tract leading to a more rapid decline in viral load. when spray vaccinated commercial broilers were examined for ibv by the real-time rt-pcr assay we found that only of birds were positive at day post-vaccination. by day , of birds were positive and by days post-vaccination of birds were positive for ibv. the dynamics of vaccine spread in commercial chicken flocks following spray vaccination in the field is not known. our data indicates that approximately half of the birds in the flock were initially exposed to the vaccine (day post-vaccination data), which then spread to other birds in the flock so that by days post-vaccination, more than % were exposed. more data will be required to verify this result, but it appears that exposure to ibv vaccine by in-house spray vaccination could be improved. initially it could be considered advantageous for ibv vaccines to spread in a marginally exposed flock providing a mechanism for all the birds in the flock to become immunized, however; vaccine spread in a flock also presents opportunities for back passage of these rapidly mutating coronaviruses potentially leading to a reversion to pathogenicity. it is important to rapidly differentiate infectious agents that cause respiratory diseases similar to low pathogenicity avian influenza and lentogenic and mesogenic newcastle disease so that veterinarians can quickly respond to outbreaks. in addi-tion, ibv remains an extremely important disease in commercial chickens and quickly diagnosing it is critical for control of this highly infectious virus. the real-time rt-pcr assay for ibv described herein can be conducted directly on tracheal swabs without the need for virus isolation. and although it does not differentiate between different types of ibv it is extremely sensitive and specific, and can be used to quantitate viral rna in clinical samples. molecular basis of the variation exhibited by avian infectious bronchitis coronavirus (ibv) infectious bronchitis, diseases of poultry infectious bronchitis, a laboratory manual for the isolation and identification of avian pathogens turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review molecular analysis of tcov, sars-cov, and ibv: how are they related further development and use of a molecular serotype identification test for infectious bronchitis virus a monoclonal antibody blocking elisa to detect serotype-specific infectious bronchitis virus antibodies production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes serotype identification of avian infectious bronchitis virus by rt-pcr of the peplomer (s- ) gene identification of avian infectious bronchitis virus by direct automated cycle sequencing of the s- gene differentiation of infectiousbronchitis virus serotypes using polymerase chain-reaction and restrictionfragment-length-polymorphism analysis origin and evolution of georgia (ga ), a new serotype of avian infectious bronchitis virus a monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes the authors would like to acknowledge the generous gifts from the following people: dr. erica spackman (usda/ars, athens, ga)-rna from aiv strains, dr. darrell kapczynski (usda/ars, athens, ga)-rna from apv and ndv strain b , dr. maricarmen garcia and sylva riblet (pdrc, university of georgia, athens, ga)-dna from iltv, adenovirus st- , and mg strains. this work was supported through specific cooperative agreement # - - - with usda/ars from department of defense supplemental homeland defense funds of cris project # - - - x. key: cord- -w pewq authors: chou, chih-fong; shen, shuo; tan, yee-joo; fielding, burtram c.; tan, timothy h.p.; fu, jianlin; xu, qiurong; lim, seng gee; hong, wanjin title: a novel cell-based binding assay system reconstituting interaction between sars-cov s protein and its cellular receptor date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: w pewq severe acute respiratory syndrome (sars), a life-threatening disease, is caused by the newly identified virus sars coronavirus (sars-cov). in order to study the spike (s) protein of this highly contagious virus, we established a clonal cell-line, cho-sg, from the chinese hamster ovary cells that stably expresses c-terminally egfp-tagged sars-cov s protein (s-egfp). the ectodomain of the s glycoprotein is localized on the surface of cho-sg cells with n-acetyl-glucosamine-terminated carbohydrate structure. cho-sg cells associated tightly with vero e cells, a sars-cov receptor (ace ) expressing cell-line, and the interaction remained stable under highly stringent condition ( m nacl). this interaction could be blocked by either the serum from a sars convalescent patient or a goat anti-ace antibody, indicating that the interaction is specific. a binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant s protein fragments expressed in escherichia coli. one of the sera obtained from the fragment encompassing amino acids - significantly blocked the interaction between cho-sg and vero e cells. the region is useful for studying neutralizing antibodies in future vaccine development. this paper describes an easy and safe cell-based assay suitable for studying the binding between sars-cov s protein and its receptor. severe acute respiratory syndrome (sars), a deadly disease, is caused by a human coronavirus named sars coronavirus (sars-cov). this highly infectious virus killed patients out of probable sars cases from november to july in countries (lingappa et al., http://www.cdc .gov/ncidod/eid/vol no / - .htm) and had a highly disruptive impact on people's lives as well as the economy. sars-cov belongs to the genus of coronavirus in the family of coronaviridae, a family of positive sense rna viruses with large envelopes that propagate in the cytoplasm of host cells and usually cause respiratory diseases in humans and animals. the complete genome of sars-cov has been sequenced from different isolates (marra et al., ; ruan et al., ) and it has a coronavirus genome organization with some unique features but is still distantly related to group coronaviruses (snijder et al., ) . the genome is nearly kb in length and encodes open reading frames (orfs) (marra et al., ; ruan et al., ) including those known coronavirus proteins, such as replicase a and b and four structural proteins, spike (s), envelope (e), membrane (m) and nucleocapsid (n). the rest of the orfs are novel and may encode for proteins with unknown functions. at least two of these proteins have been shown to be expressed during sars-cov infection (tan et al., b; yu et al., ; fielding et al., ) . antibodies against a, also termed as u , were found in the sera of sars patients (tan et al., a; yu et al., ) . aiming to control the sars epidemic, we first focused our studies on the s protein for the purpose of diagnosis, and also studied the interaction between s protein and its receptor for vaccine development. the s protein, a type i transmembrane glycoprotein, locates mainly on the outer envelope surface of sars-cov. its precursor is predicted to be amino acids in length. residues - comprise the putative signal peptide and the ectodomain is from residues to (marra et al., ) . a highly hydrophobic region from residues to is predicted to be the transmembrane domain and the rest (residues - ) are located in the cytoplasm. the s protein of coronavirus plays an important role in the initiation of the viral infection by mediating the attachment of the virus to the cell surface receptors that subsequently induces membrane fusion between the virus and the host. in some coronaviruses, the s protein (group ii mouse hepatitis virus [mhv] and group iii infectious bronchitis virus) is cleaved to generate two distinct functional subunits, s and s , by virus-encoded or host-encoded proteases (ziebuhr et al., ) . s is the nterminal fragment responsible for the attachment to the target cells; whereas, s is required for the fusion (gallagher and buchmeier, ) . the s domain of sars-cov s protein can be easily identified by sequence alignment with other coronavirus proteins but the s domain is not well conserved (spiga et al., ) . although, currently there is no evidence to show that there are cleaved s and s subunits in sars-cov-infected cells, the s equivalent domain has been cloned and demonstrated to bind the sars-cov receptor, ace , of host cells . furthermore, the binding domain of the s protein has been narrowed down to the n-terminal amino acid residues - (babcock et al., ) , - (xiao et al., ) and - (wong et al., ) . in addition to its function in viral entry, the s protein was found to be the major target of the host immune response against coronavirus as it induced neutralizing antibody (taguchi et al., ; kubo et al., ) . chinese hamster ovary (cho) cells are useful hosts to express mammalian genes and the system has been studied extensively to produce recombinant therapeutic glycoproteins. cho cells provide the advantages not only because they can be easily maintained and genetically manipulated, but also because they produce proteins with glycans similar to those native glycoproteins found in humans. in order to avoid the high risks involved in handling live sars-cov and to understand the s protein of this virus, we established a cho clone that expressed a significant amount of the recombinant s proteins on the cell surface and demonstrated that it can be used to study the properties of the sars-cov s protein. the selection of a high yield clone was facilitated by tagging the s protein with egfp at the c-terminus (s-egfp). the green fluorescence was conveniently used to monitor the expression of the protein in living cells and for observing the cell-based binding assay presented in this paper. cell-lines cho-k and vero e were purchased from american type cell collection (manassas, va, usa) and cultured at • c in % co in dmem containing g l − glucose, . mg ml − streptomycin and u penicillin and % fbs (hyclone, utah, usa). to maintain constitutive high level s-egfp expression in cho-sg, the culture medium was supplemented with m znso (tan and hong, ) . sars-cov strain sin (ruan et al., ) , an isolate from a sars patient in singapore was used in this study. the cdna for the cloning of s gene has been previously published (tan et al., a) . the vector pegfp-n as a control for the expression of egfp was purchased from clontech (palo alto, ca, usa). primers for constructing s-egfp from sars-cov strain ( - ) are sarsf ( -gcctcgaggccac-catgtttattttcttattatttcttactc- ) and sar-sr ( -gccccgggatgtgtaatgtaatttgacaccc- ), the s gene without the stop codon was pcr amplified and inserted into the xhoi/xmai sites of pegfp-n for making the fusion gene. the plasmid pegfp-n -s-egfp was further digested with xhoi and noti and then inserted into the xhoi/noti sites of pmmtc vector (tan and hong, ) . the pcr cycles comprised a denaturation step at • c for min, cycles of • c for s, • c for min, • c for min followed by a final elongation step at • c for min. the s-egfp construct was confirmed by dna sequencing. around % confluent cho cells were transfected with s-egfp and pegfp-n using dmrie-c (gibco/brl, gaithesburg, md, usa; brenner et al., ) according to manufacturer's protocol. in general, g ml − dna construct was mixed with g ml − dmrie-c in optimem (gibco/brl). to obtain stable transfectants, cho-sg (s-egfp transfected) and cho-g (pegfp-n transfected) cells were trypsinized h after transfection and then selected with mg ml − g (gibco/brl). the expression of egfp or s-egfp in transfected cells was detected by using a leica dmil-inverted fluorescence microscope fitted with fitc filter. cho-sg cells were lysed with lysis buffer ( % np in pbs, containing g ml − apotinin [sigma, st. louis, mo, usa], mm pmsf [sigma] on ice for h), and the lysate was then separated on % sds page and transferred to nitrocellulose hybond c membrane (amersham-pharmacia biotech, uppsala, sweden). prestained molecular markers were purchased from bio-rad (hercules, ca, usa). the membranes were blocked with % non-fat milk in pbst (pbs containing . % tween- ). antibodies against either gfp (monoclonal, clontech) or s (polyclonal antibod-ies from rabbit; keng et al. (submitted for publication) and horse; shen et al. (submitted for publication)) were used as primary antibody; horseradish peroxidase (hrp)-conjugated sheep anti-mouse (amersham-pharmacia biotech), goat antirabbit (pierce, rockford, usa) or goat anti-horse (bethyl, tx, usa) were used as secondary antibodies and detection was performed with a chemiluminescence kit (pierce). s-egfp was partially purified by using an agarose bound wga (wheat germ agglutinin; vector laboratories, ca, usa) column at • c. cho-sg cell lysate was first loaded onto wga column pre-washed with pbs, then the column was washed again with pbs and subsequently three column volumes of . m n-acetyl-d-glucosamine (glcnac; sigma) in pbs was used to elute the glycoproteins. s-egfp was identified by western blot analysis using rabbit anti-s antibody. non-permeable immunofluorescence staining was used to identify cell surface expression of s-egfp. cho-sg cells were dislodged with . % edta in pbs, and fixed after incubating with primary (patient's serum sample p in tan et al., a) and secondary (rhodamine-conjugated anti-human igg [sigma]) antibody reactions (both antibodies were diluted at : in growth medium and incubated on ice for h), and then loaded onto black teflon memzel slides (merck, darmstadt, germany) and fixed with % paraformaldehyde. the samples were viewed with a zeiss microscope (carl zeiss vision gmbh, hallbergmoss, germany). for all the cell-based binding assays presented in this paper, vero e cells were grown to a confluent layer and the cho-sg and cho-g cells dislodged by . % edta were used as ligands. cho-sg and cho-g cells were resuspended in growth medium and a similar amount of cells were laid over the vero e cell layer at • c for h. after the incubation, the cells were washed once with pbs and then three times with pbs containing nacl of mm, mm, mm, mm, mm or m. to determine whether ace mediates cho-sg and vero e interaction, the vero e cell layer was pre-treated with g ml − of goat anti-ace antibody (r&d systems, mn, usa) in growth medium at • c for h. then the antibody containing medium was removed, followed by incubation with cho-sg cells for two more hours. cells were washed once with pbs and three times with pbs containing mm nacl. to test whether the anti-sera from sars patients or raised against s protein fragments can block the binding between cho-sg and vero e , dislodged cho-sg cells were preincubated with the anti-serum to be tested at • c for h. then the cells were washed with growth medium and laid over the vero e cell layer for two hours. cells were washed once with pbs and then three times with mm nacl. five rabbit anti-s sera were obtained from the immunizations with recombinant s fragments, s (a.a. residues - ), s ( - ), s ( - ), s ( - ) and s ( - ), expressed in escherichia coli (keng et al., submitted for publication) and then sds page separated antigens were purified from gel slices. one mg each of the purified fragments were mixed with equal volume of complete freud's adjuvant (sigma) and used to immunize new zealand white rabbits. all anti-sera used were undiluted including normal human serum and the controls of pre-immune rabbit sera. results of the cell-based binding assay were detected by using a leica dmil-inverted fluorescence microscope fitted with fitc filter. in order to confirm the blocking effect by rabbit anti-s sera, cell lyastes (vero e and cho-sg binding cells) were analyzed by western blot using rabbit anti-s antibody. in order to clone the s portion of s-egfp, we used the viral strain (sin , accession number ay ) (ruan et al., ) isolated from a sars patient in singapore as template for pcr amplification. the primers for amplifying the full-length s gene without the stop codon were designed according to sin sequence (nucleotides to ); the amplified pcr fragment was fused with the egfp gene (fig. a) in pegfp-n . an extra octapeptide (trdppvat) from the egfp vector was generated at the junction, which should have limited effect on the transportation and expression of the fusion protein. for the expression of s-egfp in cho cells, the fusion construct was then cloned into pmmtc (tan and hong, ) . the transmembrane and cytoplasmic domains of the s protein were retained in the construct for proper anchoring and orientation of the ectodomain on the surface of cho cells as well as the intracellular localization of egfp. the expression of s-egfp was detected by using fluorescence microscope. a stable clone, cho-sg (fig. b) , expressing a relatively high level of s-egfp at the cytoplasmic membrane was selected for the study described below. next, we used western blot analysis to confirm the correct expression. s-egfp in the cell lysate of cho-sg (fig. ) was identified by a mouse anti-gfp monoclonal antibody (lane a), and two sera from rabbit (lane b) and horse (lane c) raised against the s protein. all the three antibodies recognized a common band of ∼ kda. the predicted molecular weight (mw) of sars-cov s protein is ∼ - kda (xiao et al., ) ; therefore, the mw of s-egfp is approx- imately - kda with additional kda from the egfp tag (fig. ) . these results concur with the observations from another group (xiao et al., ) that the recombinant s protein was intact in the host cells. additionally, subcellular fractionation (chou and omary, ) together with western blot analysis showed that s-egfp was present mainly in the fraction of plasma membrane (data not shown). the lectin wheat germ agglutinin (wga) is known for its ability to bind the terminal n-acetyl-glucosamine (glc-nac) of glycoproteins. in order to determine the carbohydrate characteristics of the glycoprotein s-egfp, the cell lysate of cho-sg was passed through a wga-agarose column. western blot analysis using rabbit anti-s serum was used to monitor the wga agarose chromatography. fig. shows that nearly all s-egfp was retained on the column (lane f); whereas, it was absent in the flow-through sample (lane e). this result indicates that nearly all s-egfp molecules are glycosylated with glcnac(s) at the end of the oligosaccharide side chains. although fig. b shows that the s-egfp is localized to the plasma membrane, the result did not provide information about its orientation. since the ectodomain of the s protein was expected to be outside the cell, non-permeable ifa was applied to confirm the orientation of the fusion protein. a serum sample (p ) from a convalescent sars patient (tan et al., a) was used as the primary antibody to stain the cho-sg cells followed by staining with a rhodamine-conjugated anti-human igg (fig. ) . the normal human serum was used as the negative control. the result indicates that the s portion of s-egfp was exposed extracellularly of the cho-sg cells. fig. . cho-sg expresses s-egfp at cell surface with expected orientation. non-permeable ifa using the serum (p in tan et al., a ) from a recovered sars patient showed cell surface staining of s-egfp expressing cho-sg cells. the control is normal human serum. the green fluorescence seen in the upper panels was from the egfp tag and the red fluorescence observed in the lower panels was from rhodamine labeled secondary antibody. since ace on the surface of vero e cells is responsible for the binding of and the fusion with sars-cov , we tested the binding activity between cho-sg and vero e cells as well as the binding strength between the ligand (sars-cov s protein on cho surface) and the receptor (ace on vero e ). for the control in the cell-based binding assay, a stable egfp-expressing cho clone (cho-g) was generated by introducing the plasmid pegfpn into cho cells. both cho-g and cho-sg were tested for binding to vero e cell layers and the results are shown in fig. a . the cho-g cells resisted the pbs wash but were detached by the solutions containing supplemented nacl. in contrast, the binding between cho-sg and vero e could resist up to m nacl. we sought to investigate the binding specificity of this interaction by testing whether the serum from a recovered sars patient (p in tan et al., a ) and a goat anti-ace antibody could block the interaction. to test the blocking effect, either cho-sg cells were pre-treated with human sera before incubated with vero e cell layers, or vero e cells were pre-treated with anti-ace antibody before interacting with cho-sg. all panels in fig. b were washed with mm nacl, the controls for the patient's serum was normal human serum, and for anti-ace was pbs. the results showed that both the patient's serum and the anti-ace antibody could block the binding, suggesting that this binding is specifically mediated by the s protein and ace . although, the receptor for sars-cov has been found, it is still important to localize the epitopes that induce neutralizing antibody against the virus, particularly for the purposes of vaccine development. we attempted to localize the epitopes by expressing recombinant s fragments (fig. a ) in e. coli and then immunizing rabbits with the purified fragments. to determine which anti-serum may block the binding, cho-sg cells were pre-treated with the rabbit sera individually before incubating with vero e cell layers. after the incubation, the cells were washed once with pbs and times with mm nacl. the anti-s serum demonstrated the blocking effect ( fig. b and c) indicating that some blocking epitopes are present in s . cho-sg that constitutively expresses a significant amount of s-egfp provides a convenient tool to study the recombinant sars-cov s protein. our results showed that the ectodomain of the s protein on the surface of cho cells mimics the interaction between the virus and vero e cells, presumably through the cellular receptor ace . the cellbased binding assay introduced in this paper should facilitate the screening for drugs, peptides or vaccines that block the entry of sars-cov, minimizing the use of the highly infectious live virus. the clonal cell-line cho-sg may be used for large-scale production to supply s-egfp as a diagnostic marker, and its affinity to wga is a favorable property to assist the purification of s-egfp. although, the expression of s-egfp on cho-sg was significant, self-fusion of cho-sg cells has not been observed in culture, which may be due to the absence or a low level of ace on cho (ovary) cells (donoghue et al., ) . the expressions of recombinant s protein in full-length or fragments had been demonstrated previously xiao et al., ; wong et al., ) by using human kidney cell-lines or t. we also tried two human cell-lines, t and jurkat, to express s-egfp but stable clones were not established due to the undetectable green fluorescence from g resistant colonies. the sars convalescent patient's sera (p and p in tan et al., a) blocked the interaction between cho-sg cells and vero e , confirming the authenticity of this assay. serum sample p does efficiently identify the extracellular expression of the s ectodomain on the cell surface of cho-sg by non-permeable ifa (fig. ) , but with lower potency to block the binding (data not shown in fig. b ). epitopes of sars-cov s protein responsible for the binding to ace was first demonstrated to be in the s region and then narrowed down to the amino acid residues - (babcock et al., ) , - (xiao et al., ) and - (wong et al., ) . the binding and fusion interactions were performed by using expressed soluble s fragments and the cellular receptor. we used the cell-based binding assay to test the blocking effect for five rabbit anti-s sera raised against five recombinant s fragments and three of these fragments, s , s and s (fig. a) covered the a.a. residues from to , which overlapped with the predicted s domain or the s-ace binding region. however, we were only able to identify a significant blocking effect ( fig. b and c) from anti-s (a.a. - ) rabbit serum. in order to rule out non-specific effects from undiluted serum, pre-immune rabbit sera were used as the controls for the binding assay and no blocking effect was ob- fig. . cho-sg and vero e binding assay. in all panels, vero e cells were first cultured to a confluent layer. (a) edta dislodged cho-g (upper panels) and cho-sg (lower panels) were incubated with the vero e cell layers in the cold room for h. cells were washed once with pbs and followed by three washes with pbs containing different amounts of nacl as indicated at the bottom of the figure. (b) the photos shown in the upper panels were taken under tungsten lamp and these in the lower panels were taken under green fluorescence. cho-sg cells were pre-treated with either human normal serum (a) or sars recovered patient's serum (p in tan et al., a) (b) before being added to the vero e cell layer. the cells were washed with mm nacl after the incubation with vero e cells. vero e cultures were pre-treated with goat anti-ace antibody (d) or the pbs control (c) and then incubated with the cho-sg cells followed by mm nacl washes. served. the rabbit sera produced against fragment s (a.a. - ) and s ( - ) also presented some minor blocking effects ( fig. b and c) . rabbit serum anti-s (a.a. - ) could not block the binding (or anti-s with less significant effect) may be due to the antigenicities for those modules requiring higher extent of glycosylation or the native conformation. however, although the region of s contains nine predicted n-glycosylation sites, the rabbit antiserum raised against the denatured fragment produced from e. coli was still able to block the binding. this indicates that there is a non-glycosylated and linear epitope within s that is important for s-ace binding. although the rabbit anti-s serum against s could not block the binding effectively, it was routinely used in a dilution of : as primary anti- fig. . identification of the receptor-binding region of sars-cov s gene. (a) five fragments of sars-cov s protein were expressed in e. coli. rabbits were immunized with these five recombinant s protein fragments. the rabbit anti-sera were tested for blocking cho-sg and vero e cell interaction. (b) the cho-sg cells were first treated with pre-immune serum or one of five rabbit anti-sera described in (a), and then added to the vero e cell layer. after incubation, the cells were washed with mm nacl. the rabbit serum from s had the most significant blocking effect. (c) western blot analysis using rabbit anti-s antibody to show the amount of s-egfp in (b) samples. body for western blot analysis (fig. ) . all sera from either humans or rabbits used in the binding assay were neat; however, diluted sera ( : ) were tested and revealed insignificant blocking effect. our results identified a receptor binding domain of the s protein required lesser extent of glycosylation and native conformation. the blockage of cho-sg cells to vero e cells by anti-ace antibody was nearly complete (fig. b) , indicating that ace is the main receptor on the surface of vero e cells responsible for the binding. however, we cannot exclude the possibilities of other receptors involved in the binding, such as the one closely associated with ace , which may also be blocked by an anti-ace antibody. in patients with sars, atypical pneumonia and diarrhea were common symptoms caused by sars-cov, correspondently the mrna levels of ace were comparably high in gastrointestinal system and lung (harmer et al., ) and the higher ace protein levels have been demonstrated in the epithelia of the lung and small intestine (hamming et al., ) . two human lung cell-lines (mrc and a ) and a human colon cell-line (ht ) were tested for developing the binding assay but results were unexpected due to non-specific binding between cho-sg and these cells. other human cell line of lung or digestive tissues will be examined in the future. ported by the agency for science and technology (a*star) of singapore. amino acids to of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor conserved regulation of the lymphocytespecific expression of lck in the fugu and mammals mitotic arrest with anti-microtubule agents or okadaic acid is associated with increased glycoprotein terminal glcnac's a novel angiotensin-converting enzyme-related carboxypeptidase (ace ) converts angiotensin i to angiotensin - characterization of a unique group-specific protein (u ) of the severe acute respiratory syndrome coronavirus coronavirus spike proteins in viral entry pathogenesis tissuedistribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis quantitative mrna expression profiling of ace , a novel homologue of angiotensin converting enzyme amino acids to in the s domain of sars coronavirus s protein induces neutralizing antibodies: implications for the development of vaccine and anti-viral agent localization of neutralizing epitopes and the receptor-binding site within the amino-terminal amino acids of the murine coronavirus spike protein angiotensin-converting enzyme is a functional receptor for the sars coronavirus comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group lineage molecular modelling of s and s subunits of sars coronavirus spike glycoprotein localization of neutralizing epitopes and receptor-binding site in murine coronavirus spike protein gene expression in mammalian cells profile of antibody responses against sars-coronavirus recombinant proteins and their potential use as dianostic markers a novel severe acute respiratory syndrome coronavirus protein, u , is transported to the cell surface and undergoes endocytosis a -amino-acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme the sars-cov s glycoprotein: expression and functional characterization identification of a novel protein a from severe acute respiratory syndrome coronavirus virus-encoded proteinases and proteolytic processing in the nidovirales we thank dr. eng eong ooi (environmental health institute, national environmental agency, singapore) for providing inactivated sars rna; drs. yue wang, ding xiang liu, le-ann hwang and wei-ping yu for their precious suggestions; and ms. chay boon loh for her assistance in ifa and the dna sequencing facility in imcb. this project was sup- key: cord- - lxc rj authors: soltan, mohamed a.; tsai, yun-long; lee, pei-yu a.; tsai, chuan-fu; chang, hsiao-fen g.; wang, hwa-tang t.; wilkes, rebecca p. title: comparison of electron microscopy, elisa, real time rt-pcr and insulated isothermal rt-pcr for the detection of rotavirus group a (rva) in feces of different animal species date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: lxc rj there is no gold standard for detection of rotavirus group a (rva), one of the main causes of diarrhea in neonatal animals. sensitive and specific real-time rt-pcr (rtrt-pcr) assays are available for rva but require submission of the clinical samples to diagnostic laboratories. patient-side immunoassays for rva protein detection have shown variable results, particularly with samples from unintended species. a sensitive and specific test for detection of rva on the farm would facilitate rapid management decisions. the insulated isothermal rt-pcr (rt-iipcr) assay works in a portable machine to allow sensitive and specific on-site testing. the aim of this investigation was to evaluate a commercially available rt-iipcr assay for rva detection in feces from different animal species. this assay was compared to an in-house rtrt-pcr assay and a commercially available rtrt-pcr kit, as well as an elisa and em for rva detection. all three pcr assays targeted the well-conserved nsp gene. clinical fecal samples from diarrheic animals (mainly cattle and horses) were tested. the percentage of positive samples by elisa, em, in-house rtrt-pcr, commercial rtrt-pcr, and rt-iipcr was . %, %, . %, . %, . %, respectively. the agreement between different assays was high ( . – %) in samples containing high viral loads. the sensitivity of the rt-iipcr assay appeared to be higher than the commercially available rtrt-pcr assay, with a limit of detection ( % confidence index) of – copies of in vitro transcribed dsrna. in conclusion, the user-friendly, field-deployable rt-iipcr system holds substantial promise for on-site detection of rva. rotavirus is classified as a member of family reoviridae, genus rotavirus. it is non-enveloped, - nm in diameter, and the genome length is approximately . kb. the genome is composed of segments of double-stranded rna and encodes six structural proteins (vp - , and ) and six non-structural proteins (nsp - ) (desselberger, ; zhou et al., ) . the virus capsid displays icosahedral symmetry and contains three layers, including an outer layer composed of the vp protein, with vp protein spikes, an inner or middle vp glycoprotein layer, and the core shell formed by vp (desselberger, ) . antigenic epitope analysis of the vp glycoprotein classifies the genus rotavirus into groups (a-h) (chandler-bostock et al., ; matthijnssens et al., ) . rotaviruses are characterized by relatively high antigenic and genetic diversity, as a result of accumulation of point mutations (genetic drift), and/or reassortment of genomic segments (genetic shift) (matthijnssens et al., ) . although host species barriers and host range restriction exist in rotavirus, reassortment can result in interspecies transmission, which also contributes to the diversity and evolution of rotavirus (martella et al., ; zhou et al., ) . rva is one of the main causative agents of diarrhea in young humans and animals (cho et al., ; zhou et al., ) . it is ubiq- uitous in the environment and relatively resistance to disinfectants. the adult animals are the main source of infection for newborn animals, and serological surveys revealed that - % of adult animals have an immune response against rva (schlafer and scott, ) . clinically healthy newborn animals may also shed rva, but the prevalence is lower compared to animals exhibiting diarrhea (kaminjolo and adesiyun, ) . studies have shown that detection of rotavirus in the presence of diarrhea is a significant finding in calf diarrhea (cho et al., ) . additionally, rotavirus has been shown to be significantly associated with liquid diarrhea in -to -day-old dairy calves (al mawly et al., ) and also in beef calves (cho et al., ) . there are many assays used for diagnosis of rva. historically, diagnostic laboratories routinely used electron microscopy (em) for detection of the virus. however, due to the costs of the microscope and its maintenance, as well as the required technical expertise, this method has lost favor. additionally, em lacks sensitivity, requiring ∼ viral particles/ml for virus detection (maes et al., ) . immunoassays (such as elisas) have replaced em as the diagnostic test of choice for detection of viral antigen because of their ease of use and speed of obtaining a result (desselberger, ) . most of the commercially available elisa kits are based on monoclonal antibodies against vp glycoprotein, which is expressed at a high level during infection. the vp glycoprotein is conserved among rva of different animal species, allowing cross-reactivity and detection of the virus from different hosts. therefore, the commercially available human rva elisa assays have been used for detection of rva in animals (bailey et al., ) . however, as a result of antigenic drift, there are several variants or genotypes of rva vp that have been described (mino et al., ) . differences among these vp genotypes influence the performance of various commercially available vp based immunoassays. therefore, diagnostic kit validation for each species is necessary. not all the assays have been validated for use in all animal species, and this is particularly true for horses (mino et al., ) . in fact, one human rva rapid immunoassay developed for patient-side use was shown not to work in detection of rva in horses (slovis et al., ) . recently, molecular techniques such as rt-pcr and real-time rtrt-pcr have replaced other diagnostic tests with the advantage of higher analytical sensitivity and specificity (slovis et al., ) . however, these types of assays are run in commercial or diagnostic laboratories and require expensive equipment and advanced technical skills, resulting in increasing costs to the producer, which leads to a reduction in the use of laboratory assays to support field disease investigations (izzo et al., ) . therefore, the aim of our investigation was to evaluate a recently available insulated isothermal rt-pcr (rt-iipcr) reagent set (pockit tm rotavirus a reagent set, genereach usa, lexington, ma, usa) with use of a portable pcr machine, which could potentially be used for point-of-need detection for rva in the feces of different animal species. the assay was compared to an in-house rtrt-pcr assay, a commercially available rtrt-pcr kit, a commercially available elisa, and em. a total of fecal samples from clinically affected animals, submitted to the university of tennessee, college of veterinary medicine, clinical virology laboratory, were used for comparison between different diagnostic assays. nucleic acids were extracted with an automated nucleic acid extraction system according to the manufacturer's instructions (taco tm mini, genereach usa) from all samples for molecular testing (in-house real-time rt-pcr, the commercially available real-time rt-pcr, and the rt-iipcr reagent set). briefly, mg of fecal sample was added to ml pbs and l of the supernatant was used for extraction. nucleic acids were tested immediately following extraction or were stored at − • c until tested. approximately five grams of fecal material were suspended in ml distilled water and centrifuged at , g for min. the fecal pellet was re-suspended in ml of distilled water and l of the suspension was mixed with l of % phosphotungstic acid (ph . ) in ml of distilled water. the mixture was then nebulized onto a carbon type-b, -mesh copper grid (ted pella, redding, ca, usa). the grids were examined by an electron microscope (zeiss auriga, university of tennessee, advanced microscopy and imaging center, knoxville, tn, usa). eighty four of the fecal samples were available for testing by em. a commercially available sandwich elisa, targeting the vp of human rva (premier tm rotaclone elisa kit, meridian bioscience, cincinnati, oh, usa), was used for testing the fecal samples, according to the manufacturer's instructions. eighty-five of the fecal samples were available for testing by elisa. the published nucleotide sequences of rva non-structural protein (nsp ), from different animal species ( bovine, equine, caprine, ovine and canine), were retrieved from genbank and aligned using mafft software. primers and a probe were designed, using the genscript online software, to amplify an area of bp. primer and probe sequences are shown in table . onestep real-time rt-pcr assay was performed using the superscript ® iii platinum ® one-step qrt-pcr kit (invitrogen, thermo fisher scientific, carlsbad, ca, usa) in a stepone tm real-time pcr system (applied biosystems, thermo fisher scientific, foster city, ca, usa). the proper concentrations of primers, probe and magnesium were optimized. the test was performed in -l total reaction volume containing l of the extracted nucleic acid, nm of each primer, nm of probe, mm magnesium and nm rox reference dye. following optimization, the cycle parameters were: • c for min, • c for min, followed by cycles at • c for s and • c for min. the assay sensitivity was determined and optimized by using standard rna, which was produced by cloning the pcr product from a positive clinical sample with the ta cloning ® kit with pcr tm . vector and one shot ® inv␣f' chemically competent escherichia coli (invitrogen, thermo fisher scientific, usa) according to manufacturer's instructions. the purified plasmids were sequenced to confirm correct orientation, linearized, and used as a template for synthesis of in vitro transcribed rna using the megascript t transcription kit (invitrogen, thermo fisher scientific, usa), according to manufacturer's instructions. rna copy numbers were calculated and a standard curve was generated from ten-fold serial dilutions of the rna standard at a range from × − to × − . the reproducibility of the assay was evaluated by calculation of intra-and inter-assay coefficient of variation (cv). the assay specificity was evaluated by testing dna and rna of different pathogens that are known to cause diarrhea in various animal species, including neorickettsia risticii (potomac horse the rt-iipcr test (pockit tm rotavirus a reagent set) targets the nsp gene. all the components of the rt-iipcr reaction were lyophilized in one tube. the lyophilized premix was rehydrated before reaction in l premix buffer b and l of sample rna were added to the mixture. the mixture was transferred to an rtube, centrifuged and tested by the pockit tm nucleic acid analyzer (genereach usa) as previously described (wilkes et al., ) . the assay specificity was evaluated as described for the in-house rtrt-pcr assay. double-stranded rotavirus ns rna was synthesized and used to determine sensitivity of the rt-iipcr reagent set. briefly, a plasmid containing a partial sequence of the nsp gene of the rva/horse-wt/zaf/eqrv-sa / /g p[ ] strain (genbank accession jq ) and the bovine rotavirus strain kj strain (genbank accession dq ) were used to generate positive and negative strand rna by in vitro transcription using the maxiscript ® t kit and megascript ® sp kit (life technologies, darmstadt, germany), respectively. residual dna was removed using the ambion ® turbo dna-free tm kit (life technologies). the two rna products were annealed to form double-stranded rna. residual single strand rna was removed by rnase a treatment. after phenol-chloroform extraction, integrity of the double-stranded rna preparation was confirmed by polyacrylamide gel electrophoresis analysis. concentration of rna was determined in a nanodrop spectrophotometer (nanodrop technologies, houston, tx, usa). serial dilutions of double-stranded rna were made in ng/l yeast trna. single use aliquots were stored at − • c. additionally, the sensitivity of the rotavirus rt-iipcr reagent set was evaluated by comparison with the commercially available rtrt-pcr assay using -fold serial dilutions of nucleic acid extracted from a positive bovine clinical sample. the nucleic acid samples were tested by the lsi vetmax tm triplex ruminant rotavirus & coronavirus real-time pcr kit (thermo fisher scientific, usa), according to manufacturer's instructions. the percentage of agreement between the different diagnostic assays and cohen's kappa coefficient were calculated using spss statistics software. the standard curve for the in-house rtrt-pcr assay generated from serially diluted standard rna was linear (slope = − . ) http://www- .ibm.com/software/analytics/spss/products/statistics/. the assay was linear over orders of magnitude and was able to detect as few as genomic equivalents per reaction. ( fig. ) and the coefficient of linear regression (r ) was . . the assay efficiency was estimated to be . %. the developed assay was sensitive and able to detect genomic equivalents of the target nsp gene per reaction. furthermore, it was specific, with no amplification detected from dna or rna from other pathogens known to cause diarrhea in animal species. the assay was reproducible with intra-and inter-assay cvs ranging from . to . and . to . , respectively. the limit of detection ( % confidence interval) for the rt-iipcr reagent set was and copies of dsrna, based on using log dilutions of in vitro transcribed dsrna containing target bovine and equine rotavirus sequences, respectively. the comparison between sensitivity of the rt-iipcr assay and commercially available rtrt-pcr assay using -fold serial dilutions of nucleic acid from a positive sample showed that the rt-iipcr reagent set had a fold increase in sensitivity. like the in-house assay, this assay was also specific, with no amplification in any of the samples used for specificity testing. there was variation in the percentage of positive samples detected by each assay. the rt-iipcr assay detected the most, while em detected the least. the overall percentages of positive samples by each diagnostic assay are shown in table . there was a significant difference in the number of positive samples detected with the in-house rtrt-pcr assay versus the other two molecular tests. these differences are evident when comparing the kappa coefficients (table ) . the em assay results showed substantial agreement in comparison to elisa results. this agreement becomes fair when compared to the rt-iipcr reagent set and the commercial rtrt-pcr kit, and moderate in comparison to the in-house rtrt-pcr assay. the elisa showed moderate agreement with the rt-iipcr reagent set and commercial rtrt-pcr assay but had substantial agreement with the in-house rtrt-pcr assay. a perfect agreement was found between the rt-iipcr reagent set and the commercial rtrt-pcr assay (table ) . comparison between the different assays showed a strong correlation for samples containing high viral loads (ct values ≤ ) (table ) . rva is one of the most prevalent causative agents of diarrhea in farm animals (athanassious et al., ; izzo et al., ; slovis et al., ) . it causes significant economic losses as a result of decreased weight gain, treatment costs, and high mortalities. therefore, development of a highly sensitive and specific test for point-of-need diagnosis would be beneficial to the veterinary practitioner and producer because delays associated with shipping samples to a diagnostic laboratory could be avoided, aiding in rapid control and prevention decisions (izzo et al., ) . there are many commercially available lateral flow immunochromatography assays (lat) that can be used for on-site detection of rva. one brand of lat showed favorable results in comparison to virus isolation and elisa (maes et al., ) . however, when compared to qrt-pcr, the results have been variable. the limitation of antibody-based tests for the detection of enteric pathogens is the requirement of high concentration of free antigen to generate a positive reaction, the free antigen is decreased significantly during the course of disease. therefore, these tests have lower sensitivity and could miss positive samples collected late in the course of clinical disease, when compared to rt-pcr (izzo et al., ; maes et al., ) . depending on the test used and species tested, these tests can also have low specificity (izzo et al., ) or may not work at all (slovis et al., ) . commercial elisa assays and other immunoassays, while easy to use and rapid for detection, are mostly designed for human use and care must be taken when applying these tests for animal use. the elisa used in this study was able to detect rva from three different animal species. the agreement between em and elisa was . %, which was similar to previously published reports (athanassious et al., ; benfield et al., ; reynolds et al., ) , and both of these methods lacked the sensitivity achieved with molecular assays, which has also been previously reported (izzo et al., ) . interestingly, three positive samples by elisa in this study were negative by em and all three molecular assays. the elisa assay absorbance values for these samples ranged from . to . , which indicates low viral load according to the manufacturer. these samples were considered false positives. false positive results are not uncommon for elisa assays as a result of the complex sample matrix (i.e. gut microbiota), which can increase the probability of cross-reactions. the type of antibodies (polyclonal versus monoclonal) used strongly affects their detection efficiency. the use of monoclonal antibodies is usually associated with increased specificity of the assay, but this also creates potential problems with regard to amino acid variability among the rotaviruses from different species another complication with detection of rotavirus is the fact that it can be detected in both healthy and diseased animals. establishing a causal relationship may be difficult without demonstration of classic histopathological changes (izzo et al., ) , particularly when using highly sensitive molecular detection methods. considering animals shed up to - virions/ml of feces during the acute phase of infection (izzo et al., ) , positive results obtained with less sensitive methods (em and elisa) are more likely to be associated with causality. in human medicine, elisa diagnosis is highly correlated with disease in rva infection (phillips et al., ). related to this concept is the idea that magnitude of viral shedding (based on ct value) can help determine disease etiology (phillips et al., ) . evidence of high viral load in samples tested gives the clinician more confidence that the virus is the cause of the disease process (izzo et al., ) . a high correlation between ct values ≤ and clinical disease was found in one human study that compared rva shedding in clinically healthy subjects versus those with diarrhea (phillips et al., ). we found a higher correlation between test methods when we evaluated samples with ct values ≤ . however, ct values from different protocols must be interpreted with care because the values may not equate to the same viral load per gram of feces (phillips et al., ) . this was actually seen in this study when comparing between the commercial rtrt-pcr assay and the in-house rtrt-pcr assay, particularly with the positive equine samples. the ct values obtained by the in-house rtrt-pcr assay were and . , compared to . and . , respectively, by the commercial rtrt-pcr kit. while we did not have additional equine samples to further examine this, these high ct values from equine samples are consistent with a previous report (matthijnssens et al., ) in which the same commercial molecular assay was also used for diarrheic samples from equine. ct values for positive samples from that study ranged from . - . . the authors attributed the high ct values to degradation of the viral rna in the samples or mild infection. while these are certainly possibilities, these results do raise questions about the sensitivity of this commercial rtrt-pcr assay in the diagnosis of equine rva. this disparity may potentially be attributed to mismatches in primer and probe binding areas. the designed primers and probe of the in-house rtrt-pcr assay contain several degenerate bases, in an attempt to avoid problems with base mismatches. however, as seen with the sensitivity testing and comparison between the assays, the in-house molecular test was less sensitive that the other molecular assays. based on the findings in this study when comparing ct values, it is important to note that the possibility of the significance of lower concentrations of rna may not be excluded. ct values can be affected by many factors, not just mismatches in primer/probe binding regions or inappropriate handling and storage of the sample, but also stage of the disease and the quality of the sample collected (izzo et al., ) . the commercial rt-iipcr assay performed in a portable pcr machine was shown to have higher sensitivity than the other molecular methods tested in this study. while it is possible some of the positive samples could have been false positives, we believe it is more likely associated with the increased sensitivity seen with this test, which was demonstrated with side-by-side testing, versus the commercial rtrt-pcr assay, of serial dilutions of nucleic acid from a clinical sample. high sensitivity of rt-iipcr assays has been consistently demonstrated in previous reports (ambagala et al., ; balasuriya et al., ; wilkes et al., ) . the higher sensitivity may be attributed to the performance of the reaction in gradient temperature that results from the thermal convective phenomena associated with this type of pcr (krishnan et al., ) . this allows primers and probes to anneal to sequences with mismatches (ambagala et al., ) . the commercial rt-iipcr assay incorporates a fluorescent hydrolysis probe, which increases the specificity of the assay, functioning more like a real-time pcr than a conventional pcr. however, rather than obtaining a ct value that requires interpretation, the portable machine detects the fluorescent signal before and after the reaction and automatically converts it into a positive or negative result. the numerical value for these fluorescent signals can be obtained from the machine for some determination about amount of virus present in the sample, but the sensitivity of the signal does not correlate as well as a threshold cycle value does for real-time pcr. the automated interpretation of the iipcr machine does however make the method easier to use without the need for advanced training. the portable machine is small and light weight and can be operated with a car battery. the three rt-pcr assays evaluated in the study were shown in general to have comparable performance for rva detection in fecal samples. the real-time pcr assays are excellent tools for diagnostic laboratories, and the rt-iipcr assay working in a portable pcr machine is highly sensitive and specific and shows promise for on-farm molecular detection of rva. m.a. soltan and r. p. wilkes declares no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. p.y. lee, y.l. tsai, c.f. tsai, h.f. chang, and h.t. wang are affiliated with genereach usa. however, this work does not alter our adherence to all the veterinary journal's policies on sharing data and materials. risk factors for neonatal calf diarrhoea and enteropathogen shedding in new zealand dairy farms a rapid field-deployable reverse transcription-insulated isothermal polymerase chain reaction assay for sensitive and specific detection of bluetongue virus detection of bovine coronavirus and type a 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events we wish to thank all the veterinarians who submitted samples and made this work possible. key: cord- - vf bts authors: sakurai, akira; nomura, namiko; nanba, reiko; sinkai, takayuki; iwaki, tsunehito; obayashi, taminori; hashimoto, kazuhiro; hasegawa, michiya; sakoda, yoshihiro; naito, akihiro; morizane, yoshihito; hosaka, mitsugu; tsuboi, kunio; kida, hiroshi; kai, akemi; shibasaki, futoshi title: rapid typing of influenza viruses using super high-speed quantitative real-time pcr date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: vf bts the development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. quantitative real-time pcr is often used for detecting various kinds of viruses; however, it requires more than h per run. detection assays were performed with super high-speed rt-pcr (shrt-pcr) developed according to a newly designed heating system. the new method uses a high-speed reaction ( s/cycle; cycles in less than min) for typing influenza viruses. the detection limit of shrt-pcr was copy/reaction and (− ) plaque-forming unit/reaction for viruses in culture supernatants during min. using shrt-pcr, strains of influenza viruses isolated by the tokyo metropolitan institute of public health were tested; the results showed % sensitivity and specificity for each influenza a and b virus, and swine-origin influenza virus. twenty-seven swabs collected from the pharyngeal mucosa of outpatients were also tested, showing positive signs for influenza virus on an immunochromatographic assay; the results between shrt-pcr and immunochromatography exhibited % agreement for both positive and negative results. the rapid reaction time and high sensitivity of shrt-pcr makes this technique well suited for monitoring epidemics and pre-pandemic influenza outbreaks. influenza is a highly contagious disease caused by negativestrand rna viruses of the family orthomyxoviridae. seasonal outbreaks of influenza present global health problems involving morbidity, mortality, and economic losses. since , the highly pathogenic avian influenza h n virus has spread from asia to europe and africa posing a pandemic threat of a highly lethal and contagious disease (gambotto et al., ; webster and govorkova, ) . a pandemic caused by swine-origin influenza virus (s-oiv) infection occurred in (dawood et al., ; itoh et al., ; shinde et al., ) causing more than , deaths in more than countries; the world health organization (who) announced on september , , that the pandemic had transitioned into a post-pandemic phase (who, a,b) . the occurrence of outbreaks and pandemics indicates that rapid subtyping of influenza viruses, including avian and swine-origin viruses, is a high priority for public health response systems; rapid virus detection is expected to improve the control of the pandemic spread of influenza and patient care. quantitative real-time pcr (qrt-pcr) is commonly used for detecting and subtyping viruses, including influenza viruses (van elden et al., ; who, a) . this method offers high sensitivity and selectivity, but generally requires approximately h per run; therefore, qrt-pcr is not appropriate for rapid virus detection or subtyping in outbreaks of fast-spreading and/or highly pathogenic viruses at public health centers, hospitals, airports, and other public transportation hubs. super high-speed qrt-pcr (shrt-pcr) is a recently developed version of qrt-pcr that is characterized by extremely short reaction times (less than min per run for cycles). in this assay, reaction mixtures of qrt-pcr are applied to thin compact disc (cd)-type sample containers, sealed, and rotated on heat blocks at different temperatures (for denaturing, annealing, and extension temperatures). the unique structural and thermodynamic properties of heat blocks fixed at different temperatures are critical for the super high-speed polymerase reaction because the blocks allow rapid temperature changes within the samples. while shrt-pcr is a unique variant of qrt-pcr, it can be applied to detect many kinds of microbes. in addition, the super high-speed reaction is well suited to the detection, diagnosis, and control of rapidly spreading pathogens such as those characteristic of seasonal pandemic influenza viruses. however, the clinical application of the disk-type qrt-pcr has not been reported at present. this study establishes a new method for high-speed (< min) typing of influenza viruses using shrt-pcr. the analysis targeted the nucleotide sequences of the matrix protein segment of influenza virus a (a-mp), influenza virus b (b-mp), and hemagglutinin (ha) of the pandemic s-oiv and h n avian influenza viruses. using this system, strains of influenza viruses isolated from hospitals in tokyo and the tokyo metropolitan institute of public health were rapidly analyzed and subtyped. the typing results were correlated with those using a standardized qrt-pcr method. shrt-pcr is expected to provide new strategies for controlling the transmission of influenza viruses. regular laboratory strains, including a/wsn/ (h n ), a/pr / (h n ), a/aichi/ / (h n ), and b/mass/ / were obtained from the american type culture collection (http://www.atcc.org). a/duck/hokkaido/vac- / (h n ), a low pathogenic h n subtype vaccine strain, was generated by genetic reassortment between low-pathogenic avian influenza viruses, a/duck/hokkaido/ / (h n ) and a/duck/hokkaido/ / (h n ) (soda et al., ) . a/tokyo and b/tokyo strains were isolated at tokyo metropolitan institute of public health between and seasons by the use of madin-darby canine kidney cells (mdck cells) (see table ). the institutional review boards of the tokyo metropolitan institute of medical science and the tokyo metropolitan cancer and infectious diseases center, komagome hospital, approved the procedures for use of clinical samples. samples were collected from the pharyngeal mucosa of patients who had a diagnosis of influenza-like respiratory disease on the basis of signs and symptoms such as fever. viruses were detected using espline ® (fujirebio diagnostics incorporated., chuo-ku, tokyo, japan; http://www.fujirebio.co.jp), table reaction mixtures for shrt-pcr. immunochromatographic assays, and/or qrt-pcr (cfx tm real-time pcr detection system; promega, madison, wi, usa; http://www.promega.com) using the protocols of the centers for disease control and prevention (cdc) (who, a) . total viral rna was isolated from l virus-containing cell culture medium or l phosphate-buffered saline containing resuspended pharyngeal mucosal swabs using a qiaamp ® viral rna mini kit (qiagen, hilden, germany; http://www.qiagen.com/). viral rna standard was isolated from a plaque-forming unit (pfu) virus-containing cell culture medium as pfu standard rna. lower-standard rna ( to − pfu) was prepared from the pfu standard rna by serial dilution. viral rna was isolated from each virus subtype using a qiaamp viral rna mini kit (qiagen, hilden, germany; http://www.qiagen.com/). extracted rna was transcribed into cdna using a revertra ace kit (toyobo, osaka, japan http://www.toyobo.co.jp/e/) with the uni primer (agc aaa agc agg) and cloned into the pcr -topo vector (invitrogen, carlsbad, ca, usa; http://www.invitrogen.com/) containing t promoter. standard rna was transcribed using the t ribomax express large scale rna production system (promega, madison, wi, usa; http://www.promega.com/). rna concentrations were measured table reaction conditions for shrt-pcr. step shrt-pcr was conducted using a unique qrt-pcr testing unit: ur- mk iv (trust medical, hyogo, japan; http://www.trustmedical.jp/). methodological information is presented in fig. . two reaction mixture recipes were used for the shrt-pcr. the original reaction mixture includes rush tm dna polymerase (trust medical, hyogo, japan; http://www.trustmedical.jp/) and revertra ace (toyobo, osaka, japan; http://www.toyobo.co.jp/e/). the commercial reaction mixture was from the qrt-pcr kit, rna-direct tm sybr ® green real-time pcr master mix (toyobo, osaka, japan; http://www.toyobo.co.jp/e/). the dna polymerase (derived from the thermophilic bacteria thermus thermophiles) of the kit has reverse transcriptase activity. therefore, the enzyme enables both the reverse transcription and pcr steps. the details of these recipes are described in table . the reaction mixture ( l) and extracted rna ( l) were added to a cd-type sample container with sample wells. then, the container was sealed with a sheet of film and placed in the uk- mk iv. the reaction conditions and primers are described in tables and . between and , consensus viral gene sequences and conserved bases were identified from at least viral gene sequences according to the influenza virus database of the national center for biotechnology information. typing primers were designed to correspond to the conserved regions of the matrix gene segment of influenza a and b viruses. primers for the detection of s-oiv and h subtype viruses were designed to correspond to the conserved region of the ha gene segment for the s-oiv and h subtypes. in addition, subtyping primers were checked for mismatching consensus sequences of the ha gene segment of other subtypes (< % matching). the fragment sizes of shrt-pcr were - nucleotides. multiple sets of primers were tested on shrt-pcr, and the primers which have the best sensitivity for each gene were selected (table ). the goal of this study was to establish a highly sensitive, highspeed detection system for influenza viruses. shrt-pcr was used for detecting rna segments of influenza viruses. shrt-pcr is a unique qrt-pcr technique characterized by extremely short reaction times. information about the methodology of shrt-pcr is presented in fig. . shrt-pcr was performed on duplicates to generate and measure control rna by in vitro transcription. two types of rt-pcr enzyme mixture were used in the method: original mixture and rna-direct tm sybr ® green real-time pcr master mix. the original mixture containing rush tm polymerase, whose reaction speed is slightly high, was optimized for shrt-pcr; meanwhile, the rna-direct tm sybr ® green real-time pcr master mix is a commercial qrt-pcr kit that was taken into consideration because of the possibility of other enzymes that can be used for shrt-pcr. under this condition using rna-direct tm sybr ® green real-time pcr master mix, the limits of detection (lod) for the influenza a virus (a-mp), influenza b virus (b-mp), and s-oiv (ha gene) were copy/reaction ( fig. a, c, e) ; the lod for the h subtype (ha gene) was copies/reaction (fig. g) . lods using the original mixture were similar to these results (data not shown). all analyses were performed within - min. there was no marked difference in shrt-pcr results between the mixtures. rna-direct tm sybr ® green realtime pcr master mix was prepared as a premixed solution, meaning it has improved usability in clinical practice compared with the original mixture. thus, rna-direct tm sybr ® green real-time pcr master mix was selected for use in the following experiment. ( ) % ( of ) % ( of ) % ( of ) ( ) nd nd nd a and b negative ( ) % ( of ) % ( of ) % ( of ) laboratory strains a/wsn/ (h n ), a/pr/ / (h n ), a/aichi/ / (h n ), and b/mass/ / as well as other influenza viruses isolated at the tokyo metropolitan institute of public health were tested by the system. all viruses could be detected by the appropriate primer sets. typical results using rna-direct tm sybr ® green real-time pcr master mix are illustrated for a/wsn/ by a-mp (fig. b) , b/tokyo/ / by b-mp (fig. d) , and a/tokyo/ / (h n pdm) by the s-oiv ha gene (fig. f ) as well as a/duck/hokkaido/vac- / (h n ) by the h ha gene (fig. h) ; the lod for all samples was − pfu. the number of pfus was constantly - orders of magnitude less than the number of generated rnas. influenza a virus from a total of influenza viruses ( strains of seasonal h n , strains of seasonal h n , strains of s-oiv, and strains of virus b) isolated by the use of mdck cells in the - seasons at the tokyo metropolitan institute of public health (table ). the shrt-pcr system successfully identified % ( of ) of influenza a virus (i.e., seasonal h n , seasonal h n , and s-oiv), % ( of ) of s-oiv, and % ( of ) of influenza b virus samples. no cross reactions with the incorrect type or subtype of influenza virus were identified. shrt-pcr is a variant of quantitative real-time pcr. however, the system has only sample wells at present; thus, the maximum number of target samples with quantitative analysis is only ( for duplicated assay) because negative control wells and at least wells for standard rnas are necessary. thus, based on actual clinical diagnoses, qualitative assays were conducted to identify clinical samples. a cutoff value of copies was decided upon to avoid unpredicted nonspecific peaks from being misidentified as a positive signal. this is because the fluorescence peak for copies is completely separate from the nonspecific peaks of clinical samples. this cutoff value is also used in qrt-pcr in the cdc protocol. pharyngeal mucosal swabs from patients with suspected respiratory disease were tested by shrt-pcr, standard qrt-pcr using cdc protocols, and immunochromatography using espline ® . the results are presented in table . the results of immunochromatography and shrt-pcr matched exactly (table ) . although positive sample detected by immunochromatography and shrt-pcr was identified as a negative sample by qrt-pcr, there were . copies of rna, which is less than the -copy cutoff value; the shrt-pcr reaction curve for this sample was slightly sharper than that for the -copy cutoff value. these qrt-pcr measurements indicate a similar case for the detection of very few copies of the sample. clinical diagnostic tests for influenza viruses in outpatient departments or clinics are typically based on immunochromatographic detection of influenza virus antigens (chan et al., ) . the immunochromatographic assay is easy to use and provides immediate results; however, the lod (about - pfu/ml; bai et al., ; chan et al., ; miyagawa et al., ) is insufficient for detecting influenza in preclinical stages, i.e., in the absence of signs and symptoms. additionally, immunochromatography is based on an antigen-antibody reaction, implying that it is not suitable for detecting emerging or re-emerging influenza viruses that are precursors of epidemics or pandemics. in contrast to clinical diagnoses, general surveillance of influenza viruses is usually based on genetic analyses such as qrt-pcr he et al., ; who, a) . although qrt-pcr is both highly sensitive (about - copies/reaction) and specific, it is more time consuming (> h) than immunochromatographic techniques. however, genetic analyses are more flexible than antigen-antibody-based approaches because the assays can be tailored to fit emerging and re-emerging influenza viral rna on the basis of specific primers. in this study, an innovative qrt-pcr method is described with a greatly improved reaction speed for detecting and typing influenza viruses. shrt-pcr completes the analysis within an extremely short time (< min) compared with conventional qrt-pcr systems. in addition, the lods and specificities for detecting influenza viruses are equal to those of conventional methods (table ) . the shrt-pcr system detects the highly conserved sequence of the corresponding viral genome, and the newly designed primer sets targeted for typing mp segments do not exhibit any cross reactions among other influenza viruses (table ). the lods range from to copies/reaction (fig. ) , which was sufficiently sensitive for detecting influenza viruses in of clinical cases ( table ). the results indicate that shrt-pcr provides the potential to rapidly diagnose and detect infections. furthermore, shrt-pcr is expected to be more advantageous than regular immunochromatography or qrt-pcr, especially in the emerging stages of epidemics or pandemics. the who defines phases of the pandemic stage of a disease (who, b) . phase is defined as the verification of a community-level outbreak of an animal or reassortant virus and the implementation of a pandemic containment operation. because the time for political and clinical preparation for next phase directly depends on the speed of containment of infectious patients, quick detection of the influenza virus is essential for the success of the operation. at phase , shrt-pcr can play a critical role in diagnos- table comparison of shrt-pcr and currently existing detection methods. time/run sensitivity format shrt-pcr viral rna - min - copy or − pfu well/plate regular qrt-pcr viral rna - min - copy or well/plate immunochromatography viral protein min - pfu sample/strip ing infected patients at public health centers, hospitals, and public transportation hubs (e.g., airports). shrt-pcr can also be applied to detect other rapidly spreading pathogens such as the sars coronavirus (poon et al., ; who, ) and foot-and-mouth disease virus (oleksiewicz et al., ; reid et al., ) . rapid containment is critical for limiting the spread of these viruses, and containment depends on rapid and sensitive detection. despite its advantages of rapidity and sensitivity, shrt-pct is subject to certain limitations. the shrt-pcr system used in this study is limited to a sample capacity of . it is necessary for a quantitative assay to generate a standard curve with multiple defined amounts of samples; thus, shrt-pcr may be more useful for performing qualitative rather than quantitative assays. the limitation of the number of samples can be resolved by increasing the number of sample wells on the sample container and increasing the design capacity of the testing unit. for clinical applications for public health surveillance, shrt-pcr will be more useful than immunochromatography but less useful than qrt-pcr for influenza virus typing. this is because surveillance programs should be able to deal with large numbers of clinical samples. overall, shrt-pcr is a sufficiently powerful method to provide a basis for rapid pandemic containment at the who phase stage. improvement of a rapid diagnosis kit to detect either influenza a or b virus infections rapid semiautomated subtyping of influenza virus species during the swine origin influenza a h n virus epidemic in comparative analytical sensitivities of six rapid influenza a antigen detection test kits for detection of influenza a subtypes h n , h n and h n emergence of a novel swine-origin influenza a (h n ) virus in humans human infection with highly pathogenic h n influenza virus rapid multiplex reverse transcription-pcr typing of influenza a and b virus, and subtyping of influenza a vivo characterization of new swine-origin h n influenza viruses development of a novel rapid immunochromatographic test specific for the h influenza virus development of a novel real-time rt-pcr assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues early diagnosis of sars coronavirus infection by real time rt-pcr diagnosis of foot-and-mouth disease by real-time fluorogenic pcr assay triple-reassortant swine influenza a (h ) in humans in the united states development of vaccine strains of h and h influenza viruses simultaneous detection of influenza viruses a and b using real-time quantitative pcr h n influenza-continuing evolution and spread pcr primers for sars developed by who network laboratories cdc protocol of real-time rt-pcr for swine influenza a (h n ) who pandemic phase descriptions and main actions by phase influenza updates pandemic (h n ) -update acknowledgements this work was supported by grants from the new energy and industrial technology development organization and the japan society for the promotion of science. we are grateful to trust medical co. ltd. and members of the department of microbiology, tokyo metropolitan institute of public health, and the department of infectious disease, komagome hospital, for their technical assistance. we thank dr. kohara and his laboratory members for their scientific advice as well as all members of our laboratory for their advice and assistance. key: cord- -tnwfp je authors: revilla-fernández, sandra; wallner, barbara; truschner, klaus; benczak, alexandra; brem, gottfried; schmoll, friedrich; mueller, mathias; steinborn, ralf title: the use of endogenous and exogenous reference rnas for qualitative and quantitative detection of prrsv in porcine semen date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: tnwfp je semen is known to be a route of porcine reproductive and respiratory syndrome virus (prrsv) transmission. a method was developed for qualitative and quantitative detection of the seminal cell-associated prrsv rna in relation to endogenous and exogenous reference rnas. as endogenous control for one-step real-time reverse transcription (rt)-pcr ube d mrna was selected. particularly for the analysis of persistent infections associated with low copy numbers of prrsv rna, ube d mrna is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n = ). however, the amount of ube d mrna in porcine semen varied (up to -fold), thus its use is limited to qualitative detection of prrsv rna. for quantitation, a synthetic, non-metazoan rna was added to the rna isolation reaction at an exact copy number. the photosynthesis gene ribulose- , -bisphosphate carboxylase/oxygenase large subunit (rbcl) from arabidopsis thaliana was used as an exogenous spike. unexpectedly, prrsv rna was detected in a herd of specific pathogen-free (spf) boars which were tested elisa-negative for anti-prrsv antibodies. therefore, rt-pcr for seminal cell-associated prrsv is a powerful tool for managing the spf status during quarantine programs and for routine outbreak investigations. the porcine reproductive and respiratory syndrome virus (prrsv) belonging to the arteriviridae family is an enveloped single-stranded rna virus with a plus-sense genome which replicates via a -coterminal nested set of subgenomic mrnas (dea et al., ) . the prrsv- and prrsv- strains (formerly european-and north american-type prrsv, respectively; (drew, ) ) and the monophyletic lithuanian prrsv strains (plagemann, ; stadejek et al., ) can be differentiated. in semen, prrsv may be shed from the bulbourethral gland (christopher-hennings et al., ) and can be located in immature sperm cells (sur et al., ) or macrophages (christopher-hennings et al., ) . prrsv can be detected in semen as early as - days postinoculation (p.i.) (christopher-hennings et al., ; legeay et al., ) and can be transmitted by insemination (refs. in sur et al., ) . prrsv-infected boars show no significant clinical signs, and seroconversion and/or viremia is not correlated with shedding of virus in semen (wills et al., ) . while another arterivirus, equine arteri-tis virus, is shed in semen for at least - years, a prolonged carrier state (>day p.i.) in prrsv-infected boars has not been reported (christopher-hennings et al., ) . reference rnas (internal positive controls, internal control rnas) can be classified into exogenous (spike-in rna, rna extraction control, exogenous reference transcript) and endogenous controls. exogenous controls spiked at a defined copy number to the sample before rna isolation and endogenous controls can be applied for relative quantitation of viral rnas using real-time rt-pcr. therefore, reference rna expression should be comparable with that of the target and independent from the experimental conditions, e.g. the presence of a virus. both reference types serve as controls for sample transport and storage conditions, isolation performance, normalise for differences in total rna input or contribute to the detection of false negative results caused by sample-specific inhibitors. they were applied for monitoring the course of infection or intermittent shedding of viruses and for differentiation between a viremic or persistent stage of infection (moody et al., ; pasquier et al., ) . exogenous controls are used for testing of cell-free body fluids. their copy number can be controlled precisely and adjusted easily to the copy number of the gene of interest. endogenous controls are a cost efficient alternative for the detection of viral rnas in veterinary diagnostic work. artificial insemination or conventional sexual reproduction-mediated prrs virus transmission is of great importance for prrsv epidemic. for detection of prrsv in boar semen candidate endogenous reference genes were selected among common housekeeping genes and other genes found previously to be expressed uniformely in human and mouse tissues (hamalainen et al., ; warrington et al., ) . they were chosen from different functional categories based on the panther classification system (thomas et al., ) which reduces significantly the chance that genes might be co-regulated (vandesompele et al., ) . for the development of one-step real-time rt-pcr for four endogenous reference rnas (hprt, ube d , ppia, and hmbs) appropriate target regions were selected and the assay conditions were optimised for amplification efficiency. next, their expression levels and the amplification from genomic dna contamination were examined. the ube d mrna was found to be a suitable endogenous control for qualitative molecular diagnostics of prrsv rna in the cell-associated part of porcine semen. however, due to the variable expression of ube d mrna, an exogenous reference rna derived from a plant gene was developed for quantitative detection of the viral rna. the prrsv- reference sample used for assay validation was generated as follows. a -week-old piglet of a cross-bred (landrace × large white) tested negative for antibodies against prrsv was infected artificially with a . tcid of the spanish isolate olot/ (prrsv- , genbank accession number x ) and slaughtered at day p.i. lung tissue was shipped on dry ice and kept at − • c until rna isolation. lung tissues ( prrs viral rna-negative and prrsv- -or/and prrsv- -positive) for studying ube d mrna expression originated from the breeds large white, landrace, piétrain, large white × piétrain and (large white × landrace) × piétrain. ejaculates were derived from two boar herds (set a and b boars). the set a boars (n = ; piétrain, landrace, and large white breed) were - . years old and lived in the herd since - months. the boars of set b (n = ; piétrain) which joined the herd at the age of months were between and months old. both sets of boars were housed under specific pathogen-free conditions. this was guaranteed by the quarantine and testing program for incoming boars involving two serological tests with an interval of weeks against classical swine fever virus (csfv), pseudorabies virus (prv), swine influenza virus (siv), transmissible gastroenteritis virus (tgev), porcine respiratory coronavirus (prcv), chlamydophila, brucella, and leptospira species. after quarantine, the boars are tested for these pathogens once a year. according to the management program for the set a boars the absence of prrsv antibodies was required. this was achieved by testing routinely with the idexx (herd-chek) elisa (idexx laboratories, woerrstadt, germany) for which a sample-to-positive (s/p) ratio of ≥ . is considered positive. at the time of sampling the set a boars were elisa negative (− . to . ). in addition an immunoperoxidase monolayer assay (ipma (wensvoort et al., ) ) confirmed the prrs-negative serology. the set b boars showed a high positive serostatus when semen samples were taken (idexx elisa values: . - . for n = , and . - . for n = ) indicating a recent contact with prrsv. total rna from tissue samples and viral rna contained in the porcilis ® prrs vaccine (intervet, unterschleissheim, germany) were isolated with the trizol ® reagent and the trizol ® ls reagent (invitrogen, lofer, austria) according to the instructions of the manufacturer and finally dissolved in l diethyl pyrocarbonate (depc)-treated water. the qiaamp viral rna mini kit (qiagen, hilden, germany) was used to extract total rna from l serum samples and from the modified live prrsv vaccine (ingelvac ® prrs mlv; boehringer ingelheim pharma, ingelheim am rhein, germany) applying l elution buffer. the vaccine is a cell culture-adapted derivative of the pathogenic prrsv- isolate deposited at the american type culture collection under the number atcc-vr (genbank: u ). for seminal rna isolation ejaculated boar semen was collected into a sterile container with an integrated filter (us bag tm , minitube, tiefenbach bei landshut, germany) and was immediately stored at • c to guarantee that the non-sperm cells remained intact. the interval until further processing did not exceed eight hours. rna was isolated using the genelute tm mammalian total rna kit (sigma-aldrich, vienna, austria) with modifications. from a l-semen aliquot the cellular fraction was pelleted by centrifugation at × g for min. the pellet was lysed by vortexing for min in l lysis buffer containing mm -mercaptoethanol. the rna was eluted in a volume of l and stored at − • c. the plasmid p bsk (m.p. murtaugh, university of minnesota, usa) containing a kb fragment of the prrsv- reference strain vr and an arabidopsis thaliana cdna prepared as reported previously (karsai et al., ) were used as pcr templates. amplification was carried out with the primers clus-orf -f and clus-orf -r , and rbcl f and rbcl r (rbcl gene; genbank: atu ), respectively. pcr products were gel purified and subcloned into the pgem ® -t easy vector system i (promega, mannheim, germany). plasmids were purified by a modified alkaline lysis method, verified by dna sequencing, linearised by sali digestion and recovered by phenolchloroform extraction followed by ethanol precipitation. in vitro transcription reactions were performed in l containing g linearised plasmid, × transcription buffer with mm dtt, . mm of each rntp and u t rna polymerase (mbi fermentas, st. leon-rot, germany) for h at • c. subsequently, the reaction was incubated with u rnase-free dnase i (ambion, austin, usa) at • c for min. in vitro transcripts were recovered by phenolchloroform extraction and ethanol precipitation and quantitated by spectrophotometry. the housekeeping genes hprt (genbank: af ), ube d (ubch b), ppia (cypa; genbank: ay ), and hmbs (pbgd) were selected as candidate endogenous reference mrnas considering previous reports (foss et al., ; hamalainen et al., ; steele et al., ) and the fact that in contrast to hprt, ube d and ppia there is no pseudogene for hmbs in the human genome ( (zhang et al., ) ; www.pseudogene.org). human pcr primer sequences targeting conserved regions were selected in a human/mouse-nucleotide alignment (genbank: u and m ) to determine partial porcine ube d and hmbs cdna sequences and subsequently to select primers and probes for real-time rt-pcr of ube d and hmbs. in addition to pcr product sequencing the partial porcine hmbs cdna was determined by sequence anal-ysis of subclones derived from tissue cdnas (data not shown). the amplification of cdna for pcr product sequencing was carried out in a l-reaction volume containing l of the reverse transcription reaction, × buffer, mm magnesium, . mm of each dntp, nm of each primer and u taq dna polymerase. following denaturation at • c for min, targets were amplified by cycles at • c for s, • c for s and • c for min. for sequencing the abi prism bigdye tm terminator cycle sequencing chemistry and the abi prism sequencer (applied biosystems, vienna, austria) were used. one-step quantitative real-time rt-pcr was carried out with a two-enzyme system. separate reverse transcription and dna polymerisation reactions allow testing for dna contamination by including a "minus rt" control, thus assaying for amplification from processed and non-processed pseudogenes. the two-enzyme system used (taqman ® one-step rt-pcr master mix reagents kit, applied biosystems) contains the moloney murine leukemia virus reverse transcriptase and amplitaq gold ® dna polymerase. a volume of l rna per l reaction and primer/probe concentrations of nm/ nm and nm/ nm were applied for the viral and the reference rna assays, respectively. total rna was reverse transcribed for min at • c. the hot start dna polymerase was activated by incubation at • c for min followed by amplification for cycles at • c for s and • c for min. fluorescence "real-time" measurements recorded by either the abi prism ® or ht sequence detection systems (applied biosystems) were transformed into c t values using the sds software versions . . and . , respectively. dilution series of standard rnas were made to characterise the linearity, precision, specificity, and sensitivity of the quantitation assays. standard dilutions were prepared in depc-treated water containing ng/l yeast trna carrier (invitrogen) and were amplified at least in duplicates. the c t values were plotted either against the log of the target copy number or against the log of the input rna mass to generate a regression curve with the formula y = sx + b (s, slope; b, intercept) and to determine the reaction efficiency e = − /s − . as internal quality parameter for a standard curve a regression coefficient (r ) of > . was used. optimal efficiency was concluded if e ≥ . (s < − . ). to identify conserved sites for primer and probe binding in the orf gene coding for the most conserved structural protein (m proteins of prrsv- and - show amino acid sequence identity of - % (dea et al., ) ) the following sequences were aligned using the sequence navigator software (applied biosystems): (i) seven prrsv- sequences: m , aj , x , l , af , af , af , and (ii) prrsv- sequences: u , af , af , aj , l , u , af , af , u -u , z , af , l -l , u , u -u , ab , d , af , af , af , af , af , af , and aj -aj . primers (invitrogen), taqman probes (mwg biotech, ebersberg, germany) and taqman minor groove binder (mgb) probes (applied biosystems, weiterstadt, germany) were designed using the primer express tm . software (applied biosystems, vienna, austria) and are given below as - sequence. standard taqman probes were labeled with the fluorescent quencher dye -carboxytetramethylrhodamine (tamra) and with the reporter dyes -carboxyfluorescein ( fam) or -carboxy- , , , -tetra-chlorofluorescein (tet). in the taqman mgb probe format a non-fluorescent quencher dye (nfq) and a minor groove binder (kutyavin et al., ) were applied. the complete orf and the partial orf sequences were determined by pcr product sequencing using the primer pairs eu - f (cctcgaaggggttaaagctca) and eu - r (cacgaggctccgaagtcct), and eu - f (gtagaaagtgctgcaggtctcca) and eu - r (gcactgtatgagcaaccgg), respectively. in addition, the primers cleu - f (gctcaacccttgacgag-gact), cleu - r (gctctggtttttaccggcc), clus-orf -f (ataaccagagtttcagcggaaca), clus-orf -r (tctcttctgctgcttgccgt), rbcl f (cttgaaggagacagggagtcaact) and rbcl r (catgcttccagagctactcgg) were applied. to determine the partial porcine sequences for the hmbs housekeeping version and for ube d , the primers hshmbs-f (atgtctggtaacggc, exon ), hshmbs-r (gggtac-gaggctttc, exon ) and hube d -seq f (tgaaga-gaatccacaaggaattga, exon ) and hube d -seq r (ccgagcaatctcaggcactaaa, exon ) which were deduced from the homologous human genes (genbank: nm and u ) were used. primers and probes for the endogenous control assays for ppia and hprt developed on the basis of known porcine sequences (genbank: ay and af ) were pcyc- f (gccgcgtctccttcgag), pcyc- r (gcaggaac-ctttataaccaaatcct) and pcyc-vic/mgb (vic-cagaaaacttccgtgctc-nqf-mgb), and phprt- f (tcatggactaattatggacaggactg), phprt- r (tttatatcgcccgttgactggt) and phprt-tet/tamra (tet-tgggaggccatcacatcgtagcc-tamra). oligonucleotides for quantitation of porcine hmbs and ube d mrnas based on sequences determined in this study were phmbs- f (cgcaacg-gcggaagaaa), phmbs- r (ttcagcgttgccac-cacac) and phmbs- fam/tamra ( fam-cca-gctggcccgcatacaaacg-tamra); pube d - f (aggtcctgttggagatgatatgtt), pube d - r (ttgggaaatgaattgtcaagaaa) and pube- d - fam/tamra ( fam-ccaaatgacagtccc-tatcagggtgga-tamra). real-time rt-pcr for prrsv- targeted the orf gene with the primers eu - f and eu - r (see above) and the taqman mgb probe eu -mgb ( fam-ctgtgagaaagcccggac-nfq-mgb). the orf sequence of prrsv- was quantified with the primers us - f (tccagatgccgtttgtgctt) us - r (gacgccggacgacaaatg) and the probe us -mgb ( fam-ccctgcccaccacgt-nfq-mgb). both probe binding sites were completely homologous among the respective prrsv sequences listed above. the primers rbcl f (see above) and rbcl r (taccgcggcttcgatctt) were applied in combination with the taqman probe rbcl tet (tet-tcgcgcagtaaatcaacaaagccca-tamra) to quantitate the exogenous rna standard. if possible primer and probe sequences for real-time pcr were selected to target regions with no obvious secondary structure using the rna and dna folding server (www.bioinfo.rpi.edu/∼zukerm/). a significant amplification from non-processed and processed pseudogenes of endogenous reference genes during real-time rt-pcr was prevented by primer annealing across an exon boundary (ube d and hprt assays) and by primers flanking a large intron (hmbs and ppia assays). the porcine exon-intron structure was concluded from the homologous human and mouse genes (batzoglou et al., ) . we also considered that the human and the mouse genomes contain a processed pseudogene for ube d , but not for hmbs (www.pseudogene.org). the qualitative detection of the endogenous reference ube d mrna in prrsv-infected and non-infected pigs was evaluated by the student's t-test and by the nonparametric wilcoxon rank-sum (mann-whitney) test for two independent samples included in the statistical analysis software spss for windows . . (spss inc., chicago, usa). the spearman's rank correlation test was used to analyse the relationship between ube d expression in semen rnas and the sperm cell concentration. the (porcine) ube d real-time rt-pcr assay was submitted to the real-time pcr primer and probe database (rtprimerdb id: ; (pattyn et al., ) ) and can be used for detection of human ube d mrna due to complete primer/probe homology. sequences determined within this study were submitted to the genbank under the following accession numbers: af (olot/ -g a), af (porcilis prrs vaccine), ay (hmbs), ay (ube d ), af (hprt variant) and af (ppia-like pseudogene). the sequence of the austrian prrsv- isolate ags- (af ) which mismatched in the center of the orf probe binding site targeted by a previous taqman rt-pcr assay (egli et al., ) was also submitted. endogenous control genes were designated according to the homologous human genes using locuslink (www.ncbi.nlm.nih.gov). the dynamic ranges and the amplication efficiencies of the real-time rt-pcr assays for hprt, ube d , ppia, and hmbs were analysed by generating standard curves using lung and lymphoid tissue rnas (fig. a , and data not shown). the equations for the regression lines illustrate that the reaction efficiencies for the endogenous control assays (e > . , i.e. optimal) were comparable to those of the viral assays ( e < %). a dnase i treatment step to exclude cross-amplification from genomic dna was made redundant by assay design/selection (see section ). using porcine tissue rnas we demonstrated that amplification from any contaminating dna was not significant ( c t for mrna and the corresponding minus rt control > ). for rna quantitation in semen specimens ube d and hmbs were analysed in more detail since in semen the differences between the c t value for ppia or hprt and the corresponding minus rt sample controling for genomic dna contamination were small (data not shown). the ube d and hmbs expression was analysed in native semen samples derived from two sets of boars (a, b) with negative and high positive prrsv serostatus. independently of the prrsv serostatus or presence of low prrsv rna copy numbers (see below), the c t values obtained for hmbs mrna were at or near the detection limit (table ; semen rnas for set b boars were not analysed), whereas the ube d mrna was detected in all ejaculates of the individuals analysed (table and data not shown). no correlation between ube d expression and the sperm cell num-ber was found (spearman correlation coefficient r = − . , p = . ; n = in table ). the ube d expression varied from . -fold (set b boars, n = , c t > for prrsv rna, positive prrsv serostatus), over -fold (set a boars, n = , seronegative for prrsv) to -fold (set b boars, n = , only one semen rna with detectable prrsv- orf copy numbers, c t > for prrsv- rna, high positive prrsv serostatus). to prove that ube d would be invariant in the primer and probe binding sites among different individuals of common pig breeds random porcine cdnas (n = ) were sequenced. complete homology in the binding sites was found. moreover, human and pig ube d nucleotide sequences were completely homologous in the primer/probe binding sites. since in semen the variation of ube d expression exceeded the high stringency criteria applied in gene expression studies (variation < five-fold (dheda et al., ) ), we developed a non-metazoan exogenous control rna spike. this reference rna is based on the chloroplast-encoded gene for the large subunit of ribulose- , -bisphosphate carboxylase/oxygenase (rbcl) from a. thaliana (clegg, ) . the standard curve obtained for the in vitro transcript documents optimal amplification efficiency (fig. b) . spiking the cellular fraction of a semen rna sample with , copies of the rbcl in vitro transcript, c t values of . and . were obtained. a silica membrane-based kit was selected to standardise isolation for quantitation of prrsv rna in semen specimens. different semen volumes and different sperm cell numbers were tested with a constant amount of absorbent material ( mm silica column) to determine the lowest c t value for a target mrna (ube d mrna) by one-step realtime rt-pcr. cell numbers of the twelve experimental ejaculates ranged from . to . × cells/ml. duplicate c t measurements for the , , and l semen aliquots ranged between . ± . and c t > (no amplification). the lowest c t values were obtained for the l and/or the l semen aliquots (c t s: . ± . to . ± . , and . ± . to c t > ). no correlation was observed between c t value and the number of spermatozoa. the successful removal of inhibitors for real-time rt-pcr was obtained since one-step real-time rt-pcr yielded optimal amplification efficiency (e > . for ube d mrna, prrsv- and - rnas; fig. a ). one-step real-time rt-pcr assays for prrsv- and - rna allowed quantitation with optimal efficiency (fig. a ; standard curves for two additional viremic pigs infected with prrsv- (data not shown)) as achieved for endogenous and fig. . one-step real-time rt-pcr yields optimal amplification efficiency for quantitation of prrsv rnas, endogenous (a) and exogenous reference rnas (b). c t > for minus rt controls of ube d and hmbs mrnas. prrsv- infection was mimicked by spiking virus-free semen with the ingelvac ® prrs mlv vaccine. exogenous reference rna assays ( e < %, fig. and see above) . since a variation in the detection limits for the different prrsv strains due to snps within the amplicon (barnard et al., ) or in the primer binding sites cannot be excluded, the sensitivity of the assay for a single prrsv reference strain (atcc-vr , prrsv- ) was determined. for this purpose, prrsv rna-free seminal cell fraction-derived rna (see below) was spiked with known copy numbers of the prrsv- orf in vitro transcript (serial four-fold dilutions). the resulting detection limit for the analysis of prrsv- rna in the cellular fraction of semen was orf rna copies. the specificity of the real-time rt-pcr assays for prrsv- and - was examined by using viral rna from the opposite prrsv type as a non-amplification control. viral rna contained in the porcilis prrs vaccine (prrsv- ) and the ingelvac prrs mlv vaccine (prrsv- ) was purified and used at a concentration of . × orf copies/reaction as target and non-target copy numbers (and vice versa) in the quantitation assays. these copy numbers exceeded several-fold the copy numbers found in clinical samples of serum and sperm (data not shown). the real-time rt-pcr quantitation assays (e > . ) did not detect any non-target amplification demonstrating absence of cross-amplification between the two prrsv types (data not shown). the absence of any cross-amplification with samples tested positive for classical swine fever virus (csfv), transmissible gastroenteritis virus (tgev), porcine parvovirus (ppv), pseudorabies virus (prv) and porcine circovirus type ii (pcv ) was also confirmed (data not shown). using the amplicon sequences of the prrsv real-time rt-pcr assays as query in a nucleotide-nucleotide blast (blastn) the specificity of the prrsv primer and probe sequences was demonstrated. the prrsv- and - quantitation assays were used for examination of semen samples derived from two sets of boars with negative (a) and high positive (b) prrsv serostatus (see section ). unexpectedly, semen samples with prrsv- rna copy numbers near the detection limit were detected in both sets (table and data not shown). four of the twelve sera of the set a boars were positive for prrsv- rna but negative for prrsv- rna (table ; serum rnas for set b boars were not analysed). a method was developed for qualitative and quantitative detection of the seminal cell-associated prrsv rna in relation to endogenous (ube d mrna) and exogenous (rbcl rna) reference rnas. neither rrna expression or total rna amount (reviewed by bustin, ) are applicable for normalisation of relative quantitation of prrs viral rna in the seminal cell fraction due to the large difference in expression between viral and rrna genes during persistent infections and the limited practicability of spectrophotometric analysis for routine diagnostic application. ube d mrna is an appropriate control for routine qualitative detection of prrsv rna due to its reliable and low expression in all semen samples tested. preliminary data indicate similar expression of ube d mrna in animals with prrs viral rna-positive or negative status (c t s for tissue samples: . ± . and . ± . , respectively). however, we cannot exclude that for example a selection of the individuals in the two groups based on age, breed, stage of infection or a study design testing in the same animal under the same conditions with and without prrsv would reveal differential expression of ube d depending from the status of prrsv infection. the c t values determined for ube d mrna in semen were comparable to those determined for samples with low viral rna copy numbers which is especially important for reliable virus quantitation during persistent infections. the reasons for the varying c t values in the ube d assay ( c t = . , table ) were not addressed and might involve differences in the rna quality among the samples, in the number and/or the expression level of ube d mrna expressing cells. summarising, the validation of an endogenous reference which would be a cost-efficient alternative for quantitative detection of prrsv still remains a challenge due to the heterogeneous cell population of semen and its varying cell number. the real-time rt-pcr assays for hprt, ppia, hmbs and ube d developed in this study will be useful for future gene expression studies in the pig. due to the occurrence of processed pseudogenes (or intron-less paralogs), housekeeping gene mrnas used in a previous study as endogenous references for human semen samples required the application of dnase i (juusola and ballantyne, ) . this pretreatment step is assumed to impair rna quality and increases assay-related costs and time. here we show that it is possible by a careful primer and probe design to prevent significant amplification from genomic dna without a dnase i digest. quantitation of viral rna relative to a reference rna requires knowledge of the exact copy number contained in this control. due to the considerable variation (or complete absence in the case of hmbs mrna in two samples) in expression in porcine semen rna among the candidate reference genes studied we replaced the endogenous control by an exogenous control rna. it was selected to avoid crosshybridisation with rnas from the organism studied. in the present study the photosynthesis gene rbcl from a. thaliana (clegg, ) was used. artificial sequences or sequences from other organisms are also possible as exogenous controls (baker and o'shaughnessy, ; smith et al., ) . in contrast to a homologous exogenous rna (mimic) which has the same primer but different probe binding sites as the target rna (westcott et al., ) , the use of a heterologous exogenous reference can be applied easily for detection of different target rnas (for review see freeman et al., ) . real-time rt-pcr assays for detection of viral rna in the plasma fraction of semen have been reported (balasuriya et al., ; westcott et al., ) . prrsv appears most consistently in the cellular fraction of semen and not in whole semen or seminal plasma (christopher-hennings et al., . we therefore developed a quantitation assay for seminal-cell associated prrsv rna removing the seminal plasma which has been noted to contain pcr inhibitors (refs. in bourlet et al., ) . the real-time rt-pcr assays for the endogenous and exogenous references as well as for prrsv- and - yielded optimal efficiency (e = . to . ). this allows the relative quantitation of viral rna by the c t -method (ref. in pfaffl, ) avoiding the elaborate amplification of standards in parallel. the detection limit of the prrsv real-time rt-pcr was determined for prrsv- only. future assay validation should involve (i) sequence analysis of the assay target region for new isolates, (ii) the use of primers and/or probes which are degenerated at defined nucleotide positions in order to maximise the detection of all known strains and isolates, (iii) determination of efficiency and sensitivity for isolates varying in the primer and/or probe target sites, and (iv) spiking of quantitation standards which represent these polymorphic isolates into an adequate number of semen samples before rna extraction. the occurrence of viral rna in serum and semen (set a boars in table ) of seronegative boars housed under spf conditions was unexpectedly demonstrated. the high c t values reflect low copy numbers, thus virus isolation would not be expected to be successful (wills et al., ) . the occurrence of seroconversion is highly unlikely due to these low c t s, considering the routine prrs antibody testing carried out in the spf boar stud and the fact that eight of these boars were elisa-and ipma-negative days after showing low c t s for prrs viral rna in semen (superscript b in table ). the sporadic detection of viral rna in serum after a period of negative testing was reported, and pigs which have returned to seronegative status based on elisa may still harbor infectious prrsv (batista et al., ; martelli et al., ; wills et al., ) . the serological status is also not an adequate indicator of prrsv and prrsv rna shedding in the semen (christopher-hennings et al., ) . this underlines the need to complement immunoassays with more sensitive nucleic acid-based methods for a comprehensive approach especially for the characterisation of persistent prrsv infections (batista et al., ) , e.g. during the quarantine and testing program for incoming boars to reduce later conflicts in the interpretation of the prrsv status. it is possible that our results obtained for the boars of set a, retrospectively, document only a contact between the animals and the virus before joining the herd, 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phosphoribosyltransferase, glyceraldehyde- -phosphate dehydrogenase and beta-actin mrna expression in porcine immune cells and tissues quantitative rt-pcr: pitfalls and potential identification and validation of endogenous reference genes for expression profiling of t helper cell differentiation by quantitative real-time rt-pcr messenger rna profiling: a prototype method to supplant conventional methods for body fluid identification evaluation of a homemade sybr green i reaction mixture for real-time pcr quantification of gene expression -minor groove binder-dna probes increase sequence specificity at pcr extension temperatures development of a rt-pcr test coupled with a microplate colorimetric assay for the detection of a swine arterivirus (prrsv) in boar semen intermittent shedding of prrsv in semen of seronegative boars measuring infectious bursal disease virus rna in blood by multiplex real-time quantitative rt-pcr intermittent detection of hepatitis c virus (hcv) in semen from men with human immunodeficiency virus type (hiv- ) and hcv rt-primerdb: the real-time pcr primer and probe database a new mathematical model for relative quantification in real-time rt-pcr porcine reproductive and respiratory syndrome virus: origin hypothesis exogenous reference rna for normalization of real-time quantitative pcr identification of radically different variants of porcine reproductive and respiratory syndrome virus in eastern europe: towards a common ancestor for european and american viruses variable expression of some "housekeeping" genes during human keratinocyte differentiation porcine reproductive and respiratory syndrome virus replicates in testicular germ cells, alters spermatogenesis, and induces germ cell death by apoptosis panther: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes comparison of human adult and fetal expression and identification of housekeeping/maintenance genes mystery swine disease in the netherlands: the isolation of lelystad virus use of an internal standard in a closed one-tube rt-pcr for the detection of equine arteritis virus rna with fluorescent probes duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus comparative analysis of processed pseudogenes in the mouse and human genomes we thank juan plana-duran for the infected lung, michael p. murtaugh for the gift of a plasmid containing vr prrsv cdna, marie-theres hauser for providing the a. thaliana cdna, irene sommerfeld-stur for help with statistical data analysis, birgit strobl for valuable discussion, and hanno schreiber, elisabeth hofmann, arnulf hertweck, georg walcher, kurt höller, and wolfgang sipos for assistance. we acknowledge the veterinary diagnostic laboratory labovet and the austrian agency for health and food safety for performing the elisa and the ipma tests. this work was supported by a grant of the university of veterinary medicine vienna to r.s. while s.r.-f. and g.b. declare their commercial interest in prrsv molecular diagnostics, the remaining authors have no competing financial interests. key: cord- - c vue y authors: greene, shermalyn r; moe, christine l; jaykus, lee-ann; cronin, mike; grosso, lynell; aarle, pierre van title: evaluation of the nuclisens® basic kit assay for detection of norwalk virus rna in stool specimens date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: c vue y norwalk-like viruses (nlvs) are a genetically diverse group of human caliciviruses that are the most common cause of epidemic gastroenteritis and are detected typically in stool by reverse transcription (rt)-pcr or electron microscopy (em). the application of a rapid nucleic acid sequence-based amplification (nasba) assay for the detection of nlv rna in stool is described using the nuclisens® basic kit. primers and probes for the nlv basic kit assay were based on the rna polymerase region of the prototype nlv, norwalk virus (nv) genome and could consistently detect ( ) rt-pcr detectable units of nv rna in a stool filtrate. when compared directly with rt-pcr on a dilution series of nv stool filtrate, the nuclisens® basic kit assay was equally sensitive. cross-reactivity studies with a representative panel of other enteric pathogens were negative. when applied to stool specimens from nv-challenged volunteers, the nasba basic kit application for nv detection yielded % sensitivity, % specificity, and % concordance, using rt-pcr as the ‘gold standard’. despite the specificity of the nasba primer/probe sequences for nv, other representatives from both nlv genogroups i and ii could be detected by the basic kit assay in outbreak stool specimens, although the results were inconsistent. our results suggest that the nuclisens® basic kit assay provides a rapid and sensitive alternative to rt-pcr for detecting nv rna in stool specimens. however, improvements in test specificity and primer design will be needed before the assay can be used routinely in the clinical setting. norwalk-like viruses (nlvs), also called 'noroviruses', is a genetically diverse genus that is classified in the family caliciviridae (green et al., ) . these viruses contain a positive-sense, single stranded rna genome of approximately . kb. based on sequence information for the genes encoding the viral rna-dependent rna polymerase and the capsid protein, the nlv genus is sub-divided further into two genogroups: genogroup i with norwalk virus (nv) as the prototype strain, and genogroup ii with snow mountain and hawaii virus as prototype strains. nlvs have been implicated in % of the outbreaks of acute nonbacterial gastroenteritis in the us documented by the centers for disease control and prevention (cdc) between and (fankhauser et al., ) . additionally, nlvs are a major cause of acute nonbacterial gastroenteritis in children and adults worldwide (bon et al., ; parks et al., ; taylor et al., ; vinje et al., ) . nlv outbreaks have been reported in communities, schools and day care centers, nursing homes, hospitals, recreational areas, cruise ships, military ships and bases (fankhauser et al., ; grohmann et al., ; mccarthy et al., ; sharp et al., ) . these viruses replicate in the gastrointestinal tract of the infected host and are excreted in feces. although food-borne transmission is most common, these viruses are also transmitted through fecal contamination of drinking and recreational water, as well as through contact with contaminated environmental surfaces and infected individuals. despite numerous attempts, nlv infections have not been induced in experimental animals, nor have these viruses been propagated successfully in cell culture. diagnosis has relied historically on the observation of the nm viral particles by electron microscopy (em) (glass et al., ) . however, because nlvs are shed usually in low concentrations, em is frequently not sensitive enough to detect these viruses in stool. early diagnostic tests for nv, developed in the 's and 's, were primarily immunoassays that required reagents derived from nv-infected human volunteers and were not widely available (atmar and estes, ) . the cloning and sequencing of nv in (jiang et al., ) led to the development of new molecular diagnostic assays. currently, the detection method used most widely for nlvs in stool samples is reverse transcription (rt)-pcr. the resulting rt-pcr amplicons are confirmed by probe hybridization or nucleotide sequence analysis. rt-pcr has proven to be an extremely sensitive method for detection of nlvs. however, this method has several drawbacks: the presence of amplification inhibitors in stool samples, the need to confirm amplification products to prevent false positive results, and the amount of time needed to complete the analysis. rapid identification of pathogens is important for patient care and for prompt, appropriate interventions to control outbreaks of disease. studies have demonstrated that the isothermal nucleic acid sequence-based amplification (nasba) method provides a sensitive and rapid method for detection of viral rna from human immunodeficiency virus type (hiv- ) (berndt et al., ; fiscus et al., ; segondy et al., ; van gemen et al., ) , epstein-barr virus (ebv) (brink et al., ; cruz et al., ) , human cytomegalovirus (hcmv) (blok et al., ; witt et al., ) , human rhinovirus and enterovirus (damen et al., ; lunel et al., ) . this rna amplification method utilizes three enzymes (reverse transcriptase, t rna polymerase, and rnaseh) and two target specific oligonucleotide primers. the reverse oligonucleotide primer contains the bacteriophage t promoter sequence, while the forward oligonucleotide primer contains an electrochemiluminescent (ecl) tail that is used in the detection of the amplification product. the transcription-driven nasba reaction amplifies theoretically the rna target more than -fold within min. in this study, we report the application of a rapid nasba assay for the detection of nv rna in stool using the nuclisens † basic kit. this assay was compared directly with rt-pcr and used for the detection of nv and other nlvs in fecal specimens obtained from human challenge studies and nlv outbreaks. the nasba assay described here was optimized for the detection of nv, and these nasba primers and probes were not designed to be reactive broadly for other nlv. the nv stock preparation used in this study was made from a % suspension in pbs of a liquid stool from a volunteer infected with the nv fiia inoculum (dolin et al., ) . the stool suspension was trichlorotrifluoroethane (freon)-extracted three times then filtered through a . mm filter. the first and second filtrates were pooled, aliquoted into ml portions, and stored at (/ c. virus concentration was estimated by endpoint titration rt-pcr using the heat release method (schwab et al., ) and oligonucleotide primers based on the sequence of the nv polymerase gene (nv /nv ) (moe et al., ) . estimates of nv concentration in the stock preparation ranged from )/ to )/ pcr detectable units (pdu) per ml with a mean of )/ pdu/ml. poliovirus type (pv ), strain lsc, was propagated in bgmk (african green-monkey kidney derived) cells and titered using the plaque technique (sobsey, ) . hepatitis a virus (hav, cytopathic strain hm- ) was grown and plaque assayed for enumeration using frhk- (fetal rhesus monkey kidney-derived) cells (cromeans et al., ) . other viruses in the test panel included adenovirus (ad ), astrovirus (ast ), human rotavirus (hrv ) (gifts from mark sobsey, department of environmental science and engineering, unc-ch) and coronavirus (mhv-a ) (gift from ralph baric, department of epidemiology, unc-ch). gram negative bacterial strains, including escherichia coli atcc and e. coli o : h (hc ), were cultivated overnight at c in brain heart infusion (bhi) broth (difco, detroit, mi). clostridium perfringens (nctc ) was cultivated in fluid thioglycollate (ftg) medium (difco) for h at c. aliquots of stool specimens collected from a nv human challenge study (moe et al., ) were prepared as Á/ % suspensions in sterile water and stored at c prior to use. fecal specimens collected from several nlv outbreaks were prepared in a similar manner. . . viral rna isolation from the nv stock preparation and stool specimens . . . viral rna isolation for rt-pcr assay stool specimens from the nv human challenge study and the nlv outbreaks were tested by rt-pcr using a modified heat release method (schwab et al., ) or the ultraspec (biotecx, houston, tx) rna extraction method according to the manufacturer's directions. nv stock preparation containing )/ pdu/ml was serially diluted -fold in the nuclisens † basic kit lysis buffer (biomérieux inc., durham, nc), and the rna was extracted as per manufacturer's instructions (extraction method based on that of boom et al., ) . briefly, the nv stock-lysis buffer mixture was mixed with activated silica, the bound viral nucleic acid was washed once with ml wash buffer [ m guscu, mm tris Á/hcl (ph . )], twice with ml % ethanol, and once with ml acetone. after each wash step, the suspension was briefly centrifuged, and the silica pellet was suspended in the next wash solution. following a drying step at c for min, the rna was eluted from the silica by incubation in ml of nuclisens † basic kit (biomérieux) elution buffer for min at c. purified rna was stored at (/ c prior to use in amplification reactions. for the outbreak and challenge study stool specimens, a % stool suspension (rather than %) was prepared in sterile nuclease-free water and the samples were pre-treated for removal of inhibitors by centrifugation at )/g for min. an additional ethanol wash was incorporated after the buffer wash step to facilitate removal of residual amplification inhibitors. rt-pcr on a dilution series of nv stock preparation was carried out using ml of the purified viral rna and the oligonucleotide primer pair nv /nv , that produces a -bp amplicon corresponding to nucleotides Á/ within the rna polymerase gene of nv (moe et al., ) . reactions were carried out with the rt-pcr access system kit (promega corporation, madison, wi) according to manufacturer's protocol and as previously reported (moe et al., ) . the stool specimens from the human challenge studies and the nlv outbreaks were tested by rt-pcr using the g (sr /sr ) and g primers, that produce a -bp product between nucleotides Á/ , also within the rna polymerase gene (ando et al., ) . the g and g primers are more broadly reactive than the nv / nv pair and were routinely used to screen volunteer and outbreak specimens for the presence of any nlv infection. oligonucleotide primers were prepared by the pathology oligonucleotide facility at the university of north carolina at chapel hill. briefly, rt was done at c for min. after heat inactivation of the reverse transcriptase at c for min, the samples were subjected to cycles of c for min, c for . min s, and c for min followed by a final extension at c for min. the resulting dna amplicons were electrophoresed on a % agarose gel and stained with ethidium bromide. electrophoresed rt-pcr amplicons were transferred to hybond-n ' (pharmacia amersham biotech, piscastaway, nj). hybridization was performed at c with the fluorescein- -dutp labeled ( ? oligo labeling and detection kit, pharmacia amersham biotech) using the nvint oligonucleotide probe. rt-pcr amplicons were sequenced as described previously (moe et al., ) . primer and probe sequences are listed in table . nasba oligonucleotide primers (bkp . /bkp . ) corresponded to the nv /nv pair and amplified a bp region of the nv rna polymerase gene (accession number ). these primers were designed and labeled with the t rna polymerase promoter and ecl detection sequences, respectively, through cooperation with biomérieux (table ) . nasba reactions used ml of purified viral rna, a mm primer concentration, and mm kcl, with reactions performed according to the nuclisens † basic kit instructions. briefly, ml of the premix cocktail was dispensed into thin-walled eppendorf tubes and ml of nucleic acid eluate was added to each tube. the tubes were incubated at c for min followed by cooling to c for min. a -ml aliquot of enzyme mix (rna polymerase, rnase h, and amv-reverse transcriptase) was added to each tube followed by incubation for min at c (in water bath or thermocycler). ecl detection was done immediately after amplification; alternatively, the amplified rna was stored at (/ c (for no more than days) prior to ecl detection. the nasba-generated products were detected by liquid hybridization using the electrochemiluminescence (ecl) principle employed by the nuclisens † reader. this method uses two oligonucleotides i.e. (i) a specific capture oligonucleotide immobilized onto paramagnetic beads; and (ii) a second detector oligonucleotide complexed to a ruthenium chelate. the specific capture probe used in this assay corresponded to the same nvint probe used in rt-pcr hybridizations, except that it was ? biotinylated during manufacture to facilitate its complex to streptavidin-coated magnetic beads. the detector probe sequence is generic, provided by the nuclisens † basic kit, and complementary to the ? terminal end of the amplicons which are transcribed from the stretch of nucleotides identified for primer bkp . (see table ). amplification products ( ml) were diluted in ml of detection buffer provided with the nuclisens † basic kit. hybridization was carried out using ml of a : mixture of streptavidin-coated capture probes coupled to magnetic beads and ru 'labeled oligonucleotide detection probes, and ml of the : diluted amplification product. the solution was incubated at c for min after which assay buffer ( ml) was added to the tubes followed by detection using the nuclisens † ecl reader (biomérieux). after incubation, the paramagnetic beads carrying the hybridized amplicon/ecl probe complexes were magnetically captured on the surface of an electrode that is part of the ecl reader. voltage applied to the electrode triggers the ecl reaction, such that the light emitted by the hybridized ru ' -labeled probes is proportional to the quantity of amplicon present. the ecl reader detects this luminescent energy and converts it to a digital read-out. the reader further assigns each specimen a positive or negative outcome based on the luminescent signal of the specimen relative to the positive and negative sample controls, and performance controls provided by the manufacturer. ecl results were transformed to log values for subsequent analyses using microsoft excel (redmond city, wa). rt-pcr was done using ml of nasba generated amplification product and the oligonucleotide primer pair nv /nv (moe et al., ) . reactions were performed as described above. the resulting dna amplicons were electrophoresed on a % agarose gel and visualized with ethidium bromide. for sequence analysis, the resulting bp rt-pcr products were purified (qiagen pcr, qiagen valencia ca) and the nucleotide sequences were determined at the university of north carolina at chapel hill automated sequencing facility using primers nv and nv . nucleotide sequence analyses were done using the wisconsin genetic computer group (gcg) software and basic local alignment search tool (blast) analysis (altschul et al., ) . to evaluate the limit of detection and the reproducibility of the basic kit assay for detection of nv rna, serial -fold dilutions were made of the nv stock preparation (corresponding to ( to pdu/ml) and the entire basic kit method was applied from rna isolation through nasba amplification and ecl detection. each dilution was tested in triplicate on three different days, resulting in nine replicate tests per dilution. consistent detection of nv rna was possible for dilutions corresponding to pdu/ml with very small relative standard deviations ( . Á/ . ). as the concentration of nv was reduced to the equivalent of pdu/ml, nasba detection was slightly less consistent, with eight of the nine reactions giving positive amplification results. at this dilution, larger standard deviations were observed only in those instances where one of three samples in a run was negative for viral rna. the detection endpoint occurred at approximately pdu/ml. at levels of pdu/ml and below, the nasba results were consistently negative and had small standard deviations. these results are summarized in fig. , which shows fairly reliable a uppercase letters designate genome sequences used for amplification or hybridization as designated. lowercase letters indicate t promoter sequence (bkp . ) and the sequence specific for the nuclisens † ruthenium-linked oligonucleotide detector probe (bkp . ). b nvint was ? biotinylated during its manufacture to facilitate binding to streptavidin-labeled magnetic spheres for capture of specific amplicons and subsequent detection using the nuclisens † ecl detection module. detection of nv rna by the nuclisens † basic kit assay at levels of Á/ pdu/ml from serially-diluted % stool suspensions. when the same rna preparations were tested by rt-pcr using the nv /nv primer set and followed by southern hybridization, we observed that the nasba amplification method provided Á/ -fold more sensitive detection limits. furthermore, the nuclisens † basic kit assay was completed in Á/ h compared with h for rt-pcr with southern hybridization. to ascertain whether the bkp . /bkp . amplification primers and the nvint probe were specific for nv amplification and detection, this primer/probe combina-tion was evaluated with a panel of representative bacterial and viral enteric pathogens (table ) . ecl detection was negative for the entire panel, with mean log ecl signals of / . as compared with the mean log ecl value of . for the nv-positive specimen. to compare the clinical sensitivity of the nuclisens † basic kit assay to rt-pcr for the detection of nv rna in stool specimens, stool specimens from infected and uninfected volunteers in an nv challenge study collected at pre-challenge and various times postchallenge were tested by rt-pcr and nasba (table ) . nv infection in these volunteers was also confirmed by elisa for igg seroconversion as described previously (moe et al., ) . all five specimens that were positive by rt-pcr were confirmed by a positive ecl signal, yielding a test sensitivity of % when using rt-pcr as the 'gold standard'. the log of these positive ecl values ranged from . to . with a geometric mean of . . five specimens that were negative by rt-pcr were also confirmed as negative by the nuclisens † basic kit assay. however, there were two volunteer samples (vt - , vt - ) that were negative by rt-pcr but gave inconsistently positive results using the nuclisens † basic kit. these specimens tested negative in one assay but positive in another and can be considered true false positives because they were obtained from subjects prior to nv challenge. there also were three rt-pcr negative samples that gave consistently positive results by nasba-ecl. the log ecl signals for these samples ranged from . to . , with a geometric mean of . that was more than one log lower than the geometric mean of the rt-pcr positive/nuclisens † basic kit positive samples ( . ). sequence analysis of the nasba amplicons obtained from these three samples confirmed the pre- sence of the target nv rna. two of these three specimens (vt - and vt - ) were collected from nv-infected volunteers at later post-challenge times ( Á/ days post-challenge) than the other rt-pcr positive specimens from these volunteers ( Á/ days post-challenge). we have detected nv-positive specimens at day post-challenge by rt-pcr in only two other infected volunteers (data not shown) indicating that viral shedding is possible during this time period. however, those specimens were not tested by the nuclisens † basic kit. the nasba results for vt - and vt - suggest that the nuclisens † basic kit may be more sensitive than the rt-pcr assay. however, this statement is made cautiously because a third rt-pcr negative specimen (vt - from day postchallenge) that was positive by the nuclisens † basic kit assay was from a volunteer who had no indication of viral shedding by rt-pcr and who did not seroconvert. overall specificity (using the rt-pcr gold standard) was % and concordance between the rt-pcr and nasba results was %. . . comparative specificity of the nuclisens † basic kit nasba method to rt-pcr for outbreak stool specimens to examine the ability of the basic kit assay to detect other nlvs using the oligonucleotide primer/probe set based on the nv genome, six rt-pcr positive stool specimens (cdc (gi), df- (gi), fs- (gi), lv- (gii), mc- (gii), and toronto (gii)) from nlv outbreaks were tested using the nuclisens † basic kit assay described above. only one of the three genogroup i outbreak strains (cdc with % nucleotide homology to nv) was positive by the nasba/ecl assay, although the degree of positivity was low and not consistent from run to run. the other two strains with % homology to nv were negative (df- , fs- ). of the genogroup ii outbreak strains (lv- , mc- and toronto, with Á/ % homology to nv), the toronto strain tested positive using the nuclisens † basic kit assay and nv-based primers and detection probes developed in this study. however, as seen for gi strain cdc , the degree of positivity was low and not consistent from run to run. the other two gii strains were consistently negative (data not shown). in this study, we report the application of a nucleic acid sequence based amplification assay (nasba) for the detection of the nlv prototype nv fiia strain using the nuclisens † basic kit. the nuclisens † basic kit assay compared favorably to parallel rt-pcr assays with respect to assay detection limits. however, the nuclisens † basic kit assay provides a more rapid alternative to rt-pcr, with completion in Á/ h with the former as compared with h with the latter. furthermore, the kit format for the boom et al. ( ) rna extraction method provided ease and standardization for less experienced laboratory personnel. the isothermal amplification approach used by the basic kit also simplifies equipment needs and costs compared with the use of thermocycling devices. in table comparative sensitivity of the nuclisens † basic kit assay to rt-pcr for detection of nv rna in representative stool specimens collected from nv-challenged volunteers short, the nuclisens † basic kit provides a promising nucleic acid amplification alternative that combines amplification, detection and confirmation into a simplified and streamlined protocol. nasba has proven to be a sensitive and specific assay for use in clinical diagnostic microbiology, with commercial kits available for the detection of human hiv, ebv and cmv in blood and plasma, many of which are quantitative assays. investigators have reported particularly good success using nasba methods to quantitate hiv load in blood and plasma (berndt et al., ; dyer et al., ) . more recently, the technology has been developed for the detection and/ or quantification of hcv (damen et al., ) , candida species (widjojoatmodjo et al., ) and the parasite plasmodium falciparum (schoone et al., ) , all in blood-based specimens. rna sequences from chlamydia trachomatis derived from urine and cervical scrapings (morre et al., ) , as well as rhinovirus in nasopharangeal aspirates (samuelson et al., ) have also been detected using nasba approaches. to our knowledge, this is the first published report on the use of nasba technology for the detection of an enteric pathogen in a fecal matrix. overall, the nuclisens † basic kit method for rna extraction resulted in highquality rna extracts that were capable of supporting nasba amplification with no further sample dilution. because levels of infectious virus in patients during acute phases of viral gastroenteritis can approach Á/ /g of feces (kapikian et al., ) , the potential of this method for routine diagnosis of nv infection is excellent. as with rt-pcr assays, the identification of broadly reactive primers for the detection of human caliciviruses remains a challenge. the primers and probe set used in this study were intended to demonstrate the feasibility of nasba amplification for the detection of norwalk virus in stool, and were, therefore, designed to be specific for the g nv fiia strain. although we were readily able to detect the prototype norwalk virus strain fiia with these primers, they were relatively ineffective for the detection of other genogroup i and ii nlv strains collected from food-borne disease outbreaks. even though these results were anticipated, this finding further supports the need for more broadly reactive primers to improve all nucleic acid amplification assays designed for the detection of this genetically diverse group of viral pathogens. different rna extraction methods were used for the nasba assay and the rt-pcr assay. for the nasba assay, the nuclisens † basic kit rna extraction method, based on the method of boom et al. ( ) , was used as per the manufacturer's instructions. for the rt-pcr assay, we used two methods that work well in our experience: the heat release method and the ultra-spec method. these different methods may explain in part the discrepant results from the two assays for a few specimens. it is well known that the sensitivity and specificity of nucleic acid amplification methods is affected by the efficiency of the rna isolation method. for instance, brink et al. (brink et al., ) noted that the relative sensitivity of nasba detection of ebv is more likely to be influenced by rna isolation efficiency than by amplification efficiency. others have shown that the effect of matrix-associated inhibitors on amplification efficiency is exacerbated at low levels of target template, yielding both false negative and false positive results (jaykus et al., ) . losses during rna extraction may well account for the slightly poorer detection limits noted for both the rt-pcr and the nuclisens † basic kit methods described in this paper (fig. ) , when compared with the rt-pcr detection limits we have observed for dilutions of the norwalk virus stock preparation. the norwalk virus stock solutions used in this study were titered using a heatrelease rt-pcr method (schwab et al., ) rather than the boom et al. ( ) silica-based method followed by rt-pcr. the relatively high degree of false positive amplification results noted in the basic kit assay may also be associated with residual matrix components that serve as template for non-specific amplification and hybridization. it may be possible to improve both the sensitivity and specificity of amplification by using an alternative rna extraction method, followed by amplification and detection using the latter two steps of the nuclisens † basic kit. such an approach could facilitate further removal of residual matrix associated components and thereby improve the efficiency and specificity of subsequent rna amplification. this study demonstrates the use of the nuclisens † basic kit in the application of a nasba/ecl method for the rapid and sensitive detection of nv rna in fecal samples. to our knowledge, this is the first study that has sought to apply nasba/ecl amplification and detection technology to the group of nlv. furthermore, because the virus stock used in these experiments had been titered previously by heat-release rt-pcr multiple times, we have been able to establish an approximate level of detection when applying the combined rna extraction, nasba amplification, and ecl detection of the nuclisens † basic kit. the detection limit approximates Á/ pdu per amplification reaction, which is very close to the detection limits obtained for rt-pcr. however, the ease of use and speed of detection supplied by the nuclisens † system provides a significant advantage over rt-pcr detection approaches. with further development and optimization, particularly with respect to primer design and assay specificity, we are confident that nasba/ ecl technology will continue to develop as a viable method to detect enteric pathogens in fecal matrices. basic local alignment search tool detection and differentiation of antigenically distinct small round-structured viruses (norwalk-like viruses) by reverse transcription-pcr and southern 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and monitor assays norwalk-like virus infection in military forces: epidemic potential, sporadic disease, and the future direction of prevention and control efforts application of pcr to detect norwalk virus in fecal specimens from outbreaks of gastroenteritis determination of norwalk virus dose-response in human volunteers. poster #c- outbreaks of acute gastroenteritis associated with norwalk-like viruses in campus settings rna amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of chlamydia trachomatis in cervical scrapings and urine samples genomic diversity of 'norwalk like viruses' (nlvs): pediatric infections in a brazilian shantytown development and application of a new method for amplification and detection of human rhinovirus rna detection and quantification of plasmodium falciparum in blood samples using quantitative nucleic acid sequence-based amplification use of heat release and an internal rna standard control in reverse transcription-pcr detection of norwalk virus from stool samples evaluation of the nuclisens hiv- qt assay for quantitation of human immunodeficiency virus type rna levels in plasma epidemiology of norwalk virus during an outbreak of acute gastroenteritis aboard a us aircraft carrier methods for recovering viruses in shellfish, seawater, and sediment an epidemiological investigation of norwalk virus infection in south africa quantification of hiv- rna in plasma using nasba during hiv- primary infection the incidence and genetic variability of small round-structured viruses in outbreaks of gastroenteritis in the netherlands analytical performance and clinical utility of a nucleic acid sequence-based amplification assay for detection of cytomegalovirus infection we would like to thank biomérieux (formerly organon teknika corporation) for providing reagents and equipment for this study and lisa lindesmith, hannah cluck and erin-joi collins mcneal for assistance with this manuscript. this research was supported in part by a grant from the national institutes of health p dk . key: cord- -m rlmlun authors: pearson, morgan; lavoy, alora; chan, leo li-ying; dean, gregg a. title: high-throughput viral microneutralization method for feline coronavirus using image cytometry date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: m rlmlun feline coronaviruses (fcov) are members of the alphacoronavirus genus that are further characterized by serotype (types i and ii) based on the antigenicity of the spike (s) protein and by pathotype based on the associated clinical conditions. feline enteric coronaviruses (fecv) are associated with the vast majority of infections and are typically asymptomatic. within individual animals, fecv can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (fip), the leading infectious cause of death in domestic cat populations. there are no approved antiviral drugs or recommended vaccines to treat or prevent fcov infection. the plaque reduction neutralization test (prnt) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput and typically requires – days for the formation and manual counting of cytolytic plaques. host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. in addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. to address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. subsequently, this fcov viral neutralization assay was used to explore immune correlates of protection using plasma from naturally fecv-infected cats. we demonstrate that the high-throughput viral neutralization assay using the celigo image cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds and vaccines. this method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development. feline coronaviruses (fcov), present in most domestic cat populations worldwide, are members of the alphacoronavirus genus that are further characterized by serotype (type i and ii) based on the antigenicity of their spike (s) proteins (an et al., ; kummrow et al., ; li, ; pratelli, ) . more recently, designation as clade a or b based on the sequence and biological properties of s protein has been proposed and would correlate with fcov type i and ii viruses, respectively (whittaker et al., ) . viruses from both types and clades are additionally characterized by distinct pathotypes based on the associated clinical conditions. feline enteric coronaviruses (fecv) are associated with the vast majority of infections and are typically asymptomatic or present as a mild enteropathy. within individual animals, fecv can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (fip) . fip is the leading infectious cause of death in domestic cats (hartmann, ) , affecting . % and % of seropositive kittens every year (addie et al., ; rohrbach et al., ) . currently, there are no approved antiviral drugs or recommended vaccines to treat or prevent fcov, although recent reports provide some promise (addie et al., ; dickinson et al., ; pedersen, a; pedersen, b; pedersen et al., ; takano et al., ) . the complex and variable pathogenesis of fip has been studied for decades. the clinical presentation, often vague, consists of fever, weight loss, and depression. diagnosis can be difficult and is based on a preponderance of evidence as there is no single definitive ante mortem test (felten and hartmann, ) . histological lesions are characterized by granulomatous j o u r n a l p r e -p r o o f inflammation and vasculitis that may or may not result in proteinaceous effusions in body cavities. neurologic symptoms with associated inflammatory lesions in the central nervous system are also relatively common (foley et al., ) . the complexity of the virus itself along with the variability of the clinical manifestation has confounded efforts to determine immune correlates of protection that should be targeted with vaccine strategies. we recently reported that control of fecv replication in naturally infected cats was associated with mucosal and systemic antibody responses while cell-mediated responses were minimally apparent (pearson et al., ) . therefore, assays to determine effector functions of antibodies will be crucial for the development and evaluation of a successful vaccine. traditionally, plaque-reduction neutralization tests (prnt) have been the preferred neutralization assays involving fcov (shiba et al., ) . in prnt assays, plasma or serum is first mixed with infectious virus. after incubation, the mixture is added to susceptible host cells and incubated until cytopathic effects (cpe) appear. subsequently, the host cells are fixed and stained with crystal violet to reveal the fcov infected plaques. previously, fcov neutralizing antibodies have shown approximately % or more plaque reduction (shiba et al., ) . the prnt assay for fcov is time-consuming, requiring up to - days for the formation of cytolytic plaques (supplementary figure ) . additionally, the host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, making it extremely difficult to manually count accurately in bright field (masci et al., ) . manual plaque counting is low-throughput and has operator-to-operator variation, and digital records are not always captured (masci et al., ) . therefore, it is crucial to develop improved methods for high-throughput measurement of viral infection and neutralization. j o u r n a l p r e -p r o o f recent advancements in image cytometry systems have enabled the development of novel high-throughput cell-based assays performed directly in standard -to -well microplates (jorquera et al., ; masci et al., ; shambaugh et al., ; yang et al., ) . the celigo image cytometer has demonstrated automated enumeration of viral plaques, foci, and individual virus-infected cells in a -well microplates using bright field or fluorescence imaging in less than or min, respectively, which can significantly reduce the time required for counting and analysis as well as eliminate operator-to-operator variation (ramos et al., ; rosen et al., ; viedma and pickett, ) . furthermore, it has been shown that fluorescence detection of plaques, foci, or individual infected cells via immunostaining or fluorescent protein reporter can be detected earlier than the traditional cytolytic plaques, where assay time duration can be effectively reduced by % (masci et al., ) . in this work, we developed a high-throughput fcov microneutralization assay employing the celigo image cytometer for rapid quantification of virus-infected cells. multiple parameters were optimized including host cell seeding density, microplate surface coating, virus concentration and incubation time, fluorescent labeling, and assay buffers. the optimized assay was employed to test feline plasma for the presence of fcov-neutralizing antibodies. the proposed high-throughput viral microneutralization assay using celigo image cytometer provides a robust and efficient method for virologists to quickly identify potential therapeutic antibodies or antiviral compounds for various viral infectious diseases, which could have a significant impact on efficiency of drug discovery and vaccine development. allowed to adhere to the plate over night at °c for the infectivity and neutralization assays. feline coronavirus strain fcov-wsu- - was generously provided by dr. niels pedersen (university of california, davis) (herrewegh et al., ) . crfk cells were added to a collagen type i-coated t- flask ( - , greiner bio-one) and incubated for h. the medium was then removed and the cells were washed with dulbecco's phosphate buffered saline (dpbs, sh .fs, ge healthcare, chicago, il). an untitered stock of fcov-wsu- - type ii virus was first diluted : in dpbs and then added to the cells and incubated for h at °c. next, the viral inoculum was removed and the cells were washed x with dpbs. subsequently, culture medium was added and cells were incubated for h. the supernatant was collected into a -ml tube, centrifuged for min at x g, aliquoted and stored at - °c for single use. crfk infections in -well plates followed the same method unless noted. (chan, ; kessel et al., ; zhang et al., ) . the celigo software application "confluence " was used to measure the host cell confluence percentages using the acquired bright field images. the preset analyze parameters were optimized to automatically calculated the area of host cells covering the well surface to determine the confluence percentages. the results were used to select optimal crfk seeding density, as well as wash buffer and plate coating. the celigo software application "target + + mask" was used to identify the total number of dapi-positive host cells in the blue channel and quantify the percentages of green fluorescent infected cells. the celigo instrument was set up to acquire images in the target (bf), target (green), and mask (blue) channels, where the exposure times for alexa fluor and dapi were , and , µs, respectively. hardware-based autofocus was used to focus in the bf channel, and the focus offsets were applied for the green (+ µm) and blue (- µm) channels. the preset analyze parameters were optimized to identify the dapi-positive host cells above an intensity threshold of , then the gating feature was used to analyze the fluorescence intensities to determine af -positive infected cell population percentages. image acquisition and analysis were performed simultaneously. in addition, the af fluorescence j o u r n a l p r e -p r o o f intensities were measured to determine the optimal primary and secondary antibody concentrations for staining the infected host cells. crfk seeding density was optimized so cells would reach high confluency by the end of the post-infection incubation. the number of cells seeded ranged from to cells per well in increments of and was tested in conjunction with growth time of and h (n = ). at each time point, bright field images were acquired and analyzed using the image cytometer to automatically measure the confluence percentages of the host cells. immunostaining of fcov-infected cells was optimized by testing the antibody concentrations above and below manufacturer recommendations using cells infected with a : dilution of the viral stock. the primary antibody was tested at . , . , . , and . µg/ml. the secondary antibody was tested at . , . , . , and . µg/ml. after acquiring images of the infected cells using the image cytometer, the lowest concentrations that generated the highest signals with the lowest background were selected. the dapi staining solution was used at . µg/ml following the manufacturer's protocol. the viral supernatant inoculation concentration and incubation time were optimized to achieve the desired infection rate of - %. approximately , crfk cells were seeded into a -well plate ( , greiner bio-one) for h. next, the host cells were infected with a serial dilution of infectious supernatant ( : , : , : , : , : , : , : , : , : , : , and : ) and post-inoculation incubation time points were analyzed at , , , and h using image cytometry with fluorescent immunostaining (n = ). cats were cared for in accordance with the association for the assessment of laboratory animal care standards and with approval from the colorado state university institutional j o u r n a l p r e -p r o o f animal care and use committee (protocol - a, approved july, ). edta plasma of the type i fecv-infected cats was collected and stored at - °c as part of a previous study in which cats were monitored monthly for plasma fcov antibodies and fecal fcov rna over seven months (pearson et al., ) . five representative feline plasma samples (table ) were vortexed, centrifuged, and heat-inactivated at °c for h (pearson et al., ) . next, serial dilutions in dpbs were prepared directly in a -well titration plate ( , greiner bio-one). the plasma samples were first diluted : in row "a" by adding µl of plasma into µl of dpbs ( µl total) in triplicate. rows "b" through "h" were then filled with µl of dpbs, and subsequently, µl of row "a" was added to row "b" and mixed. the procedure was repeated from rows "b" through "h" to generate the plasma dilutions of : , : , : , : , : , : , and : . the extra µl at row "h" was discarded. the celigo image cytometer was used to screen the selected plasma samples for neutralization of fcov using the optimized assay parameters. first, , crfk cells per well were seeded in collagen type i-coated -well plates ( , greiner bio-one) and cultured for h. during this time, the viral supernatant ( µl) at the optimal concentration ( : dilution of the viral stock solution in dpbs) was added to the plasma samples in the prepared plasma titration plate described above and uniformly mixed with a multi-channel pipette. the plasma/virus mixtures were then incubated for at rt for h. in addition, wells with only viral supernatant or only plasma were prepared as positive and negative controls. during the plasma/virus incubation time, the host cell microplate was prepared by removing the crfk medium and washing once with µl of dpbs. after the plasma/viral j o u r n a l p r e -p r o o f supernatant incubation, the mixtures ( µl) were added to the host cells and incubated for h at °c and % co . after the incubation the plasma/virus mixtures were removed, the wells were washed x with µl of dpbs. next, µl of crfk medium was added to each well and the plates were incubated for h. the infected cells were then fluorescently stained and immediately scanned and analyzed using the image cytometer. the scanning and analysis settings for every experiment were optimized to generate proper images and results (figure ). the scanning parameters were selected to acquire bright field and fluorescent images that were in focus for the entire plate. the analysis parameters were selected so the software can accurately identify and count the total host cells and infected cells. after the settings were established, they were saved as experimental presets and applied to subsequent plates without the need for further adjustments. development of the image cytometry-based high-throughput fcov infection detection method involved the optimization of key variables: host cell seeding density, fluorescent labeling, assay buffer, microplate surface coating, virus concentration, and incubation time. the first step in developing the method was to optimize the host cell seeding density. the seeding density-dependent bright field images and measured confluence percentages are shown in figure , which showed decreasing confluence as cell density decreased as expected. an optimal seeding density of , cells per well and an incubation time of h post-seeding allowed the host cells to reach approximately % confluence at the end of viral infection, where the cells can be fixed and stained for image cytometry analysis. although the -h time point showed cell confluence of % or higher, we observed that hours of incubation caused the host cells to formed layers near the well edges that were easily washed off. the acquired green fluorescent images at different primary and secondary antibody concentrations for antibody optimization are shown in figure a . primary antibody at g/ml in combination with or g/ml of secondary antibody generated uniform fluorescence bright enough for the celigo software to count accurately. similarly, the measured mean af fluorescence intensities were higher with the same antibody concentrations (figure b ). although the af fluorescence intensity was the highest at g/ml of the secondary antibody, the background fluorescence was measured by examining the cell-free well area, which was higher than the µg/ml (data not shown). therefore, µg/ml was selected as the optimal concentration for both the primary and secondary antibodies. it was also determined that enumeration of crfk cells required the use of a dapi nuclear stain. while the celigo can count many cell types by detection of cell edges in a bright field image, the contrast of the crfk is too low to accurately identify individual cells in confluent areas. in addition, we observed that black-walled microplates reduced auto-fluorescence and light scattering near the edge of the wells and enabled % of the well to be included in the analysis, compared to less than % with clear plates (supplementary figure ) . two assay buffers as well as four microplate surface coatings were compared by using the confluence analysis (figure a) . dpbs demonstrated minimum cell loss during the wash and dilution steps, and the collagen type i surface coating allowed the highest adhesion of crfk cells to the well surface. in addition, the infection rates were slightly higher with dpbs than with dmem (figure b ). it is important to note that the isotype control for the primary antibody showed no nonspecific staining. the low number of infected cells counted in the dpbs-isotype j o u r n a l p r e -p r o o f wells represents the level of fluorescent artifact that could be used to establish the lower limit of quantification in viral titration assays (figure b) . the af and dapi overlay fluorescent images in respect to viral titration (figure a) show a decrease in virus-infected cells as viral concentrations decreased. the : dilution of viral stock solution yielded our desired - % infected cells (figure b ) and no infected cells were observed beyond : . the amount of cell loss during washing and staining steps increased at the -, -, and -h time points. in order to minimize host cell loss during subsequent processing steps, a viral incubation time of h was selected. five feline plasma samples were obtained from the colorado state university closed cat colony, which had naturally circulating feline coronavirus in the population and was considered to be otherwise specific pathogen-free. the fluorescence-based high-throughput fcov microneutralization assay was performed twice with the plasma samples at concentrations each in triplicates ( figure ) . a high infection rate ( - %) was measured and no infection was detected with only plasma. the plasma titrations showed no neutralization effects; however, the infection rates did show a slight inverse dose-dependent trend for each feline plasma. the high-throughput microneutralization assay described here represents an important advancement for the evaluation of vaccine candidates, testing of antiviral drugs, and investigation of fcov pathogenesis. the method can easily be modified for other virological assays such as label-free cytolytic plaque counting, cytopathic effect detection, and antibody-j o u r n a l p r e -p r o o f viral antigen binding inhibition assays (masci et al., ; rosen et al., ; viedma and pickett, ; yang et al., ) . in this work, the development and optimization of the fluorescence-based virus-infected cell counting method was challenging due to the complex interactions of different variables. when optimizing the host cell seeding density, it was discovered that if the crfk cells were not close to - % confluency during the viral inoculation, the percentage of infected cells was inconsistent (data not shown). in addition, if confluency was greater than %, cells were more likely to lift off the well surface during subsequent processing steps. for the host cell seeding incubation, it was also important to select the shortest possible time frame after -h host cell adherence to minimize the assay time, as well as increase efficiency and throughput. filtration of the primary and secondary antibodies was determined to be beneficial; without filtration, fluorescent aggregation particles can be observed throughout the wells, which can cause nonspecific detection of infected cells. there were no differences observed between filtered and unfiltered dapi. while it is possible to combine antigen and dapi staining, we observed that the sequential staining of af and dapi generated stronger fluorescent signals. it was observed that crfk medium inhibited infection during the -h viral inoculation (data not shown), thus removing and washing the culture medium from the wells was critical. dpbs was selected for this step because it demonstrated minimal disruption to the adherence of the crfk cells in comparison to dmem. when washing with dmem, most of the host cells lost adhesion to the bottom of the wells and disrupted their surface coverage. therefore, dpbs was used for all wash steps as well as for the dilution of virus, plasma, and staining reagents. the viral titer results revealed that the - % infection rate optimal for the neutralization assay was achieved with a : dilution of the viral stock. a higher percentage j o u r n a l p r e -p r o o f could mask the neutralization effects, and a lower percent could lead to more well-to-well variation. it is important to note that, different dilutions of virus stock were used to optimize conditions for virus propagation and fluorescence testing. after the optimization of each parameter for the high-throughput virus-infected cell counting method, the image cytometer performed the neutralization assay at approximately min per -well plate for simultaneously acquiring and analyzing whole well images in bright field and fluorescence, equivalent to ~ sec per sample. the high-throughput functionality can be achieved with the developed method, allowing rapid screening for neutralizing antibodies in plasma or serum samples. in a -well plate, columns may be dedicated to samples at dilutions, and the th column can be dedicated to positive and negative controls. furthermore, the assay could be conducted in a -well plate to further increase the throughput of the screening assay. the high-throughput neutralization assay was conducted two times and no fcov neutralization was observed, however, the lack of neutralization was not surprising. the virus used in this assay, fcov-wsu- - , is a type ii virus while the cats evaluated in this study were infected with a type i fecv (pearson et al., ) . the spike proteins of type i and ii viruses differ significantly in sequence, structure and function (jaimes et al., ) . furthermore, while feline aminopeptidase n (fapn) is known to be the target cell receptor used by type ii viruses, the type i receptor remains unknown (dye et al., ; hohdatsu et al., ) . these differences explain why antibodies against either serotype may not cross-neutralize (pedersen et al., ; terada et al., ) . unfortunately, type i viruses do not readily grow in cell culture, which creates an obstacle to assessment of antibody neutralization in an appropriate homologous assay format using plasma from type i infected cats against type i virus infection in vitro. recent j o u r n a l p r e -p r o o f advances that may eventually address this conundrum include the generation of an infectious type i molecular clone and knock-out of the type i alpha-interferon receptor in feline cells (mettelman et al., ; terada et al., ) . the latter may allow more robust replication of natural type i fecv isolates. the use of the celigo image cytometer reduces assay time while providing richer, quantitative readouts to accelerate assessment of vaccine and antiviral compound candidates. importantly, the optimization strategy can be employed to adapt other cell types to the assay format or to create entirely new virus-host cell combinations. the author llc declares competing financial interests. the research instrument used in this manuscript is a product of nexcelom bioscience, llc. the work was performed to demonstrate a novel high-throughput viral neutralization screening method for feline coronavirus using the celigo image cytometer. feline infectious peritonitis. abcd guidelines on prevention and management oral mutian®x stopped faecal feline coronavirus shedding by naturally infected cats prevalence of korean cats with natural feline coronavirus infections high-throughput direct cell counting method for immuno-oncology functional assays using image cytometry antiviral treatment using the adenosine nucleoside analogue gs - in cats with clinically 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cats examined at veterinary medical teaching hospitals a high-throughput inhibition assay to study mers-cov antibody interactions using image cytometry development of a high-throughput respiratory syncytial virus fluorescent focus-based microneutralization assay differentiation of feline coronavirus type i and ii infections by virus neutralization test antiviral effects of hydroxychloroquine and type i interferon on in vitro fatal feline coronavirus infection establishment of a virulent full-length cdna clone for type i feline coronavirus strain c emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses characterizing the different effects of zika virus infection in placenta and microglia cells improving virus taxonomy by recontextualizing sequence-based classification with biologically relevant data: the case of the alphacoronavirus species il- ameliorates acute lung injury in influenza virus infection novel high-throughput cell-based hybridoma screening methodology using the celigo image cytometer the authors wish to acknowledge the support of the morris animal foundation award #d fe- . the author, mp, was supported by nih grant number t od .j o u r n a l p r e -p r o o f key: cord- -tgowzrqo authors: li, yong-hua; li, jie; liu, xue-en; wang, ling; li, tong; zhou, yi-hua; zhuang, hui title: detection of the nucleocapsid protein of severe acute respiratory syndrome coronavirus in serum: comparison with results of other viral markers date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: tgowzrqo a capture enzyme-enhanced chemiluminescence immunoassay (eclia) based on three specific monoclonal antibodies to detect the nucleocapsid (n) protein of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in the serial serum samples from sars patients was developed. the anti-sars-cov igg and the viral rna were also detected in the sera by elisa and rt-pcr, respectively. during the first days after onset, anti-sars-cov igg, sars-cov rna and the n protein were detected in . , . , and % of the patients’ sera, respectively. the detection rate of the n protein during days – of the disease was still significantly higher than those of anti-sars-cov igg and sars-cov rna. the data demonstrated that detection of the n protein with the capture eclia appears to be more useful than detection of other viral makers for rapid diagnosis of sars in patients. severe acute respiratory syndrome (sars), an infectious disease which occurred firstly in the guangdong province, southern part of china, in november , caused outbreaks in countries and areas in spring with clinical cases and deaths, resulting in an overall mortality rate of . % (who, b) . after the outbreaks were contained in mid-july , three events of laboratory-associated infections caused a total of clinical sars cases (normile and vogel, ; normile, a normile, , b . again in guangdong, four clinical sars cases occurred sporadically without contact histories from december , to january , (liang et al., ) . on the other hand, the origin of the etiological agent of sars, sars-cov , remains elusive although sars-cov-like virus was isolated from civet cats . the fact that initial clinical sars cases occurred independently without any contact history in different areas of guangdong and the find-ings that the viral sequences isolated from these patients were grouped in different subtype clusters (chinese sars molecular epidemiology consortium, ) suggest that the patients in china were infected by sars-cov from multiple infectious sources, rather than from a single source. therefore, sars may be recurrent in human beings. sars-cov is an enveloped, single-stranded positivesense rna virus classified in a new group of the coronaviridae family. the viral genome is approximately kb in length with open reading frames. sars-cov contains four major structural proteins, including spike (s), membrane (m), small envelope (e), and nucleocapsid (n) (marra et al., ; rota et al., ) . the s protein mediates attachment of the virus to cellular receptors (li et al., b) and virus entry by fusion with cell membranes (hofmann et al., ; yang et al., ) . both m and e proteins are integral membrane proteins and form the minimum protein units for virus assembly. the n protein is an extensively phosphorylated, highly basic protein, which interacts with viral rna and makes up the viral core and nucleocapsid (lai, the diagnosis of sars depends basically upon detecting sars-cov rna by rt-pcr and/or testing specific antibodies directed against sars-cov by assays based on cultured virus or recombinant viral antigens. although rt-pcr is a reliable technique for the diagnosis of sars in the early phase, the detection rate for probable sars was only . - % wu et al., ) . antibodies directed against sars-cov, examined by immunofluorescent assay, immunochromatographic test, enzyme-linked immunosorbent assays (elisa), or western blot, can detect over % of the cases (wu et al., ; guan et al., ; he et al., a; li et al., a) , however, it takes - weeks after the onset for the measurable specific antibodies to develop. therefore, detecting anti-sars-cov igg is not a practical strategy for rapid diagnosis. since sars-cov is highly contagious and the disease is severe with a high mortality rate, rapid diagnosis of sars has significant importance both for treating patients and for preventing the spread of the disease. recently, assays based on monoclonal antibodies (che et al., a (che et al., , b or polyclonal antibodies (lau et al., ) directed against the n protein were developed to detect the n protein of sars-cov in serum samples and in other clinical specimens. in the present study, a capture eclia was developed based on three monoclonal antibodies directed against the n protein of sars-cov, and the n protein in the longitudinal serum samples from the sars patients were detected with this method. anti-sars-cov igg and sars-cov rna were also detected in the serial sera from probable sars patients. the usefulness of the n protein, sars-cov rna, and anti-sars-cov igg in identifying the infection at the early phase was evaluated. three hundred and thirty-six serial serum samples, collected from probable sars patients in xiongke hospital of beijing, were used in this study. of the probable patients, were female, and were male. the patients were aged between - years. all patients had clinical features of probable cases with sars defined by who (who, a) . in addition, serum samples from the close contacts that did not develop any symptom and samples from healthy blood donors were also used in this study. the study to review the clinical, radiological and laboratory features of the patients was approved by the ethics committee of the beijing xiongke hospital. anti-sars-cov igg was detected using a commercial indirect elisa kit (bdi-gbi biotech, beijing), which was developed based on sars-cov lysates as coating antigens. briefly, l/well of each serum diluted -fold in . % tween in pbs (pbs-t) was added to the viral protein-coated plates and incubated at • c for min. following five washes with pbs-t, l/well of peroxidase-conjugated mouse anti-human igg (fc-gamma specific) antibody diluted -fold in pbs-t was added. following incubation at • c for min, the plates were washed as above and l/well of the substrate tetramethylbenzidine solution was added. after incubation at room temperature for min, the color development was stopped by adding m h so . optical density (od) at nm was measured with an elisa reader. blank, negative, and positive controls were always included in each plate. the cut-off value was . , which was calculated as the mean plus s.d. of normal serum controls. total rna from l of each serum from the sars patients was extracted using the qiaamp virus rna mini kit (qiagen). the extracted rna was subjected to a single step rt-pcr, followed by a second round of pcr to amplify the target viral sequences essentially the same way as described elsewhere yam et al., ) . the resultant products were electrophoresed through a % agarose gel in tris-borate buffer. target bands were visualized by staining with ethidium bromide. positive and negative controls were included in each run, and all precautions to prevent cross-contamination were observed. a capture eclia was developed in collaboration with beijing weixiao biotech to detect the n antigen of sars-cov in the sera from the sars patients based on the assay developed by che et al. ( a) with modifications. microtiter plates were coated with l/well of a mixture of three anti-n protein monoclonal antibodies, n e , n e , and n e (kindly provided by dr. che, zhujiang hospital, south medical university, china) (che et al., ) each at a concentration of g/ml in carbonate-bicarbonate buffer ( . m, ph . ) and incubated at • c overnight. the plates were washed five times with pbs-t and then blocked with l of % casein in pbs-t for h at • c. following five washes with pbs-t, l of each serum diluted two-fold in pbs-t was added to each well and then incubated at • c for h. after five washes, l/well of peroxidase-conjugated rabbit anti-sars-cov polyclonal antibodies (beijing weixiao biotech) diluted -fold in pbs-t was added and again incubated at • c for h. after five washes, l/well of chemiluminescence substrate solution was added and incubated for min at room temperature. finally, the relative luminescence value (rlv) was read by the microplates single photon counter (beijing weixiao biotech). the cutoff value was . times of the mean rlv of negative controls. all serum samples from probable sars patients were tested for anti-sars-cov igg by an indirect elisa which was developed based on cultured sars-cov lysates as coating antigens. as shown in table , during the first days of the disease, anti-sars-cov was detected only in three of patients. thereafter, more patients seroconverted and the positive rate of anti-sars-cov igg increased from . % during - days after onset to . % during - days. all patients had detectable specific antibodies at days after onset. to observe at least how long the antibodies lasted, anti-sars-cov in the subsequent sera collected afterwards from patients was also measured. all these sera were still positive for anti-sars-cov, indicating that anti-sars-cov igg lasted at least days after the onset. in addition, the result also showed that the concentration of anti-sars-cov igg increased during the convalescent phase since the od values of the convalescent sera were higher than those of the acute samples (data not shown). on the other hand, all serum samples from the subjects who had close contacts with probable sars patients but did not develop any symptom were negative for anti-sars-cov igg. similarly, none of the serum samples from healthy blood donors collected during the period of sars outbreak in was positive for these antibodies. a total of serum samples collected during different phases of the disease from sars patients were tested for viral rna of sars-cov by rt-pcr. table shows the positive rates of the viral rna in the circulation at various stages of the disease. during the first days, six ( . %) of the patients were positive for sars-cov rna. in the subsequent phases, the positive rates of viral rna in the the n protein of sars-cov was tested in a total of sera from sars patients with an antigen-capture eclia based on monoclonal antibodies directed against the n protein of sars-cov. as shown in table , the n protein was detected in nine of the ten patients in the first days after onset. the positive rate remained to be % during days - . on the other hand, during the first days, sars-cov rna was detected only in four of the ten patients, and the positive rate was only % during days - (table ). the n antigen still remained to be positive in eight of the and one of the samples during days - and days - of the disease, respectively. however, sars-cov rna became negative during these periods. the n protein was not detected in serum samples collected during days - after onset while anti-sars-cov igg was positive in all these serum samples. the n protein was not detected in the sera from normal blood donors (data not shown). in this study, the anti-sars-cov igg, sars-cov rna, and the n protein were examined in consecutive serum samples from the probable sars patients. the detection of the n protein of sars-cov in serum samples by eclia appears to be superior to the detection of the viral rna by rt-pcr in rapid diagnosis of sars patients. all probable cases in this study were confirmed retrospectively to be infected with sars-cov by detecting anti-sars-cov igg in their convalescent serum samples. the elisa for anti-sars-cov igg used in this study was shown to be highly specific (li et al., a; chen et al., ) . the findings that none of the sera from healthy blood donors was positive for anti-sars-cov also indicated that the antibodies detected in the probable cases were directed specifically against sars-cov. in addition, the anti-sars-cov igg was not detected in any serum from the people with close contact histories to sars patients, suggesting that occult infection of sars-cov occurs rarely. usually, human beings develop specific igm antibody earlier than igg after being infected with microorganisms. however, based on an elisa which was also used in this study, anti-sars-cov igg and igm antibodies almost simultaneously appeared in the patients' sera (li et al., a) , therefore, only anti-sars-cov igg antibody was detected, and anti-sars-cov igm was not measured in the patients' sera in this study. since the genome of sars-cov was sequenced completely (marra et al., ; rota et al., ) , rt-pcr has been used widely for the detection of the viral genome in clinical specimens as the most sensitive method for rapid diagnosis of sars. however, the positive rate in the early phase of the disease was generally about % or less. in the present study, sars-cov rna was only detected in . % of the patients during the first days after the onset ( table ). this is in agreement with the results reported by others wu et al., ) . apparently, about half of sars patients cannot be diagnosed at the early phase based on detection of the viral genome. the n protein of coronavirus forms the viral core and nucleocapsid and probably is the most abundant component during replication since all transcripts of the virus may carry the nucleotide from the n gene. based on a specific monoclonal antibody, the n protein of coronavirus infectious bronchitis virus was detected in tracheal smears and sliced tracheas from chickens infected experimentally (yagyu and ohta, ) . it is likely that the n protein of sars-cov is also abundant during replication since the n protein is detected easily in the sars-cov cell culture lysates (lau et al., ) . this feature makes it possible to detect the n protein of sars-cov as a rapid diagnostic strategy. recently, it is reported that most of sars patients had detectable n protein in their sera at the early stage of the disease (che et al., a (che et al., , b . the other clinical samples including nasopharyngeal aspirate, urine, and stool samples from sars patients also had detectable n protein (lau et al., ) . in the present study, the n protein was tested in consecutive sera from sars patient with an assay based on specific monoclonal antibodies. in the meantime, the sars-cov rna was also amplified in the same samples. nine sera from patients collected during days - after onset were positive for the n protein but only four of the patients were positive for the viral rna. of those sera collected during days - , % were positive for the n protein, however, the positive rate of sars-cov rna was only % (table ). fig. shows that the detection rates of the n protein, the viral rna, and the anti-sars-cov igg at the different times after the onset. the data demonstrated that detection of the n protein appeared to be more sensitive than detection of the viral rna and the specific antibodies during the early phase, although the sample number in this study was relatively small. generally, the n protein and viral rna circulate together in the peripheral blood as sars-cov particles, thus both components should have been detected at a comparable rate; however, the detection rate of the n protein was higher than that of the viral rna. it is likely that the current rt-pcr is not sensitive enough to detect the low concentration of sars-cov rna. indeed, only half of the sars patients are positive for the viral rna wu et al., ) , which was also observed in the present study. instability of viral rna, inefficient extraction of rna, unoptimized primers, and potential existence of inhibitory substances may contribute to the less sensitivity of the rt-pcr. indeed, the sensitivity of rt-pcr may be improved by optimizing the reaction conditions yam et al., ) . since the n protein of sars-cov shares low homology with nucleocapsid proteins of other members of the coronavirus family (marra et al., ; rota et al., ) , the monoclonal antibodies directed against the n protein of sars-cov should not capture the n proteins of other coronaviruses. it is indeed the case that the antibodies directed against the n protein of sars-cov did not recognize the nucleocapsid proteins of two human coronaviruses oc and e (che et al., b) . in this study, all healthy blood donors were negative for the n protein of sars-cov, indicating that the assay was highly specific. therefore, the n protein, which was detected in the sera of the sars patients, should be derived from sars-cov. notably, of fourteen sera collected during days - after the onset, eight were positive for the n protein but none was positive for viral rna (table ), indicating that the n protein of sars-cov tends to persist in the circulation longer than the viral rna. in chickens infected experimentally with coronavirus infectious bronchitis virus, the n protein also lasts longer than the virus in tracheal smears and sliced tracheas (yagyu and ohta, ) . this is likely because the n protein is not as degraded easily as viral rna in the circulation since the n protein, when expressed in mammalian cells, can self-interact to form dimers or oligomers (he et al., b) , which may stabilize its helical structure. therefore, the eclia for the n protein can detect the viral maker in sars patients in whom rt-pcr shows negative. recently, a competition elisa based on two monoclonal antibodies against sars-cov developed by berry et al. ( ) appears to be more sensitive than the current indirect elisa in detecting anti-sars-cov. that method may be an alternative to rapidly diagnose sars-cov infection. the comparison of the eclia used in this study and berry et al.'s elisa in identifying sars-cov infection remains to be further studied. in conclusion, although the number of the patients included in the present study was limited, the data demonstrated that the eclia for the n protein of sars-cov is more sensitive than rt-pcr for the viral rna in identifying sars-cov infection at early stage of the disease. in addition, eclia has the advantages of avoiding special expertise, time-consuming procedures, and cross-contamination over rt-pcr and can be used easily in clinical laboratories. therefore, the n antigen-capture eclia is a promising method for early diagnosis of sars-cov infection. development and characterization of neutralizing monoclonal antibody to the sars-coronavirus sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome nucleocapsid protein as early diagnostic marker for sars rapid and efficient preparation of monoclonal antibodies against sars-associated coronavirus nucleocapsid protein by immunizing mice antibody response and viraemia during the course of severe acute respiratory syndrome (sars)-associated coronavirus infection molecular evolution of the sars coronavirus during the course of the sars epidemic in china identification of a novel coronavirus in patients with severe acute respiratory syndrome recombinant protein-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin g antibodies to severe acute respiratory syndrome (sars) coronavirus in sars patients isolation and characterization of viruses related to the sars coronavirus from animals in southern china development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome analysis of multimerization of the sars coronavirus nucleocapsid protein s protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients sars virus: the beginning of the unraveling of a new coronavirus detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay profile of specific antibodies to the sars-associated coronavirus angiotensin-converting enzyme is a functional receptor for the sars coronavirus laboratory diagnosis of four recent sporadic cases of community-acquired sars infectious diseases. early indications point to lab infection in new sars case infectious diseases. mounting lab accidents raise sars fears infectious diseases. second lab accident fuels fears about sars coronavirus as a possible cause of severe acute respiratory syndrome early diagnosis of sars coronavirus infection by real time rt-pcr ontario laboratory working group for the rapid diagnosis of emerging infections case definitions for surveillance of severe acute respiratory syndrome (sars) (revised summary of probable sars cases with onset of illness from serologic and molecular biologic methods for sars-associated coronavirus infection detection of infectious bronchitis virus antigen from experimentally infected chickens by indirect immunofluorescent assay with monoclonal antibody clinical evaluation of real-time pcr assays for rapid diagnosis of sars coronavirus during outbreak and post-epidemic periods evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign this study was supported by the grants from national major projects of national committee of science and technology of china ( aa z ). we thank dr. che for generously providing three monoclonal antibodies against the n protein of sars-cov. key: cord- - e vjtop authors: schildgen, oliver; jebbink, maarten f.; de vries, michel; pyrc, krzysztov; dijkman, ronald; simon, arne; müller, andreas; kupfer, bernd; van der hoek, lia title: identification of cell lines permissive for human coronavirus nl date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: e vjtop six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus nl . two monkey epithelial cell lines, llc-mk and vero-b , showed a cytopathic effect (cpe) and clear viral replication, whereas no cpe or replication was observed in human lung fibroblasts mrc- s. in rhabdomyosarcoma cells, madin–darby–canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked the results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus nl . the human coronavirus nl was detected in (van der hoek et al., ; fouchier et al., ) and meanwhile appeared to be a serious pathogen causing infection of the upper and lower airway in children and elderly (e.g. bastien et al., ; van der hoek et al., ) . although the nl infections occur frequently, the virus remained undetected until the usage of a new virus discovery strategy (vidisca), which led to its detection. the reason for this late detection despite the use of classical virus isolation techniques remains unclear. difficulties to initiate an nl -infection in the cell lines routinely used for diagnostics, or lack of cpe in these cells may be responsible. in order to address these questions with the aim of the development of a more sensitive diagnostic, we consequently tested several cell lines commonly used for routine diagnostic purposes for their ability to support coronavirus nl replication. the following cell lines were cultivated with mem hanks'/earle's medium (invitrogen, breda, the netherlands) with % bovine fetal calf serum (fcs) at • c: human lung fibroblasts (mrc- : atcc-ccl- ), rhabdomyosarcoma * corresponding author. cells were inoculated with moi . (i.e. , tcid /ml) in mem hanks'/earle's medium with % fcs for h at • c. in order to avoid subsequent detection of dead input virus or free input nl -rna, the inoculation medium was removed, cells were washed twice with phosphate buffered saline, and fresh pre-warmed medium ( • c) was added. aliquots ( l) from the supernatant were sampled at inoculation and then every h up to day post inoculation (p.i.). cells were viewed under a microscope daily and the occurrence of cpe was recorded. total rna was extracted from the supernatant by the silicaaffinity based boom extraction method (boom et al., ) and eluted in l water. reverse transcription was performed with moloney murine leukemia virus reverse transcriptase (mmlv-rt) (invitrogen; u per reaction) and ng of random hexamers (amersham biosciences) in mm tris ph . , mm kcl, . % triton-x , mm of mgcl and m of each dntp at • c for min in a total volume of l. virus yield was determined by real time pcr using the platinum quantitative pcr supermix-udg (invitrogen) (fig. ) . a l of cdna was amplified in l × platinum quantitative pcr super mix-udg (invitrogen) with . mm of mgcl , m of specific probe labeled with fam and tamra and m of each primer. the following primers were used for hcov-nl -sense: -gcgtgttcctaccagaga gga- ; anti-sense: -gctgtggaaaacctttggca- ; probe: -fam-atgttattcagtgctttggtcctcgtgat-tam-ra- . the reaction was carried out on an abi prism ® sequence detection system (applied biosystems foster city, california). following the udg treatment for min at rna from a titrated virus culture was used as input for the standard curve. the concentration in the viral rna stock was determined using in vitro transcribed rna. the viral growth kinetics examined by real time pcr revealed that llc-mk cells as well as vero-b cells, both monkey kidney cell lines, supported the amplification of viral genomes, resulting in high titers with an up to -fold increase in virus yield. in both cell lines an initial decrease in the number of genomes in the supernatant was observed that was most obvious for vero-b cells inoculated with coronavirus nl . the number of genomes increased from days to post inoculation and reached a plateau on day , resulting in equal kinetics. this observation confirmed and extended the earlier finding by fouchier et al. ( ) and kuiken et al. ( ) who found vero permissive for nl and sars coronavirus, respec-tively, consequently giving rise to the assumption that vero cells in general are permissive for nl . only marginal increases in the number of genomes that were in the range of the intra-assay variability were observed for mdck cells, ms cells, and rd cells, giving raise to the hypothesis that these cell lines were not permissive or at most not fully permissive for coronavirus nl . taking into account that these cell lines also displayed a cpe (see below) one may speculate that inoculation of mdck cells, ms cells, and rd cells with coronavirus nl is toxic for these cell lines, probably due to the temporarily expression of viral genes of nl that was not earlier. however, it remains to be investigated whether there is indeed no intracellular genome amplification or whether these cell lines solely cannot excrete viral particles into the cell culture supernatant, both of which is possible in case of llc-mk and vero-b cells. most interestingly, lung fibroblasts were not permissive for nl at all, suggesting that these cell lines have de-differentiated in cell culture. cpe were observed in all cell lines from days to (mdck, ms, vero-b , llc-mk ) or at days - (rd). this effect was virus-specific since negative control cells displayed no cytopathicity until day . the observed nl -cpe was virtually indistinguishable from cytopathic effects caused by other viruses; thereby, from the morphological changes observed, the observed cpe resembles the cpe induced by picornaviruses. infected cells became small and detached from the cell culture flask. thereby, no plaque formation but a diffuse initiation of cpe formation was observed. neither a cpe nor an increase in the number of genomes was observed for mrc- lung fibroblast. the results indicate, in analogy to earlier observations with human metapneumovirus (deffrasnes et al., ) , that some cell lines routinely used for viral laboratory diagnostics are permissive for coronavirus nl infection. the lack of a specific cpe but also the limited number of fully permissive cell lines may be the reason for the "late" identification of the virus, as the cpe, if it occurred, may have been interpreted as cpe from known viruses. this latter observation that the cpe was unspecific or may have been absent at all may explain why isolation of new nl isolates has not been described since the initial studies by van der hoek et al. ( ) and fouchier et al. ( ) . as llc-mk and vero are very common cells in diagnostic facilities, the original isolation of nl may thus have been an exceptional event and diagnostic culture on these cells may be very unrewarding. thus, also in case of a lacking cpe it may be worth to monitor cell culture supernatants by pcr assays in case of a given clinical evidence for a viral respiratory infection. in summary, in order to improve the laboratory diagnosis of coronavirus nl , we would recommend first to perform the pcr analysis of the specimen and then to subsequently inoculate a broad spectrum of cell lines, followed by an additional pcr analysis of the cell culture supernatant. by this algorhithm there would be no delay in the diagnosis, because in routine diagnostics time is often a critical issue, but additional information may be available after all procedure were finished. although we have not compared the differences in the sensitivity of the combined pcr/culture/pcr protocol versus a pcr-only protocol, a number of earlier publications describing the identification of "new" viruses clearly demonstrate that with respect to the spectrum of viruses the combined approach appears to be superior (e.g. van den hoogen et al., ; van der hoek et al., ; fouchier et al., ) . rapid and simple method for purification of nucleic acids human coronavirus nl infection in canada analysis of replication kinetics of the human metapneumovirus in different cell lines by real-time pcr a previously undescribed coronavirus associated with respiratory disease in humans newly discovered coronavirus as the primary cause of severe acute respiratory syndrome a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus croup is associated with the novel coronavirus nl this work was supported by grants from the else kröner-fresenius-foundation (bad homburg, germany; grant-number a / //f ) and the medical faculty's research support program bonfor (o- . ). lvdh is supported by vidi grant . . from netherlands organization for scientific research (nwo). we thank christian drosten for design of the primers used in real time pcr. key: cord- - wn ivq authors: berry, jody d; jones, steven; drebot, michael a; andonov, anton; sabara, marta; yuan, xin y; weingartl, hana; fernando, lisa; marszal, peter; gren, jason; nicolas, brigitte; andonova, maya; ranada, francesca; gubbins, michael j; ball, t.blake; kitching, paul; li, yan; kabani, amin; plummer, frank title: development and characterisation of neutralising monoclonal antibody to the sars-coronavirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: wn ivq there is a global need to elucidate protective antigens expressed by the sars-coronavirus (sars-cov). monoclonal antibody reagents that recognise specific antigens on sars-cov are needed urgently. in this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mabs) against the sars-cov is presented, based upon their specificity, binding requirements, and biological activity. initial screening by elisa, using highly purified virus as the coating antigen, resulted in the selection of mabs to the sars virus. subsequent screening steps reduced this panel to seventeen igg mabs. a single mab, f g , is specific for the nucleoprotein as seen in western immunoblot while five other mabs react with the spike protein. two of these spike-specific mabs demonstrate the ability to neutralise sars-cov in vitro while another four western immunoblot-negative mabs also neutralise the virus. the utility of these mabs for diagnostic development is demonstrated. antibody from convalescent sars patients, but not normal human serum, is also shown to specifically compete off binding of mabs to whole sars-cov. these studies highlight the importance of using standardised assays and reagents. these mabs will be useful for the development of diagnostic tests, studies of sars-cov pathogenesis and vaccine development. the sars-coronavirus (sars-cov) is recognised as the causal agent of severe acute respiratory syndrome (sars) in humans. this virus caused nearly deaths and infected more than people in various affected countries throughout the world (stadler et al., ) . the sars-coronavirus spike protein has only - % pairwise identity at the amino acid level to the spike proteins of other previously characterised coronaviruses. recently, the genomes of sars-cov isolates, implicated in the toronto outbreak, were sequenced in their entirety (marra et al., ; rota et al., ) . the production of mabs to the sars-cov virus is critical for diagnostic development, vaccine research and studies of viral pathogenesis. assays that detect the presence of virally encoded proteins or nucleic acids may be preferable for diagnosis of sars infections as the development of serum antibodies in infected individuals is quite protracted (li et al., a) . coronaviruses are enveloped, single-stranded rna viruses that replicate in the host cell cytoplasm (fields et al., ) . the coronaviruses form a single genus of the family coronaviridae and the virions are large ( - nm in diameter) pleomorphic, but generally spherical, particles. virions of most coronaviruses contain three major proteins: the phosphorylated nucleocapsid protein (n); a small membrane-embedded glycoprotein (m); and a large club-shaped peplomer glycoprotein (s) which appears in em micrographs as protruding spikes nm in length. the m protein is synthesised on ribosomes bound to the endoplasmic reticulum and accumulates in the golgi apparatus. the subcellular localisation of m protein to the golgi is believed to influence the site of virus budding in the infected cell. the s-protein mediates many of the biological properties of the virus, including attachment to cell receptors, penetration, and cell-fusion, and it is the major target for virus-neutralising antibodies (collins et al., ; talbot et al., ; wege et al., ; jimenez et al., ; laude et al., ; godet et al., ) . a portion of the s glycoprotein that is not incorporated into budding virions is transported to the plasma membrane of the cell where it remains bound to the cell surface (gerna et al., ) . coronaviruses infect a wide range of mammalian hosts to produce a variety of disease outcomes including respiratory disease, enteritis and encephalitis. antigenic similarities between various coronaviruses have been demonstrated to reside in the s-protein and have been used to study the evolution of this virus family (brian et al., ) . for most coronaviruses causing enteric and respiratory diseases the pathophysiological events leading to clinical symptoms are due to the acute cytocidal infection of the target cells. these infections can be limited by the local immune response resulting in the production of secretory antibodies specific for the s-protein (enjuanes et al., ) . in contrast, many coronaviruses are maintained and spread in the population as inapparent and subclinical infections. the sequence of events leading to chronic versus acute disease is unknown but likely depends on the expression of viral genes, the functional impairment of host cells, and the interaction with the host immune response. there is a critical need to elucidate the immunologic basis for protection against sars-cov infection. recently the sars s-protein was shown to be a functional fusogen and is about - kda in size (xiao et al., ) . a host cell receptor, angiotensin-converting enzyme (ace- ) was recently identified as a functional receptor for the sars-cov, and mediated infection of t cells in vitro (li et al., b) . therefore, antibody responses to the s-protein may neutralise the infectivity of the sars-cov. the immunogenetics of antibody responses to protective epitopes is of particular importance and will lead to a clearer understanding of the nature of protective antibody responses to sars. lastly, the production of protective monoclonal antibodies may lead to the development of new recombinant therapeutic antibodies in order to provide rapid protection in sars patients. in the present work, a description of the development of murine mabs against the sars-cov involved in the toronto sars outbreak is presented. the mabs were analysed for pertinent immunochemical properties and for their ability to neutralise sars-cov in vitro. for preparation of partially purified whole-virus antigen, sars-cov was expanded after plaque purification in vero- cell monolayers and partially purified through a sucrose cushion. highly purified sars-cov (tor- strain, isolated from a patient infected in the toronto sars outbreak; krokhin et al., ) was prepared in the same way, except the viral particles were further purified using gradient centrifugation. briefly, ml of supernatant from sars-cov infected vero- cells was concentrated first on top of a cushion of iodixanol in a beckman sw rotor (mississauga, on). the virus was subsequently mixed to form a suspension of % iodixanol and subjected to centrifugation in a beckman nvt rotor (mississauga, on) for . h at , × g. fractions were collected from the bottom of the self-generated gradient, tested by western immunoblot with sars-cov-infected, convalescent human patient serum, and the sars-cov positive fractions were pooled and dialysed against phosphate buffered saline (pbs). the dialysed virus preparation was further concentrated by ultracentifugation for . h at , × g. immunisation of mice was performed according to ncfad standard operating procedures under iso . five-to six-week-old female balb/c mice (charles river, wilmington, ma) were injected subcutaneously (sc) with g of beta-propiolactone-inactivated, partially purified sars-cov (tor- strain) with an equal part of complete freund's adjuvant (h -ra, cfa) from difco (bd, oakville, on) on day . on day the mice received g of partially purified sars-cov s.c. in incomplete freund's adjuvant (ifa) in a total volume of l. on days and , the mice received g of the same antigen in a total volume of l sc with ifa. the mice received a final booster injection with g of highly purified sars-cov in l pbs to the intra-peritoneal cavity days prior to hybridoma fusion. mice were euthanised by anaesthesia overdose and exsanguinated by cardiac puncture. the spleens were subsequently excised under aseptic conditions. infected vero cells were scraped off of cm corning tissue culture flasks (corning, ny) and clarified by centrifugation. a borate saline mixture ( . m boric acid, . m, nacl, . m naoh) was used to wash the cell pellet twice and the pellet was resuspended in ml borate saline + % triton x- for each t flask. the pellet was kept at • c using a water bath and sonicated for ten minutes at % power. the debris was pelleted via centrifugation at , × g for min and the supernatant was collected and stored at − • c in aliquots for later use. removal of mouse spleens, preparation of spleen and myeloma cells, and the fusion for hybridoma production were performed according to ncfad standard operating procedures under iso . ampoules of the myeloma cell line p x ag . (atcc, rockville, md) were thawed week prior to fusion and grown in bd cell mab quantum yield medium in the presence of -azaguanine (sigma, oakville, on). cells were in log-phase growth at the time of fusion. hybridoma fusion was performed essentially as originally described (kohler and milstein, ) with the following modifications. briefly, spleens were harvested days after a final boost with a given antigen and the splenocytes were prepared by splenic perfusion as follows. under aseptic conditions, the spleens were perforated with a cm syringe with a gauge sterile disposable needle. the spleen cells were perfused out of the spleen with injections of serum free bd cell mab quantum yield medium (bd-pharmingen, oakville, on). two identically immunised mouse spleens were used to produce these hybridoma clones. the fusion was performed using the p x ag . myeloma line in log-phase growth. peg ( ml; roche, basel, sw) was added drop-wise over min while gently tapping the tube containing the thoroughly washed myeloma-splenocyte pellet. the peg was slowly diluted out over three minutes with serum free bd-cell mab quantum yield medium. the cells were resuspended and mixed into ml of stemcell clonacell medium d (hat) (vancouver, bc) containing ml origen hybridoma cloning factor (hcf) (igen, gaithersburg, md) and plated out according to the manufacturer's instructions. the plates were incubated at • c under a % co overlay for - days in humidified chambers. visible colonies were picked from the plates after approximately weeks growth and placed into -well plates containing - l of complete hybridoma medium supplemented with × hypoxanthine thymidine (sigma, oakville, on), % hcf and % fbs (wisent). supernatants were screened days later via elisa using purified virus as antigen. isotyping was performed using a commercial murine isotyping dipstick test (roche, basel, sw) according to the manufacturer's instructions. hybridoma culture supernatants were concentrated - fold using amicon stirred cell nitrogen concentrators with kda cutoff millipore (ym- ) membranes (both from millipore, billerica, ma). hybridoma culture supernatants were assayed for binding to highly purified sars-cov in an elisa assay when the cultured cells were confluent in the culture plates. the costar -well / well elisa plates (corning, ny) were coated with either bovine serum albumin (bsa) or highly purified sars-cov ( - ng/well) in pbs overnight at • c and then blocked with . % bsa in pbs, for h at • c. the supernatant ( l/well) was incubated neat for h at • c. the elisa plates were washed times with distilled water and patted dry on a paper towel. a pan-goat anti-mouse igg-hrp antibody (southern biotechnology associates, birmingham, alabama) was diluted to : in . % bsa in pbs, applied to the elisa plates for min at • c, and then washed as described above. positive binding was detected with commercial abts used according to the manufacturer's instructions (roche, basel, sw). the od was read at nm at and min intervals after addition of the developing reagent. mouse immune and preimmune sera were diluted : with %-bsa in pbs for use as positive and negative controls, respectively, and for the establishment of the hybridoma screening assay. competition elisa (c-elisa) measured the binding of murine mabs to highly purified sars-cov in the presence of human serum. infected serum was from confirmed infected patients with well-established sars. these samples were from the initial set of patients from which the virus was isolated. plates were coated with highly purified sars-cov at ng per well, and normal human serum or serum from convalescent sars patient s , serially diluted in % bsa-pbs, was allowed to react on the pre-blocked plates for min. mab f g (spike specific) or f g (nucleoprotein specific) were pre-diluted to a concentration that gives approximately half-maximum optical density after h development in the presence of no competing serum. the diluted mab preparations were then applied to the wells, and the incubation and development of the c-elisa was performed as described above. whole virions and sars-cov-infected vero cell lysates, at a final total protein concentration of g per lane, were boiled in sds-loading buffer for min. the samples were loaded in criterion pre-cast gels (biorad, mississauga, on) and electrophoresed at v for min. the proteins were transferred to immobilon nylon membranes (millipore, billerica, ma) for h at room temperature at v, or overnight at v at • c. blots were blocked with % bsa in tbs, rinsed three times with tbs, and reacted with monoclonal antibody overnight at • c. the antibody supernatants were reacted neat and the concentrated supernatants were diluted : in . % bsa in pbs. blots were washed three times with tbs-tween- ( . %) for min before being incubated with secondary antibody (same as above) at : in tbs, . % bsa for h. the blots were washed as above and developed using dab insoluble substrate (pierce, rockford, il). monolayers of sars-infected vero cells were stained as follows. glass slides were coated with infected vero cell monolayers and fixed with acetone. the slides were irradiated with kilogreys from a cobalt gamma irradiator, removed from biocontainment, and then stored at − • c. dilutions of antibodies and test sera were made initially in -well plates (bd-falcon, oakville, on) in sterile phosphate-buffered saline (ph . ). samples were allowed to incubate for min in a • c incubator, and were washed with distilled water. fluorescein labelled goat anti-mouse secondary antibodies (sigma, oakville, on) diluted in pbs were added to the slides and incubated for min at • c, washed as above, and air dried. slides were coated with mounting medium and stored at • c until examined. immuno-dotblot analysis was performed using immobilon nylon membranes (millipore, billerica, ma). a total of l of sars-cov antigen (infected vero cell lysate) or g of highly purified virus is coated (per spot) for h at • c. in an attempt to further characterise the immunochemical properties of the individual mabs, antigen was pre-treated in several different conditions as follows: untreated native antigen; denatured by heat treatment at • c for min; denatured by sodium dodecyl sulphate treatment (sds) % prior to coating on the membrane; denatured by both heat treatment and sds as above together; reduced with beta-mercaptoethanol ( %) prior to coating on the membrane; denatured by temperature as above and reduced as above, together; denatured by both heat and sds in the presence of beta-mercaptoethanol. antigen-coated blots were blocked with % bsa in tbs for h at • c, and rinsed three times with tbs, prior to incubation with mabs. the concentrated mab supernatants were diluted : in . % bsa in tbs (tris-buffered saline, ph . ) and reacted overnight at • c on a rotary platform shaker with gentle agitation. blots were washed three times with tbs-tween- ( . %) for min before being incubated with secondary antibody (goat anti-mouse igg-hrp, southern biotech, same as above) at : in tbs, . % bsa for h. the blots were washed as above and developed using dab insoluble substrate (pierce, rockford, il). the elisa positive monoclonal antibodies were screened for neutralisation of sars-cov using two independent formats. the first was a standard plaque reduction assay and the second measured reduction of cytopathic effect (cpe) in a microtiter format. a standard plaque reduction neutralisation test was performed as previously described (beaty et al., ) using highly purified sars-cov. briefly, mixtures of pre-titred ( pfus) sars-cov and serial two-fold dilutions of hybridoma supernatant were incubated at • c for h and added to six well plates containing vero cell monolayers. after incubation at • c for h, a nutrient-agar overlay was added and the plates were placed in a co incubator at • c for approximately days. a second overlay was then added which contained neutral red as a vital stain. plates were then checked periodically over the next few days for plaque formation. the highest dilution tested that produced a plaque reduction of at least % was defined as the titration end point. a microtiter format cpe reduction assay was used to test the sars-cov reactive mabs for neutralisation of sars-cov and transmissible gastroenteritis virus diamond strain (kindly provided by dr. susy carman, lsd, university of guelph). briefly, concentrated hybridoma supernatants were diluted : in cell culture medium and incubated with tcid of either highly purified sars-cov (tor- strain), or tgev, for h at • c, for a final dilution of : . the virus-antibody mix was then transferred onto cell monolayers in -well plates (costar, corning, ny). vero v- cells were used for the sars-cov, st cells for the tgev. the plates were incubated until cpe developed in virus back titration controls. elisa screening on highly purified sars-cov identified a panel of mabs reactive to the sars-cov antigen preparation. negative screening on bsa reduced this number to a panel of igg/k type mabs ( fig. ; table ). in general, binding reactivity of these mabs is greatly affected by both the conformation and purity of the antigen as illustrated by the decreased binding of these mabs, when tested in elisa, to heat denatured pure sars-cov as antigen compared to native virus (fig. ) . this clearly shows the importance of selecting suitable antigen for screening assays. western immunoblot analysis identified mabs to the sars-cov spike and nucleoprotein. five mabs (f g , g , g , g and g ) react specifically with the sars-cov spike protein in western immunoblot. these mabs recognise spike in western immunoblot on both highly purified virus and infected cell lysates (fig. ) , but do not react with mock-infected cell lysates (data not shown). this result suggests that these mabs target linear epitopes within the spike protein. another mab, f g , bound fig. . elisa reactivity of mabs with whole, inactivated sars-cov. hybridoma supernatants were tested at a : dilution in pbs + . % bsa on pre-blocked plates, coated with ng per well of inactivated, highly purified sars-cov. positive clones were identified as having positive binding (color) in wells that were at least four-fold higher than the background level reactivity on bsa. antigen legend: black bars-native, highly purified sars-cov; white bars-heat denatured, highly purified sars-cov; grey bars-bsa control. a virus neutralisation tests were performed independently in separate containment laboratories (nml, national microbiology laboratory; ncfad, national centre for foreign animal disease). the last six rows denote neutralising clones. b only a single dilution of / was tested in microwell format. c protein specificity tests shown here were determined by western immunoblot with purified virus and infected cell lysate under denaturing conditions (fig. ) . u, unknown target antigen. d immuno-dotblot was performed using antigen treated under various conditions described in section . n, native; h, heat denatured, • c for min; d, sds treated ( %); h+d, heated in the presence of sds ( %); r, treated with reducing agent, beta-mercaptoethanol ( %); h + r, heated in the presence of reducing agent, beta-mercaptoethanol ( %); a, treated with heat, sds ( %) and reducing agent beta-mercaptoethanol ( %). e immune-fluorescence on whole cell slides infected with sars-cov (see fig. ); ++, strong positive reaction; +, positive reaction; ±, weak positive reaction; −, negative reaction. f epitope properties described as follows: l, linear or continuous; e, surface exposed; c, conformational; p, protective in vitro; nd, not determined. to the nucleoprotein in western immunoblot assays. interestingly, the majority of the identified mabs bound to the spike protein and not the nucleoprotein, despite the strong sero-reactivity of the polyclonal serum of the immunised mice for the nucleoprotein (fig. ) . the identity of the target antigen of eleven other mabs could not be determined by western immunoblot analysis. this observation suggests that these mabs likely target conformational epitopes that are sensitive to the conditions employed in such analyses. work is planned to identify the specific targets of these mabs. immuno-dotblot analysis reveals a spectrum of conformational requirements for binding (summarised in table ). the effects of different denaturing treatments on the binding activity of a subset of neutralising and some non-neutralising mabs were examined using immuno-dotblot assays on infected lysates compared to uninfected lysates. the series of conditions tested include exposure to heat, detergent, a reducing agent, and combinations thereof. interestingly, mab f g , which binds to the nucleoprotein in western immunoblot, does not bind to the sars-cov antigen in immuno-dotblot under any of the conditions tested. the inherent charge of the highly phosphorylated nucleoprotein, or of the immobilon membrane used in the assays, may provide an explanation for these results. immuno-dot blots were not performed with mabs f g , f g , f g , f g , or f g , however the binding of these mabs is considered conformational as they do not bind to sars-cov proteins in western immunoblot (fig. ) . the binding of spike protein specific mabs f g , f g , and f g is inhibited in immuno-dotblot assays when antigens are treated with heat plus detergent, or heat plus detergent plus reducing agent. when the immuno-dotblot assay was instead performed using purified virus particles, the result was identical for nucleoprotein specific mab f g , and spike specific mabs f g , f g , and f g (data not shown). antigen pre-treatment for western immunoblot, which depends on the application of an electrical current, differs when compared to passive adsorption in elisa and immuno-dotblot assays, and clearly do not always correlate with one another. these studies illustrate the need to use multiple assays for epitope characterisation and reveal limitations of current epitope classification schemes. the spike protein is an immunodominant antigen when inactivated sars-cov is used as an antigen. murine antibody responses to inactivated whole sars-cov are focused upon the sars spike and np protein as shown by the specificity of the recovered mabs. while multiple sars-cov proteins appear to be the target of mouse serum antibody as shown in western blot (fig. , immune sera) , the majority of the mabs recognise the spike protein in western immunoblot. in a sars-cov infection, the host immune system is exposed to a large load of nucleoprotein antigen, due to the presence of replicating virus in infected tissues. this would suggest that a competitive elisa format based upon the np as the target antigen might provide fig. . competition elisa measuring the binding of murine mabs to highly purified sars-cov in the presence of human patient serum. dilutions (as indicated) of a normal human serum control (white bar), or serum from convalescent sars patient s were applied to wells coated with highly purified whole sars-cov. mab f g (spike specific; black bars) or f g (nucleoprotein specific; grey bars) were then added to the reactions. the results depicted for the pooled normal human serum (nhs) represent the mean of three replicate tests performed in the presence of mab f g combined with three replicate tests performed in the presence of mab f g . results are representative of identical assays performed in duplicate with gamma-irradiated patient sera ( mrad) (*p = . , **p = . , student's t-test). increased sensitivity for early detection of infection. towards this goal, the ability of sars convalescent serum to compete for binding of mabs to the nucleoprotein or to spike on whole sars-cov was tested via elisa. the serum samples were gamma-irrradiated prior to use. antibodies in the serum of patient s clearly inhibit binding of both the nucleoprotein mab f g and the spike protein specific mab f g (fig. ) . in contrast, antibody in pooled normal human sera does not compete for binding by either mab. while not a statistical analysis these data show that these mabs are useful for the development of serological competition assays which can be subjected to validation tests. the sera from several other sars-cov infected patients demonstrate a similar ability to inhibit binding by the f g (nucleoprotein specific mab) and f g (spike specific mab). however, in some samples there is inhibition of only the binding to nucleoprotein without inhibition of the mab to the spike protein suggesting that spike antibody responses take longer to develop in infected humans (data not shown). the predominance of mabs to the spike protein in mice immunised with intact viral particles led us to test for biological activity in virus neutralisation assays. the sars-cov spike protein is the target of neutralising antibodies. neutralisation positive mabs bind both to linear epitopes in the spike protein and to unknown conformational epitope(s) either in the spike protein or in other proteins. a total of six mabs were identified that could neutralise sars-cov infectivity: f g , g , g , g , g , and g (table ) . significantly, two of these mabs, f g and , were positively identified as being specific for spike, as determined by western immunoblotting. the specific targets of the remaining neutralising mabs remain to be elucidated. no cross-neutralisation was observed for the animal coronavirus tgev. this shows that these mabs are specific for sars-cov epitopes and do not cross-neutralise via tgev epitopes. the remaining mabs were unable to prevent viral growth when they were applied to the neutralisation assays. these data suggest that vaccines capable of engendering neutralising antibody responses to the spike protein may be effective in blocking infection with sars-cov. the four western immunoblot negative, virus-neutralising mabs were tested for their ability to bind native sars-cov in infected cells by immunofluorescence assay. nonneutralising mab f g was used as a positive control, since this mab recognises spike protein in immunohistochemical staining of infected vero cells (data not shown). immunofluorescence analysis reveals that the neutralising mabs f g , g , g , and g specifically recognise sars-cov infected but not uninfected vero cells (fig. ) . irrelevant, isotype matched mabs, produced in an identical fashion, do not react with sars-cov infected vero cells. the sars-neutralising mabs bind epitopes with higher conformational requirements than the non-neutralising mabs as they are less tolerant to denaturation of the epitopes. the method of antigen preparation and quality of the antigen greatly affect the interpretation of mab binding results obtained from immuno-dotblot assays. importantly, none of the mabs react with mock-infected lysates as assayed in immuno-dotblots (data not shown). this observation suggests that the majority of the neutralising mabs likely target surface exposed, protein epitopes on the native viral particle. one such putative protective antigen has been identified as the spike protein by western immunoblot analysis with neutralising mabs f g and f g . antigen quality and conformation affects the binding of the anti-sars-cov mabs in elisa. while purified virus is clearly the optimal antigen tested in this series of experiments, the lower quality sars-cov-infected vero cell lysates are, however, much easier to prepare for diagnostic assays. therefore, the limits of mab binding to sars-cov infected vero cell lysates were further examined via elisa. the majority of these mabs exhibit decreased binding when the antigen is heat denatured ( table ) . heat denaturation has very little effect on the binding of non-neutralising mab f g , which also maintains a high level of binding in elisa using infected vero cell lysates. f g does, however, show higher background on the irrelevant antigen, bsa, and has inconsistent reactivity in western immunoblots with heat denatured viral lysate (fig. , table ). the combination of lower quality antigen in infected vero cell lysates, along with heat denaturation of the antigens, has a stronger negative effect on binding by the neutralising mabs compared to non-neutralising mabs (table ) . indeed, regardless of western immunoblot reactivity, the non-neutralising clones retain a greater ability to bind heat denatured antigen compared to the neutralising mabs (lower mean percent reduction in od per group p < . , student's t-test, table ). this supports the earlier observation in elisa on purified sars-cov (fig. ) that shows that the neutralising mabs have higher requirements for native epitope conformation compared to non-neutralising mabs. the higher conformational requirement by neutralising mabs may help to explain some discrepancies observed in the immuno-dotblot methods. the immuno-dotblot is, overall, a less sensitive method, and the results are more difficult to quantify, compared to elisa. for example, mab f g binds to the sars-cov spike protein in western immunoblot and neutralises sars-cov infection in vitro. while binding in western immunoblot generally suggests the epitope is linear in nature, nonetheless, heat treatments clearly affect binding of some mabs to the whole virion. the immuno-dotblot data reveal that with the lower quality antigen of the infected vero cell lysate, under the conditions of heat-plus detergent, or heat-plus detergent and reducing agent, mab f g cannot bind (table ) . this paper describes the development of murine mabs which recognise sars-cov antigens in elisa, immuno-dotblot, western immunoblot, on the surface of infected cells, and in neutralisation assays. these data are consistent with the appearance of coronavirus antigens on the surface of the infected cell during replication (talbot et al., ) , although the fixation process may allow for reactivity of these mabs with internal antigens as well. the conformational sensitivity of most of the sars-cov neutralising mabs is consistent with properties of neutralising mabs raised against other enveloped viruses, which generally require more native conformation for binding (wilson et al., ; zwick et al., ) . the immuno-dotblot assays contradict the classification of several putative epitopes as being linear as is suggested by positive western immunoblot reaction (for example with neutralising mab f g ). indeed, it appears that the strict traditional classification of epitopes as being linear or conformational must account for a broader spectrum of conformational requirements, especially when dealing with antigens of variable quality in different immunological tests. however, in general the neutralising mabs can be considered to have a higher requirement for correct epitope conformation compared to non-neutralising mabs in elisa. it will be important to verify, under optimised conditions (opstelten et al., ) the use of viral lysates designed for maximal recovery of coronavirus proteins, and to this end the production of high quality recombinant protein antigens will provide useful insights. unfortunately, preparation of highly purified viral antigen requires enormous efforts under bio-containment conditions, which emphasises the need for a quality recombinant antigen assay. collectively, these data demonstrate that development of mab-based diagnostic tools for the detection of sars-cov infection is well within reach. the spike protein of the sars-cov is a target of neutralising mabs. epitopes on the spike protein provide neutralising targets on sars-cov in vitro, and this is consistent with the spike being the target of neutralising antibodies for other coronavirus strains (godet et al., ) . moreover, these mabs may be useful for the identification of protective epitopes for vaccine formulations (enjuanes et al., ) . studies are underway to determine the identity of the antigen(s) recognised by the neutralising, western immunnoblot negative mabs. preliminary analysis of the nucleotide and predicted amino acid sequences of the cloned v h and v l coding regions of these mabs suggests that the mabs are distinct. this observation implies that the hybridoma clones expressing the anti-sars-cov neutralising mabs were derived from individually rearranged and clonally selected b cells in vivo. it also reveals that there is no apparent consensus sequence within the complementarity determining regions that is required for the mabs to exhibit virus neutralisation activity. this finding makes it feasible to engineer multiple mabs with high specificity and avidity for various sars-cov epitopes for preparations of defined cocktails of therapeutic mabs. a detailed description of the immunogenetic properties of these mabs is in preparation (berry et al., manuscript in preparation) . the np specific mab, f g , is useful in competitive elisa with patient sera. this is important as early detection of sars infections is key to risk management of this disease. this is the first description of neutralising mabs from a host immunised with whole sars-cov and these antibodies should prove useful for the development of new diagnostic tests, studies of antigenic variation, and vaccine development in the global fight against sars. arboviruses proceedings of the fourth international symposium on neonatal diarrhea. s.d. acres monoclonal antibodies to murine hepatitis virus- (strain jhm) define the viral glycoprotein responsible for attachment and cell-cell fusion tropism and immunoprotection in transmissible gastroenteritis coronaviruses fields virology reactivity of human coronavirus oc and neonatal calf diarrhoea coronavirus membrane-associated antigens major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein critical epitopes in transmissible gastroenteritis virus neutralization continuous cultures of fused cells secreting antibody of predefined specificity mass spectrometric characterization of proteins from the sars virus: a preliminary report antigenic structure of transmissible gastroenteritis virus. i. properties of monoclonal antibodies directed against virion proteins profile of specific antibodies to the sars-associated coronavirus angiotensin converting enzyme is a functional receptor for the sars coronavirus envelope glycoprotein interactions in coronavirus assembly sars-beginning to understand a new virus topographical mapping of epitopes on the glycoproteins of murine hepatitis virus- (strain jhm): correlation with biological activities hybridoma antibodies to the murine coronavirus jhm: characterization of epitopes on the peplomer protein (e ) epitopes involved in antibody-mediated protection from ebola-virus the sars-cov s glycoprotein: expression and functional characterization identification and characterization of a peptide that specifically binds the human broadly neutralizing anti-human immunodeficiency virus type antibody b the authors would like to thank ms. nicole beausoleil, mr. daryl dick, mr. darrell johnstone, ms. kathy frost, ms. hilary holland, and mr. richard nickel for expert technical assistance. mg is supported by a health canada ocs postdoctoral fellowship. thanks to dr. john copps (nc-fad) for expert veterinarian services and for assisting in the design of the emergency animal use document. thanks also to dr. susy carman (university of guelph, canada) and dr. lorne babiuk (vido, canada) for providing animal coronavirus strains. funding for this work was provided by health canada and the canadian food inspection agency. this work is dedicated to the memory of lloyd d. berry. key: cord- -lhe ws authors: wong, sallene; pabbaraju, kanti; wong, anita; fonseca, kevin; drews, steven j. title: development of a real-time rt-pcr assay for detection of resistance to oseltamivir in influenza a pandemic (h n ) virus using single nucleotide polymorphism probes date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: lhe ws resistance to oseltamivir in pandemic (h n ) influenza a virus is linked to an amino acid change from histidine (h) to tyrosine (y) at position in the neuraminidase protein (na). a real-time one step rt-pcr assay using single nucleotide polymorphism (snp) probes was developed to detect this mutation in respiratory specimens. the limit of detection was . copies/reaction for wild-type h rna and . copies/reaction for the mutant h y rna. the assay did not cross-react with other respiratory pathogens. the clinical sensitivity and specificity of the assay was compared to the gold standard sanger sequencing method using sensitive, resistant and negative samples. the sensitivity and specificity was . % and % respectively with the soiv_osel_sen probe designed to detect the h allele and % for the soiv_osel_res probe detecting the y allele. the sensitivity of the assay using nine admixtures of sensitive and resistant alleles was . % and . % with the soiv_osel_sen probe and soiv_osel_res probe respectively. the presence of mixed sensitive and resistant alleles in patient samples and mixtures of in vitro rna were detected reproducibly. this assay can be used for screening of original samples for oseltamivir resistance without the need for culture and phenotypic testing. the influenza pandemic of saw the emergence of a new influenza a (h n ) strain that showed initial resistance to adamantanes and susceptibility to oseltamivir (dawood et al., ) . however, by the summer of , there were reports of isolates with an amino acid change from histidine (h) to tyrosine (y) at position in the neuraminidase (na) glycoprotein that was linked to oseltamivir resistance (chen et al., ; harvala et al., ; janies et al., ; thabet et al., ; who, a) . some of these cases were identified in alberta, canada starting in july . although the prevalence of oseltamivir resistance in the - respiratory season has been low, there have been instances in the recent past of dramatic increases in oseltamivir and adamantane resistance in seasonal influenza a (ifva) over a relatively short time frame in the absence of selective pressure by anti-viral usage (deyde et al., ; dharan et al., ; hauge et al., ; hurt et al., ; janies et al., ; nelson et al., ; pabbaraju et al., ; sheu et al., ) . resistance to oseltamivir has been reported to result from point mutations in several regions of the na protein of the virus (e.g., d g, s g or s n, and h y) (sheu et al., ) . phylogenetic studies have revealed that the oseltamivir-resistant seasonal ifva (h n ) strain evolved from a susceptible strain (clade b) which circulated in the - season by acquiring the h y change in the na gene . currently, the gold standard method for detection of the c t nucleotide polymorphism that leads to the h y amino acid change is dideoxy sanger sequencing of the na gene. neuraminidase inhibition assays including commercial kits such as the na-star ® influenza neuraminidase inhibitor resistance assay (applied biosystems, foster city, ca, usa) have been described for phenotypic resistance testing (hurt et al., ). these phenotypic assays require culturing of the virus, which would not be possible in the case of novel sub-type variants such as the pandemic (h n ) prior to characterization of the virus. both sequencing and phenotypic testing are labor intensive, time-consuming, and expensive. the use of pyrosequencing for monitoring nucleotide changes has also be described, however this requires specialized instrumentation (deyde et al., (deyde et al., , . a reverse-transcriptase pcr coupled with a restriction fragment length polymorphism assay (rtpcr-rflp) has been described for seasonal h n viruses but it is a lengthy process and not practical for high volume testing (guo et al., determination of oseltamivir resistance for seasonal and pandemic h n influenza a viruses has been reported recently using liquid microarrays on the luminex platform (mahony et al., ) . realtime reverse-transcriptase (rt)-pcr assays using single nucleotide polymorphism (snp) probes for allelic discrimination have been described for the detection of h y mutation in seasonal h n viruses (bolotin et al., ; carr et al., ; operario et al., ) and pandemic (h n ) viruses (van der vries et al., ) . rapid methods for the detection of mutations leading to oseltamivir resistance allow diagnostic laboratories to monitor the development and transmission of resistance especially in high risk populations such as immuno-compromised patients and patients requiring antiviral treatment. this study describes the validation of a high throughput, sensitive and specific rt-pcr for detection of the c t nucleotide polymorphism leading to the h y amino acid change in the na protein of pandemic (h n ) virus. this assay could be used in surveillance of tamiflu resistance, detection of a resistant virus and monitoring response to therapy. respiratory specimens submitted to the provincial laboratory for public health (provlab), alberta, canada, between july th and january th for respiratory virus testing, were included in this analysis. the current diagnostic algorithm for testing of influenza a in our laboratory includes primary screening using a real-time rt-pcr assay (infa) targeting the matrix gene of all human influenza a viruses (dawood et al., ) . samples that test positive for ifva are tested subsequently for h , h and pandemic (h n ) sub-types (dawood et al., ; pabbaraju et al., ) . specimens from patients with a significant travel history, epidemiological links to pandemic (h n ) virus, severe respiratory infection, immuno-compromised/bone marrow transplant cases and intensive care unit patients were identified based on the requisitions submitted by the physician. additionally, patients from the community with none of the above predisposing factors for development of resistance were also included in the analysis. selected pandemic (h n ) virus confirmed samples were tested for oseltamivir resistance using the gold standard sanger sequencing method (n = ). sixty-nine specimens were selected for the assay validation, of which were upper respiratory specimens including nasopharyngeal and throat swabs, were lower respiratory specimens including endotracheal aspirate, bronchoalveolar lavage and auger suction. extraction of total nucleic acid from all clinical specimens was performed on the easymag ® automated extraction system (biomérieux canada inc., québec, canada) following the manufacturer's instructions and as described previously (pabbaraju et al., ). primers and two minor-groove-binding (mgb) hydrolysis probes were designed based on the available na gene sequences from genbank (as of september , ) using the primer express tm software [version . . , applied biosystems (abi), foster city, ca, usa]. the two mgb probes were designed for discrimination of sensitive and resistant alleles. the probe specific for detection of the h allele that has been reported to lead to oseltamivir sensitivity (soiv osel sen) was labelled with carboxyfluorescein (fam); and a probe specific for the h y allele that has been reported to lead to oseltamivir resistance (soiv osel res) was labelled with vic at the end; both probes had a non-fluorescent black hole quencher (bhq) at the end. nucleotide sequences of the primers and probes are indicated in table . optimal primer concentrations were determined by checkerboard titrations using concentrations ranging from nm to nm. the optimal probe concentration was determined by testing concentrations ranging from nm to nm. primers were synthesized at the core dna services, university of calgary (alberta, canada) and the probes were purchased from abi. full length amplification of the na gene with the h and h y alleles from pandemic (h n ) virus was performed using primers described previously (mak et al., ) . these amplified fragments were cloned into the plasmid vector pcrii-topo ® and transformed into one shot ® top- chemically competent escherichia coli cells (invitrogen, carlsbad, ca, usa). the recombinant plasmids with cloned na fragments were purified using the qiaprep ® kit (qiagen), linearized using the restriction enzyme hind iii and used as templates for transcription. rna was transcribed using the t ribomax tm express kit (promega, madison, wi, usa) and treated with turbo ® dnase (applied biosystems canada, streetsville, ontario, canada) for degradation of template dna. the purified rna was quantified using the nanodrop tm spectrophotometer (thermo fisher scientific, wilmington, delaware, usa) and the absorbance value was used to calculate the copy number of transcribed rna. amplification was carried out using superscript tm iii platinum ® one-step quantitative kit (invitrogen) in a final volume of l with l of extracted rna as template, nm each of the forward and reverse primers, and nm of each probe. amplification and detection was performed on the sequence detection system ( sds, applied biosystem tm (abi), foster city, ca, usa). the table primer and probe sequences designed for detection of the c t mutation. the snp position is highlighted in bold and underlined. oligonucleotide name oligonucleotide sequence ( amplification conditions were: reverse transcription at • c for min, taq activation for min at • c (there was no difference in ct values with activation for min), followed by cycles of amplification comprising of denaturation for s at • c, annealing and primer extension for min at • c. a threshold of . was used for analysis of amplification curves and the baseline was calculated using default settings on the software (sds version . ). . . sensitivity, dynamic range, specificity and reproducibility of the rt-pcr analytical sensitivity of the real-time rt-pcr was determined by testing serial dilutions of quantified in vitro transcribed rna for both the h and h y alleles in eight replicates. serial dilutions of rna spanning eight logs of template concentration were tested in triplicate to determine the dynamic range of the assay. the primers and probes were designed to be specific for the pandemic (h n ) virus sequence, however, they were evaluated experimentally for cross-reactivity with the following targets using extracted dna/rna from high titre viral and bacterial cultures or positive respiratory samples as template: ifva (seasonal h n , n = ; seasonal h n , n = ; h n , n = ; recombinant h n , n = ), influenza b (ifvb) (n = ), parainfluenza (piv) (types , , , b), respiratory syncytial virus (rsv) (a and b), human coronaviruses (hcov) ( e, oc , nl and hku ), human metapneumovirus (hmpv) (lineages and ), human rhinovirus b, coxsackievirus a , echovirus , human adenovirus type , human bocavirus, chlamydophila pneumoniae, legionella pneumophila and mycoplasma pneumoniae. the inter-assay and intra-assay variability was assessed using three clinical samples each with the h and h y alleles at three levels of viral load as determined by the cycle threshold (ct) values. the samples were tested in triplicate on three independent runs. sixty-nine samples that had been confirmed previously as positive for pandemic (h n ) virus by real-time rt-pcr and sequenced for the na gene were used for testing assay accuracy. these included sensitive (c ) and resistant ( t) samples. nine samples containing admixtures of c and t alleles as determined by close observation of the electropherogram were also included. twenty samples which had tested negative for ifva viruses were included as negative controls. in vitro transcribed rna with the c and t markers were selected at different concentrations to represent high and low viral titre samples. the copy numbers for high titre rna were . × and . × per reaction for c and t, respectively. the copy numbers for low titre rna were . × and . × per reaction for c and t, respectively. at each concentration, a mixture of rna was prepared ranging from % to % of each allele. the rna mixtures were tested in triplicate for the detection of mixed clones and estimation of sensitivity for detection when both alleles are present. conventional sanger sequencing of the region of interest in the na gene was used as the gold-standard method for this study. three hundred and six clinical samples that tested positive for pandemic (h n ) virus from immuno-compromised, intensive care unit cases, patients who had received oseltamivir treatment, had a travel history and from the community were selected for sequencing of the partial na gene including the h y (c t) region. a bp fragment of the na gene was amplified using primers (na f nested sw and na r nested sw) (table ) , and the amplified product was purified using the ez- pcr purification kit (bio basic inc., markham, ontario, canada). sequencing was performed on both strands using the bigdye ® terminator v . cycle sequencing kit (abi) in the abi prism ® -avant genetic analyzer. careful manual examination of the electropherogram at base pair was also performed for the presence of mixed bases. . . dynamic range, sensitivity, specificity and reproducibility of the assay this rt-pcr assay showed a broad dynamic range using in vitro transcribed rna. linear amplification of template was observed between . × to . × and . × to . × copies per reaction for h and h y alleles, respectively ( fig. a and b) . the assay efficiency was . % and . % for the soiv osel sen probe and soiv osel res probe respectively, and the r values comparing template concentrations and ct values were . for both probes. limit-of-detection limit for the assay was determined by testing -fold serial dilutions of quantified rna in triplicate, resulting in . copies and . copies for the h and the h y alleles per reaction, respectively. the primers and probes did not cross-react with high titre respiratory viruses and bacteria listed in the materials and methods section resulting in % specificity. the intra-assay coefficient of variation in ct values for three clinical samples representing a range of viral loads was tested in triplicate on three independent runs. this parameter ranged from . % to . % with the h allele and . - . % for h y samples. the inter-assay variability ranged from . % to . % and . - . % with the h and h y alleles, respectively. accuracy of the rt-pcr assay was determined using samples with c , with t and nine with a mixture of c and t nucleotides at position by the gold standard sanger sequencing method. additionally, samples that were negative for ifva were also tested. the specimens containing the h y allele and h /h y admixtures populations were from patients belong to immuno-compromised and intensive care unit cases identified in the described screening process previously. the ifva negative samples were not detected by the assay indicating good primer and probe specificity. of the samples with the h allele, the soiv osel sen probe resulted as positive and three samples tested negative (table a) . these samples had been tested previously by a screening assay for influenza targeting the matrix gene (infa) (dawood et al., ). the ct values by this screening assay for the three discordant samples were . , . , and . . a negative result suggests either a low viral load or sequence variation in the primer or probe binding region. the sequence of the assay detection region was determined for these samples and analyzed for the presence of mutations. the sample with a ct value of . had no change in the probe binding region indicating that the sample possibly tested negative as a result of low viral load. the samples with ct values of . and . were found to have a base pair mutation at different positions in the probe binding region (fig. ) . researchers using this region for the design of probes to detect oseltamivir resistance should be aware of the presence of these mutations in some isolates. these samples were not detected by the soiv osel res probe as expected by fig. . (a) dynamic range of the developed rt-pcr assay. amplification curves (delta rn vs. cycle number) and standard curve (ct vs. log concentration) for serial dilutions of the plasmid with c nucleotide. assay dynamic range was linear between template concentrations ranging from . × copies/reaction to . × copies/reactions. (b) dynamic range of the developed rt-pcr assay. amplification curves (delta rn vs. cycle number) and standard curve (ct vs. log concentration) for serial dilutions of the plasmid with c t nucleotide. assay dynamic range was linear between template concentrations ranging from . × copies/reaction to . × copies/reactions. performance of rt-pcr assay using soiv osel sen probe in comparison to the gold standard sanger sequencing method. performance of rt-pcr assay using soiv osel res probe in comparison to the gold standard sanger sequencing method. the probe design. fig. shows an alignment of the two snp probes and mutations detected in this region. samples with the h y allele were present either as t (n = ) or admixtures of c and t (n = ). all of the samples with only the t were detected by the soiv osel res probe and not by the soiv osel sen probe. of the admixtures, the soiv osel sen probe detected eight as positive and the soiv osel res probe detected seven as positive. one admixture that was not detected by the soiv osel sen probe (but detected by the soiv osel res probe) showed a major t population and a minor c population based on peak height in the electropherogram and also had a ct value of . for detection of ifva, indicating a low viral load (table a) . two samples with admixtures that had minor populations of the t nucleotide present as determined by sequencing were not detected by the soiv osel res probe (table b) . assay sensitivity for the detection of samples with c by the soiv osel sen probe was % and detection of t by the soiv osel res probes was %. sensitivity for the detection of samples with mixtures of c and t by soiv osel sen probe and soiv osel res probe was . % and . %, respectively. there was no non-specific detection of the h or h y alleles, thus both probes were % specific (tables a and b) . representative amplification curves for the detection of c , t and a mixture of both nucleotides are shown in fig. . the sensitive and resistant samples showed a single amplification curve; whereas the admixtures had two detectable amplification curves as indicated in fig. . to test the ability of the assay for detection of both sequence variants when they are present in various ratios and at different viral loads, a mixture of known template concentration of resistant and sensitive in vitro rna was tested. two mixtures were prepared, one at a low copy number with . × and . × copies/reaction and the other mixture with . × and . × copies/reaction for the sensitive and resistant rna, respectively. in vitro rna with the two sequence variants was mixed in ratios from % to % at both viral loads. in the mixture prepared using higher copies of in vitro rna, the soiv osel res probe detected the presence of t when its proportion ranged from % to % of the total template concentration; this probe did not give a positive result when t was present at concentrations less than %. in the same mixtures, the presence of c was detected by soiv osel sen probe when present at a concentrations ranging from % to % of total template as indicated in table a . for mixtures with low copies of in vitro rna, the soiv osel res probe detected the presence of t when its proportion ranged from % to % of the total template concentration; this probe did not give a positive result when t was present at concentrations less than %. in the same mixtures, the presence of c was detected by soiv osel sen probe when present at a concentrations ranging from % to % of total template and presence of c at lower than % was not detected (table b) . resistance to oseltamivir in seasonal and pandemic influenza a (h n ) has been linked primarily to an h y polymorphism in na glycoprotein sheu et al., ) . the h y amino acid change was first reported in pandemic (h n ) virus in june in an immuno-compromised patient treated with oseltamivir (who briefly note ), and more cases have since been reported sporadically worldwide (baz et al., ; chen et al., ; le et al., ) . as there are still some questions as to whether or not this h y polymorphism impairs viral fitness and transmission (seibert et al., ) , we must be prepared for the possibility that oseltamivir-resistant isolates of the pandemic (h n ) virus could expand in a manner similar to that seen in the previous seasonal h n viruses besselaar et al., ; hurt et al., ; lackenby et al., ; meijer et al., ; sheu et al., ) . screening tools should take into account the low prevalence of oseltamivir-resistance in the pandemic (h n ) strain and ideally detection should be approached with cost effective and simple screening methods (dawood et al., ; harvala et al., ; pabbaraju et al., ; thabet et al., ) . the current gold standard methods for detection of resistance include phenotypic detection using the na-star ® assay from applied biosystems or genetic detection of relevant mutations using sanger sequencing. both of these methodologies are labor intensive, time-consuming and expensive. additionally, phenotypic methods require viral culture which may not be feasible for novel sub-type influenza viruses that are restricted to high-level biosafety environments. other methods such as reverse-transcriptase pcr coupled with restriction fragment length polymorphism assay (rtpcr-rflp), pyrosequencing, microarray and multiplex rt-pcr with luminex detection technologies have also been reported for detection of resistance but require specialized equipment and training (bolotin et al., ; carr et al., ; deyde et al., deyde et al., , guo et al., ; hindiyeh et al., ; mahony et al., ; operario et al., ; van der vries et al., ) . real-time rt-pcr using probe technology is now commonplace in molecular diagnostic laboratories; these assays are highthroughput and amenable to large scale testing of patient samples with a quick turn-around time for results. many laboratories are currently equipped to utilize snp hydrolysis probes for the detec-tion of oseltamivir resistance in pandemic (h n ) virus. this approach has been reported previously for the detection of the c t mutation in seasonal h n (bolotin et al., ; carr et al., ; operario et al., ) and pandemic (h n ) viruses (hindiyeh et al., ; van der vries et al., ) . the possible caveats of using snp probes to detect h and h y include the presence of mutations in the probe region adjacent to the snp positions. this may result in false negative results; the presence of such mutations was detected in this study and has been published previously (hindiyeh et al., ) . to address this issue degenerate probes have been utilized by other investigators, however, the issue of cross reactivity and sensitivity was not investigated thoroughly (hindiyeh et al., ) . snp assays may also be unable to detect minor populations in heterogenous mixtures of h and h y alleles within some specimens. given the concerns about how h y snp assays perform in the context of mixed viral populations and mutations in the probe-binding region, the authors feel that the results of a snp assay should be interpreted with caution and ambiguous results should be confirmed by sequencing. in conclusion, this manuscript describes the development and validation of a snp assay for the detection of h y mutations associated with oseltamivir resistance in pandemic (h n ) virus. the strength of this assay is its ability to identify h y mutations rapidly without specialized sequencing equipment. this tool is still relevant in the post-pandemic period as the pandemic strain is currently the circulating seasonal h n influenza a strain of the - respiratory season. emergence of h y oseltamivirresistant a(h n ) influenza viruses in japan during the - season emergence of oseltamivir-resistant pandemic h n virus during prophylaxis widespread oseltamivir resistance in influenza a viruses (h n ) development of a novel real-time reversetranscriptase pcr method for the detection of h y positive influenza a h n isolates rapid molecular detection of the h y oseltamivir resistance gene mutation in circulating influenza a (h n ) viruses oseltamivir-resistant influenza a pandemic (h n ) virus emergence of a novel swine-origin influenza a (h n ) virus in humans surveillance of resistance to adamantanes among influenza a(h n ) and a(h n ) viruses isolated worldwide pyrosequencing as a tool to detect molecular markers of resistance to neuraminidase inhibitors in seasonal influenza a viruses detection of molecular markers of drug resistance in pandemic influenza a (h n ) viruses by pyrosequencing infections with oseltamivir-resistant influenza a(h n ) virus in the united states rapid identification of oseltamivir-resistant influenza a(h n ) viruses with h y mutation by rt-pcr/restriction fragment length polymorphism assay the emergence of oseltamivir-resistant pandemic influenza a(h n ) virus amongst hospitalised immunocompromised patients in scotland oseltamivirresistant influenza viruses a (h n ), norway rapid detection of influenza a pandemic (h n ) virus neuraminidase resistance mutation h y by real-time reverse transcriptase pcr emergence and spread of oseltamivir-resistant a(h n ) influenza viruses in oceania selection for resistance to oseltamivir in seasonal and pandemic h n influenza and widespread co-circulation of the lineages emergence of resistance to oseltamivir among influenza a(h n ) viruses in europe a community cluster of oseltamivir-resistant cases of h n influenza development of a novel bead-based multiplex pcr assay for combined subtyping and oseltamivir resistance genotyping (h y) of seasonal and pandemic h n influenza a viruses longitudinal analysis of genotype distribution of influenza a virus from oseltamivir-resistant influenza virus a (h n ) the origin and global emergence of adamantane resistant a/h n influenza viruses neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective highly sensitive and quantitative detection of the h y oseltamivir resistance mutation in seasonal a/h n influenza virus design and validation of real-time reverse transcription-pcr assays for detection of pandemic (h n ) virus adamantane resistance in seasonal human influenza a viruses from calgary oseltamivir-resistant variants of the pandemic h n influenza a virus are not attenuated in the guinea pig and ferret transmission models surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from evaluation of a rapid molecular algorithm for detection of pandemic influenza a (h n ) virus and screening for a key oseltamivir resistance (h y) substitution in neuraminidase first oseltamivir-resistant pandemic (h n ) influenza from emr oseltamivir resistance in immunocompromised hospital patients pandemic (h n ) breifing note genetic makeup of amantadine-resistant and oseltamivir-resistant human influenza a/h n viruses we would like to thank the staff in molecular diagnostics and virology departments at provincial laboratory for public health (provlab) for their technical assistance in undertaking routine respiratory virus testing. in particular, we thank ms. kara gill and ms danielle zarra for their excellent technical support for sequencing of samples. key: cord- -ujo w authors: bennett, susan; carman, william f.; gunson, rory n. title: the development of a multiplex real-time pcr for the detection of herpes simplex virus and , varizella zoster virus, adenovirus and chlamydia trachomatis from eye swabs date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: ujo w infectious conjunctivitis can be difficult to distinguish clinically due to the considerable overlap in clinical presentation so clinical diagnosis of conjunctivitis is often insufficient. it is therefore necessary to have a rapid diagnostic test that differentiates between the different causes of infectious conjunctivitis. screening clinical samples by sample type/syndrome based multiplex real time pcr would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient. a multiplex real-time pcr assay for rapid and simultaneous detection of hsv and , vzv, adenovirus and chlamydia trachomatis (c. trachomatis) from eye swabs was developed and evaluated. the multiplex assay was shown to be sensitive, specific and robust. reductions in sample turn around times have been achieved by reducing the amount of separate tests needed to be carried out. infectious conjunctivitis is common and can be caused by viruses, bacteria, and parasites. infections are usually mild; however complications can arise and can be sight-threatening. infectious conjunctivitis can be difficult to distinguish clinically due to the considerable overlap in clinical presentation so clinical diagnosis of conjunctivitis is often insufficient (o'brien et al., ) . this can lead to misdiagnosis resulting in misuse of antibiotics, antibiotic resistance, and subsequently unnecessary costs (thanathanee and o'brien, ) . it is therefore necessary to have a rapid diagnostic test that differentiates between the different causes of infective conjunctivitis. traditional detection methods for virus detection rely on often slow, laborious and insensitive cell culturing, which has now been largely replaced by nucleic acid amplification tests. pcr is usually carried out as a single test for each pathogen and tests for all pathogens are not always available at the same site. screening clinical samples by sample type/syndrome based multiplex real time pcr would allow for rapid detection of a variety of pathogens simultaneously, which will in turn aid in the treatment and clinical management of the patient. a multiplex real-time pcr assay for the rapid and simultaneous detection of hsv and , vzv, adenovirus and c. trachomatis from eye swabs has been developed and evaluated. the hsv / , vzv and adenovirus assays used in the multiplex were published previously (ryncarz et al., ; van doornum et al., ; hawrami and breuer, ; heim et al., ) . these assays have been shown to be sensitive and specific, and to detect all strains. the c. trachomatis assay was developed in-house (see below). a c. trachomatis assay was designed in-house using primer express tm (applied biosystems). primers and probes were developed to target the cryptic plasmid (sequences given in table ). primers and probes were then compared to all sequences available in blast (www.ncbi.nlm.nih.gov/blast) and shown to only detect all serovars of c. trachomatis including the swedish variant (sw c. trachomatis) discovered in . no interfering table primer and probe sequences. this publication secondary structures were observed using the mfold algorithm (www.bioinfo.rpi.edu). . . assessment of the c. trachomatis real time assay . . . endpoint sensitivity the c. trachomatis assay was compared to a fully automated commercial extraction and real-time pcr system, the abbott realtime chlamydia trachomatis/neisseria gonorrhoea (ct/ng) pcr assay (abbott, maidenhead, uk), by comparing endpoint sensitivity using a dilution series of a positive clinical sample. the c. trachomatis assay was further evaluated using the quality each probe was labelled with a different fluorescent dye (table ); in the case of hsv the hsv and probes were labelled with the same fluorescent dye, atto . this enabled the detection of all pathogens in one multiplex screen and also reduced the risk of cross-talk between dyes. the inability to differentiate between hsv and was discussed with our users prior to development. our users agreed it was sufficient to detect both hsv and on the same dye. optimisation of the hsv probes had to ensure that both probes produced similar levels of fluorescence. the primer probe concentrations for all assays, singleton and multiplex, were individually optimised using in-house protocols (gunson et al., ) , all primers and probes are shown in table . the vzv probe and all primers where purchased from applied biosystems (cheshire, uk). the remaining probes were purchased from eurogentec (seraing, belgium). the optimised concentration for each probe was m and for each primer m. the endpoint detection limits of each component in the multiplex were directly compared to the current routine single assays using a dilution series of positive controls for each target. these were carried out to ensure that multiplexing the assays did not result in a loss of sensitivity at the endpoint of detection. the the inter-assay and intra-assay variability of the multiplex was also assessed. the inter-assay variability (reproducibility or longterm precision) was assessed by monitoring positive run controls over pcr runs. this assesses the whole testing system by including extraction and pcr runs with different users. the intra-assay (repeatability or short-term precision) variability was assessed by testing a positive control in wells on one pcr run. all samples were extracted either on the qiagen mdx using the qiaamp viral rna kit (qiagen, crawley, uk) or the nuclisens easymag (biomérieux, hampshire, uk) according to manufacturer instructions. both extraction platforms have comparable sensitivity and are used interchangeably. the abbott commercial ct/ng assay uses the abbott m automated extraction platform for sample extraction. pcr was performed on l of dna extract with platinum qpcr pcr mastermix pcr kit (invitrogen) on an abi prism sds realtime platform (applied biosystems) in a l reaction volume. the following thermal profile was used: min at • c, min at • c for dna polymerase activation followed by amplification cycles of sec at • c and sec at • c each (annealing-extension step). a -fold dilution series was assessed through the c. trachomatis assay and the abbott commercial system ( table ). the results suggest that sensitivity of the c. trachomatis assay is similar to the abbott system, the c. trachomatis assay detected down to the − dilution, whereas the abbott system detected the − dilution, however this was detected as "beyond the cut-off" without a ct value. when a sample is detected as "beyond the cut-off" the sample is repeated through the system (extraction and pcr). if the sample is determined "beyond cut-off" on repeat, the sample is reported as positive. the abbott system detected the − dilution as "beyond cut-off" on repeat, therefore these results suggest that the abbott system may be slightly more sensitive at the endpoint of detection. the c. trachomatis assay was assessed using the qcmd c tracho- the assay was also evaluated using a panel of sda positive clinical samples; the assay detected all sda positive samples as positive. . . . endpoint sensitivity comparing the endpoint detection limit of each component of the multiplex assay to the single assays showed that multiplexing had no detrimental effect on the endpoint detection limit of each component (table ). the adenovirus single assay detected the − dilution in out of occasions, the multiplex detected down to the − dilution. when testing the dilution series of hsv- , the hsv- single assay detected the − dilution; the multiplex assay detected the − dilution in out of occasions. the hsv- single assay and multiplex detected the − dilution in out of occasions. when testing the dilution series of vzv, the single vzv assay detected the − dilution in out of occasions; the multiplex detected the − dilution. the c. trachomatis assay detected − ; the multiplex assay detected − in out of occasions. the specificity of the assay was confirmed by testing a panel of other commonly encounter pathogens and no false positive results were encountered. the inter-assay variability was assessed by monitoring positive run controls over pcr runs (table a) , and intra-assay variability by testing a positive control in wells on one pcr run (table b ). the results suggest that there is little inter-assay variability as the ct of each component was similar over different runs with low standard deviation values and low co-efficient of variation (c v ) values. the results also suggest that the intra-assay variability of the assay is also good for each component as little variation was observed when the positive control was repeatedly tested and low (c v ) values. overall these results suggest that the duplex assay is precise and robust. this paper describes the development and validation of a multiplex real-time pcr assay, which will allow rapid and simultaneous detection of hsv / , vzv, adenovirus and c. trachomatis on eye swabs. the validation showed that the newly designed c. trachomatis assay was sensitive and specific. when compared to the abbott system the in house may be slightly less sensitive at the endpoint of detection. the in-house assay therefore may occasionally miss very weak positive c. trachomatis samples. multiplexing the five assays had no effect on the performance of any of the individual components by assessment of dilution panels. the multiplex assay was shown to be specific by assessment of quality control panels and a panel of commonly encountered pathogens. the multiplex assay was also shown to be precise and robust by assessment of the inter-assay and intra-assay variability. multiplexing by sample type/disease syndrome simplifies the routine service; overall costs are reduced when compared to single testing as rapid diagnosis can reduce unnecessary antibiotic usage and investigations. reductions in sample turn around times have been achieved by reducing the amount of separate tests needed to be carried out. this ensures more samples can be tested within the working day (gunson et al., ) . since the introduction of the real-time pcr multiplex in the turn around time (per sample) has reduced from an average of . - working days (table ). in addition the percentage of positive samples also increased since this time. the reason for this increase in detection rate is unclear. it may indicate better sampling by clinicians or could be due to the implementation of the new c. trachomatis real time pcr method. multiplex pcr allows for the rapid differentiation between mild and potentially serious causes of conjunctivitis that may be difficult to distinguish clinically. for example, hsv may result in keratoconjunctivitis that is indistinguishable from adenovirus and is the leading cause of viral-induced blindness in the western world. in addition multiplex pcr allows the screening of both viral and bacterial causes of conjunctivitis which can be important as an individual may have to stay off work/school for up to weeks if the infection is viral, whereas in the case of a bacterial infection, such as c. trachomatis, they could return to work/school - hours after initiating antibiotic treatment. although c. trachomatis may not be a serious cause of conjunctivitis, diagnosis is important in aiding management of the patient as it indicates a possible genital infection. the patient will be treated and referred to a genitourinary medicine (gum) clinic where further testing can be offered. in the case of a baby being infected the mother would also be referred for testing and treatment. optimisation of pcr reactions using primer chessboarding using multiplex real time pcr in order to streamline a routine diagnostic service development of a fluorogenic polymerase chain reaction assay (taqman) for the detection and quantitation of varicella zoster virus rapid and quantitative detection of human adenovirus dna by real-time pcr acute conjunctivitis: truth and misconceptions development of a high-throughput quantitative assay for detecting herpes simplex virus dna in clinical samples conjunctivitis: systematic approach to diagnosis and therapy diagnosing herpesvirus infections by real-time amplification and rapid culture key: cord- - cu alm authors: zheng, yuan zhi; hyatt, alex; wang, lin-fa; eaton, bryan t.; greenfield, paul f.; reid, steven title: quantification of recombinant core-like particles of bluetongue virus using immunosorbent electron microscopy date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: cu alm immunosorbent electron microscopy was used to quantify recombinant baculovirus-generated bluetongue virus (btv) core-like particles (clp) in either purified preparations or lysates of recombinant baculovirus-infected cells. the capture antibody was an anti-btv vp monoclonal antibody. the clp concentration in purified preparations was determined to be . × ( ) particles/l. clp concentration in lysates of recombinant baculovirus-infected cells was determined at various times post-infection and shown to reach a value of × ( ) particles/l of culture medium at h post-infection. the results indicated that immunosorbent electron microscopy, aided by an improved particle counting method, is a simple, rapid and accurate technique for the quantification of virus and virus-like particles produced in large scale in vitro systems. bluetongue virus (btv) is the prototype virus of the genus orbi irus in the family reo iridae. the virus contains ten double-stranded rna genome segments each of which codes for a single protein. seven of the proteins are structural and form a double-shelled particle. proteins vp and vp constitute an icosahedral inner capsid that is surrounded by an outer layer composed of proteins vp and vp . in addition to the structural proteins, infected cells contain three nonstructural proteins the roles of which remain obscure. french and roy ( ) constructed a dual-recombinant baculovirus containing genes that encode vp and vp and their expression in insect cell culture resulted in the synthesis of large quantities of vp and vp and the concomitant formation of core-like particles (clp) that lack rna. clp have the potential to be used as vaccines against bluetongue and other diseases. other antigens linked to the vp protein, have demonstrated increased antigenicity when presented as part of clp (belyaev and roy, ; pearson and roy, ) . however, if clp are to be considered seriously as vaccines, it is important to be able to quantify the yield of such particles. quantification of vp and vp (or vp -fusion proteins) is not sufficient to indicate the yield of intact clp present in lysates of infected cells. existing electron microscopy (em)-based procedures are cumbersome and difficult to optimise. in this paper, we report the development of an immunosorbent em procedure for quantifying clp particles present in crude samples and which precludes a requirement to purify clp particles prior to quantification. immunosorbent electron microscopy (isem) is a useful method for the identification and quantification of viruses. derrick ( ) used parlodion-filmed, carbon-coated grids with anti-potato virus y (pvy) or anti-tobacco mosaic virus (tmv) antisera to quantify pvy and tmv in crude leaf extracts. he found a linear relationship between the log of the number of tmv particles attached to serologically-specific grids and the dilution of a crude extract of tmv-infected tobacco tissue. other researchers have also used isem to detect and differentiate between different viruses. lewis ( ) investigated serological differences between strains of norwalk-like virus (nlv) from five different outbreaks using anti-nlv-coated grids. dea and garzon ( ) identified coronaviruses from various species in clarified faecal specimens and gupta et al. ( ) demonstrated that a close relationship between brinjal necrotic mosaic virus and other strains that were trapped by antisera on a collodionfilmed and carbon-coated grid. isem was evaluated for the quantification of recombinant baculovirus-generated btv clp (hereafter referred to as clp) using monoclonal (mab) and polyclonal antibodies to the core proteins vp and vp . spodoptera frugiperda cells (sf ) were obtained from the american type culture collection (atcc crl ) and adapted for growth in sf ii serum-free medium (gibco, ny, usa). the cells were grown at °c either in monolayer culture or in suspension in ml medium/ ml duran erlenmeyer flask placed on an orbital shaker operating at rpm. recombinant autographa californica nuclear polyhedrosis virus (acnpv) co-expressing btv core proteins vp and vp (clp-acnpv) was obtained from the nerc institute of virology and environmental microbiology, oxford, uk (french and roy, ) . recombinant autographa californica nuclear polyhedrosis virus expressing b-galactosidase (b-gal-acnpv) was purchased from amrad, australia. stock preparations of clp-acnpv were propagated by infecting monolayer cultures of sf cells at a multiplicity of infection (moi) of two plaque forming units (pfu) per cell. progeny virus ( × pfu/ml) was harvested days post-infection. anti-vp mab e , a and d were raised in mice to sodium dodecyl sulphate-treated btv- , using the procedure described by lunt et al. ( ) . hybridoma culture supernatants were used at dilutions of : ( e , a ) and : ( d ). polyclonal anti-vp antiserum was raised in sheep against yeast-expressed vp (martyn et al., ) and used at a dilution of : . ovine polyclonal antiserum was raised to core particles of btv- isolated from virus-infected cells and purified by equilibrium density centrifugation in cesium chloride (personal communication, b.t. eaton). the ovine antiserum was used at a dilution of : . polyclonal anti-vp antiserum was raised in rabbits to polyacrylamide gel-purified vp of btv- expressed in escherichia coli as described previously (wang et al., ) . after affinity purification using a protein-g column, the anti-vp serum was used at a dilution of : . sf cells were suspended in ml sf ii medium at a concentration of - × cells/ml. cell density and viability were monitored by counting in the presence of . % trypan blue. at a density of × per ml, cells were infected with clp-acnpv at a moi of two pfu/cell. aliquots were removed at regular intervals postinfection and stored at − °c. the method used to purify clp was adapted from that described by french and roy ( ) . sf cells in suspension ( × cells/ml) were infected with clp-acnpv at a moi of two pfu/ cell and incubated at °c for h. cells were pelleted by low-speed centrifugation ( × g) and resuspended at a concentration of × cells/ml in mm tris-hcl (ph . ) containing . % np- . after min on ice, the lysate was spun at rpm ( ×g) for min to remove nuclei. clp in the lysate were purified by ultracentrifugation in self-forming % (w/v) cesium chloride density gradients at × g for h (beckman l ). clp banded at a density of . g/cm and were analyzed by electrophoresis in % sds-page (french and roy, ) , western immunoblotting and electron microscopy. airfuge analysis was carried out as described by zheng et al. ( ) . briefly, the sector core of the em- rotor (beckman instruments, palo alto, ca) was lubricated with silicon vacuum grease and ml aliquots of serially diluted solutions of clp were centrifuged ( × g, min) onto thin-bar mesh copper grids that had been filmed with . % collodion pre-treated with polylysine. the supernatants were removed and grids stained for ultrastructural examination. . . immunosorbent electron microscopy (isem) electron microscope grids ( mesh thin-bar copper) were filmed with either % formvar or % collodion and carbon-coated. other grids were filmed with evaporated carbon and contained no plastic films. grids were coated with mab e at dilutions of : , : , : , : , : , : and : in either phosphate buffered saline (pbs) (ph . ), mm tris-hcl (ph . ) or mm carbonate buffer (ph . ). volumes ( ml) of antibody were deposited onto grids and incubated for , or min at room temperature, and °c. normal mouse igg, buffer only, and grids only were used as controls. the grids were washed with double distilled water, mm tris (ph . ) or pbs (ph . ) ( × min). grids were then blocked with ml of % bovine serum albumin (bsa) in pbs (ph . ) for min. cesium chloride gradient-purified clp and crude clp in lysates of acnpv-clp-infected cells were analysed by isem. purified clp were serially diluted six times in a lysate of uninfected sf cells starting at : . the lysate was generated by treating uninfected cells with mm tris-hcl (ph . ) containing . % np- . infected cells were lysed by addition of np- to . % and lysates were stored at − °c. following thawing, the lysates were centrifuged at low speed ( rpm, × g) and ml aliquots added to pretreated grids prepared as described above for , or min at room temperature, and °c. uninfected and b-gal-acnpv-infected cell lysates and media only were used as controls. after incubation with shaking, the grids were washed in double distilled water and stained with ml of % aqueous uranyl acetate or % sodium phosphotungstate (containing mg/ml bacitracin) for min. preparations were examined at a magnification of with a jeol- transmission electron microscope. areas for examination were selected at random and recorded in the form of electron micrographs. three to five micrographs were recorded per sample and each sample was prepared in duplicate. quantification of clp was performed by counting ('handy counter') the number of clp per negative film (imaged in a microfiche at× magnification). the concentration of purified clp was determined by isem ('binding concentration') and by the airfuge technique ('original concentration') and the capture efficiency was determined by dividing the 'original concentration' by the 'binding concentration'. the number of crude clp per negative film was determined by the isem method and the concentration of particles calculated by multiplying the number of clp by the capture efficiency. purified clp were used to generate a calibration standard. the concentration of clp was calculated using the airfuge technique (zheng et al., ) . fig. shows a linear relationship between the number of particles counted and the dilution of clp. using the formula described previously to calculate particle numbers (zheng et al., ) , the concentration of purified clp was determined to be . × per ml. serial dilutions of purified clp were made in uninfected sf cell lysates and ml aliquots of each dilution added to grids coated with a : dilution of antibody e . fig. (a) shows that clp were distributed evenly on the antibodycoated grids. a linear relationship was obtained between the number of particles and their dilution from : to : (fig. b) . regression analysis indicated that there were particles bound to the grid/ml of purified clp. the ratio of bound to total clp (binding efficiency) was . × / . × or . × . several factors can affect the quantification of virus particles by isem. these include nature of the support film, specificity and concentration of antibody, washing procedure and staining solution. in our study, results indicated that there was no difference in the clp capture efficiency with either which carbon-coated, formvar-filmed or carbon-coated, collodion-filmed grids that had been coated with mab e (data not shown). carbon-filmed grids were found to be unstable as they frequently broke during incubation and washing steps. to determine the optimum antibody dilution, the number of clp bound was determined at a range of antibody dilutions. for mab e , dilutions of : to : were equally effective in capturing clp (fig. ) . of the various antibodies tested, mab e captured the largest number of clp (fig. ) . the best mab e coating and clp adsorption times were found to be from to min. adsorption times greater than min generated background that was most likely attributable to adsorbed hybridoma cell debris. in virus capture experiments, min was found to be optimum. incubation periods beyond min generated background that could be attributed to the impurity of the preparations i.e. non-specific adsorption of cell debris to the substrate. in the washing steps, various buffers and water were tested to assess whether the washing buffer facilitated removal of clp from the grids. we found that the type of buffer used did not have a deleterious effect and thereafter we used double distilled water. aliquots of ml of : dilutions of crude clp culture samples, prepared at various times postinfection, were added onto mab e -coated grids. the numbers of clp were determined as described for purified clp. fig. (a) shows negative-contrasted clp from a crude sample captured on a grid coated with mab e . the concentration of mab e -captured clp increased with time post-infection (fig. b) . the maximum yield of clp ( . × per l) was obtained at h post-infection. isem is a technique that captures specific antigens and can be used to quantify viruses from a range of biological samples. other techniques such as adsorption, centrifugation (e.g. the use of airfuges) and the spraying of particles directly onto grids all have the disadvantage that both target and non-target particles/antigens become adsorbed to the grid substrate. of the factors/variables that have been reported to influence the efficiency of isem (support films, dilution of antibody, incubation time and temperature, washing media, and staining solution), support films and washing media were found to be of little to no consequence. we found that carbon- coated, formvar-filmed grids had good stability and adsorption characteristics for antibody binding. this result is consistent with the results of milne and luisoni ( ) but different to those obtained by derrick and brlansky ( ) . in the latter study the authors stated that formvar-filmed grids, with or without carbon-coating, were unsuitable for isem. the authors reported the best results were obtained with collodion-filmed, carbon-coated grids. the results from this study indicated that either film substrate could be used with equivalent efficiency. potassium phosphotungstate (pta) and uranyl acetate (ua) are commonly used as negative contrast stains. in this study pta resulted in the disruption of clp whereas ua maintained their integrity in addition to generating high contrast images. the choice of antibody was critical to the overall efficiency of the isem technique. the best antibody for capturing clp was mab e and its efficiency may be attributable to its specificity and the accessibility of the e epitope (wang et al., ) . the anti-vp polyclonal antibody would be expected to recognise multiple epitopes but some are almost certainly inaccessible in clp, thereby decreasing the capture efficiency of the technique. vp is not expressed on the surface of clp or core particles and therefore anti-vp polyclonal antibody would be expected to be inefficient as a capture antibody for clp. some reports (e.g. milne and lesemann, ; lesemann et al., ) state that the concentration of antibody affects the efficiency of particle capture. the data from this study indicated that mab e dilutions of : - : yielded similar results for purified clp while dilutions of and beyond : led to a reduction in the number of captured clp. these results may be explained in terms of coating density and steric hindrance. at dilutions of and beyond : steric hindrance may no longer be an obstacle to clp binding and the density of antibody adsorption is reduced.the data presented in this paper indicate that the isem technique may be useful for quantification of viral antigens in diagnosis and the production of virus vaccines. accurate quantification is dependent upon determining the capture efficiency. presentation of hepatitis b virus pres epitope on bluetongue virus core-like particles quantitative assay for plant viruses using serologically specific electron microscopy assay for viruses and mycoplasmas using serologically specific electron microscopy identification of coronaviruses by the use of indirect protein a-gold immunoelectron microscopy synthesis of bluetongue virus (btv) core-like particles by a recombinant baculovirus expressing the two major structural core proteins of btv immunosorbent electron microscopic studies on a tobamovirus causing brinjal necrotic mosaic the trapping of tymovirus particles on electron microscope grids by adsorption and serological binding three serotypes of norwalk-like virus demonstrated by solid-phase immune electron microscopy evaluation of a monoclonal antibody blocking elisa for the detection of group-specific antibodies to bluetongue virus in experimental and field sera an immunoelectron microscopic investigation of oat sterile dwarf and related viruses high level expression of the major core protein vp and the nonstructural protein ns of bluetongue virus in yeast: use of expressed vp as a diagnostic, group-reactive antigen in a blocking elisa serological relationships among maize rough dwarf-like viruses genetically engineered multicomponent virus-like particles as veterinary vaccines. immunol topography and immunogenicity of bluetongue virus vp epitopes improved method for counting virus and virus like particles we would like to thank john white, csiro, australian animal health laboratory for the supply of antibodies, and prof. polly roy and nerc institute of virology and environmental microbiology (oxford, uk) for the supply of acnpv-clp baculovirus strains. this study received financial support from the australian research council. key: cord- -m otfdjw authors: wang, pei title: combination of serological total antibody and rt-pcr test for detection of sars-cov- infections date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: m otfdjw the purpose of this study was to investigate the feasibility of serological total antibody tests combined with rt-pcr for detection of sars-cov- . we conducted a retrospective study in which patients were enrolled during the outbreak of sars-cov- from th january to th march . patients were divided into a covid- group (n = ) and a control group (n = ). serum samples and throat swabs were collected from patients for total antibody testing against sars-cov- and rt-pcr analysis, respectively. the results indicated that diagnostic sensitivity and specificity were . % and . %, . % and % by total antibody tests and rt-pcr, respectively. the sensitivity and specificity of total antibody tests combined with rt-pcr were . % and . %. the sensitivity of the combined method was significantly higher than rt-pcr (x( ) = . , p < . ), and similar to that of total antibody tests (x( ) = . , p > . ). this study supported the advantage of the combined method for detection of sars-cov- with a high degree of sensitivity and specificity, as a useful tool for accurate diagnosis and timely treatment of suspected patients, epidemiological investigation, as well as monitoring ongoing outbreaks of infections with sars-cov- . as of may , , more than , people died from covid- worldwide, and the estimate of future deaths will number in the millions (roberton et al., ) . the sars-cov- is now quickly spreading across the world after being reported in wuhan first , and has become a global health concern (wang et al., a) . sars-cov- is an enveloped non-segmented positive-sense rna virus, which is highly contagious with high mortality ranging from % to . %, depending on the cohort characteristics (wang et al., a) . currently, virus rna detection conducted by rt-pcr has become the standard assessment for the confirmation of sars-cov- infection . however, virus rna detection has some limitations in terms of accuracy (xie et al., ) . technically, rna testing works with throat swabs or nasopharyngeal swabs as testing samples. the rt-pcr test comprises rna extraction and amplification procedures. it usually takes to hours to accomplish an rt-pcr testing circle. the rna detection results rely on the quality of the sample, extracted rna, the source of the rt-pcr reagents and the multiple steps in rna preparation. moreover, different sample types yield different positive detection rates varying from % to % (wang et al., b) , and the viral load fluctuates at different infectious phases (cai et al., ) . taking the above into account, the seventh edition of the medical guideline for sars-cov- infections from the national health commission of the people's republic of china added that serological testing could be used for confirmed diagnosis of covid-j o u r n a l p r e -p r o o f (china national health commission., ) . to this end a commercially available kit (wantai, xiamen, china) using a chemiluminescence microparticle immunoassay (cmia) for the determination of total antibody in serum samples can be used. this kit was applied for diagnosing suspected patients of covid- and for epidemiological study . in this study, we presented the results of two diagnostic methods: serum total antibody assays against sars-cov- by cmia and the rt-pcr for detection of viral rna. in addition, the combination of the results of the total antibody test and rt-pcr was discussed for detection of sars-cov- infections. j o u r n a l p r e -p r o o f . materials and methods this study was performed in the first people's hospital of jingmen, hubei province, china, which is a comprehensive public hospital with beds located in central area of china with the capability of serving , inhabitants. we retrieved the data of patients which was recorded during the outbreak of sars-cov- from th of january to th of march . for all enrolled patients, the clinical data included the date of illness onset, clinical features, chest ct during the hospitalization period. personal demographic information was obtained from the clinical records. the laboratory data included results of total antibody test against sars-cov- and rt-pcr detection. all patients who visited the hospital with respiratory complaints were included. of the patients, were confirmed to have sars-cov- infection . this group consisted of male and female patients with a median age of years ). the need for informed consent was waived. the throat swab specimens were collected by using a foam swab with transport medium (sigma virocult, uk). specimens were then put in a biosafety transport box and sent to the laboratory located at the hospital for rt-pcr detection immediately. the blood samples ( ml) were collected before patients were discharged from the hospital. sample taking time varied from - , - , > days after illness onset. specimens of covid- patients and controls were collected at one of the aforementioned three time periods. the samples were centrifuged at - g for minutes, and the serum was aliquoted and tested to determine the total antibody against sars-cov- . virus rna was extracted from throat swabs with a nucleic acid kit (roche, mannheim, germany) on an automatic workstation magna pure system (roche, mannheim, germany). the whole process of extraction was performed according to the guidelines. in addition, next to the wantai kit two samples were tested with another antibody kit (shenzhen, yhlo biotech co.,ltd.). a database was established and statistical analysis was performed with spss . . sensitivity, specificity for detection of sars-cov- by rt-pcr, and the total antibody test method as well as the combined methods were analysed. sensitivity and specificity were calculated with the rate of positive test results and the rate of negative of the covid- patients throat swabs were taken several times till day after admission or until the rt-pcr became positive. samples were taken for the first time between day - after admission to the hospital ( table a number of patients was transferred from a local hospital to our hospital. they were admitted a few days after illness onset. the illness onset date was taken from the patients records. the illness period varied from - days, - days, > days. samples for antibody testing were only taken before discharge of the patients. data of total antibodies against sars-cov- were grouped according to these three time periods. in a total of the covid- patients, became positive by the total antibody test j o u r n a l p r e -p r o o f during the three time periods, out of cases were tested positive by rt-pcr assay and cases were rt-pcr negative. nine out of the rt-pcr-negative cases were detected positive by the total antibody assay. of the non-covid- patients, none was positive by the rt-pcr test and were positive by the total antibody assay. to obtain the results for the combined method, results of the rt-pcr (negative and positive) were supplemented with the positive results of the antibody test (table ) the joint method was found to be more sensitive than rt-pcr alone ( . % vs . %, x = . , p< . ). there is no significant difference in sensitivity between the joint method and total antibody test alone( . % vs . %, x = . , p> . ). j o u r n a l p r e -p r o o f successful control of sars-cov- spread will need an accurate, rapid and costeffective detection method. the aim of this study was to evaluate the performance of total antibody test by using cmia, the rt-pcr, and explore the feasibility of the combination, serological total antibody tests and rt-pcr, as a possible diagnostic tool for detection of sars-cov- . the presence of sars-cov- infection can be detected by rt-pcr in samples from nasopharyngeal or throat swab. a number of patients show progressive multiple peripheral ground-glass opacities in lungs but with negative rt-pcr results (ai et al., ) . swabs had to be taken - times from a number of patients to get a positive rt-pcr, and never had a positive rt-pcr. all rt-pcr negative patients had ctscan changes of ground-glass opacities in the lungs. a negative rt-pcr for covid- patients is not uncommon. a prior study reported only % positives among specimens from fever clinics . a number of factors may affect this lack of sensitivity of the rt-pcr testing, like the sample (nose or throat swabs), the sampling procedure, the rna extraction. also, the time of sampling may be important. the results showed that detection of sars-cov- by rt-pcr was often late while clinical symptoms were already present. our study proved that rt-pcr had a high specificity ( %) but relatively low sensitivity ( . %). to solve this diagnostic problem, the th edition of guideline for covid- issued by the national health commission of the people's republic of china, recommends serological testing as supporting proof for covid- diagnosis (china national j o u r n a l p r e -p r o o f health commission, ). several groups determined the antibody response to sars-cov- and compared new commercial serologic assays , geurtsvankessel et al., , krüttgen et al. , lassaunière et al., . the total antibody test of wantai has good sensitivity and specificity as compared to other tests (lassaunière et al., ) . the wantai antibody test provides a semi-quantitative result. total antibodies are determined by cmia, which is an automated, rapid and high throughput assay, objective and quantitative, but it requires an expensive instrument carris (lou, et al., ) . the results show a satisfactory quality for sensitivity ( . %) and specificity ( . %) of total antibodies against sars-cov- by cmia. of the covid- patients, had antibodies against sars-cov- . six patients out of the covid- patients were found to be antibody negative, of them were rt-pcr positive. three of these patients had kidney disease and ongoing hemodialysis therapy. additional samples were taken from these patients. after discharge from the infectious disease department ward, they were transferred to the hemodialysis ward. samples were taken before and after hemodialysis. the s/co values for these three patients before hemodialysis were . , . , and . , respectively. after hemodialysis these values decreased to . , . , and . , respectively. the effect of hemodialysis on the presence and/or absence of antibodies has also been described for the detection of anti-hepatitis-hcv antibodies (el-sherif et al., ) . the fourth patient with a positive rt-pcr had severe anemia and his s/co value was . . the remaining two without antibodies in the wantai assay were re-j o u r n a l p r e -p r o o f tested and found to be positive by using another brand of antibody assay (shenzhen yhlo biotech co., ltd.). in the non-covid patients, were found positive with the total antibody test. among three positive cases, patients were pregnant, the s/co values of the antibody test were . and . , respectively. after birth, the results changed from weak positive to negative. the third patient suffered from colon cancer and the antibody test had an s/co value . . heterophile antibodies present in elderly, pregnant women, and cancer patients can interfere with immunoassays by a non-competitive mechanism and lead to false positive results (tate and ward, ) . the combination of the results of both methods, the rt-pcr and cmia antibody test, does improve the sensitivity to . %. a high sensitivity is beneficial for screening and confirming covid- patients. no doubt, for covid- diagnosis, rt-pcr played an important role at an early stage. antibodies against sars-cov- appear around - days after the onset of the disease. therefore, the total antibody test displays next to the rt-pcr a powerful diagnostic efficiency at a later stage. a combination of the two assays is superior and results in the diagnosis of more covid- patients. the presence of pre-existing antibodies to sars-cov- due to a prior infection cannot be excluded. by testing only one sample, the laboratory should carefully interpret these test results, and use additional blood samples, and/or other criteria like rt-pcr, ct-scans and disease history, to prove a recent infection. in conclusion, this study supported the use of a combined method for detection of sars-cov- infections with a high degree of sensitivity and specificity, which was shown to be a useful tool in the diagnosis of suspected patients. next to diagnostic purposes, serological testing will be needed for epidemiological investigations, as well as monitoring of ongoing outbreaks of infection with sars-cov- and testing the effects of future vaccines. the author declares that there is no conflict of interest related to this article content. the author of this paper has no financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper. **, the second samples were taken at day and after admission of the patients of whom the first samples were negative. patients whose samples were negative in the second test were re-tested by samples taken at days or . when still negative, a th and th testing was performed. *** cumulative number of patients tested positive with the rt-pcr. table the detection of sars-cov- infections by the rt-pcr ,the total antibody assay and a combination of both methods j o u r n a l p r e -p r o o f t table the detection of sars-cov- infections by the rt-pcr ,the total antibody assay and a combination of both methods correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases a peptide-based magnetic chemiluminescence enzyme immunoassay for serological diagnosis of coronavirus disease (covid- ) china national health commission. china national health commission. china national health commission, . diagnosis and treatment of -ncov pneumonia in china high false-negative rate of anti-hcv among egyptian patients on regular hemodialysis towards the next phase: evaluation of serological assays for diagnostics and exposure assessment ct screening for early diagnosis of sars-cov- infection comparison of four new commercial serologic assays for determination of sars-cov- igg evaluation of nine commercial sars-cov- immunoassays positive rate of rt-pcr detection of sars-cov- infection in cases from one hospital in serology characteristics of sars-cov- infection since the exposure and post symptoms onset early estimates of the indirect effects of the covid- pandemic on maternal and child mortality in low-income and middle-income countries: a modelling study. the lancet. global health interference in immunoassay a novel coronavirus outbreak of global health concern detection of sars-cov- in different types of clinical specimens clinical characteristics of hospitalized chest ct for typical -ncov pneumonia: relationship to negative rt-pcr testing & china novel coronavirus investigating and research team, . a novel coronavirus from patients with pneumonia in china at days - after onset, patients were discharged and of these were antibody positive at - days, out of were antibody-positive and these are rt-pcr-neg. this makes the total rt-pcr-pos.+ antibody-pos.= positives. at - days, out of were antibody-pos and rt-pcr-neg. the total is rt-pcr-pos. + antibody-pos.= positives the author would like to thank drs gjalt w. welling and sytske welling-wester, university of groningen, the netherlands, for critical reading of the manuscript.j o u r n a l p r e -p r o o f key: cord- -dash udv authors: decaro, nicola; amorisco, francesca; desario, costantina; lorusso, eleonora; camero, michele; bellacicco, anna lucia; sciarretta, rossana; lucente, maria stella; martella, vito; buonavoglia, canio title: development and validation of a real-time pcr assay for specific and sensitive detection of canid herpesvirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: dash udv a taqman-based real-time pcr assay targeting the glycoprotein b-encoding gene was developed for diagnosis of canid herpesvirus (chv- ) infection. the established assay was highly specific, since no cross-reactions were observed with other canine dna viruses, including canine parvovirus type , canine minute virus, or canine adenovirus types and . the detection limit was ( ) and . × ( ) dna copies per μl(− ) of template for standard dna and a chv- -positive kidney sample, respectively: about -log higher than a gel-based pcr assay targeting the thymidine kinase gene. the assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. chv- isolates of different geographical origins were recognised by the taqman assay. tissues and clinical samples collected from three pups which died of chv- neonatal infection were also tested, displaying a wide distribution of chv-l dna in their organs. unlike other chv- -specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of chv- dna in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs. canid herpesvirus (chv- ) is a member of the family herpesviridae, subfamily alphaherpesvirinae, genus varicellovirus. chv- host range is restricted to domestic and wild canids. serological studies have revealed close antigenic relationships among chv- , felid herpesvirus and phocid herpesvirus (rota and maes, ) . chv- has a worldwide distribution and its pathogenic potential appears to be related to the age of the infected animals. adult dogs are susceptible to chv- , but the infection is usually asymptomatic or subclinical, or localized to the genital tract (decaro et al., d ). in contrast, primary infection or reactivation of latent infection in pregnant bitches may cause reproductive disorders, including infertility, abortion, fetal resorption or mummification, stillbirth or perinatal infection, with systemic disease and neonatal death (decaro et al., d) . recently, chv- infection has also been associated with canine ocular disease (ledbetter et al., (ledbetter et al., , a . diagnosis of chv- infection usually relies on virus isolation in canine cell culture. the polymerase chain reaction (pcr) has been established for detection of chv- nucleic acid (burr et al., ; schulze and baumgärtner, ; miyoshi et al., ; ronsse et al., ; reubel et al., ) . a real-time pcr assay has also been developed to monitor virus shedding in the ocular secretions after experimental reactivation of latent chv- (ledbetter et al., b) . however, the assay was not fully validated by assessment of analytic sensitivity, specificity, linearity and reproducibility of the method. the development and validation of a taqman-based real-time pcr assay are described for rapid diagnosis of chv- infection and for quantitation of the viral load in tissues and/or body fluids of infected animals. to validate the assay chv reference isolates provided by other researchers and clinical samples from dogs with chv infection were used: chv- reference strains dk , dk and as-d (usa) were kindly provided by dr. l.e. carmichael, james a. baker institute for animal health, cornell university, ithaca, ny. the italian strain chv-mirri was kindly provided by dr. a. guercio, istituto zooprofilattico della sicilia, palermo, italy. the viruses were cultivated at - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.jviromet. . . (decaro et al., b) and in (decaro et al., unpublished data) , respectively. a vaginal swab collected from the dam of pups / -t and / -n was also tested. dna was extracted from l of viral suspension or mg of tissue sample using the dneasy tissue kit (qiagen, milan, italy), following the manufacturer's instructions. dna of each sample was eluted in l of ae buffer (elution buffer) and diluted : in distilled water prior to molecular analysis in order to decrease residual inhibitors of dna polymerase activity to an ineffective concentration (decaro et al., a (decaro et al., , a ,b,c). in order to establish a real-time pcr assay specific for chv- , sequences of the glycoprotein b (gb) gene of chv- strains were retrieved from genbank and aligned using the bioedit software package (www.mbio.ncsu.edu/bioedit/bioedit.html). a target region useful for real-time pcr was identified by visual inspection of the sequence alignment. primers and probe were designed using the software beacon designer (bio-rad laboratories srl, milan, italy), to amplify a -bp fragment of the gb gene. primers and probe were synthesised by mwg biotech ag (ebersberg, germany), with the taqman probe labeled with the fluorescent reporter dye -carboxyfluoroscein ( -fam) at the end and with blackhole quencher (bhq ) at the end. the position and sequence of the primers and probe used for real-time pcr amplification are shown in table . for construction of chv- standard dna, the -bp fragment generated by the primers used for real-time pcr was cloned into a pcr ® -topo ® vector (topo ta cloning ® kit for sequencing, invitrogen srl, milan, italy) and propagated in chemically competent escherichia coli one-shot top cells, following the manufacturer's instructions. plasmid dna was purified from transformed cells using wizard plus midiprep (promega italia, milan, italy) and quantified by spectrophotometrical analysis at nm on the basis of plasmid size and of the corresponding dna mass. ten-fold dilutions of the plasmid, representing to copies of dna/ l of template, were spiked into a kidney homogenate ( %, w/v), that tested negative to chv- -by a gel-based pcr targeting the thymidine kinase (tk) gene (schulze and baumgärtner, ) . aliquots of each dilution were frozen at − • c and used only once. real-time pcr for simultaneous detection and quantitation of chv- dna was performed on a real-time pcr system (applied biosystems, foster city ca) with itaq tm supermix added with rox (bio-rad laboratories srl, milan, italy). the reaction mixture ( l) contained . l of itaq tm supermix, each primer at a concentration of nmol l − and the probe at a concentration of nmol l − , and l of template or plasmid dna. the thermal cycling consisted of activation of itaq dna polymerase at • c for min and cycles of denaturation at • c for s and annealingextension at • c for min. the taqman assay was carried out in duplicate for each unknown and standard sample and two chv- -negative samples (a canine vaginal swab and a kidney sample from a dead pup) and a template control were included in each assay. the increase in the fluorescent signal was registered during the extension step of reaction and the data were analysed with the appropriate sequence detector software ( system software v. . . ). the increase of pcr product is proportional to an exponential increase in fluorescence ( rn). the application software produces an amplification curve resulting from a plot of rn versus cycle number. the threshold cycle number (c t ) for each sample tested was regarded as the cycle number at which the amplification curve crossed the threshold which is usually selected automatically from the average of the rn of the samples between cycles and . lower c t values corresponded to a greater amount of initial template and a negative result was considered to have a c t value of or more cycles. in order to exclude losses of dna during the extraction step and of pcr inhibitors in the dna extracts, an internal control (ic), consisting of ovine herpesvirus type (ovhv- ) dna (decaro et al., ) , was added to the lysis buffer (al buffer, quagen) as described previously (decaro et al., a) . the fixed amount of the ic added to each sample had been calculated to give a mean c t value in a real-time pcr assay (hüssy et al., ) of . with a sd of . as calculated by separate runs. real-time pcr for ic detection was carried out in a separate run and samples in which the c t value for the ic was > . (average plus sd) were excluded from the analysis. in order to exclude cross-reactivities between chv- and other canine viral pathogens, the assay was evaluated for specificity by testing dna extracts from the following viruses: canine parvovirus types , a, b and c (decaro et al., b) , canine minute virus (decaro et al., a) , canine adenovirus types (decaro et al., a) and (decaro et al., a) . tissues samples collected from the bodies of chv- -uninfected pups as well as sterile water were also included in the analysis as negative controls. to evaluate the detection limits of the real-time pcr assay, fold dilutions of the plasmid dna, ranging from to copies, were made in a chv- -negative kidney homogenate and tested subsequently. serial -fold dilutions of plasmids containing from to copies of standard dna and corresponding c t values were used to plot the standard curve for quantitation of chv- dna. the reproducibility of the assay was evaluated by testing repeatedly tissue samples containing various amounts of chv- dna, spanning the whole range covered by real-time pcr, as previously described (decaro et al., a (decaro et al., , a . since it was not possible to collect field samples containing less than copies of viral dna, coefficients of variation were not calculated for dna copies. a conventional pcr assay, targeting the tk gene and based on ethidium-bromide staining, was performed for specific detection of chv- dna as described previously by schulze and baumgärtner ( ) , with minor modifications. pcr amplification was conducted using la pcr kit ver. . (takara bio., shiga, japan) in a -l reaction containing mol l − of primers chv and chv (table ) to compare the sensitivity of real-time and conventional pcr, -fold dilutions of a kidney sample containing . × copies of chv- dna were carried out on a chv- -negative kidney homogenate and tested subsequently by both real-time and gelbased amplifications. the template controls, negative samples and other canine viruses did not produce any detectable fluorescence signal, confirming that the real-time pcr was highly specific for the detection of chv- dna. the detection limit of the assay was estimated as and . × dna copies per l − of template for standard dna and for chv- -positive kidney sample, respectively. the gel-based pcr was slightly less sensitive: it detected . × dna copies per l − of template of the positive field sample. ten-fold dilutions of standard dna were tested and used to construct a standard curve by plotting the plasmid copy number logarithm against the measured c t values. the generated standard curve covered a linear range of nine orders of magnitude (from to copies of standard dna) and showed linearity over the entire quantitation range (slope = − . ), providing accurate measurement over a very large variety of starting target amounts. to determine the reproducibility of the assay, intra-assay and inter-assay studies were undertaken (fig. ) . intra-assay cvs ranged from . % (samples containing dna copies) to . % ( dna copies), while the inter-assay cvs ranged between . % ( dna copies) and . % ( dna copies). the ic was detected in all the examined samples (the viral isolates and the clinical samples from naturally infected dogs), with c t values below the threshold value of . . therefore, significant dna losses did not occur during nucleic acid extraction. also, dna polymerase inhibition was not observed. the titres of the four chv- strains isolated in cell cultures ranged from . × to . × dna copies/ l − of template (table ). in real-time pcr chv- was detected in the vaginal swab of the dam of pups / -n and / -t and in all the tissues of the three infected pups. chv- load was . × dna copies l − in the vaginal swab, and ranged from . × to . × copies l − of template in the three pups infected naturally, with the highest viral loads being detected in the kidneys. as all the samples contained high viral loads, far above the detection limit of conventional pcr, a % concordance was demonstrated between real-time and gel-based amplifications (table ) . real-time pcr assays based on the taqman technology have been established for the principal viruses infecting the dog, including canine parvovirus (decaro et al., a) , canine enteric coronavirus (decaro et al., b) , canine respiratory coronavirus (decaro et al., c; mitchell et al., ) , and canine distemper virus (elia et al., ) . also, real-time protocols have been designed to reliably predict viral variants (decaro et al., b (decaro et al., , b , or to discriminate between vaccine and field strains (decaro et al., a,c) . precise quantitation of the viral load by real-time pcr has contributed greatly to the knowledge on viral pathogenesis (decaro et al., b (decaro et al., , a and the efficacy of commercial vaccines (decaro et al., b (decaro et al., , a . a real-time pcr assay has also been proposed for assessment of chv- shedding by ocular secretions of dogs infected latently after treatment with corticosteroids (ledbetter et al., b) . however, the analytic performance of the assay such as sensitivity, specificity, linearity and reproducibility was not evaluated thus preventing its extensive use as a standardised test in diagnostic laboratories. in addition, quantitation of chv- was obtained by comparative c t method, thus being expressed as viral load per cell and not as absolute dna copy number, which can be obtained by processing simultaneously standard dnas represented by -fold dilutions of a plasmid containing the pcr target. the development and validation of a real-time pcr assay for detection and absolute quantitation of chv- dna in tissue samples and body fluids of dogs are described. the assay was highly sensitive and able to detect as few as and . × dna copies l − of template for standard dna and chv- -positive kidney sample, respectively, which was -log higher than a gelbased pcr assay targeting the thymidine kinase gene (schulze and baumgärtner, ). the precise chv- dna copy numbers (absolute quantitation) were calculated in field samples and in cell lysates of cell-propagated reference viruses by a standard curve generated by analysis of -fold dilutions of a plasmid dna incorporating the target of the assay (a fragment of the gb protein gene). the intra-assay and inter-assay cvs were low. intra-assay cv was . % for the samples containing copies of chv- dna. no crossreactivity was observed with the dna of other common canine viruses, showing a high specificity. in addition, a % concordance was demonstrated between real-time and gel-based pcr assays. compared with the molecular methods established previously, the real-time pcr assay described above has several advantages, such as increased laboratory throughput and simultaneous processing of several samples. compared to classical pcr protocols, the processing time required by real-time pcr is shorter, the contamination risks are lower because of the lack of post-pcr steps and the specificity is increased by the probe hybridisation. these advantages make real-time pcr an attractive method for the laboratory diagnosis of chv- infection and for a precise evaluation of the extent and duration of viral shedding in animals infected naturally. detection of canine herpesvirus in a wide range of tissues using the polymerase chain reaction evaluation of the innate immune response in pups during canine parvovirus type infection infezione erpetica del cane e mortalità neonatale dei cuccioli: descrizione di un focolaio first two confirmed cases of malignant catarrhal fever in italy canine distemper and related diseases: report of a severe outbreak in a kennel quantitation of canine coronavirus rna in the faeces of dogs by taq-man rt-pcr a real-time pcr assay for rapid detection and quantitation of canine parvovirus type dna in the feces of dogs genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs a minor groove binder probe real-time pcr assay for discrimination between type -based vaccines and field strains of canine parvovirus characterisation of the canine parvovirus type variants using minor groove binder probe technology diagnostic tools based on minor groove binder probe technology for rapid identification of vaccinal and field strains of canine parvovirus type b infectious canine hepatitis: an "old" disease reemerging in italy tissue distribution of the antigenic variants of canine parvovirus type in dogs experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia evidence for immunisation failure in vaccinated adult dogs infected with canine parvovirus type c detection of bovine coronavirus using a taqman-based real-time rt-pcr assay canine adenoviruses and herpesvirus severe parvovirosis in a repeatedly vaccinated -yearold dog genetic analysis of canine parvovirus type c detection of canine distemper virus in dogs by real-time rt-pcr quantitative fluorogenic pcr assay for measuring ovine herpesvirus replication in sheep corneal ulceration associated with naturally occurring canine herpesvirus- infection in two adult dogs outbreak of ocular disease associated with naturally-acquired canine herpesvirus- infection in a closed domestic dog colony experimental reactivation of latent canine herpesvirus- and induction of recurrent ocular disease in adult dogs development of a quantitative real-time pcr for the detection of canine respiratory coronavirus detection of canine herpesvirus dna in the ganglionic neurons and the lymph node lymphocytes of latently infected dogs suitability of canine herpesvirus as a vector for oral bait vaccination of foxes canine herpesvirus- (chv- ): clinical, serological and virological patterns in breeding colonies homology between feline herpesvirus- and canine herpesvirus nested polymerase chain reaction and in situ hybridization for diagnosis of canine herpesvirus infection in puppies this work was supported by grants from the italian ministry of health, ricerca corrente , project izs ve / rc "definizione di una procedura validata per la selezione di cani per programmi di interventi assistiti dagli animali (iaa)". the authors are grateful to dr. diane addie (feline institute pyrenees, france) for the english revision of the manuscript. key: cord- -pj p x l authors: pratelli, annamaria; martella, vito; decaro, nicola; tinelli, antonella; camero, michele; cirone, francesco; elia, gabriella; cavalli, alessandra; corrente, marialaura; greco, grazia; buonavoglia, domenico; gentile, mattia; tempesta, maria; buonavoglia, canio title: genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: pj p x l the sequence of the s gene of a field canine coronavirus (ccov), strain elmo/ , revealed low nucleotide ( %) and amino acid ( %) identity to reference ccov strains. the highest correlation ( % nt and . % aa) was found with feline coronavirus type i. a pcr assay for the s gene of strain elmo/ detected analogous ccovs of different geographic origin, all which exhibited at least – % nucleotide identity to each other and to strain elmo/ . the evident genetic divergence between the reference ccov strains and the newly identified elmo/ -like ccovs strongly suggests that a novel genotype of ccov is widespread in the dog population. canine coronavirus (ccov) is an enveloped, positive stranded rna virus of dogs associated with moderate to severe enteritis in young pups. the genome contains two large open reading frames (orfs), a and b , encoding two polyproteins leading to the viral replicase formation. downstream to the orf b, there are Á/ smaller orfs encoding for the structural proteins s (orf ), e (orf ), m (orf ) and the nucleocapsid (n) protein (enjuanes et al., ) . the small membrane protein (e) has been found recently to be important for viral envelope assembling (raamsman et al., ) . the m protein is a type iii glycoprotein consisting of a short amino-terminal ectodomain, a triple-spanning transmembrane domain, and a long carboxyl-terminal inner domain (rottier, ) . the orf encodes for a glycosilated protein (s) ranging from to amino acids (aa) in length (enjuanes et al., ) , constituting the large, petal-shaped spikes on the surface of the virion. this large protein can be divided into three structural domains. the large external domain at the n-terminus is divided further into two subdomains s and s . the s sub-domain includes the n-terminal half of the molecule and forms the globular portion of the spikes. it contains sequences that are responsible for binding to specific receptors on the membrane of susceptible cells. s sequences are variable, containing various degrees of deletions and substitutions in different coronavirus strains or isolates. mutations in the s region have been associated with altered antigenicity and pathogenicity. in contrast, s sequences are more conserved and contain two heptad repeat motifs that suggest a coiled-coil structure (lai and holmes, ) . on the basis of phylogenetic analysis and antigenic cross reactivity, three groups can be distinguished in the coronaviridae family. group i includes ccov, the transmissible gastroenteritis virus of swine (tgev), the porcine epidemic diarrhoea virus (pedv), the porcine respiratory coronavirus (prcov), the feline coronaviruses (fcovs) and the human coronavirus e (hcov e). fcovs can be distinguished into two serotypes, i and ii, on the basis of a virus neutralization assay in vitro using both type-specific feline sera and monoclonal antibodies directed against the s protein (herrewegh et al., ) . in the field, fcovs type i are predominant and fcovs type ii are detected only sporadically. differences in the s gene of fcovs type i and that of fcovs type ii may also account for the different properties observed in vitro, as indeed fcovs type i grow poorly in tissue culture cells (pedersen et al., ) while type ii strains grow well. in a previous study, sequence analysis of ccovs detected in faecal samples collected from dogs with diarrhoea revealed multiple nucleotide substitutions accumulating over a fragment of the m gene (pratelli et al., ) . a genetic drift to fcov type ii was also observed in the sequence of ccovs detected in the faeces of two pups infected naturally during the late stages of long-term viral shedding. it was thus hypothesized that (i) the dogs might have been infected by a mixed population of genetically different ccovs, or (ii) the viruses detected in both the pups were the result of mutation/recombination events (pratelli et al., b) . subsequently, extensive sequence analysis on multiple regions of the viral genome, including orf a, orf b and orf , of several ccov positive faecal samples provided strong evidence for the existence of two separate genetic clusters of ccov. the first cluster includes ccovs intermingled with reference ccov strains, such as insavc- and k , while the second cluster segregates separately from ccovs and, presumably, represents a genetic outlier referred to as fcovlike ccov (pratelli et al., ) . the aim of the present study was to evaluate the genetic differences between the fcov-like and the 'typical' ccovs in the sequence of the gene encoding for the s protein. twenty faecal samples, collected in four kennels in southern italy from Á/ month-old pups affected with diarrhoea, were tested. three of the kennels were sited in different areas of puglia, about km from each other, while the fourth shelter was located in abruzzo, more than km far from the other three. the faecal samples were stored at (/ c until tested. all the samples were negative by a haemoagglutination test for canine parvovirus and positive for ccov when submitted to a pcr assay targeting a fragment of the m gene (primers ccov Á/ccov ) (pratelli et al., ) . the presence of fcov-like ccovs in the same samples was detected by means of a differential pcr assay, using primers (ccov a Á/ccov ) able to recognise nucleotide substitutions conserved in the m gene across all the fcov-like ccovs (pratelli et al., a) . the sequence of the primers and their positions in the m gene are shown in table . comparative sequence analysis of the s gene have revealed a higher degree of variation at the n-terminus rather than at the c-terminus of the s protein (jacobs et al., ; motokawa et al., ; horsburgh and brown, ; wesley, ) . taking into account the sequence drift to fcovs observed in the m gene, we designed a pair of primers, ucd f Á/ucd r, amplifying a bp fragment at the very ? end of the sequence of the s gene of fcovs type i (strains ucd , ku- and black), which encodes for the highly conserved c-terminus of the spike protein. all the canine samples were tested with this primer pair and yielded an amplicon of the expected size. the sequence of the amplicons obtained was determined by direct sequencing of the pcr products and displayed Á/ % nucleotide identity to fcov type i strains. to verify the extent of genetic variation between the two clusters of ccov in the s gene, we determined the nearly-full length sequence of the orf of one (elmo/ ) of the samples that had tested positive to fcov-like ccov. degenerate primers were designed with the code-hop strategy (rose et al., ) , using a wide selection of coronaviruses belonging to group i of the coronaviridae. this strategy is based on the identification of blocks of homology in the amino acid sequences of distantly related organisms. hybrid oligonucleotides, with a short ? degenerate core region and a longer ? consensus clamp region, are selected by retro-translation on the blocks individuated. codehop sense primers with low degeneracy index were selected to amplify overlapping fragments of orf . one-step reverse transcription and pcr amplification were carried out using superscript tm one-step rt-pcr for long tem-plates (life technologies, invitrogen. milan, italy) . to select against any ccov-like virus during pcr amplification, reverse primers specific for the s gene of fcovlike ccov elmo/ were used in combination with the forward degenerate primers. reference ccov strains, s , k , / (buonavoglia et al., ) , usda, / and se, were used as controls in the pcr reactions, to verify that no ccov was amplified by the primers. the amplicons were cloned into pcr † . -topo † vectors (topo ta cloning † , invitrogen, milan, italy) and the recombinant clones individuated by blue/white screening. plasmid dna was extracted and subjected to sequence analysis (genome express: labo grenoble, france) . following this strategy, the fragments of the fcov-like virus elmo/ were inserted into the clones. a consensus of the sequences obtained was determined and the overlapping fragments were manually edited. alignments and sequence analysis were performed using the bioedit software package (hall, ) . the guidelines of the strategy used to determine the sequence of the s gene of the fcov-like ccov are schematised in fig. . the position and sequence of the degenerate primers are reported in table . the nucleotide sequence of elmo/ will appear in the ddbj/embl/genbank databases under accession no. ay . the amino acid sequence of the fcov-like ccov elmo/ was inferred and aligned with a selection of coronaviruses of the group i. phylogenetic and molecular evolutionary analyses were conducted using mega version . (kumar et al. ) and paup version . b (swofford, ) . a parsimony tree was elaborated using a heuristic algorithm and supplying statistical support by bootstrapping over replicates. the primer pair, v f Á/v r, designed on the sequence of virus elmo/ , was chosen to selectively amplify fcov-like ccovs. the sequences and positions of the primers are shown in table . all the samples previously characterised as fcov-like by two separate primer pairs targeted to the m gene (ccov a Á/ccov ) (pratelli et al., a) and to the s gene (ucd f Á/ucd r) were screened with the new primers specific for virus elmo/ . the rna was reverse transcribed with random hexamers using mulv reverse transcriptase (applied biosystems, roma, italy) and then amplified with amplitaq dna polymerase (applied biosystems, roma, italy ), by cycles at c for min, c for min and c for min. to assess the intra-genotypic variability in the s gene of fcov-like ccovs, four strains, each representative of a different geographical area, were selected and subjected to sequence analysis. all the faecal samples characterised previously as fcov-like ccovs were recognised by the primer pair ucd f Á/ucd r, yielding an amplicon of the expected size of bp. about % ( nucleotides) of the orf encoding for the s protein of strain elmo/ was determined. using the orf of strain ucd as a reference sequence, the fragment sequences between nt and and between aa and may be approximately localised. the highest nucleotide identity was to fcov type i strains ku- , ucd and black (Â/ %), whereas identity to fcovs type ii and ccovs was about %. comparison of the inferred amino acid sequences revealed . Á/ . % identity to fcovs type i, . Á/ . % to fcovs type ii and . % to reference ccov strains (table ). in accordance with previous observations, the sequence of the s protein was much more conserved at the c-terminus rather than at the nterminus. for instance, amino acid identity to the bestmatching sequence (strain ku- ) ranged from . to . % and to strain insavc- from . to . % in the n-and c-terminus, respectively. the inferred amino acid sequence of the s protein of strain elmo/ is shown in fig. . similar to other coronaviruses, there were several potential n-glycosilation sites, asn Á/x Á/ser (nxs) or asn Á/x Á/thr (nxt). most of the glycosilation sites were conserved between strain elmo/ and feline/ccovs, in particular with respect to the most closely related fcovs type i. interestingly, a potential cleavage site, the stretch of basic amino acids, arg Á/arg Á/ala Á/arg Á/arg (rrarr), was found. the basic stretch is about at the same position as in the s protein of group ii and group iii coronaviruses, but it is absent in the s protein of all the other group i coronaviruses. parsimony analysis on the s protein of group i coronaviruses revealed that strain elmo/ is much more related to fcovs type i rather than to typical ccovs. conversely, typical ccovs tightly segregate with fcovs type ii and the porcine coronaviruses tgev and prcov (fig. ) . the new pair of primers, v f Á/v r, specific for strain elmo/ , successfully amplified all the samples tested, yielding an expected pcr product of bp. the sequence of the v f Á/v r amplicon of four strains representative of different geographical areas was determined, revealing a nucleotide variability of Á/ %. genetic divergence within the coronavirus group i is accounted for by linear evolution as well as by a sudden, dramatic shift generated by rna deletions or recombination. for instance, the s protein of pedv occupies an intermediate position between hcov e and tgev (kocherhans et al., ) , while the s protein of prcov is closely related to tgev but has a large deletion in the n-terminus (more than aa) that may explain the change in the pathobiology of the virus (vaughn et al., ) . comparative sequence analysis of the genome of fcovs type i and type ii and ccov has demonstrated that fcov type ii has arisen from a template switch between fcov type i and ccov, which took place between the s and m genes. an additional template switch has been mapped in the orf b region for strain fcov - and in the orf a b region for strain fcov - . the double recombination event deter-mined the introduction of a large genome fragment, encompassing the ccov-like s-encoding gene, into the background of a fcov genome (herrewegh et al., ) . the s gene of ccov is closely related to fcovs type ii, tgevs and prcovs, and is more divergent from fcovs type i, pedvs and hcov e (wesseling et al., ) . so far, little evidence has been provided for genetic drifts or shifts affecting ccov. wesley ( ) has described a canine strain displaying a higher sequence identity to tgev in the n-terminus of the s protein, explained as a possible recombination between ccov and tgev, and related to improved growth in swine testicular cells. the findings in the present study clearly indicate that a novel ccov type, highly divergent from the reference ccov strains, and more closely related to fcovs type i, circulates among dogs. indeed, by means of rt-pcr, elmo/ -like strains were successfully detected in all the samples tested. all the samples had been characterised as 'atypical' ccovs when screened with a rt-pcr targeted to the m gene and able to distinguish between the two genetic lineages previously identified (pratelli et al., a) . extensive sequence analysis of multiple regions in the orf a and b , as well in the m-encoding gene, has confirmed the existence of a distinct genetic lineage of ccov, evolutionarily localised between ccov and fcov. many amino acid residues observed in the m protein of fcovlike ccovs are the same as in fcovs and presumably represent a retention of the sequence of an ancestral virus (pratelli et al., ) . re-considering these data in the light of the findings of the present study and considering the analogies with closely related viruses, we have concluded that the extent of genetic variation observed within the ccovs is limited in the orf a and slightly greater in the orf b and orf , though it still accounts for a clear pattern of segregation into a distinct genetic lineage. the two genotypes of ccov diverge dramatically in the orf , where there is more than . % nucleotide and about . % amino acid variation from reference ccovs. analysis of the s gene of the elmo/ -like ccovs revealed a little degree of variation ( Á/ %), which may be explained by their different geographical origin. the majority of the sequence changes observed are conservative, demonstrating that there is some heterogeneity in the orf of elmo/ -like ccovs. in conclusion, the findings suggest that the two canine genotypes underwent a linear evolution rather than a sudden shift originating from a recombinant event analogous to those leading to the appearance of fcovs type ii. finally, recombination with an ancestral coronavirus from which fcovs type i and elmo/ -like ccovs directly evolved may not be excluded. whether the elmo/ -like ccovs have phenotypic properties different from those of typical ccovs, similarly to fcovs type i and ii, will be interesting to evaluate. the high divergence in the amino acid composition and the loss and gain of potential glycosilation sites, compared to the most closely related coronaviruses (fcov type i, fcov type ii and typical ccov), strongly suggest that the elmo/ strain is poorly correlated antigenically with the other coronaviruses of dogs and cats. moreover, the presence of the stretch of basic residues rrarr is indicative of a potential cleavage of the protein (wesseling et al., ) . a similar basic motive is present, approximately in the same position, in all the coronaviruses identified to date of both group ii and iii, but it is absent in all the coronaviruses of group i. cleavage of the s protein of coronaviruses has been correlated to cell-fusion activity in vitro (hingley et al., ) but the potential implications in viral pathobiology have not been determined. on the basis of the significant genetic differences between the reference and the elmo/ -like ccovs our tentative proposal is to designate the new genotype identified as ccov type i, and to designate the reference strains, such as insavc- and k , as ccovs type ii. this new designation does not take into account the order of discovery of the viruses, but it is based on the genetic similarity between ccovs type ii and fcovs type ii and between ccov type i and fcov type i. l'infezione da coronavirus del cane: indagine sulla presenza del virus in italia virus taxonomy, classification and nomenclature of viruses bioedit: a user-friendly biological sequence alignment and analysis program for windows / /nt feline coronavirus type ii strains - and - originate from a double recombination between feline coronavirus type i and canine coronavirus the spike protein of murine coronavirus mouse hepatitis virus strain a is not cleaved in primary glial cells and primary hepatocytes cloning, sequencing and expression of the s protein gene from two geographically distinct strains of canine coronavirus the nucleotide sequence of the peplomer gene of porcine transmissible gastroenteritis virus (tgev): comparison with the sequence of the peplomer protein of feline infectious peritonitis virus (fipv) completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence mega : molecular evolutionary genetics analysis software coronaviridae: the viruses and their replication comparison of the amino acid sequence and phylogenetic analysis of the peplomer, integral membrane and nucleocapsid proteins of feline canine and porcine coronaviruses pathogenic differences between various feline coronavirus isolates development of a nested pcr for the detection of canine coronavirus variation of the sequence in the gene encoding for transmembrane protein m of canine coronavirus (ccv) pcr assay for the detection and the identification of atypical canine coronavirus in dogs m gene evolution of canine coronavirus in naturally infected dogs identification of coronaviruses in dogs that segregate separately from the canine coronavirus genotype characterization of the coronavirus mouse hepatitis virus strain a small membrane protein e consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences the coronavirus membrane protein paup*, phylogenetic analysis using parsimony (*and other methods) three new isolates of porcine respiratory coronavirus with various pathogeni cities and spike (s) gene deletions the s gene of canine coronavirus, strain ucd- , is more closely related to s gene of transmissible gastroenteritis virus than to that of feline infectious peritonitis virus nucleotide sequence and expression of the spike (s) gene of canine coronavirus and comparison with the s proteins of feline and porcine coronaviruses this study was supported by grants from cegba (centro di eccellenza di genomica in campo biomedico e agrario) and from ministry of university, italy (project: enteriti virali del cane). key: cord- -k hlf authors: sun, dongbo; shi, hongyan; guo, donghua; chen, jianfei; shi, da; zhu, qinghe; zhang, xin; feng, li title: analysis of protein expression changes of the vero e cells infected with classic pedv strain cv by using quantitative proteomic technique date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k hlf recent outbreaks of porcine epidemic diarrhea virus (pedv) have caused widespread concern. the identification of proteins associated with pedv infection might provide insight into pedv pathogenesis and facilitate the development of novel antiviral strategies. we analyzed the differential protein profile of pedv-infected vero e cells using mass spectrometry and an isobaric tag for relative and absolute quantification. a total of proteins were identified that were differentially expressed between the pedv-infected and mock-infected groups (p < . , quantitative ratio ≥ . ), among which the expression of proteins was up-regulated and that of proteins was down-regulated in the pedv-infected vero e cells, involving in integrin β /β , cystatin-c. the gene ontology analysis indicated that the molecular function of the differentially expressed proteins (deps) was primarily related to binding and catalytic activity, and that the biological functions in which the deps are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. among the disease-related functions, certain anti-viral pathways and proteins, such as the rig-i-like receptor, rap , autophagy, mitogen-activated protein kinase, pi k-akt and jak-stat signaling pathways, and integrin β /β and cystatin-c proteins, represented potential factors in pedv infection. our findings provide valuable insight into pedv-vero e cell interactions. the porcine epidemic diarrhea virus (pedv) is an enveloped, single-stranded positive-sense rna virus that causes porcine epidemic diarrhea (ped), an acute and highly contagious enteric disease in pigs. ped is characterized by severe diarrhea, vomiting, dehydration, and a mortality rate of up to % in suckling piglets (pensaert and debouck, ) . ped was first reported in belgium and the united kingdom in , and frequent outbreaks have occurred in various asian countries . since , acute ped outbreaks have continually occurred in thailand, china, and the usa, which have resulted in substantial economic losses (puranaveja et al., ; li et al., ; chen et al., ; huang et al., ; marthaler et al., ; stevenson et al., ; yang et al., ; chen et al., ) . the continued outbreaks of ped, despite control efforts, have caused widespread concern. the pedv belongs to the genus alphacoronavirus, in the family coronaviridae and order nidovirales (belouzard et al., ) . previous studies have investigated various control measures to protect against pedv infection, such as vaccines, diagnostic tools, and therapeutic drugs (sun et al., ; ren et al., ; sun et al., ; zhu et al., ; guo et al., ; kim and lee, ) . various aspects of pedv infection remain unclear, for example, swine testis (st) cells expressing porcine aminopeptidase n of pedv receptor were not susceptible to pedv infection. african green monkey kidney (vero) cells are highly susceptible to pedv infection, and are widely used for the primary isolation and cultivation of pedv guo et al., ) . therefore, vero lineages are suitable hosts for understanding the mechanisms of pedv infection. proteomics techniques are effective tools for characterizing protein expression profiles, and have been used widely to investigate disease-associated proteins (hondermarck et al., ; boja et al., ; he et al., ; sun et al., ) . among current proteomics methods, quantitative high-throughput proteomics approaches are useful for the analysis of infection-associated proteins of pathogens (linde et al., ; papachristou et al., ; ye et al., ; zeng et al., ) . in our current study, we used a quantitative proteomics approach based on an itraq tandem mass spectrometry (ms/ms) technique to identify proteins differentially expressed between pedv-infected and mock-infected vero e cells. the functions of the differentially expressed proteins (deps) were analyzed to determine whether they might be associated with pedv infection. our findings provide valuable insight into the changes in cellular processes that occur during pedv infection. the cv strain of pedv, kindly provided by maurice pensaert at ghent university (merelbeke, belgium), was used in all of our experiments after being adapted to vero e cells, as previously described (hofmann and wyler, ) . the vero e cell-adapted pedv, the vero e cells, and the monoclonal antibody against the nucleocapsid protein (np) of pedv were stored at the diarrhea-related viruses section, division of swine infectious diseases, national key laboratory of veterinary biotechnology, harbin veterinary research institute of the chinese academy of agricultural sciences. the vero e cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum (fbs) in -cm flasks at • c in a % co atmosphere. when the cells reached - % confluence, they were inoculated with the pedv at a multiplicity of infection of in presence of g/ml trypsin. at h postinoculation, the cells began to exhibit cytopathic effects (cpes) of viral infection, but no cells lysis or shedding had occurred. the cells were washed three times with cold phosphate-buffered saline (pbs, ph . ). a . -ml aliquot of lysis buffer containing % sds, mm dtt, and mm tris-hcl (ph . ) was added to each flask, and the flasks were incubated at • c for min. the cell lysates were collected using a cell scraper, and boiled for min. three cell lysate replicates were prepared for the pedv-infected (v -v ) and mock-infected (c -c ) vero e cells, and stored at − • c. western blotting was performed to confirm pedv infection by detecting the presence of the np of pedv in the vero e cells. aliquots of the cell lysates were subjected to sds-page on a % acrylamide gel, and the protein bands were transferred to a nitrocellulose membrane using a semi-dry transfer device (bio-rad, hercules, ca, usa). the membrane was blocked using % (w/v) nonfat dried milk in pbs at • c for h, before incubation in pbs containing the anti-np monoclonal antibody ( : dilution) at • c for h. after washing three times with % tween in pbs (pbst), the membrane was incubated in pbst containing a horseradish peroxidase-conjugated goat anti-mouse igg ( : dilution) at • c for h. after washing three times with pbs, the membrane was incubated with enhanced chemiluminescence detection reagents (biotopped, beijing, china) at room temperature for min, and the peroxidase-mediated luminescence was digitally captured using the molecular imager chemidoc xrs+ system (bio-rad) and the image lab software (bio-rad). to verify the differential expression of the selected deps, equivalent volumes of the cell lysate replicates from the pedv-infected (v -v ) and mock-infected (c -c ) vero e cells were pooled into the v and c samples, respectively, and western blotting was performed as described above, with the following exceptions: a : dilution of the polyclonal antibodies anti-␤ tubulin, anti-integrin-␤ , anti-cystatin-c, anti-protein s -a , anti-apolipoprotein e , and anti-centrin from rabbit (beijing biosynthesis biotechnology, beijing, china) was used as the primary antibody, and a : dilution of the hrp-conjugated goat anti-rabbit igg (sigma-aldrich, st. louis, usa) was used as the secondary antibody. protein digestion of the samples was performed according to the fasp procedure described by wiśniewski et al. ( ) . an aliquot of each cell lysate containing g of protein was combined with l of std buffer containing % sds, mm dtt, and mm tris-hcl (ph . ). the detergent, dtt, and other low-molecular-weight components were removed by dilution in ua buffer containing m urea and mm tris-hcl (ph . ) and repeated ultrafiltration using microcon ( kda) ultrafiltration units. the reduction of cysteine residues was blocked by the addition of l of . m iodoacetamide to the ua buffer. the samples were incubated for min in darkness before ultrafiltration. the microcon filters were washed three times with l of ua buffer, followed by two washes with l ds buffer containing mm triethyl ammonium bicarbonate (ph . ). the final protein suspensions were digested using g of trypsin (promega, madison, wi, usa) in l of ds buffer overnight at • c, and the digested peptides were collected as the filtrate. the peptide content was quantified based on absorbance at nm using an extinction coefficient of . for a . mg/ml solution. the digested peptide mixture was labeled using the -plex itraq reagent (life technologies, carlsbad, ca, usa), according to the manufacturer's instructions. each itraq reagent was dissolved in l of ethanol, and added to the digested peptide mixture. the samples were labeled as c - , c - , c - , v - , v - , or v - . the samples were multiplexed, and vacuum dried. the itraq labeled peptides were fractionated by scxc using the akta purifier system (ge healthcare, waukesha, wi, usa). the dried peptide mixture was reconstituted, and acidified by the addition of ml of buffer a containing mm kh po in % acetonitrile (ph . ). the samples were loaded onto a . mm × mm column packed with polysulfoethyl ( m, Å) chromatography resin (polylc, columbia, maryland, usa). the peptides were eluted at a flow rate of ml/min using a gradient of - % buffer b containing mm kcl and mm kh po in % acetonitrile (ph . ). the gradient elution consisted of - % buffer b for min, - % buffer b for min, and - % buffer b for min. the absorbance of the eluate was monitored at nm, and fractions were collected at -min intervals. thirty fractions were combined into ten pools, and desalted using empore standard density spe c cartridges (sigma-aldrich, st. louis, mo, usa) with a bed diameter of mm and a volume ml. each fraction was concentrated by centrifugation in a vacuum, and reconstituted in l of . % (v/v) trifluoroacetic acid. all samples were stored at − • c until the ms analysis was performed. the lc-ms/ms experiments were performed using a q exactive mass spectrometer coupled to a proxeon biosystem easy nanolc (thermo fisher scientific, waltham, ma, usa). ten microliters of each fraction was injected for nanolc-ms/ms analysis. the peptide mixture ( g) was loaded onto a c -reversed phase column ( cm × m) packed with rp-c ( m) resin in buffer a containing . % formic acid, and eluted with a linear gradient of buffer b ( % acetonitrile and . % formic acid) at a flow rate of . l/min for min using the intelliflow technology. the eluate underwent electrospray ionization for the ms/ms analysis. the ms/ms instrument was run in the peptide recognition mode, and the spectra were acquired using a data-dependent top- method based on the selection of the most abundant precursor ions from the survey scan ( - m/z) for hcd fragmentation. the determination of the target value was based on the predictive automatic gain control, and the dynamic exclusion duration was s. survey scans were acquired at a resolution of , at m/z , and the resolution for the hcd spectra was set to , at m/z . the normalized collision energy was ev, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as . %. the ms/ms spectra were compared to the uniprot cercopithecidae database ( sequences, downloaded november , ) and a decoy database using the mascot search engine, version . (matrix science, london, uk), embedded in the proteome discoverer . software (thermo electron, san jose, ca). the following parameters were used for protein identification: a peptide mass tolerance of ppm; an ms/ms tolerance of . da; trypsin digestion; a missed cleavage value of ; the fixed modifications included carbamidomethyl, itraq plex(k), and itraq plex(n-term); the variable modification was oxidation; and an fdr value ≤ . . protein quantification was performed using the proteome discoverer . software based on the centroided reporter ion peak intensity. the average quantitative value of each protein in samples c , c , and c (mock-infection group) was used as the internal reference. the value of the quantitative ratio for each protein relative to the internal reference was calculated, and averaged to obtain the quantitative ratio (v/c) of the proteins identified in the treatment groups (unwin et al., ) . a protein was considered to be differentially expressed between the pedv-infected and mock-infected groups based on the following criteria: the protein had to be present in three replicates of both groups, the difference in the level of the protein between the two groups had to be statistically significant (p < . ), and the change ratio for the protein had to be ≥ . (yuan et al., ) . the expression of a protein with a v/c > . was considered to be up-regulated, and those with a v/c < . were considered to be down-regulated. the data were analyzed using a two-tailed, paired student's t test. the statistical analysis was performed using the excel software (microsoft, redmond, wa, usa). the deps were annotated using the blast go, version . . , program (ashburner et al., ; quevillon et al., ; götz et al., ) . the deps were blasted against the kegg genes database (human). the gene ontology categories (gocs) were retrieved, and mapped to pathways in the kegg database (kanehisa et al., ) . table the proteins identified from pedv-infected and mock-infected groups. the vero e cells inoculated with pedv displayed distinct cpes at h postinoculation, including cell shrinkage, cell fusion, and a rounded cell morphology, but no cells lysis or shedding was observed (fig. a) . the immunoblotting analysis confirmed that the vero e cells were pedv-infected. the band corresponding to the np of pedv was detected in samples v , v , and v , whereas none was detected in samples c , c , and c (fig. b) . the identified peptides, identified proteins, quantified proteins, known/uncharacterized proteins, and the goc annotations are showed in table . a total of proteins, including peptides, were identified in the pedv-infected and mock-infected groups using the itraq-ms/ms approach, among which ( . %) were quantified, ( . %) were known proteins, and ( . %) were uncharacterized/putative proteins. based on the gocs, ( . %) of the proteins were annotated as biological process, ( . %) were annotated as molecular function, and ( . %) were annotated as cellular components. the quantification and significance of the identified proteins are shown in fig. . the changes in the levels of expression between the two groups were analyzed based on statistical significance. of the proteins identified, ( . %) were not differentially expressed (p > . ), and ( . %) were expressed at statistically different levels between the pedv-infected and mockinfected vero e cells (p < . ), including proteins ( . %) with a p-value between . and . , proteins ( . %) with a p-value between . and . , and proteins ( . %) with a p-value < . . the proteins with a p-value < . were also filtered based on whether the v/c or c/v was ≥ . . based on these criteria, a total of ( . %) of the identified proteins were determined to have been differentially expressed between the pedv-infected and mock-infected groups (table ) . among the deps, . % ( / ) were up-regulated, and . % ( / ) down-regulated. the known proteins and uncharacterized/putative proteins accounted for . % ( / ) and . % ( / ) of the deps, respectively. the dep displaying the greatest increase in expression in the pedv-infected vero e cells was isoform of the ovarian cancer immunoreactive antigen domaincontaining protein ( : . ), and the dep displaying the greatest decrease in expression in the pedv-infected vero e cells was cystatin-c ( : . ). the gene ontology (go) database has been widely used for describing protein function in a standardized format. according to their gocs, the deps were annotated as cellular component, biological process, or molecular function. the go annotations are shown in table , and distributions of the go annotations are shown in fig. . seventy-eight deps were distributed among groups of biological processes (fig. a) . the metabolic process (go: ), cellular process (go: ), single-organism process (go: ), and biological regulation (go: ) groups contained the highest proportions of the biological process deps. there were more up-regulated proteins in the cellular component organization group (go: ) than down-regulated proteins. seventy-four deps were distributed among eight cellular component groups (fig. b) , among which the organelle (go: ) and cell (go: ) groups contained the highest proportion of cellular component deps. there were more down-regulated deps in the membrane group (go: ) than up-regulated deps, and there were more up-regulated deps in the macromolecular complex group (go: ) than downregulated deps. ninety-seven deps were distributed among eight molecular function groups (fig. c) , among which the binding (go: ) and catalytic activity (go: ) groups contained the greatest proportion of molecular function deps. the kyoto encyclopedia of genes and genomes (kegg) pathway is a collection of pathway maps that represent molecular interactions and reaction networks in cells. seventy-five of the deps identified were annotated, and mapped to a total of six kegg pathway categories, which included the metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases pathway categories (fig. ) . the annotations in the metabolism, organismal systems, and diseases pathway categories represented , , and pathway groups, respectively (fig. a, b and f). the annotations in metabolism pathways category included the carbohydrate, energy, lipid, nucleotide, amino acid, glycan biosynthesis, cofactors and vitamins, biosynthesis of other secondary metabolites, and xenobiotics pathway groups (fig. a ). the annotations in the organismal systems category included the tolllike receptor (tlr) signaling (ko ), rig-i-like receptor (rlr) signaling (ko ), and natural killer cell mediated cytotoxicity (ko ) pathway groups (fig. b) , which represent pathways related primarily to the immune response to virus infection. the largest number of deps in the cellular process category were mapped to the lysosome (ko ) pathway group, all ten of which were down-regulated deps (fig. c ). the annotations in the genetic information processing category included pathway groups related to dna replication and repair, transcription, translation, and the folding, sorting, and degradation of proteins (fig. d ). the annotations in the environmental information processing proteins and one up-regulated protein. overall, more disease pathway groups were assigned to a single down-regulated dep than those assigned to up-regulated deps. the integrin (␤ and ␤ subunits) protein was annotated to the largest number of pathway groups ( ), which included the organismal systems, environmental information processing, cellular processes, and diseases categories. the ␤ tubulin as loading control, three down-regulated deps cystatin-c, apolipoprotein e and centrin- , two up-regulated deps integrin-␤ and protein s -a , were selected to verify differential expression between the pedv-infected and mock-infected vero e cells. the immunoblotting analysis showed that the ratios of these proteins between the pedv-infected and mock-infected groups were consistent with those obtained using the quantitative proteomics analysis (fig. ) . in our study, pedv infection significantly alters protein expression in vero e cells. the differentially expressed proteins (deps) annotated to virus infection-associated signaling pathways, autophagy, and virus entry-associated proteins were analyzed further to assess their potential roles in pedv infection. in mammals, the first line of defense against virus infection is the innate immune system. early antiviral responses are initiated upon the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs), resulting in the production of interferons for the innate immune response and the maturation of dendritic cells for establishing acquired immunity (yokota et al., ) . the prrs are grouped into the tlrs, rlrs, and nucleotide binding-oligomerization domain-like receptors. our results showed that pedv infection induced the deps that participated in six signaling pathways involved in viral infection, including the rlr, rap , pi k-akt, mapk, jak-stat, and tlr signaling pathways. the pedv is an enteric virus that infects the intestinal epithelial cells (iec) of swine, causing severe diarrhea. hirata et al. ( ) reported the rig-i signaling pathway plays an important role in antiviral innate immunity mechanisms in iecs. sheikh et al. ( ) reported the rap a signaling pathway was associated with secretory diarrhea. the jak-stat signaling pathway regulates the adaptive and innate mechanisms related to mucosal immunity (heneghan et al., ; wang et al., ) . our results showed that deps induced by pedv infection in vero e cells involved in the rlr, rap , and jak-stat signaling pathways. it has been reported that the tlr, mapk, and pi k-akt signaling pathways play roles in host cell responses to coronaviruses (mizutani et al., ; integrins are cell surface ␣/␤ heterodimeric glycoproteins that contribute to a variety of cellular functions (stewart and nemerow, ) . combinations of the various isotypes of the ␣ and ␤ subunits of integrins generate more than different integrin proteins. previous studies have shown that various integrin molecules are used as receptors for virus attachment (stewart and nemerow, ; sun et al., ) . in our current study, the expression of integrin-␤ and -␤ was down-regulated and up-regulated, respectively, in response to pedv infection. the upregulation of integrin-␤ expression is consistent with that observed in response to dengue virus infection (zhang et al., ) . our pathway analysis revealed that both integrin-␤ and -␤ are involved in pathways that contribute to organismal systems, environmental information processing, cellular processes, and diseases. the integrin ␣v␤ protein has been shown to serve as an entry receptor for various viruses (guerrero et al., ; neff et al., ; chu and ng, ; parry et al., ; wang et al., ) , some of which bind the integrin through an rgd sequence in a viral structural protein to initiate infection (stewart and nemerow, ) . the s protein of pedv is a glycoprotein peplomer on the viral surface that plays an important role in receptor-mediated binding and cell membrane fusion. in our study, the integrin recognized sequences of pedv s protein was analyzed based on ruoslahti's ( ) report. the results indicated that four conserved integrin-recognized amino acid motifs (asp-gly-glu, lys-gly-glu, arg-leu-asp, and leu-asp-val) were found in the s proteins of various pedv strains (data not shown). these data suggest that integrin proteins may act as an infection associated protein for the attachment and entry of pedv. autophagy is an essential component of host defenses against viral infection (dong and levine, ) . maier and britton ( ) reported that ␤-coronaviruses induced autophagy. in our study, more deps were mapped to the autophagy pathway group than any of the other pathway groups. fifteen deps were mapped to the lysosome and phagosome pathways. of the proteins, ( %) were down-regulated deps. although the autophagy pathway plays an antiviral role in virus-infected cells, the autophagy machinery is exploited by certain viruses for viral evasion and propagation. our results showed that pedv infection induced the downregulation of the expression of many autophagy-associated proteins. therefore, pedv infection might inhibit autophagy in vero e cells, thus facilitating virus replication. previous studies have shown that the microtubule-associated protein b is a useful biomarker protein for autophagy (dong and levine, ) . we found that the expression of map b was up-regulated . -fold in the pedv-infected vero e cells. these results suggest that the pedv induces autophagy. cystatin-c has been shown to reduce the replication of certain viruses, including the poliovirus, rhinovirus, and human coronaviruses oc and e (korant et al., ; collins and grubb, ) . the cleavage of s protein has been shown to be essential for the induction of cell-to-cell fusion and coronavirus entry into cells (sturman et al., ) . shirato et al. ( ) reported the transmembrane type ii serine protease enhanced infection of pedv in vero cells by increasing virus release. in our study, the reduced expression of cystatin-c might facilitate pedv replication and release through the activation of cysteine-associated proteases in vero e cells. apolipoprotein e , galectin, clusterin, and transferrin receptor have also been shown to be associated with virus infection (hishiki et al., ; peng et al., ; martin and uprichard, ; tripathi et al., ) , and may therefore function as infectionassociated proteins in pedv-infected vero e cells. additionally, the decreased in vitro expression of the adherens junction protein, such as cadherin, might be associated with a reduced integrity of pedv-infected intestinal epithelial cells in vivo. to the best of our knowledge, our study represents the analysis of the interactions between pedv and vero e cells using a quantitative proteomics technique. pedv infection-associated pathways and proteins are described and discussed based on the bioinformatics analysis of the differentially expressed proteins. our analysis of vero e cell responses to pedv infection identified relevant targets for subsequent in-depth studies of pedv pathogenesis, expand the current knowledge base regarding the interaction between the pedv and the host cell, and provide useful basic information about other coronaviruses. although the vero e cells are highly susceptible to pedv infection and facilitate experimental design and performance for proteomics, the vero e cell line is an interferondeficient cell line and not a pig cell line. so, the detailed functions of these pathways and proteins in pedv infection require further verification in the actual host cells of pedv. gene ontology: tool for the unification of biology mechanisms of coronavirus cell entry mediated by the viral spike protein evolution of clinical proteomics and its role in medicine detection and molecular diversity of spike gene of porcine 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two novel b cell epitopes on porcine epidemic diarrhea virus spike protein influenza a virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein clusterin simultaneous analysis of relative protein expression levels across multiple samples using itraq isobaric tags with d nano lc-ms/ms porcine reproductive and respiratory syndrome virus nsp ␤ inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-␣ degradation integrin alphavbeta is a coreceptor for human cytomegalovirus universal sample preparation method for proteome analysis genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central china from quantitative proteomics by amino acid labeling in foot-and-mouth disease virus (fmdv)-infected cells the battle between virus and host: modulation of toll-like receptor signaling pathways by virus infection cytotoxicity evaluation of oxidized single-walled carbon nanotubes and graphene oxide on human hepatoma hepg cells: an itraq-coupled d lc-ms/ms proteome analysis proteome analysis of porcine epidemic diarrhea virus (pedv)-infected vero cells up-regulated expression of beta integrin induced by dengue virus serotype infection associated with virus entry into human dermal microvascular endothelial cells expression and purification of the scfv from hybridoma cells secreting a monoclonal antibody against s protein of pedv this work is supported by the national natural science foundation of china (grant no. ), the state national key laboratory of veterinary biotechnology (grant no. sklvbf / ), and the program for new century excellent talents in heilongjiang provincial university (grant no. -ncet- ). key: cord- -j vajdi authors: pratelli, annamaria; elia, gabriella; martella, vito; palmieri, alessandra; cirone, francesco; tinelli, antonella; corrente, marialaura; buonavoglia, canio title: prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of italy date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: j vajdi an enzyme-linked immunosorbent assay (elisa), using as antigen canine coronavirus-infected crfk cell supernatant, was developed to detect antibodies against canine coronavirus (ccov). out of a total of dog serum samples, which were positive by routine virus neutralisation test were also elisa positive. seventeen samples which were negative by the virus neutralisation test, were positive by elisa and by the confirmatory western blotting test. the elisa was substantially more sensitive than the virus neutralisation test in detecting antibodies to ccov and may be used as an alternative technique to virus neutralisation. coronaviruses are large viruses that cause respiratory, enteric and generalised disease in humans and domestic animals. canine coronavirus (ccov) belongs to one of the major antigenic groups of coronaviruses (siddell et al., ; spaan et al., ) and is related serologically and genetically to transmissible gastroenteritis virus of pigs, porcine epidemic diarrhoea virus, feline coronaviruses (fcovs) and human coronavirus e (hcov- e) (sanchez et al., ; horsburgh et al., ; wesseling et al., ) . the viruses have enveloped virions containing a nonsegmented, positive plus-stranded rna genome that is - kb in length (siddell, ) and is packaged by the nucleoprotein n ( kda) into a helical nucleocapsid. the ribonucleoprotein is surrounded by a lipid envelope which contains three viral glycoproteins: the spike s glycoprotein forming the long club-shaped surface projections on the virion surface, the integral m glycoprotein ( kda) and the small membrane (e) protein ( kda). ccov was first isolated from faecal specimens of american military dogs with diarrhoeal disease (binn et al., ) . ccov infects dogs of any breed or age, causing depression, anorexia, vomiting and diarrhoea in young animals. the dogs generally recover spontaneously - days after infection, but the diarrhoea may persist for more than weeks. death may occur - days after the onset of disease, especially in young pups (carmichael and binn, ) . definitive identification of ccov-induced disease may be achieved by detection of ccov shed in faeces at electron microscopy or by virus isolation in cell culture. the common presence of coronavirus-like particles in faeces makes the diagnosis of ccov by electron microscopy arduous and requires confirmation by other diagnostic methods (athanssious et al., ) . on the other hand, many investigators have experienced difficulties in cultivating coronaviruses in vitro (de groot and horzinek, ; tennant et al., ; pratelli et al., pratelli et al., , wesley, ) . recently, nested pcr assay (n-pcr) for the detection of ccov with primers to the transmembrane protein m gene, has been described (pratelli et al., ) . assessment of antibodies by the virus neutralisation assay (mochizuki et al., ) , or by indirect enzyme-linked immunosorbent assay (elisa) (rimmelzwaan et al., ; tuchiya et al., ) provides an indication of the exposure of an animal to ccov. detection of immunoglobulin m (igm) and igg against ccov by indirect elisa (tennant et al., ; naylor et al., ) determines current or previous exposure of an animal to ccov. the aim of this study was to improve the detection of ccov-specific antibodies in canine sera, by using an elisa that was compared to the virus neutralisation test and western blotting assay. crandell feline kidney (crfk) cells were grown in dulbecco modified eagle's medium supplemented with % foetal bovine serum. a cell culture adapted ccov strain / , isolated from a dog with enteritis (buonavoglia et al., ) , was used throughout this study. a total of serum samples, collected from dogs of the small animal clinic, faculty of veterinary medicine, bari, italy, were employed. the supernatants of crfk cell cultures infected with ccov strain / , or mock infected cultures, were harvested h postinfection and clarified at ×g for min at °c. subsequently, the supernatants were centrifuged for h at × g at °c. the pellets were resuspended in phosphatebuffered saline (pbs, ph . ) at / the initial volume and used as positive and negative antigens for elisa and western blotting tests. serial twofold dilutions starting from / of each sample were mixed with tcid of ccov / strain in -well microtitre plates. the plates were kept at room temperature for min and then crfk cells were added to each well. the plates were read after days of incubation at °c when the cytopathic effect was complete in the virus control cultures. the titre was expressed as the highest serum dilution neutralizing the virus. microtitre plates (costar) were coated with ml per well of antigen diluted in carbonate buffer ( mm na co , mm nahco , [ph . ]) and incubated overnight at °c with shaking. the plates were washed four times in pbs containing . % tween (pbs-t), then treated with blocking solution ( . % gelatin in carbonate buffer) for min at °c and washed four times with pbs-t. dilutions of / in pbs-t of each canine serum were added in duplicate and the plates were incubated for min at °c. the washing cycle described above was then repeated and ml of peroxidase-conjugated caprine igg, specific for canine igg (sigma chemicals, st. louis, mo), diluted in pbs-t were added to each well, and the plates were incubated for h at °c. after another washing cycle, ml of freshly prepared substrate were placed in each well. the solution consisted of mg , %azino-di-[ -ethylbenzthiazoline sulfonate] diammonium salt (abts, sigma) in ml . m phosphate citrate buffer, ph . , containing ml/ ml hydrogen peroxide and the optical densities at nm (od ) were determined. the adjusted od values of each sample were obtained by subtracting the absorbance of the mock antigen-coated well from that of the corresponding virus antigen-coated well. the antigen preparations diluted : in laemmli sample buffer were heated at °c× min, subjected to electrophoresis in sodium dodecyl sulphate (sds)-polyacrylamide minigel ( - %) and transferred onto nitrocellulose membrane (immobilon p, pore size . mm) with a biorad transblot cell apparatus at v for h. non-specific binding sites were blocked overnight at °c with % non-fat dry milk (blotting grade blocker, biorad) in tris buffered saline (tbs; tris mm, nacl mm, ph . ) containing . % tween (tbs-tm). all the subsequent steps were conducted with shaking at room temperature. after washing three times with tbs tween (tbs-t), the membrane was probed with canine serum samples diluted : in tbs-tm for h. the membrane was then washed three times with tbs-t ( min per wash) and incubated for h with peroxidase labeled caprine igg specific for canine igg (sigma chemicals, st. louis, mo). after being washed extensively in tbs-t, dab ( , %-diaminobenzidine tetrahydrochloride [sigma] in tbs [ph . ], . % hydrogen peroxide) was used in the chromogenic reaction. a total of of the samples examined were negative by the virus neutralisation test and were examined subsequently by western blotting. ten of these sera were found concomitantly to be free of ccov specific antibodies and used to adjust the elisa cut-off value (three standard deviations higher than the arithmetic mean of the absorbance of concordantly negative samples). samples with value exceeding than . were considered to be positive. as shown in fig. a , of the serum samples proved to be positive at the virus neutralisation test. nineteen samples, which were found to be free of ccov neutralising antibodies, gave a positive signal by the elisa. in of these, ccov specific antibodies were also found by the western blotting test. these sera, therefore, were recorded as positive concomitantly by elisa (fig. b) . two discrepant sera remained which gave a positive result exclusively by elisa. with the western blotting test, most serum samples showed reactivity to the n and m proteins of ccov and only a few samples also reacted against the s protein (data not shown). considering virus neutralisation as a 'gold standard' test, elisa had a sensitivity of % and a specificity of . %, with an overall agreement of . %. however, when the virus neutralisation test combined with the confirmatory western blotting test, were used as the 'gold standard', the elisa showed an improved specificity ( . %), while the sensitivity remained unchanged ( %), with an overall agreement of . %. in this study an elisa was developed to detect ccov-specific antibodies. to determine whether the elisa could be used for this purpose, sensitivity and specificity were evaluated, considering the virus neutralisation test and the virus neutralisation combined with western blotting tests as 'gold standards'. out of sera, false-positives and no falsenegative samples were detected initially with the elisa, using virus neutralisation as the standard test. in / of the false-positive samples, however, ccov antibodies were also detected by western blotting and these samples were thus considered to be positive. three of the samples, which were negative only by the virus neutralisation test, yielded consistently high od values by elisa; this apparently strange result is not easy to explain, but since western blotting showed both specific and nonspecific reactivity for the same samples (cellular antigens?), this may account for the high elisa od values. recently, ccov infection has attracted scientific interest especially concerning the pathogenesis of infection in dog (bandai et al., ; naylor et al., ) , viral genome variability (pratelli et al., ) and the development of rapid and sensitive diagnostic tests (pratelli et al., ; bandai et al., ; naylor et al., ) . routine measurements of ccov-specific antibodies are still based on the virus neutralisation test, which is costly as well as time-consuming (at least days) and requires specialised laboratories. we carried out a preliminary comparison of the virus neutralisation and the elisa and the findings clearly revealed a discrepancy between the results of the two tests, especially in evaluating ccov seronegativity: out of the samples that were negative in virus neutralisation and positive at elisa were confirmed to be positive by the western blotting. antibody determination by different methods does not necessarily give parallel results. whereas, elisa and western blotting are able to detect antibodies to all major viral proteins, the virus neutralisation test only measures the neutralising antibodies and, as a result, may lack sensitivity. the lower sensitivity of the virus neutralisation test for detecting the antibodies induced by ccov, may provide misleading information on the epidemiological features of the infection. above all, the virus neutralisation test may impede evaluation of the pre-existing immunological status of the dogs used in pathogenesis studies or in immunogenicity/potency trials on the vaccines employed for immunisation of dogs against ccov infection. because of its advantages, such as rapidity and greater sensitivity, the elisa described may be considered more attractive than the virus neutralisation test and prove a useful tool for the serological diagnosis of ccov infection. detection of bovine coronavirus and type a rotavirus in neonatal calf diarrhea and winter dyssentery of cattle in quebec: evaluation of three diagnostic methods canine coronavirus infections in japan: virological and epidemiological aspects recovery and characterization of a coronavirus from military dogs with diarrhea l'infezione da coronavirus del cane: indagine sulla presenza del virus in italia new canine enteric viral infection feline infectious peritonitis analysis of a . kb sequence from the % end of canine coronavirus genomic rna micro-neutralisation test with canine coronavirus for detection of coronavirus antibodies in dogs and cats identification of canine coronavirus strains from feces by s gene nested pcr and molecular characterisation of a new australian isolate diagnosis of canine coronavirus infection using nested-pcr variation of the sequence in the gene encoding for development of a nested-pcr assay for the detection of canine coronavirus the use of enzyme-linked immunosorbent assay systems for serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in the netherlands antigenic homology among coronaviruses related to transmissible gastroenteritis virus the coronaviridae: an introduction the biology of coronaviruses coronaviruses: structure and genome expression studies on the survival of canine coronavirus under different environmental conditions canine coronavirus infection in the dog following oronasal inoculation enzyme-linked immunosorbent assay for the detection of canine coronavirus and its antibody in dogs the s gene of canine coronavirus, strain ucd- , is more closely related to the s gene of transmissible gastroenteritis virus than to that of feline infectious peritonitis virus nucleotide sequence and expression of the spike (s) gene of canine coronavirus and comparison with the s proteins of feline and porcine coronaviruses this study was supported by grants from cegba (centro di eccellenza di genomica in campo biomedico e agrario) and from ministry of university, italy (project: enteriti virali del cane). we thank dr athina papa for revising the english of the manuscript. key: cord- -tjy f rk authors: park, sang-ik; park, da-hae; saif, linda j.; jeong, young-ju; shin, dong-jun; chun, young-hyun; park, su-jin; kim, hyun-jeong; hosmillo, myra; kwon, hyung-jun; kang, mun-il; cho, kyoung-oh title: development of sybr green real-time rt-pcr for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: tjy f rk unclassified bovine enteric calicivirus (becv) is a newly recognized bovine enteric calicivirus that differs from bovine norovirus, and which causes diarrhea in the small intestines of calves. to date, methods such as real-time reverse transcription-polymerase chain reaction (rt-pcr) have not been developed for the rapid detection, quantitation and diagnosis of becv. presently, a becv-specific sybr green real-time rt-pcr assay was evaluated and optimized. diarrheic specimens (n = ) collected from to were subjected to rt-pcr, nested pcr and sybr green real-time rt-pcr. by conventional rt-pcr and nested pcr, ( . %) and ( %) samples tested positive, respectively, whereas the sybr green assay detected becv in ( . %) samples. using becv rna standards generated by in vitro transcription, the sybr green real-time rt-pcr assay sensitively detected becv rna to . × ( ) copies/μl (correlation coefficiency = . ). the detection limits of the rt-pcr and nested pcr were . × ( ) and . × ( ) copies/μl, respectively. these results indicate that the sybr green real-time rt-pcr assay is more sensitive than conventional rt-pcr and nested pcr assays, and has potential as a reliable, reproducible, specific, sensitive and rapid tool for the detection, quantitation and diagnosis of unclassified becv. caliciviruses are small, nonenveloped viruses that are - nm in diameter and possess a single-stranded, plus-sense rna genome with a size of . - . kb, encoding a single-structural protein of - kda (green et al., ) . the family caliciviridae is classified into four genera: norovirus (nov), sapovirus (sav), vesivirus and lagovirus (green et al., ) . among these genera, nov and sav are associated with enteric diseases in humans and animals (bridger, ; guo et al., ; saif et al., ) . the bovine enteric calicivirus (becv) newbury agent- (na ), which is different from bovine norovirus (bnov), was associated with calf diarrhea in the united kingdom in (woode and bridger, ) . data from electron microscopy, animal cross-protection experiments and solid-phase immune electron microscopy has revealed that na is unrelated to bnov dastjerdi et al., ; woode and bridger, ) . recent genomic data has revealed the circulation in cows of the virus strains bo/nebraska/ /us (nb) and bo/newbury / /uk (na ) * corresponding author. tel.: + ; fax: + . e-mail address: choko@chonnam.ac.kr (k.-o. cho). (han et al., ; oliver et al., ; smiley et al., ) . nb-like becv is genetically most similar to sav and lagovirus, and causes pathological lesions in the small intestine of gnotobiotic calves (smiley et al., ) . furthermore, nb and na formed a distinct clade that is independent of the four recognized genera (oliver et al., ) . since this potentially new genus, which includes nb-and na -like unclassified becvs, has not been named by the international committee on viral taxonomy, the present study used the terms "unclassified becvs" and "nb-and na -like viruses" to designate the strains. real-time reverse transcription-polymerase chain reaction (rt-pcr) is relatively easy to conduct and has a high-throughput capacity. it has become the method used most widely for gene detection and quantitation (oka et al., ; yaper et al., ) . in recent years, real-time rt-pcr has emerged as a highly reproducible and sensitive method for detection and diagnosis of novs and savs, proving to be more sensitive than methods used previously such as electron microscopy and conventional rt-pcr (atmar and estes, ; glass et al., ; trujillo et al., ) . various realtime rt-pcr assays have been reported for nov and sav detection including taqman assays (chan et al., ; hohne and schreier, ; jothikumar et al., ; kageyama et al., ; oka et al., ; trujillo et al., ; vainio and myrmel, ) table list of primers used for conventional rt-pcr, nested pcr, and sybr green real-time rt-pcr assay for the detection and quantification of unclassified bovine enteric calicivirus in the fecal specimens from the diarrheic calves. this study becr c r: gcacgggcttcttctagaga park et al. ( ) a f: forward primer for conventional rt-pcr, nested pcr, and sybr green real-time rt-pcr; r: reverse primer for conventional rt-pcr, nested pcr, and sybr green real-time rt-pcr. b all the procedure of rna extraction, conventional rt-pcr and nested pcr were performed as described previously (cho et al., ; park et al., park et al., , a park et al., ,b, . c becr primer sequence for sybr green real-time rt-pcr was same with nr primer sequence for nested pcr. based on the sybr green chemistry (pang et al., ; richards et al., ) . although the quantitation of the four caliciviridae genera including nov, sav, largovirus and vesivirus has been reported worldwide (green et al., ) , there are no reports of real-time rt- fig. . sybr green real-time rt-pcr assay for the quantitation of becv crna standard. (a) amplification of × , × , × , × , × , × , × , × and × copies of crna standard used in parallel with each sybr green-based real-time rt-pcr assay. (b) sybr green real-time rt-pcr products using serially diluted in vitro transcripts. m: molecular marker; lanes - : . × , . × , . × , . × , . × , . × , . × , . × and . × viral copies/l; n: negative control. (c) standard curves of the real-time rt-pcr based on serial dilutions of becv crna standards. in the standard curve of these dilutions each dot represents the result of duplicate amplification of each dilution. the coefficient of determination (r ) and the slope (s) of the regression curve are indicated. pcr assays for the detection and quantitation of unclassified becv in bovine stool samples. the present study evaluated, optimized and validated a sybr green real-time rt-pcr assay for detecting and quantitating unclassified becvs with archived stool samples. from the same samples previously evaluated by conventional rt-pcr and nested pcr assays, sybr green real-time rt-pcr proved to result in more specific and sensitive quantitation. bovine stool samples (n = ) were selected from archived fecal samples collected by local veterinary clinicians in korea during [ ] [ ] . all bovine samples were tested for the presence of unclassified becv by conventional one-step rt-pcr and nested pcr, and were sequenced for confirmation (park et al., ) . fifty-nine samples tested positive for becvs and samples were negative for becv by conventional rt-pcr and nested pcr, respectively. the stool specimens were stored at − • c. rna was extracted from a centrifuged l starting volume of % fecal suspension using trizol-ls (gibco-brl, grand island, ny). the recovered total rna was suspended in l of rnase free water and stored at − • c until analysis. the oligonucleotide primers and optimal conventional rt-pcr and nested pcr conditions for the detection of unclassified becv (table ) are described elsewhere (cho et al., ; park et al., park et al., , a park et al., ,b, . a one-step real-time rt-pcr was developed based on sybr green detection. the primers were designed based on the published sequence of the becv rdrp gene (table ). all reactions were performed using a corbett research rotor-gene real-time amplification system (corbett research, mortlake, australia) and sensimix one-step rt-pcr kit with sybr green (quantace, london, uk). titration of primers was achieved using rna from positive fecal samples. reactions were run using primer concentrations from . to . m. a concentration of . m of each primer gave the highest sensitivity, together with a limited formation of primer dimers. real-time rt-pcr was performed in a final volume of l that contained l of rna template, l sensimix one-step mixture, . m each of forward and reverse primers, . l of sybr green solution, . l of rnase inhibitor and l of rnase free water. reverse transcription was carried out at • c for min, followed by the activation of the hot-start dna polymerase at • c for min and three-step cycles: • c for s, • c for s and • c for s. samples were considered positive if both an exponential increase of fluorescence and a becv-specific melting peak were observed. the amplicon of becv rdrp gene ( bp) obtained from rt-pcr was used as the source of dna for the preparation of in vitro rna transcripts. after cloning the amplified pcr products (yeastern biotech, taipei, taiwan), a clone was selected based on sequencing of the insert. the plasmid dna of the recombinant clone was digested with the restriction enzyme psti, electrophoresed using a % agarose gel and the purified digested plasmid was recovered from the gel using the qiaquick gel-extraction kit (qiagen, valencia, ca). the gel-purified linearized dna clone served as the template for the in vitro transcription using the megascript kit (ambion, auston, tx) according to the manufacturer' instructions. after h of incubation at • c, the dna template was removed by digestion with dnase treatment with a turbo dna free kit (ambion). crna was then purified with a rneasy mini kit (qiagen). after dilution, the concentrations were calculated by measuring the absorbance at nm with a nanodropnd (nanodrop technologies, wilmington, de, usa). crna samples were stored at − • c until use. the regression lines between the logarithms of the input amounts of crnas and the corresponding mean threshold cycle (ct) values were calculated using the rotor-gene software version . . (corbett research). statistical analyses were performed by spss version . . for windows (spss, chicago, il). the two-tailed fisher's exact test was used to assess the statistical significance of the detection rate between real-time rt-pcr, conventional rt-pcr and nested pcr. a p-value < . was considered statistically significant. forward and reverse becv primers for real-time rt-pcr were designed in conserved stretches of a portion of the rdrp gene to allow a broad reactivity to unclassified becv. the standard rna was diluted in a -fold dilution series ranging from undiluted to . × − , and was tested in duplicate in each run in the sybr green real-time rt-pcr assay. quantities used for each dilution corresponded to . × - . × viral copy numbers of stranded becv rna (fig. ) . the sybr green real-time rt-pcr assay detected as little as . × copies of becv in vitro transcripts per reaction and displayed linearity over a wide dynamic range of copy number ( . × - . × copies) (fig. a) . amplicons of expected size by sybr green real-time rt-pcr were visualized by gel electrophoresis (fig. b) . standard curves with a higher correlation coefficient (r > . ) were generated using a serial dilution of in vitro transcripts of becv rna from . × to . × , and the slope value of the standard curves with these transcripts was . (fig. c) . the standard rna subjected to sybr green real-time rt-pcr presented a specific fluorescence signal and ct values between . and . (data not shown). real-time rt-pcr was successful in fig. . sensitivity of conventional rt-pcr and nested pcr with in vitro transcripts. (a) one-step conventional rt-pcr was performed in the same tube with serially diluted in vitro transcripts. (b) nested pcr products with one-step conventional rt-pcr products. m: molecular marker; lanes - : . × , . × , . × , . × , . × , . × , . × , . × and . × viral copies/l, respectively; n: negative control. detecting positive samples and quantifying the unclassified becv viral load. to assess the sensitivity and end point of sybr green real-time rt-pcr as compared to conventional rt-pcr and nested pcr assay, -fold serial dilutions of the in vitro transcripts described above were tested. conventional rt-pcr was performed in the same tube. nested pcr was also performed using the amplified products from the conventional rt-pcr. the lowest detection limits of the conventional rt-pcr and nested pcr as determined using serial dilutions were . × and . × viral copies/l, respectively ( fig. a and b) . analysis of the dilution series showed that the sybr green real-time rt-pcr assay could detect a quantity , and times lower than conventional rt-pcr and nested pcr, respectively. nonspecific reactions were not evident with any of the three pcr approaches (data not shown). the detection rates of becv by real-time rt-pcr, conventional rt-pcr and nested pcr were compared using stool samples (table ) . nine samples ( . %) were positive by both conventional rt-pcr and real-time rt-pcr, and samples ( . %) were positive by real-time rt-pcr and negative by conventional rt-pcr. no samples that were negative by real-time rt-pcr were positive by conventional rt-pcr, and samples ( . %) were negative by both tests (table (a) ). the percentage of agreement between these two assays was . %. the sensitivity and specificity of real-time rt-pcr compared with conventional rt-pcr were . % and . %, respectively. the agreement beyond chance was calculated with a kappa statistic, which was . . the detection rate of real-time rt-pcr was slightly higher than that of conventional rt-pcr (p > . ). comparing nested pcr and real-time rt-pcr, samples ( . %) were positive by both approaches, samples ( . %) were positive by real-time rt-pcr and negative by nested pcr, one ( . %) sample was negative by real-time rt-pcr and positive by nested pcr and ( . %) were negative by both tests (table (b) ). the percentage of agreement between the two assays was . %. the sensitivity and specificity of real-time rt-pcr compared with nested pcr were . % and . %, respectively. the agreement beyond chance calculated with a kappa statistic, which was . . the detection rate table comparison of the detection of unclassified becs by the sybr green real-time rt-pcr, conventional rt-pcr, and nested pcr assay. of real-time rt-pcr was moderately higher than that of nested pcr (p < . ). the efficacy of sybr green real-time rt-pcr for the quantitation of becv in stool samples was evaluated. all positive samples subjected to sybr green real-time rt-pcr presented a specific fluorescence signal and ct values between . and . (data not shown). becv amplicons displayed a melting temperature (t m ) between . and . • c (data not shown). range of t m values for unclassified becv using real-time rt-pcr using sybr green chemistry in vitro transcripts for previously positive and negative fecal samples tested by conventional rt-pcr and nested pcr, was - . , - and . - • c, respectively. t m values depend on different factors including the initial concentration and size of the template, gc content and the sequence of the amplified fragment (ririe et al., ) . the viral copy numbers in sybr green realtime rt-pcr positive samples ranged from . × to . × . nine samples that tested positive in real-time rt-pcr and conventional rt-pcr contained . × - . × viral copies, whereas samples that tested positive by real-time rt-pcr and nested pcr had . × - . × viral copies. however, the fecal samples that tested positive only by real-time rt-pcr possessed comparatively lower viral copies, ranging from . × to . × viral copies. the specificity of real-time rt-pcr was assessed using fecal samples that had tested positive for other enteric pathogens including groups a, b and c bovine rotaviruses (brv a-c), bovine torovirus (btov), bovine coronavirus (bcov), bnov, bovine viral diarrhea virus (bvdv), salmonella spp., clostridium spp., campylobacter spp., shiga-toxin-producing escherichia coli, coccidium spp. and cryptosporidium spp. (asakura et al., ; park et al., a, b) . no positive signal was recorded for any of these pathogens. there are no reports concerning the use of real-time rt-pcr for the detection and quantitation of unclassified becvs in bovine stool samples. in this study, a one-step sybr green real-time rt-pcr is described. this method allows the detection and quantitation of unclassified becvs in a shorter period of time than has previously been obtained. in addition, the sensitivity and specificity of the developed sybr green real-time rt-pcr was evaluated and compared to conventional rt-pcr and nested pcr assays reported previously (park et al., ) . the ideal method for detection of becvs in stool samples should have a high degree of sensitivity and consistency of performance in the laboratory. presently, the one-step sybr green quantitative real-time rt-pcr assay was significantly superior to the other pcr assays in terms of sensitivity, specificity and quantitative linearity. rt-pcr and nested pcr using serially diluted becv crna could detect . × and . × viral copies/l, respectively, whereas the lower detection limit of sybr green real-time rt-pcr was . × viral copies/l. therefore, the sybr green rt-pcr assay is and , times more sensitive than conventional rt-pcr and nested pcr, respectively. the short amplicons in the real-time pcr assays used in this study likely resulted in more efficient amplification and higher sensitivity. taken together, the observations indicate the utility of the sybr green-based rt-pcr assay in the detection of becvs. in this study, a primer pair for sybr green-based real-time rt-pcr assay was designed to target sequences centered on the conserved rdrp gene. since nov, sav and becv belong to family caliciviridae, the rdrp gene appears to be highly conserved within the family and is a region of choice in the design of new detection methods. in addition, the new sybr green real-time rt-pcr assay could be used to screen human fecal samples to ascertain further the role of unclassified becvs in human diarrhea outbreaks, with a particular emphasis on human diarrhea cases from developing countries where cattle-human contact is more common (park et al., ; smiley et al., ) . to date, the reported fecal prevalence of becvs in calf diarrhea by conventional rt-pcr or nested pcr has ranged from . % to . % in uk (oliver et al., ) and south korea (park et al., ) to . % in united states (smiley et al., ) . in the present study, bovine stool samples were selected from archival diarrheic fecal samples. fifty-nine tested positive for becv by both conventional rt-pcr and nested pcr, and the remaining samples tested negative by conventional rt-pcr and nested pcr (park et al., ) . among these diarrheic stool samples, one-step sybr green real-time rt-pcr not only detected becvs in all fecal samples that had tested positive by nested pcr but also detected becvs in fecal samples that had tested negative by nested pcr. these findings support the above suggestion that sybr green real-time rt-pcr is more sensitive than conventional rt-pcr and nested pcr. in addition, these observations indicate that the fecal prevalence of the becvs in calf diarrhea might be higher than that reported previously in korea (park et al., ) . becv is more pathogenic than bnovs, inducing anorexia, diarrhea and xylose malabsorption and severe pathology in the small intestine smiley et al., ) . interestingly, fecal samples that tested positive only by sybr green real-time rt-pcr contained lower numbers of becv rna ( . × - . × copies/l). these fecal samples of diarrhea with lower number of becvs might be coinfected with other enteric pathogens. indeed, these samples positive for other enteric pathogens including bcov, brva and bvdv (data not shown). therefore, it is presumed that becv alone can induce diarrhea in calves or can play a role in accelerating the clinical and pathological presentation of diarrhea in the field calves. in conclusion, this study reports the development of a rapid, sensitive, specific and reproducible one-step sybr green real-time rt-pcr assay for detection, diagnosis and quantitation of unclassified becv in stool samples. this high-throughput assay may be useful in the investigation of possible sporadic and nosocomial gastroenteritis outbreaks in humans, in epidemiological and etiological studies and even for routine surveillance. detection and genetical characterization of shiga toxin-producing escherichia coli from wild deer diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses characterization of calici-like virus (newbury agent) found in association with astrovirus in bovine diarrhea small viruses associated with gastroenteritis in animals sapovirus detection by quantitative real-time rt-pcr in clinical stool specimens crossprotection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and rt-pcr and nested pcr for their detection the bovine newbury agent- is genetically more closely related to human srsvs than to animal caliciviruses taxonomy of the caliciviruses human caliciviruses the epidemiology of enteric caliciviruses from humans: a reassessment using new diagnostics detection and molecular characterization of cultivable caliciviruses from clinically normal mink and enteric caliciviruses associated with diarrhea in mink lesions of gnotobiotic calves experimentally infected with a calicivirus-like (newbury) agent genetic recombination between two genotypes of genogroup iii bovine noroviruses (bonvs) and capsid sequence diversity among bonvs and nebraska-like bovine enteric caliciviruses detection and characterization of norovirus outbreaks in germany: application of a one-tube rt-pcr using afluorogenic real-time detection system rapid and sensitive detection of noroviruses by using taq-man-based onestep reverse transcription-pcr assays and application to naturally contaminated shellfish samples broadly reactive and highly sensitive assay for norwalklike viruses based on real-time quantitative reverse transcription-pcr detection of human sapovirus by real-time reverse transcription-polymerase chain reaction genomic characterization of the unclassified bovine enteric virus newbury agent- (newbury ) endorses a new genus in the family caliciviridae evaluation and validation of real-time reverse transcription-pcr assay using the lightcycler system for detection and quantitation of norovirus molecular epidemiology of bovine noroviruses in south korea molecular detection and characterization of unclassified bovine enteric caliciviruses in south korea detection and characterization of bovine coronaviruses in fecal specimens of adult cattle with diarrhea during the warmer seasons detection and molecular characterization of calf diarrhea bovine coronaviruses circulating in south korea during genogroup i and ii noroviruses detected in stool samples by real-time reverse transcription-pcr using highly degenerate universal primers product differentiation by analysis of dna melting curves during the polymerase chain reaction rotavirus-like, caliciviruslike, and -nm virus-like particles associated with diarrhea in young pigs characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus reverse transcription-pcr assays for detection of bovine enteric caliciviruses (bec) and analysis of the genetic relationships among bec and human caliciviruses use of taqman real-time reverse transcription-pcr for rapid detection, quantification, and typing of norovirus molecular epidemiology of norovirus outbreaks in norway during to and comparison of four norovirus real-time reverse transcriptase pcr assays isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves rapid and quantitative detection of crimean-congo hemorrhagic fever virus by one-step real-time reverse transcriptase-pcr this study was supported by funds from the national veterinary research and quarantine service (nvrqs) of the ministry of agriculture and forestry, and the regional technology innovation program (rti - - ) of the ministry of commerce, industry and energy (mocie), republic of korea. the authors acknowledge a graduate fellowship provided by the korean ministry of education and human resources development through the brain korea project. key: cord- -ujabzic authors: yuk, seong-su; kwon, jung-hoon; noh, jin-yong; hong, woo-tack; gwon, gyeong-bin; jeong, jei-hyun; jeong, sol; youn, ha-na; heo, yong-hwan; lee, joong-bok; park, seung-yong; choi, in-soo; song, chang-seon title: comparison between dot-immunoblotting assay and clinical sign determination method for quantifying avian infectious bronchitis virus vaccine by titration in embryonated eggs date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: ujabzic a sensitive and specific method for measuring the vaccine titer of infectious bronchitis virus (ibv) is important to commercial manufacturers for improving vaccine quality. typically, ibv is titrated in embryonated chicken eggs, and the infectivity of the virus dilutions is determined by assessing clinical signs in the embryos as evidence of viral propagation. in this study, we used a dot-immunoblotting assay (dia) to measure the titers of ibv vaccines that originated from different pathogenic strains or attenuation methods in embryonated eggs, and we compared this assay to the currently used method, clinical sign evaluation. to compare the two methods, we used real-time reverse transcription-pcr, which had the lowest limit of detection for propagated ibv. as a clinical sign of infection, dwarfism of the embryo was quantified using the embryo: egg (ee) index. the dia showed . % higher sensitivity and . % higher specificity than the clinical sign determination method. the dia was particularly useful for measuring the titer of ibv vaccine that did not cause apparent stunting but propagated in embryonated chicken eggs such as a heat-adapted vaccine strain. the results of this study indicate that the dia is a rapid, sensitive, reliable method for determining ibv vaccine titer in embryonated eggs at a relatively low cost. infectious bronchitis virus (ibv) is a gamma coronavirus that causes a highly contagious disease in chickens. the virus causes severe economic losses to the poultry industry worldwide because it can affect the upper respiratory and reproductive tracts, and some strains can cause nephritis in chickens (jackwood, ) . despite intensive vaccination using both live attenuated and killed vaccines to prevent the disease, the emergence of new variant strains that do not serologically cross-react has complicated disease control and demonstrates the importance of vaccinating chickens with the disease-causing ibv types (cavanagh, (cavanagh, , . vaccination is considered the most cost-effective approach for controlling ibv infection (meeusen et al., ) . to prevent the economic losses caused by ibv, live attenuated vaccines and inactivated oil-emulsion vaccines containing both the km and massachusetts (m ) strains are widely used in korea. more recently, vaccine strains providing broad cross-protection have been developed, including the k and k / strains (kim et al., ; lim et al., lim et al., , . in korea, the viral content of a vaccine preparation is quantified by ibv titration according to standard procedure approved by the animal and plant quarantine agency. for this procedure, the chicken embryos are inoculated with serial dilutions of the virus preparation, and then the embryos are examined for the presence of specific lesions caused by the virus, i.e., dwarfing, curling, and stunting (doherty, ) . however, as a variety of live-attenuated vaccines have been developed for variant field strains, it is unclear whether the clinical sign determination method reflects viral propagation in ibv-infected embryos. furthermore, as there are no specific standards for measuring clinical signs, the measurements of specific lesions can differ among observers. our previous study revealed that a novel dot-immunoblotting assay (dia) using monoclonal antibodies against several ibvs could detect viruses propagated in embryonated chicken eggs (song et al., ) . to accurately, reproducibly, and efficiently measure vaccine titers, we used the dia to detect ibv propagated in inoculated http://dx.doi.org/ . /j.jviromet. . . - /© elsevier b.v. all rights reserved. embryonated eggs. the aim of this study was to evaluate and compare the sensitivity and specificity of the dia to the clinical sign determination method for detecting ibv in inoculated embryonated eggs during titration of ibv vaccines. a respiratory strain belonging to the mass group (m ), a nephropathogenic strain belonging to the km -like subgroup (km ), and a recombinant nephropathogenic strain belonging to korean new cluster (k / ) were used to evaluate the titer of ibvs used in killed vaccines as described by kim et al. ( ) . two nephropathogenic strains that were attenuated by serial passages (k p ) or heat-adapted passages (k / hp ) in chicken embryos were used to evaluate the live-attenuated vaccine strains (lee et al., ) . all viruses were propagated in -day-old specific-pathogen-free embryonated chicken eggs (ece; hy-vac, adel, ia) at • c for h. allantoic fluid from each egg was harvested, aliquoted, and frozen at − • c until use. all animal protocols used in this study were reviewed, approved, and supervised by the institutional animal care and use committee of konkuk university. to confirm the detection limit of the dia, each vaccine strain was serially diluted -fold in phosphatebuffered saline (pbs; invitrogen, carlsbad, ca) and analyzed by dia and real-time reverse transcription (rt)-pcr. concurrently, triplicate -fold serially diluted samples were used to determine the detection limit of real-time rt-pcr. to measure vaccine titer based on the infectivity of chicken embryos, -fold serial dilutions of a virus solution ( − - − ) were generated by mixing ml of the virus with ml of pbs containing g/ml gentamicin sulfate (sigma-aldrich, st. louis, mo); all dilutions were kept on ice. next, . ml of the − through − dilutions were inoculated into five -day-old spf ece (hy-vac), and the eggs were incubated at • c. no eggs had to be discarded due to non-specific death of embryos within one day of inoculation. after three days of incubation, l of allantoic fluid was extracted from the inoculated eggs using a ml syringe and was used to detect propagated ibv by the dia and real-time rt-pcr. eggs were then resealed with paraffin for further incubation. seven days after inoculation, the embryo:egg (ee) index was calculated for all eggs. propagation of the inoculated virus was determined using real-time rt-pcr, dia, and the ee index method, and the % egg infectious dose (eid ) of the five vaccines was calculated based on the method of reed and muench ( ) . rna was extracted from each inoculated allantoic fluid sample using the exiprep viral rna/dna extraction kit (bioneer co., daejeon, korea) according to the manufacturer's instructions, and eluted in a l volume. real-time rt-pcr analysis of the extracted rna samples was conducted as previously described (callison et al., ) . the primers and taqman dual-labeled probe were synthesized by macrogen (seoul, korea). the primers and probe were utilized in a l reaction containing . l of quantitect probe rt-pcr × mix (qiagen, hilden, germany), . l of rt enzyme (qiagen), . mol of primers, . mol of probe, and l of the rna sample from l of extracted rna. amplification was performed in an abi prism real-time pcr system using the following program: • c for min, • c for min, and cycles of • c for s and • c for s, with emitted fluorescence measurement. cycle threshold (ct) values below the detection limit were considered positive for ibv. the dia was conducted as described previously with slight modifications (song et al., ) . allantoic fluids from eces inoculated with the serially diluted vaccines were centrifuged at × g for min. next, l of the supernatant was dotted onto a nitrocellulose membrane ( . m pore size) using a hybri-dot -well filtration manifold (bio-rad laboratories, hercules, ca). uninfected normal allantoic fluid was used as a negative control. the membrane was then blocked with % bovine serum albumin in tris-buffered saline (tbs; mm tris, . % sodium chloride, ph . ) at • c overnight. the primary monoclonal antibodies ( f ) were diluted : with tris tween-buffered saline (ttbs; mm tris, . % tween , . % sodium chloride, ph . ) and incubated with the membrane for min at • c. after washing the membrane in ttbs three times for min each with gentle agitation, the membrane was incubated for min at • c with biotinylated anti-mouse immunoglobulin g (vectastain abc kit; vector laboratories inc., burlingame, ca) diluted in ttbs. following washing, the membrane was incubated for min at • c with biotin and avidin-conjugated peroxidase complex (vectastain abc kit) diluted in ttbs. after the final washing, the membrane was developed using mg of diaminobenzidine (pierce, rockford, il) in ml of mm tris and l h o for min. the reaction was then stopped by rinsing with distilled water (three times) and the membrane was allowed to air-dry. dark brown-colored dots were considered positive for ibv. dwarfing of the infected chicken embryos was detected by determining the ee ratio. the weights of the eggs were measured days after inoculation of the serially diluted viruses. the respective eggs and embryos were weighed using an electronic balance (ohaus, florham park, nj). the ee ratio was defined as the weight of embryo divided by the weight of respective egg. a total of spf eggs (hy-vac) were used to determine the ee ratio of control ece (mock-inoculated). briefly, we inoculated -day-old eggs with l of sterile pbs as a mock-inoculated control. the ee ratio of eggs was determined at days post-inoculation. the average ee ratio and the standard deviation of the mock control were calculated. the ee index was determined by dividing the ee ratio of inoculated eggs by the mean ee ratio of mock-inoculated eggs (dhinakar raj et al., ) . to assess the receiver operating characteristics (roc) using medcalc ® version . statistical software (mariakerke, belgium), positive or negative results confirmed by real-time rt-pcr were assigned as "true-positive" or "true-negative," respectively. the area under the roc-curve (auc index) was then calculated for both the dia and clinical sign detection methods. high values (close to ) indicate a highly accurate test (greiner et al., ) . the concordance of both assays to the properly classified samples as positive or negative was estimated by calculating the weighted kappa statistic (Ä test). Ä test values of . - . indicate moderate agreement, values of . - . indicate substantial agreement, and values of . - . indicate nearly perfect agreement (viera and garrett, ) . the -fold diluted virus was detected concurrently by dia and real-time rt-pcr (supplementary material fig. ). the highest dilution that showed dark-brown dots in the dia was − for k p and k / and − for m , km , and k / hp . the -fold diluted viruses were also examined by real-time rt-pcr (supplementary material fig. ). for the tested pathogenic or attenuated ibvs, there were no detectable ct values at the − dilution. therefore, the detection limit of the real-time rt-pcr assay was set to the ct value of the − dilution. the real-time rt-pcr method was found to be approximately -fold more sensitive than the dia method. thus, real-time rt-pcr is suitable for detecting propagated viruses and can be used to compare the dia and ee ratio detection methods for propagated ibv during titration. because the dia does not distinguish between infectious and noninfectious virus, we had to confirm that the assay does not detect inoculated virus in the eggs. as shown by the detection limit of the dia, we observed that the input virus was not detected at dilutions higher than − - − . thus, none of the dilutions used in the titering ( − - − ) would give a positive result without viral replication, especially after further dilution in the allantoic fluid. after days of incubation, the mean ee ratio and standard deviation of mock-inoculated eggs was . ± . . to detect dwarfism, the ee index was defined as the ee ratio of ibv-inoculated ece divided by the mean ee ratio of mock-inoculated ece. embryos with ee indices below the lowest individual ee index for mock-inoculated ece ( . ) were classified as positive for dwarfism. supplementry material related to this article found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . based on the results of real-time rt-pcr, the titer of respiratory the type ibv m strain was . eid /ml ( table ) . four of the five eggs inoculated with the − dilution showed positive results. the dia results were identical to the real-time rt-pcr results, even the individual egg results. in contrast, only three of the five eggs inoculated with the − dilution were positive by the ee index. the weight of the other two eggs exceeded the lowest value of the individual ee index of mock-inoculated ece ( . , . ). because one embryo inoculated with the − dilution died days after inoculation, we excluded this egg from the ee index measurements. one egg inoculated with the − dilution showed a positive result for the ee index ( . ). the titer of m obtained using the ee index was lower than that obtained using the other assays ( . eid /ml). according to real-time rt-pcr analysis, the titer of the korean nephrogenic type ibv (km ) and its attenuated form (k p ) were both . eid /ml. based on the individual values, four of the five eggs inoculated with the − dilution and three of the five eggs inoculated with the − dilution showed positive results for both viruses. the dia results were identical to the real-time rt-pcr results, including the individual egg results. unexpectedly, in the km titration, one egg at the − dilution showed a negative result ( . ) for the ee index, despite showing positive results in the other assays. because one embryo inoculated with the − dilution died days after inoculation, we excluded this egg from the ee index measurements. ee indices of two of the eggs inoculated with the − virus dilution ( . , . ) were slightly below the lowest individual ee index of the mock-inoculated ee index. the titer of km determined using the ee index was higher than that determined using the other assays ( . eid /ml). in the k p titration, the results according to the ee indices were identical to those obtained by real-time rt-pcr, except for one egg inoculated with the − dilution ( . ). accordingly, the titer of k p determined using the ee index was higher than that determined using other assays ( . eid /ml). based on the real-time rt-pcr results, the titers of the recombinant nephrogenic type ibv k / and its heat-attenuated form k / hp were . and . eid /ml, respectively ( table ) . two of the five eggs inoculated with the − dilution of the k / were positive. however, the ct value of one egg inoculated with the − dilution of k / hp ( . ) was higher than the detection limit of the dia ( . ) but lower than that of real-time rt-pcr ( . ). thus, the egg was negative according to the dia. in the k / titration, two embryos inoculated with the − and − dilutions died at four and five days after inoculation, respectively; therefore, we excluded these eggs from the ee index measurement. the ee index of one egg inoculated with the − dilution ( . ) was positive. in the k / hp titration, one embryo inoculated with the − dilution died at two days after inoculation and was excluded from the ee index measurement. the ee indices of two eggs inoculated with the − dilution ( . , . ) and one egg inoculated with the − dilution ( . ) were negative. two eggs inoculated with the − dilution ( . , . ) and one egg inoculated with the − dilution ( . ), which were positive according to real-time rt-pcr, did not show clear dwarfism. consequently, the three assays showed three different titers. when compared to the titer measured by real-time rt-pcr ( . eid /ml), the titers measured by dia ( . eid /ml) and ee index ( . eid /ml) were -fold and -fold lower, respectively. the performance of the two methods was also compared ( table ) . dia detected propagated ibv in ( . %) of positive samples, with perfect specificity ( %) and an auc index of . %. the value for this test was . , indicating that the values were highly consistent with the real-time rt-pcr results. of the true-positive samples, two samples in k / and k / hp were false-negatives by dia, which was also observed using the clinical signs method. the results of the ee index, which was defined by the cut-off value of the lowest ee index of the mockinoculated ece ( . ), showed propagated ibv in ( . %) of positive samples, with relatively low specificity ( . %) and an auc index of . %. the Ä value for this test was . , indicating moderate agreement with the real-time rt-pcr results. of the nine false-negative results obtained using the clinical sign method, two samples were m , four samples were km , two samples were k p , and one sample was k / . of the false-positive results obtained using the clinical sign method, three samples were m , two samples were km , one sample was k p , and five samples were k / hp . for the ee index results, which were obtained using the cut-off value of the lowest individual ee index of the mock-inoculated eces, we compared the cut-off value to the optimum value calculated based on the ee index distribution. the optimum cut-off ( . ) calculated based on the ee indices distribution suggested that the cut-off value of the lowest individual ee index of the mock-inoculated eces ( . ) was reliable and could be used to classify clinical signs by weight. using this new cut-off, only one false-negative sample was corrected to a positive result, and the assay showed . % sensitivity and . % specificity (fig. ) . we compared the clinical signs and dia results to the real-time rt-pcr results and found that the dia results were highly consistent with the real-time rt-pcr results. among the eggs inoculated with serially diluted ibv samples, only two samples showed false-negative results. however, results obtained using the clinical signs method did not match the real-time rt-pcr results. for the m respiratory strain, the titer calculated by the clinical signs method were lower than the titer calculated by real-time rt-pcr. the nephropathogenic strain km and its attenuated form k p showed higher titer when measured by the clinical signs method. compared to km , k p showed clearer signs of stunting. this demonstration that egg-adapted strains induce clearer embryo dwarfism than wild strains is in agreement with those of dhinakar raj et al. ( ) . however, the recombinant nephrogenic strain k / and its attenuated counterpart k / hp , which was egg-passaged with heat adaptation, showed opposite results. the overall ee indices were higher for k / hp than for k / . moreover, the k / hp titer measured by the clinical signs method was approximately -fold lower than the titer measured by real-time rt-pcr. these results suggest that the clinical signs induced by this heat-adapted virus might not be sufficient to produce dwarfism in eces. the extensive genetic diversity and high mutation rate of ibv generate many different virus types that the existing ibv vaccine strains do not protect against. thus, there is great demand for a multiple serotype ibv vaccine that provides broad protection. in addition, several research groups have attempted to develop a multivalent vaccine against not only multiple ibv serotypes but also other avian infectious diseases such as newcastle disease, avian influenza, and infectious laryngotracheitis, as well as nonrespiratory diseases such as marek's disease and fowl pox (ndegwa et al., ; sharma et al., ; vagnozzi et al., ; winterfield, ) . for such multivalent vaccines, which must be mixed accurately to induce a protective immune response, it is thought that the higher or lower titers observed by the clinical sign method (e.g., for the nephropathogenic strain or heat-adapted strains) could prompt manufacturers to add less or more antigen than required, respectively. this is especially important for live vaccines, because the virus first infects the host and then induces an immune response via innate and acquired immunity, the virus quantitation must represent the true propagating ability within the host. in korea, the currently approved methods for quantifying freeze-dried live ibv vaccine or killed ibv vaccine before virus inactivation state that quantification should be determined by observing infectious bronchitis-induced lesions at seven days after inoculation of serially diluted vaccine into -day-old ece (qia, ) . generally, the disease-induced lesions are clinical signs, including stunting, curling, and clubbed down. in the us, the method for titrating ibv vaccines also states that on the seventh day after virus inoculation, all eggs should be examined for ibv lesions (usda, ) . in this method, specific lesions, such as bile stasis and kidney urates, are listed as general clinical signs. however, assessing specific lesions is laborious and time consuming. moreover, well-trained observers who can recognize the clinical signs and can skillfully approach without damaging them are required. in this study, we choose stunting as a typical clinical sign because it is the most objective and quantifiable clinical sign, and it can be easily distinguished without observer error. real-time rt-pcr is a reliable method because its detection limit is lower than that of other methods. however, the fluorescence thermal cycler requires continuous maintenance and trained operators who can perform the experiments without introducing cross-contamination between samples, which is very costly. accurate measurement of vaccine titer could help manufacturers increase vaccine production or prevent vaccine failure resulting from antigen content that is insufficient to induce an immune response. the dia method could be used to measure the titer of ibv vaccines that do not elicit clear stunting signs but propagate in eces, such as the heat-adapted vaccine strain. in addition, this method is faster than clinical signs method, and could reduce inter-observer differences. the results indicate that the dia presented in this study is a relatively low cost, timesaving, sensitive, and reliable method for detecting ibv during ibv vaccine titration in embryonated eggs. development and evaluation of a real-time taqman rt-pcr assay for the detection of infectious bronchitis virus from infected chickens coronaviruses in poultry and other birds coronavirus avian 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massachusetts serotype vaccination suppresses replication of arkansas vaccine virus korean standard assay of veterinary biological products, chicken infectious bronchitis freeze-dried live vaccine standard examination a simple method of estimating fifty per cent endpoints field trial in commercial broilers with a multivalent in ovo vaccine comprising a mixture of live viral vaccines against marek's disease, infectious bursal disease, newcastle disease, and fowl pox detection and classification of infectious bronchitis viruses isolated in korea by dot-immunoblotting assay using monoclonal antibodies united states department of agriculture center for veterinary biologics testing protocol. in: supplemental assay method for the titration of newcastle disease vaccine, infectious bronchitis vaccine, and combination newcastle disease-infectious bronchitis vaccine in chicken embryos. united states department of agriculture animal and plant health inspection service protection induced by infectious laryngotracheitis virus vaccines alone and combined with newcastle disease virus and/or infectious bronchitis virus vaccines understanding interobserver agreement: the kappa statistic respiratory signs, immunity response, and interference from vaccination with monovalent and multivalent infectious bronchitis vaccines this work was supported by a grant from the agricultural biotechnology development program, ministry of agriculture, food and rural affairs, republic of korea (no. - ). key: cord- -n xp mlz authors: hall, richard j.; wang, jing; todd, angela k.; bissielo, ange b.; yen, seiha; strydom, hugo; moore, nicole e.; ren, xiaoyun; huang, q. sue; carter, philip e.; peacey, matthew title: evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: n xp mlz the discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. the preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. with the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. a review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. these were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as dna (adenovirus) and rna viruses (influenza a and human enterovirus), being either non-enveloped capsid or enveloped viruses. the effect of the enrichment method was assessed by both quantitative real-time pcr and metagenomic analysis that incorporated an amplification step. reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qpcr, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. a -step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. this study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. this study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated. since the proliferation of high-throughput sequencing technologies, the search for viruses has entered a new era (lipkin, (lipkin, , . these technologies are capable of producing millions of sequence reads without a priori knowledge of the sample. sequence data generated from a sample is compared to known sequence databases in order to identify viruses. previously, virus metagenomics was accomplished by cloning and sanger-based sequencing of randomly amplified nucleic acid (breitbart et al., ; djikeng et al., ) . despite the small amount of sequence data produced, it was still possible to detect viruses due to either high concentrations, or some process of prior enrichment being applied that removes host cells and exogenous nucleic acid. the relative abundance of a virus (or viral nucleic acid) in a sample, compared to that of other organisms such as bacteria or host cells (or their genomes), is a critical factor for the discovery of viruses when using metagenomics. a higher proportion of viral sequence increases the probability that ( ) viral sequences will be represented in a metagenomic dataset and ( ) larger contigs can be assembled, increasing the likelihood of a match in the database. it is has been shown that without some type of physical enrichment method, viruses may not be present in high enough concentrations to be detected (daly et al., ) . this problem is somewhat overcome as high-throughput sequencing technologies advance in both read length and sequencing depth. nevertheless, gains in sensitivity are still possible by the application of a physical enrichment process for viruses, and may also avoid the cost of generating and analysing additional data. in the field of viral ecology there have already been significant advances in the validation of physical enrichment methods for viruses (duhaime and sullivan, ; john et al., ) , such as the methods used to concentrate and purify viruses from seawater for virus discovery by metagenomics (hurwitz et al., ) . however, only a few methodological studies have been applied to evaluate the efficiency of viral enrichment methods in metagenomics that seek to diagnose animal or human disease. a study on human liver tissue compared enrichment techniques of freeze-thaw, centrifugation and nuclease-treatment for the detection of hepatitis c virus using both roche and illumina high-throughput sequencing platforms (daly et al., ) . the abundance of viral sequences in each treatment group was compared to results obtained by quantitative real-time pcr detection of transcripts, and the effect of each treatment method on viral genome coverage was also determined. such studies show that physical enrichment methods do increase the sensitivity of detection for viruses in metagenomics. for new researchers looking to perform work on human or animal samples for the purposes of detecting or diagnosing new or unexpected viruses, it can be difficult to ascertain which virus enrichment method may be applicable to a given sample type. methods described for viral ecology studies are unlikely to be applicable. a review was undertaken of published metagenomic studies that sought to describe viruses present in human or animal samples (excluding virus ecology studies) and provide details on the enrichment methods used (table ) . most of these studies incorporate the use of low-speed centrifugation and/or filtration to remove host cells or other micro-organisms, with a final nucleasetreatment step, where dnase or rnase will destroy exogenous nucleic acid but is not thought to affect nucleic acid protected by the viral capsid or envelope. ultracentrifugation also features as a common method for the concentration of viruses from samples. the application of virus discovery methods using metagenomics has been considered for routine use in diagnostic and reference laboratories to aid in the diagnosis of human (svraka et al., ) and animal disease (belak et al., ) . the application of these techniques in a clinical setting will require that any virus enrichment methods are simple to perform, fast, robust, effective, standardised and do not require significant capital expenditure. it is noted that the vast majority of the published studies in table apply the simple enrichment techniques without any a priori justification for the selection of the technique. this study sought to examine the rapid and simple enrichment techniques for viruses that appear to be in routine use in the literature for diagnosing animal and human diseases, but for which the effects on metagenomic data have not been studied. this was achieved by examining the effect of these enrichment methods on the relative abundance of viruses in a metagenomic dataset derived from a simple and well-characterised artificial sample. human enterovirus was cultured in human rhabdomyosarcoma cell line in hanks mem (life technologies, carlsbad, ca, usa) supplemented with % foetal bovine serum (thermofisher scientific, waltham, ma, usa). human adenovirus was also cultured in the human rhabdomyosarcoma cell line. influenza a(h n )pdm was cultured in mdck-siat cells (canine) in r-mix (diagnostic hybrids, athens, oh, usa). all virus cultures were composed of cell culture supernatant and monolayer present after freeze-thaw. escherichia coli o was cultured in brain heart infusion broth (bhi) and incubated at • c overnight. human a cells were cultured in dmem (life technologies, carlsbad, ca, usa) supplemented with % foetal bovine serum (thermofisher scientific, waltham, ma, usa). an artificial sample was formulated to consist of known amounts of e. coli o , a human epithelial lung carcinoma cells (atcc ccl- ), human enterovirus , human adenovirus and influenza a(h n )pdm . aliquots of the final dilution were subjected to three freeze-thaw cycles and were frozen and stored at − • c. based upon a review of enrichment methods presented in table , five combinations of three simple methods of enrichment were selected and performed on ml aliquots of the artificial sample as follows; low-speed centrifugation in a microfuge at × g for min at • c, sterile syringe filtration at . m, nuclease treatment using . u l − turbo dnase (life technologies, carlsbad, ca, usa), . u l − rnase one (promega, fitchburg, wi, usa) and x dnase buffer (life technologies, carlsbad, ca, usa) and incubation at • c for min, or combinations of these being a -step method (centrifugation followed by filtration), or -step (centrifugation, filtration then nuclease-treatment). independent duplicates for each treatment were performed and used in all subsequent experiments. the extraction of rna was achieved using the iprep purelink virus kit (life technologies, carlsbad, ca, usa), where l of the artificial sample was extracted and eluted into l of molecularbiology grade water. all real-time quantitative pcr assays (qpcr) were performed on a stratagene mx p real-time pcr system (agilent technologies, santa clara, ca, usa). qpcr on extracted rna was used to quantify a human cells, influenza, adenovirus, e. coli o and enterovirus present in the artificial sample. the human rnase p (rnp) gene was used as a target for the detection of human a cellular rna. the nucleoprotein gene target was used for the detection of influenza a(h n )pdm rna. both assays were performed using the agpath one step rt-pcr kit reagents (life technologies, carlsbad, ca, usa) and the primers and probes for these assays have been previously described (who, ) . each l reaction contained l of nucleic acid, . l of rt-pcr buffer, l of x rt-pcr enzyme mix, . m probe and . m primers. following an initial min reverse transcription step at • c and min denaturation step at • c, a -step cycling procedure of denaturation at • c for s with annealing and extension at • c for s over cycles was used. adenovirus dna was detected using a previously published assay (brittain-long et al., ) and the agpath one step rt-pcr kit (life technologies, carlsbad, ca, usa). each l reaction contained l of dna, . l of rt-pcr buffer, l of x rt-pcr enzyme mix, . m probe and . m primers. after an initial min reverse transcription step at • c and min denaturation table virus enrichment process prior to sequencing in metagenomic studies on human and animal samples. author year journal aim of study sample step step step step step step step step step step step step step step step at • c, a -step cycling procedure of denaturation at • c for s with annealing and extension at • c for min over cycles was used. enterovirus rna was detected using a previously described assay (oberste et al., ) step system (life technologies, carlsbad, ca, usa). each l reaction contained l of rna, . l of x invitrogen reaction mix, . l of mm mgso , . l of superscript ® iii rt/platinum ® taq mix, . m probe and . m primers. following an initial min reverse transcription step at • c and min denaturation step at • c, a -step cycling procedure of denaturation at • c for s, annealing at • c for s and extension at • c for s over cycles was used. for e. coli o a one-step assay was performed using the lightcycler ® probes mastermix (roche, indianapolis, in, usa) as previously described (paton and paton, ; thomas et al., ) . each l reaction contained l of dna, l of lightcycler ® probes master, . m probe (stx ) and . m primers (o and o ). following an activation step of • c for min, a -step cycling procedure of denaturation at • c for s, annealing at • c for s and extension at • c for s over cycles was used. every assay included negative and positive controls, and rnase-free reagents and handling procedures. the real-time pcr assays were made quantitative by including a dilution series of a plasmid with known copy number, which contained concatenated primer-probe-primer target sequences for each of the five assays. dna was co-purified with rna during the nucleic acid extraction method, and thus dna was removed using ambion dna-free (life technologies, carlsbad, ca, usa), then l of this rna was reverse transcribed into cdna using a first-strand cdna synthesis kit primed by random hexamers as per the manufacturer's instructions (life technologies, carlsbad, ca, usa) including the recommended rnase h digestion. the minimum g amount of dna required for input into the illumina truseq dna library preparation protocol was not achieved. amplification of the cdna was achieved by using a whole transcriptome amplification kit (qiagen, valencia, ca, usa) as described previously (berthet et al., ; cheval et al., ) . briefly, the reverse transcription step required in the kit was not utilised, but the ligation and amplification steps were followed as per the manufacturer's instructions except that the ligation reaction was terminated by heating to • c for min, and the amplification step was performed for h followed by termination of the reaction at • c for min. for each sample more than g of dna was produced and this was sequenced on an illumina miseq instrument (new zealand genomics limited, massey genome service, massey university, palmerston north, new zealand) using an illumina truseq dna library preparation (illumina, san diego, ca, usa). water-only negative controls failed to amplify any dna. illumina miseq sequence data consisted of bp paired-end reads. quality checking and redundant-read collapsing was performed and sequence reads were then compared to the ncbi non-redundant nucleotide database (downloaded from ncbi ftp the % confidence interval. the grey line represents the copy number of the target gene when no enrichment method is applied. site in february ) using blastn (blast+ . ). an e value of . was used as the cut-off threshold value for significant hits. the blastn output files were imported and parsed in megan (huson et al., ) for taxonomic assignment. the abundance of nucleic acid from the model organisms in the artificial sample (human cells, bacteria, influenza, adenovirus and enterovirus) was determined by using quantitative real-time pcr. the effect of different enrichment methods on target gene copy number was assessed (fig. ) . in general, all enrichment methods decreased the quantity of every model organism when compared to no treatment at all. human rna was removed to a limited extent by all the methods, with the -step treatment and nuclease-only treatments being the most effective, resulting up to -fold reduction in copy number. similarly, some bacterial nucleic acid was removed by all of the enrichment methods, with the nuclease-only or the -step treatment being the most effective. the three viruses used in this experiment represent a dna virus (adenovirus), and two rna viruses one of which is enveloped (influenza) and the other non-enveloped (enterovirus). the subsequent metagenomic analysis was targeted at the detection of rna viruses, but a dna virus was included to assess the potential to detect dna viruses using this methodology. each virus also represented a different level of concentration ( ) a high concentration at × copies (enterovirus), ( ) a moderate concentration (adenovirus) at , copies and ( ) a low concentration (influenza) at , copies. all viruses showed a decrease in copy number when an enrichment method was applied. this decrease was consistent across all enrichment methods, with most showing no greater than a -fold reduction in copy number except the -step method when applied to enterovirus, where the virus copy number was reduced by -fold. the same rna extraction that was used for the qpcr was also used for a metagenomics experiment ( fig. and table ). the first replicate sample of each treatment was indexed and run on one illumina miseq run producing , , sequence reads of bp in length, the second replicate set was indexed and run on an independent illumina miseq run and produced , , sequences reads of bp in length. after submission to blastn, each sequence read was given a taxonomic assignment using megan. all five model organisms were represented in the untreated samples ( fig. and table ) and for simplicity of data representation the family level of enterobacteriaceae was chosen to represent the e.coli organism in the sample, which was the most abundant taxa identified accounting for . % of the total sequences ( , , reads; table ). the kingdom level of metazoa was chosen to represent the human cells within the untreated sample, which accounted for % of the total reads ( , reads; table ). the decision to use metazoa and enterobacteriaceae was made to facilitate simple representation of the data, but was arbitrary, as the aim of this experiment was to compare variations in the proportions of bacteria, human cells and viruses between the enrichment methods. viruses were present in the untreated sample in the following proportions; enterovirus . % ( , reads), adenovirus . % ( reads) and influenza virus . % ( reads). it is interesting that the dna virus, adenovirus, was identified in the dataset, given that the method was targeted at rna viruses. a combined total number of sequence reads for two independent physical replicates which were also run on different illumina miseq flowcells. this figure represents the collapsed sequencing data, therefore redundant reads are not represented more than once. b serial applications of treatment methods. the -step method consisted of centrifugation then filtration. the -step method consisted of centrifugation, filtration then nuclease-treatment. ( . %) that were not assigned to these aforementioned taxa were accounted for as either ( ) no hit in the blast search, ( ) no clear taxonomic assignment from the blast search due to the stringency of parameters required by megan for taxonomic assignment ( ) low complexity sequence ( ) assignment to a taxonomic level that was not captured by enterobacteriaceae, metazoa or viruses. the enrichment techniques did not greatly change the relative abundance of enterobacteriaceae, at best there was a % reduction in the number of reads assigned to this taxon when applying the -step treatment. there was an increase in the proportion of metazoa sequences when filtration was applied increasing from % to . %, with only the nuclease-treatment showing the greatest effect reducing metazoan sequence to . % (a -fold reduction). for viruses, the -step treatment was the only treatment to show a significant increase in the proportion of viral sequence, by -fold for influenza (from . % to . %) and -fold for enterovirus. the proportion of adenovirus hits appeared unaffected by all enrichment methods (fig. ) . this study compares the effect of five different viral enrichment methods on the ability to detect viruses in a metagenomic approach. enrichment techniques were deliberately chosen that have been commonly referenced in previous metagenomic studies which seek to identify new or rare viruses (table ) . these enrichment methods are often selected without prior justification. the effect of these enrichment techniques was examined by application to a highly specific artificial sample. this study does not provide a full validation of the enrichment methods, but highlights some possible risks if an enrichment method is selected based solely upon methods published by others and without consideration for the sample being examined. validation of enrichment techniques in the field of virus ecology is well developed (duhaime and sullivan, ; john et al., ) , but there is a paucity of data on the validation of enrichment methods applied to the detection of viruses in animal or human samples for the purpose of diagnosis. in the present study, an artificial sample was composed which represents the type of organisms that could possibly be observed in clinical samples i.e. a rectal swab taken from a human patient. of course, the artificial sample is unlikely to have similar characteristics to complex biological samples from humans or animals. bacteria are often a very abundant organism in de novo metagenomic datasets, and host sequence is also often present. to this end, e.coli and a human cell line were chosen, as well as two rna viruses (influenza virus, enterovirus) and a dna virus (adenovirus). the dna virus was included to assess the potential for detection when using an rna virus targeted approach, as many virus discovery projects have to create two workflows to independently target dna or rna viruses. differing amounts of each virus were placed into the artificial sample so as to represent varying concentrations. this artificial sample represents a starting point to evaluate simple and rapid viral enrichment methods for use in virus metagenomics studies that seek to detect a virus that is causing disease in humans or animals. at present, there is little guidance for researchers seeking to work in this area and published studies have often selected these simple enrichment techniques with no justification for their inclusion in the method. in general, it was observed that the choice of enrichment method such as low speed centrifugation, syringe-based filtration, nuclease treatment (dnase and rnase) or combinations of these methods, did not substantially increase the relative abundance of viruses in this metagenomics dataset, except in selected cases. despite reductions in the quantity (copy number) of bacterial and human rna gene targets as shown in the qpcr data, this did not translate into a substantially increased relative abundance for viral sequences in the metagenomic data. the qpcr method detects the absolute quantity of genome target present, whereas the metagenomic data is proportional (relative abundance). even though large gains can be made in reducing bacteria and human nucleic acid, the proportion of viral sequences in the metagenomic datasets still remained relatively low (i.e. generally less than %) except for when a -step enrichment method was applied. nevertheless, individual enrichment methods did have some effect on relative abundance of the model organisms' representation in the metagenomics data. nuclease treatment alone was successful in reducing the proportion of human (metazoa) sequences by -fold. it is hypothesised that the initial freeze-thaw processing of the artificial sample has lysed human cells and thus liberated human genome. this exogenous nucleic acid is more susceptible to digestion by nuclease than the protected bacterial and viral genomes, which are protected by membranes or capsids, which are more resistant to freeze-thaw action. the -step treatment was particularly effective at increasing the abundance of both influenza and enterovirus, both in absolute concentration as measured by real-time pcr, and also relative abundance as measured in the metagenomics data. this finding supports the use of the -step procedure for virus enrichment, and similar -step procedures have previously been employed in published virus metagenomic studies (table ) . regarding sensitivity, without an enrichment method × copies of influenza virus genome were detected using a metagenomic approach ( sequence reads; table ), and the detection of the dna genome of adenovirus present at × copies ( sequence reads; table ) was also possible. this experiment was targeted at the discovery of rna viruses but included the dna virus (adenovirus) to determine if an rna virus detection method could also co-detect dna viruses. given that the starting material used for this experiment is rna, it is possible to surmise that adenovirus mrna expressed during infection was detected. however, this may not be the correct explanation as the dnase treatment used before cdna synthesis is known to be less than % efficient, and some dna is likely to have been carried right through into the sequencing library preparation. the ability to detect dna viruses when using an rna-targeted method will also no doubt be influenced by the specific replication cycle of any given virus. it is noted that the viral metagenomic method chosen uses multiple displacement amplification, and therefore there is likely to be a bias in the amplification of larger genomes e.g. bacteria and host, which could confound results when considering the relative abundance of sequences in the metagenomic data. there are many different methods of amplification that are available and this represents only one. however, this amplification method is one that is in practical use, and has been applied in other virus discovery studies (cheval et al., ) , therefore the examination of enrichment techniques using this specific amplification method are still relevant. there is a certainly a need for future studies to expand this work into a full validation, so as to examine the effect of enrichment when using other amplification methods i.e. sispa, nextera, lamp, lasl. it would also be interesting to include a greater array of enrichment methods, sample types, sequencing platforms and bioinformatics approaches which may include the incorporation of sequence assembly methods, or the use of other search algorithms. the findings presented here should provide a starting point for those considering the use of rapid or simple enrichment methods for the purposes of diagnosing new viral diseases in human or animal samples by using metagenomics. this study also highlights the possible risks of arbitrarily selecting an enrichment method purely based upon previously published studies. rjh and mp designed the study with assistance from pec. qsh provided the design of the real-time pcr assays, and akt, sy, hs, xr, nem performed the experiments. jw, ab, and rjh analysed the data. rjh, mp, xr, nem, jw and pec interpreted the findings. rjh wrote the manuscript draft while all authors edited the manuscript. a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species new viruses in veterinary medicine, detected by metagenomic approaches phi polymerase based random amplification of viral rna as an alternative to random rt-pcr metagenomic analyses of an uncultured viral community from human feces multiplex real-time pcr for detection of respiratory tract infections evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples a viral discovery methodology for clinical biopsy samples utilising massively parallel next generation sequencing viral genome sequencing by random priming methods ocean viruses: rigorously 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pathog. , e . who, . who information for molecular diagnosis of influenza virus in humans -update metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (ftls) in henan province, china: discovery of a new bunyavirus this study was funded by the esr core research fund provided by the new zealand ministry of business, innovation and employment. we are thankful for the support from the esr technical staff in the clinical virology laboratory and enteric reference laboratory for providing the model organisms and protocols for real-time pcr. we also wish to acknowledge new zealand genomics limited for the provision of high-throughput sequencing services and staff at the massey genome service, in particular lorraine berry and dr patrick biggs. our kind thanks also go to the esr information technology staff ned rajanayagam and phillip mitchell for developing the computing infrastructure to support this study. key: cord- -fpqwzf k authors: ulloa, s.; bravo, c.; parra, b.; ramirez, e.; acevedo, a.; fasce, r.; fernandez, j. title: a simple method for sars-cov- detection by rrt-pcr without the use of a commercial rna extraction kit date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: fpqwzf k the world health organization (who) has declared a pandemic caused by a new coronavirus named sars-cov- . the growing demand for commercial kits used for automated extraction of sars-cov- rna, a key step before rrt-pcr diagnosis, could cause a shortage of stocks that hinders the rapid processing of samples. although the recommendation is to use automated methods for nucleic acid extraction, alternatives are necessary to replace commercial kits. however, these alternatives should be as reliable as automated methods. this work describes a simple method to detect sars-cov- from specimens collected in different preservation media. samples were previously inactivated by heating and precipitating with a peg/nacl solution before rrt-pcr assays for orf ab, n and s genes. the new method was compared with an automated protocol of nucleic acid extraction. both procedures showed similar analytical results. consequently, this simple and inexpensive method is a suitable procedure for laboratory diagnosis of sars-cov- infection. sars-cov- is a virus that causes the ongoing covid- pandemic, an acute respiratory distress syndrome which represents the most serious problem for global health (cascella et al., ) . most cases are mild, usually with self-limiting symptoms and recovery within two weeks. severe patients progress rapidly with an acute respiratory distress syndrome and septic shock, eventually ending in multiple organ failure (wu and mcgoogan, ) . who and the centers for disease control and prevention (cdc) published reverse transcription-quantitative polymerase chain reactions protocols (rrt-pcr) for sars-cov- diagnosis. this included an rna extraction step to purify the viral rna from a nasopharyngeal swab (cdc, ) . however, since march , some manufacturers reported the shortage of supplies related to covid- driven by the sudden increase in global demand for rna extraction kits. different approaches avoiding viral nucleic extraction have been tested such as diluting samples and using a heat-processed method. however, heating oropharyngeal swabs is not as sensitive or accurate as rt-qpcr reactions performed on purified samples, detecting . % of the covid- -positive patients with no false positives results (fomsgaard and rosenstierne, ) . sars-cov- detection by direct rrt-pcr without rna extraction and inactivating samples at °c for minutes, was showed from specimens placed in utm and molecular water, but not from samples in hanks medium and saline buffer (merindol et al., ) . moreover, sars-cov- detection without rna extraction was described by mixing respiratory samples in a : (v/v) ratio with sputasol (oxoid, basingstoke, england) before adding it directly to the rrt-pcr reaction mix (wee et al., ) . in this research, a simple, inexpensive and reliable alternative method that uses heat inactivation and nucleic acid precipitation was used to process samples placed in different media, without the requirement of using commercial kits. this new procedure showed similar results compared to an automated method. thus, this new technique is proposed for sars-cov- laboratory diagnosis. one-hundred and four nasopharyngeal swab samples (npss), previously extracted using an automated nuclisens ® easymag ® (biomérieux) and tested for covid- at the institute of public health of chile. ninety-four positive samples, with a ct range from . to . , and negative samples were used for this study. samples were collected in tubes containing utm-rt mini transport media (copan diagnostics inc.) (n= ), pbs x solution (n= ), hanks medium (n= ) or dna/rna shield tm (zymo research) (n= ). in a class ii type a laminar flow biosafety cabinet (nuaire), µl of a solution % (w/v) chelex- (sigma aldrich, usa) prepared in nuclease-free water was added to a . ml microtube using a p micropipette (range - µl). when chelex- was precipitated, the supernatant was removed. then, µl of a solution % (w/v) peg (sigma aldrich, usa), . m nacl (ambion, usa) and µl of npss were added. tubes were incubated at °c for minutes (thermomixer comfort, eppendorf) and centrifuged at , g for minutes. supernatant was discarded and µl of % ethanol was added once for pellets washing. samples were centrifuged at , g for minutes and ethanol supernatant was discarded. pellets were allowed to dry for minutes at rt and resuspended in µl of nuclease-free water, pipetting up and down using a p to avoid blocking the pipette tip with chelex- . supernatants ( µl) were used directly for rrt-pcr cov- detection. for all samples, taqman™ -ncov assay kit v (thermofisher) was used for viral detection, using duplicates for each, following the manufacturer's instructions in a quantstudio real time pcr system (thermofisher). one-hundred and four npss ( positives and negatives) were extracted with an automated easymag ® and peg/nacl precipitation methods. then, three viral genes (orf ab, n and s) and one internal control gene (rnase p or rnp) were amplified by rrt-pcr. viral inactivation at ºc for min did not affect rrt-pcr amplification (n= ). all samples showed a concordant result ( positive and negative samples) between the easymag ® extraction and the peg/nacl precipitation method. results of rrt-pcr after easymag ® extraction were considered as being true. consequently, the sensibility and specificity for peg/nacl method were % and %, respectively. furthermore, the accuracy was % with each viral gene. moreover, the internal rnp gene was successfully amplified in all samples, corroborating extraction effectiveness (data not shown). thus, the four different media used in this study (utm, pbs x solution, hanks medium and dna/rna shield tm ) did not affect analytical results, because all precipitated samples were able to be detected by rrt-pcr using the whole viral panel (orf ab, n and s genes) and the internal control gene. performance of both easymag ® and peg/nacl extraction methods was evaluated in positive samples (n= ) by rrt-pcr. the mean of ct values was compared for each viral gene. orf ab and n values were lower when easymag ® was used and differences between both methods were significant (wilcoxon test, p< . ). on the other hand, the mean of ct value for s was lower with easymag ® extraction and differences were not significant (wilcoxon test, p= . ) ( fig. and table ). when ct values from each gene were compared within each extraction method, significant differences were observed (friedman test, p< . ). the best performance was achieved with the n gene (ct mean . ± . ) for automated extraction, and with the s gene (ct mean . ± . ) for the peg/nacl precipitation (fig. ). in a pandemic context, it is relevant to obtain rapid and reliable results to diagnose patients and take rapid public health actions. here, a simple protocol to detect sars-cov- from npss using rrt-pcr after a heat inactivation and a precipitation/concentration step is proposed. in order to inactivate sars-cov- present in contaminated objects, a treatment for minutes at above °c or minutes at above °c or minutes at above °c is recommended (abraham et al., ) . moreover, a cov- isolate (usa-wa / ), inactivated for minutes at °c, is being commercialized for different purposes (bei resources). in this research, a minutes incubation step at °c was used to obtain safe samples to be manipulated in the laboratory, without previous rna extraction using commercial kits. also, chelex- was incorporated as a protective agent during extraction at high temperatures because this reagent favors chelating groups to bind to cellular components (lounsbury et al., a) . moreover, this resin stabilizes the rna during the thermal shock and protects it from degradation (fontaine and guillot, ) . in this study a precipitation step using a peg/nacl solution was used, after heat inactivation of npss to concentrate samples and remove unwanted inhibitors, before the rrt-pcr. this solution was previously used for selective precipitation of large rnas (lounsbury et al., b; nilsen, ) . also, it is broadly used for phage precipitation and purification (yamamoto et al., ) . samples collected in different media (utm, pbs x, hanks and dna/rna shield) were tested using this protocol. all analytical results obtained by this protocol were concordant with those obtained by an easymag ® procedure. this is an advantage over a direct rrt-pcr without rna extraction described, where it was possible to detect sars-cov- only from samples collected in utm or molecular water, but not from samples collected in phosphate-buffered saline or hanks medium (merindol et al., ) . a protocol that used samples heat-processed for minutes at °c was reported (fomsgaard and rosenstierne, ) . this protocol allowed detecting only . % of covid- -positive patients in comparison to peg/nacl precipitation that detected % of the positive cases. performance of viral gene amplification (orf ab, n, s) was evaluated and differences in ct values were slightly better using easymag ® extraction. however, the three genes were identified in all samples and, remarkably, s gene did not show significant differences in amplification between both extraction methods. it is important to highlight that npss input using heat to kill sars-cov - . reviews in medical virology features, evaluation and treatment coronavirus (covid- ), in: statpearls. statpearls publishing covid- ) [www document an alternative workflow for molecular detection of sars-cov- -escape from the na extraction kit-shortage study of s rrna and rdna stability by real-time rt-pcr in heat-inactivated cryptosporidium parvum oocysts comparison of commercial realtime reverse transcription pcr assays for the detection of sars-cov- an enzyme-based dna preparation method for application to forensic biological samples and degraded stains an enzyme-based dna preparation method for application to forensic biological samples and degraded stains sars-cov- detection by direct rrt-pcr without rna extraction selective precipitation of large rnas rapid direct nucleic acid amplification test without rna extraction for sars-cov- using a portable pcr thermocycler characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application we thank all investigators from molecular genetics sub department and viral diseases sub department, both from institute of public health of chile. the authors declare that there are no conflicts of interest associated with this work. key: cord- -z wf wli authors: wang, leyi; zhang, yan; byrum, beverly title: development and evaluation of a duplex real-time rt-pcr for detection and differentiation of virulent and variant strains of porcine epidemic diarrhea viruses from the united states date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: z wf wli porcine epidemic diarrhea virus (pedv) has caused significant economic losses in the us swine industry since may . a new variant strain of pedv emerged in the us in the late december, . this variant strain of pedv differs from the virulent strain of pedv currently circulating in the us in nt of the ’end of the s domain in the spike gene. importantly, the variant pedv caused significantly less mortality in piglets than the virulent pedv, based on clinical observations. this suggests it may be a potential vaccine candidate for ped. variant pedv has been detected in samples from multiple states by our laboratory as well as other laboratories in the us. it is critical to detect and differentiate variant pedv from the virulent pedv during outbreaks to enhance control and to prevent ped associated disease. in this study, the development and validation of a duplex real-time rt-pcr assay for detection and differentiation of the variant and the virulent strains of pedv currently circulating in the us was reported. porcine epidemic diarrhea (ped) virus is a member of the order nidovirales, family coronaviridae, subfamily coronavirinae, genus alphacoronavirus. ped is a highly contagious diarrheal disease, characterized by severe watery diarrhea and high mortality in piglets. ped was originally identified in england in (oldham, ) . since then, it has been reported in several european and asian countries including china and korea. since , a highly virulent strain of pedv emerged in china and caused significant loss in the pig industry (sun et al., ) . in may , this virulent strain of pedv was recognized in the united states (us). by march of , ped had been detected in us states and a total cases were confirmed (http://www.aasv.org/pedv/pedv weekly report .pdf). the disease has caused severe economic losses to the swine industry in the us. recently the virus was found in canada. studies have shown that pigs either naturally or experimentally infected with virulent strain of pedv developed characteristic gross (thin and dilated intestinal walls) and histologic lesions (severe atrophy of villi) (stevenson et al., ; jung et al., ) . complete genomic analysis of the virulent pedv strains from the us showed * corresponding author. tel.: + ; fax: + . e-mail address: yzhang@agri.ohio.gov (y. zhang). that they cluster in a single clade and are closely related with the ah strain reported in china (stevenson et al., ; huang et al., ) . recently, we reported a new variant pedv detected in ohio . clinical observation indicated that this new variant virus (oh ) caused mild clinical disease with low mortality in newborn piglets (unpublished data), making it a potential vaccine candidate. strain oh is distinct from the virulent strains of pedv in the us and is most closely related to ch/hbqx/ reported in central china (zheng et al., ) , based on the phylogenetic analysis of the full-length spike gene . further analysis showed that the strain oh differs from the virulent strains of pedv in the first nucleotides (nt) of spike gene, indicating that at least two genotypes of pedv are circulating in the us pigs (stevenson et al., ; huang et al., ; wang et al., ) . since effective vaccines are not currently available in north america, accurate diagnosis combined with biosecurity is the only reliable method for control and prevention of ped. electron microscopy was widely used in the diagnosis of the initial outbreaks of pedv (pospischil et al., ) . since then, several methods have been developed for laboratory diagnosis of pedv, including the direct immunofluorescence test for detection of pedv antigen (guscetti et al., ) , enzyme-linked immunosorbant assays (elisa) for detection of either pedv antigen or antibodies (carvajal et al., ) , and reverse transcription-polymerase chain reaction (rt-pcr) (kwon et al., ; ishikawa et al., years, real-time rt-pcr has increasingly been used to detect viral pathogens because of its advantages including high specificity and sensitivity, fast turnaround, and quantification of pathogen loads. currently, the animal disease diagnostic laboratory in the ohio department of agriculture uses a real-time rt-pcr method which targets the membrane (m) gene for detection of pedv. however, since the m gene is highly conserved between the virulent and the variant strains of pedv in the us, this real-time rt-pcr assay cannot differentiate between the two strains of pedv. therefore, the present study sought to develop and evaluate a duplex real-time rt-pcr method to distinct between the virulent and the variant strains of pedvs. multiple-sequence alignments with the virulent and the variant strains of pedv sequences detected in the us were carried out with the mega . program. primers were designed by targeting the conserved regions between virulent and variant pedv, while the probes were designed by targeting the location where variant pedvs have two deletions. for multiplexing, the probe for the virulent pedv was labeled with the -reported dye cy and the -quencher bhq , and the probe for the variant pedv was labeled with the -reported dye -carboxyfluorescein (fam) and double quencher of the internal zen and 'iowa black ® fq ( iabkfq). the sequences and amplicon sizes of the primers and probes are listed in table and fig. . sequences of primers and probe for the real-time rt-pcr targeting m gene were listed in table . rna was extracted with the trizol reagent (invitrogen, carlsbad, ca, usa). amplification was performed with the qiagen one step rt-pcr kit (valencia, ca, usa) in a smartcycler ii instrument. the amplification conditions were • c for min; • c for min; and cycles of • c, s, • c, s, and • c, s. primers (integrated dna technologies, coralville, iowa, usa) at nm and each probe (integrated dna technologies, coralville, iowa, usa) at nm were used for one reaction. the primer set and individual probe were tested first in a single real-time rt-pcr assay and then in a duplex real-time rt-pcr assay. intra-specificity of the duplex rt-pcr assay was determined using single probe of either virulent pedv probe for variant pedv strain or variant pedv probe for virulent pedv strain and two probes for both types of pedvs for positive control. inter-specificity of duplex rt-pcr assay was examined using various swine virus strains available in our lab. these viruses include porcine reproductive and respiratory syndrome virus, swine influenza virus (h n ), transmissible gastroenteritis virus, encephalomyocarditis virus, porcine coronavirus hku , porcine parvovirus, and pseudorabies virus. for dna virus porcine parvovirus and pseudorabies virus, dna samples were extracted using dneasy blood & tissue kit (qiagen, valencia, ca, usa). in the assay, . l rna or dna samples were used, . l each of virulent pedv oh strain and variant pedv oh were used as positive control in the duplex rt-pcr and . l distilled water was used as negative control. the pcr products amplified by using rnas from oh (variant pedv) and oh (virulent pedv) and the primer set p -p covering the region where contains the majority of sequence variations between the virulent and variant pedvs were cloned into the pcr . vector (invitrogen, carlsbad, ca, usa). the plasmids with the oh (pcr . -oh ) or oh (pcr . -oh ) genes were confirmed by sequencing. the detection limit of the real-time rt-pcr assay was determined through serial dilutions of each plasmid. duplicates for each dilution were examined for separate and duplex reactions. clinical fecal and intestinal samples submitted to the animal disease diagnostic laboratory in ohio department of agriculture were processed for rna extraction. rna samples were first tested for pedv by a real-time rt-pcr targeting the m gene. if positive, the duplex real-time rt-pcr was then used to differentiate the variant and the virulent strains of pedv. based on the sequence alignment and analysis of both virulent and variant pedv partial s region, in addition to several sequence variations, there were deletions and one insertion present in the variant pedv as compared with virulent pedv (fig. ) . the primers were designed by targeting the conserved regions between the two viruses whereas the probes targeting the region where the first two-deletion regions were located in the variant strain of pedv. the duplex rt-pcr assay can detect specifically the virulent strain of pedv by the cy probe or the variant strain of pedv by the fam probe. in contract, the duplex rt-pcr did not cross-react with any other pig viruses used in the study, the cy probe did not cross-react with variant pedv strain, and the fam probe did not cross-react with virulent pedv strain (table ) . table sequences of primers and probes used in this study. primer/probe sequence amplicon size (bp) pedv s forward -aggcggttcttttcaaaatttaatg- pedv s reverse -gaaatgccaatctcaaagcc- for virulent pedv virulent pedv s probe -/ cy /tattggtgaaaaccagggtgtcaat/ bhq /- for variant pedv variant pedv s probe - the sensitivity of the duplex real-time rt-pcr assay was validated through serial dilutions of pcr . -oh and pcr . -oh constructs. the detection limit was copy for both variant and virulent strains of pedvs. standard curves were plotted using -fold serial dilutions of plasmid dna of virulent and variant pedv for the duplex real-time rt-pcr. as shown in fig. , there is a strong linear correlation (r > . ) between c t values and the corresponding amount of plasmid copy numbers for both virulent and variant pedv. the standard curves of virulent and variant pedv were plotted with slopes of − . and − . , respectively ( fig. a and b) . the duplex real-time rt-pcr detected genomic copy for both virulent and variant strain of pedvs. a total of positive samples tested by the real-time rt-pcr targeting on m gene were run again by the duplex real-time rt-pcr. forty five samples tested positive for the variant pedv and the remaining samples were positive for the virulent pedv. the results were confirmed by sequencing using p -p primer set. pedv causes diarrhea in pigs and high mortality in piglets. since may of , pedv has been identified in the us resulting in severe economic losses to the us swine industry. data on ped outbreaks have been collected and complied by the us national animal health laboratory network each week since june of . since there is no pedv vaccine available in north america, it is important to take biosecurity strategies to control ped. the findings of a recent study demonstrated that pedv was found in . % of trailers used to transport pigs, highlighting the importance of strict biosecurity (lowe et al., ) . in the late december , a variant strain of pedv was detected in ohio by our laboratory . genetic analysis showed that this virus differed from the virulent strains of pedv currently circulating in the us in the end of the s domain (mainly located in the first nt of spike gene), thus the real-time rt-pcr targeting on the m gene does not provide differentiation of the variant pedv from virulent strain of pedv. therefore, the aim of this study was to develop a duplex real-time rt-pcr which would detect and differentiate the virulent strain from the variant strain of pedv. to optimize the ability of the assay to detect both variant and virulent strains of pedv, the two primers were located in the conserved region of s region. to differentiate specifically the variant pedv from the virulent strains of pedv, probes were designed by targeting the highly variable region containing the first two deletions present in the variant pedv (fig. ) . it is possible that the third deletion and insertion site may also be used as a target for designing primers and probes (fig. ) . the assay is specific for pedvs, since cross-reaction with non-ped viral genomes used in this study was not detected. in addition, the assay is highly sensitive, being able to detect copy in l reaction of either variant or virulent strains of pedv. the efficiency of the duplex real-time rt-pcr assay was determined by testing clinical samples which were positive by the real-time rt-pcr assay targeting the m gene. of the clinical samples that tested positive for the variant pedv, all of them were confirmed by sequencing of the spike gene. in conclusion, we have developed a duplex real-time rt-pcr assay that reliably detects and differentiates the virulent strain and variant strain of pedv. this assay may be used by veterinary diagnostic laboratories to detect the new variant strain and the virulent strains of pedv currently circulating in the us. evaluation of a blocking elisa using monoclonal antibodies for the detection of porcine epidemic diarrhea virus and its antibodies immuno-histochemical detection of porcine epidemic diarrhea virus compared to other methods origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states direct and rapid detection of porcine epidemic diarrhea virus by rt-pcr pathology of us porcine epidemic diarrhea virus strain pc a in gnotobiotic pigs rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction role of transportation in spread of porcine epidemic diarrhea virus infection, united states letter to the editor. pig farming light microscopy and ultrahistology of intestinal changes in pigs infected with epizootic diarrhoea virus (edv): comparison with transmissible gastroenteritis (tge) virus and porcine rotavirus infections emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets new variant of porcine epidemic diarrhea virus, united states molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus field strains in central china during - outbreaks key: cord- - xyjrjpf authors: kim, yong kwan; lim, seong-in; choi, sarah; cho, in-soo; park, eun-hye; an, dong-jun title: a novel assay for detecting canine parvovirus using a quartz crystal microbalance biosensor date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: xyjrjpf rapid and accurate diagnosis is crucial to reduce both the shedding and clinical signs of canine parvovirus (cpv). the quartz crystal microbalance (qcm) is a new tool for measuring frequency changes associated with antigen–antibody interactions. in this study, the qcm biosensor and prolinker™ b were used to rapidly diagnosis cpv infection. prolinker™ b enables antibodies to be attached to a gold-coated quartz surface in a regular pattern and in the correct orientation for antigen binding. receiver operating characteristics (roc) curves were used to set a cut-off value using reference cpvs (two groups: one cpv-positive and one cpv-negative). the roc curves overlapped and the point of intersection was used as the cut-off value. a qcm biosensor with a cut-off value of − hz showed . % ( / ) sensitivity and . % ( / ) specificity when used to test field fecal samples compared to pcr. in conclusion, the qcm biosensor described herein is eminently suitable for the rapid diagnosis of cpv infection with high sensitivity and specificity. therefore, it is a promising analytical tool that will be useful for clinical diagnosis, which requires rapid and reliable analyses. canine parvovirus (cpv), a member of the genus parvovirus within the family parvoviridae, first emerged as a canine pathogen in the late s (appel et al., ; kelly, ) . a few years after the first outbreak, cpv- was completely replaced by two antigenic variants, termed cpv- a and cpv- b (parrish et al., ) . in , a novel mutant of cpv, harboring an amino acid substitution at position (cpv- c), emerged in italy and rapidly spread to several countries (buonavoglia et al., ; decaro and buonavoglia, ) . infection by canine cpv results in a highly contagious enteric disease with a high rate of morbidity and mortality (larson and schultz, ; parrish, parrish, , . cpv is a small, non-enveloped single-stranded dna (ssdna) virus with a genome of approximately . kb that encodes two structural (vp and vp ) and two non-structural (ns and ns ) proteins (cotmore and tattersall, ) . vp is the major capsid protein, and plays an important role as an antigenic determinant (chang et al., ; horiuchi et al., ) . * corresponding author. tel.: + ; fax: + . currently, a diagnosis of cpv is based on clinical signs, which include vomiting and diarrhea; however, a definitive diagnosis is difficult because these symptoms are common to other enteric diseases (elia et al., ; hirasawa et al., ) . conventional detection methods such as electron microscopy (teramoto et al., ) , virus isolation (mochizuki et al., ) , latex agglutination (veijalainen et al., ) , hemagglutination (mochizuki et al., ; teramoto et al., ) , and enzyme-linked immunosorbent assay (drane et al., ; teramoto et al., ) have been developed for the detection of cpv, and all are effective and accurate. molecular detection techniques, including pcr (mochizuki et al., ) , real-time pcr (decaro et al., ) , nested pcr (hirasawa et al., ) , and loop-mediated isothermal amplification (cho et al., ; mukhopadhyay et al., ) have also been used to diagnose cpv, although the sensitivity and specificity of these techniques are variable; however, a major drawback is that these methods are both laborious and time-consuming. the key to preventing the spread of cpv is early and rapid diagnosis. infected dogs can then be isolated and given supportive treatment to reduce both morbidity and mortality. therefore, a rapid, on-site diagnostic method is required for the early detection of cpv. the quartz crystal microbalance (qcm) is a nanogram-sensitive technique that utilizes acoustic waves generated by oscillating a piezoelectric, single crystal quartz plate to measure mass (dixon, ). the piezoelectric effect is due to the electrical charge that accumulates in certain solid materials, such as crystals, in response to an applied mechanical stress. the qcm biosensor measures mass per unit area by measuring the change in the frequency of a quartz crystal resonator, and is a convenient method for detecting antigen-antibody interactions. the qcm biosensor is a "label-free technology" because binding is detected directly without the need for labeled reagents. many studies have used the qcm biosensor to detect pathogenic microorganisms such as vibrio cholerae, bacillus anthracis, malaria, salmonella typhimurium, leishmania infantum, and canine influenza virus (cabral-miranda et al., ; carter et al., ; cooper et al., ; hao et al., ; ittarat et al., ; kim et al., ; salam et al., ) . the aim of the present study was to use the qcm biosensor in conjunction with prolinker tm b (used to immobilize cpv-specific antibodies onto a gold-coated quartz surface) to develop a suitable method for the rapid and accurate diagnosis of cpv. the change in mass at the quartz crystal surface induced by antigenantibody interactions generates a measurable change in frequency. the qcm biosensor described herein constitutes a rapid and sensitive method for diagnosing cpv infection in dogs. the cpv-positive references (virus isolation) were obtained from the korea veterinary culture collection (kvcc) (http://kvcc.kahis.go.kr) (supplemental table ). there were also cpv-negative references, which comprised of cpv-negative fecal samples, two bacterial (escherichia coli and salmonella) and two viral (canine coronavirus and canine rotavirus) isolates. all of the cpv-positive and cpv-negative reference samples were confirmed as positive and negative by pcr, respectively. the field samples comprised fecal samples taken from dogs with suspected cpv which were being treated at animal hospitals in seoul and gyeonggi province in south korea from to . dna was extracted using a qiagen dna extraction kit (hilden, germany), according to manufacturer's instructions. all samples were identified as cpv-positive or -negative by pcr using the following cpv-specific primers: forward, -caaatagagcattgggcttacc- and reverse, -caatctccttctggatatcttc- . the pcr reaction mixture comprised l of dnase-free water, l of × pcr buffer, l of each primer, l of dntp mix, l of pcr enzyme mix, and l of extracted dna as a template (total volume l). the pcr conditions were as follows: • c for min, followed by cycles of min at • c, min at • c, and min at • c, with a final extension at • c for min. supplementary table related to this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . a commercial immunochromatography ag kit was purchased from the bionote inc (hwaseong, south korea). the test device that comes in the kit shows the letters "t" (test line) and "c" (control line) on its surface. the control line should always be visible if the test has been performed properly. a purple "test line" is visible if sufficient cpv antigen is detected. briefly, fecal samples were placed in sample collection tubes containing assay diluent and mixed vigorously. the tube was then left to stand for min at room temperature to allow any particles to settle. finally, four drops of sample solution were loaded into the sample hole using a disposable dropper and the results read after min. an endpoint dilution assay was used to measure virus titer. serial dilutions of kvcc-vr (supplemental table ) were prepared and inoculated onto replicate crandall ress feline kidney (crfk) cell cultures in -well plastic plates. the number of infected cell cultures at each virus dilution was then determined by looking for a cytopathic effect (cpe). after an incubation period for days, wells in plates that displayed a cpe were scored as positive. the tcid was calculated using the reed & muench endpoint method. the quartz was soaked in a solution containing meoh and % hcl ( : ) for min. a freshly prepared piranha solution (a : mixture of concentrated h so and % h o ) was then added for min. the quartz was then thoroughly washed with distilled water and dried under a stream of nitrogen (n ) gas. the precleaned quartz was then gold-coated by thermal evaporation in a magnetron sputtering system (cliotech, seoul, south korea) before immersion in a mm prolinker tm b solution (proteogen, chuncheon, south korea), which contains exposed sh groups, for h. the quartz was then rinsed sequentially with chcl , etoh, and distilled water before drying under a stream of n gas. a monoclonal anti-cpv igg antibody was purchased from median diagnostics inc (chuncheon, south korea). the monoclonal anti-cpv igg antibody ( g/ml) was mixed with pbs ( ml containing % glycine) and l of the mixture added to the gold-coated quartz and incubated overnight at • c. the quartz was then rinsed twice with pbs and deionized water, and dried under a stream of n gas. the antibody-coated quartz was stored at • c until use. a x-delic xq instrument (ubtgen, seoul, south korea) was used to measure frequency changes in the gold-plated quartz. briefly, the monoclonal cpv igg antibody-coated quartz was placed horizontally and loaded with l of pbs. when the frequency reached equilibrium, l of fecal sample was loaded onto the quartz surface. real-time changes in resonant frequency were recorded until the signal again reached equilibrium. each experiment was performed at room temperature and repeated three times. all of the cpv-positive (n = ) and -negative reference samples (n = ) were classified according to their measured frequency changes (supplemental table ) and receiver operating characteristics (roc) curves constructed (fig. ) . reference samples were divided into two groups, namely, cpv-positive and cpv-negative. although both groups showed clear separation, there was a point of overlap and this point of intersection was used as the cut-off value. the cut-off value was the threshold value that determined whether a sample was identified correctly as positive or negative. according to the roc, the cut-off value was set at a frequency change of − hz. the diagnostic sensitivity and specificity were calculated as follows: sensitivity = true positive/(true positive + false negative); specificity = true negative/(true negative + false positive). as a cpv-positive reference sample registered with the kvcc (kvcc-vr ) was used to set the detection limit. the tcid /ml for this sample, as measured by a commercial immunochromatography ag kit (ciak) and pcr, was and , respectively (table ) . using the test sample in the qcm biosensor revealed that frequency changes increased gradually as the concentration of the test sample increased (fig. ) . frequency changes exceeding the cut-off value (− hz) were generated at a minimum tcid /ml value of . if the qcm biosensor is to be of practical use in the field, the antibodies immobilized on the surface must retain long-term activity. therefore, we stored the cpv igg antibody coated quartz at • c for , , , and months. the diagnostic accuracy at each time point was then tested using kvcc-vr (supplemental table ) at tcid /ml. as shown in supplemental table , the coefficient of variation (cv) at , , , and months increased gradually ( . %, . %, . %, . % and . %) in a time-dependent manner. the frequency change and cv at each time point are shown in fig. . a cv < % was maintained for months and thus quartzimmobilized cpv igg antibody can be stored at • c for up to year, making them eminently suitable for field use. supplementary table related to this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . all suspected cpv-positive fecal samples were confirmed as cpv-positive or -negative by pcr before application to the qcm biosensor. of the field samples tested, . % ( / ) were cpv-positive and . % ( / ) were cpv-negative; of the cpv-positive samples confirmed by pcr, cpv- a, b and c comprised to . % ( / ), . % ( / ) and % ( / ), respectively. the qcm biosensor identified / as positive ( . % sensitivity) and / as negative ( . % specificity). however, the sensitivity and specificity of the ciak were . % ( / ) and . % ( / ), respectively. the gold-coated quartz surface is a relatively new class of device to which biological recognition elements can be attached. when measuring the frequency change that caused the antigen-antibody interaction, it is important that the frequency is highly stabile and that background noise is kept to a minimum; this will ensure maximum sensitivity. several factors may cause frequency drift, including the electrode surface and the viscosity of the liquid (buttry and ward, ; tsionsky et al., ) . previous studies used silver and gold electrodes as qcm biosensors (kim et al., ; su et al., ) ; however, gold electrodes produce more consistent results because the surface is less rough than that of silver (su et al., ) . the roughness of the electrode surface is critical if frequency drift is to be minimized. frequency drift is also associated with temperature (rocklein and george, ; martin et al., ; wang et al., ) . if the temperature fluctuates, both real mass changes over time and apparent mass changes must be measured concurrently; thus temperature-dependent apparent mass changes must be characterized and minimized to allow the qcm biosensor to provide an accurate readout of the frequency change (rocklein and george, ) . over the last decade, the use of the qcm has been extended to the liquid phase (bruckenstein et al., ) . the resonance frequency of the crystal responds to the properties of a contacting liquid. inconsistences in liquid viscosity are easily overcome by using pbs or water; thus the electrode surface, the temperature, and the viscosity of the liquid must all be taken into account if frequency drift is to be minimized. if a qcm biosensor is to have both high sensitivity and specificity, important issues such as antibody orientation on the sensor surface must be resolved. there are two main approaches to preparing the surface on an antibody-based sensor: random antibody immobilization and site-directed antibody immobilization. the simplest technique is to allow random adsorption of the antibody onto the quartz surface. although simple, this method has major drawbacks, including low stability and lower activity due to random orientation (buijs et al., ; makaraviciute and ramanaviciene, ) . direct antibody immobilization via a self-assembled monolayer has multiple advantages, namely, that the technique is simple and the covalent bonding means that the surface is both stable and reusable; however, a major drawback is the relatively low sensitivity due to random antibody orientation and the low availability of antibody binding sites (kausaite-minkstimiene et al., ; tsai and pai, ). therefore, site-directed antibody immobilization methods have been developed to address these short-comings (della ventura et al., ; hussack et al., ; kwon et al., ; um et al., ; yang et al., ; yoon et al., ) . a previous study showed that prolinker tm b, a calixcrown- derivative, is able to orient antibodies in a site-directed manner, leading to a more consistent position on the surface and a significant increase in sensitivity for the target antigen (lee et al., ) . as mentioned above, gold electrodes are the preferred carrier for use in qcm biosensors because they generate a stable frequency (steegborn and skládal, ) ; however, the major drawback is the high unit price of gold and thus gold-coated electrodes must be reusable to minimize the cost of each experiment. the antigen-antibody interaction is usually irreversible; however, the interaction can be disrupted by chaotropic reagents, organic solvents, or even ultrasonic radiation (marazuela and moreno-bondi, ) . prolinker tm b can easily be detached from quartz surfaces using piranha solution (lee et al., ) . the gold-coated quartz crystal described herein is reusable, which significantly reduces the cost of the qcm biosensors used to detect cpv. in conclusion, the qcm biosensor described herein represents a promising analytical tool for use in clinical diagnosis, where rapid and reliable analyses are needed. therefore, the qcm biosensor is a potential sensitive and specific assay for the rapid and early diagnosis of cpv infection. the authors have no conflicts of interest to declare. status report: canine viral enteritis dual quartz crystal microbalance oscillator circuit. minimizing effects due to liquid viscosity, density, and temperature changes in the secondary structure of adsorbed igg and f(ab ) studied by ftir spectroscopy evidence for evolution of canine parvovirus type in italy measurement of interfacial processes at electrode surfaces with the electrochemical quartz crystal microbalance detection of parasite antigens in leishmania infantum-infected spleen tissue by monoclonal antibody-, piezoelectric-based immunosensors quartz crystal microbalance detection of vibrio cholerae o serotype direct and sensitive detection of a human virus by rupture event scanning the autonomously replicating parvoviruses of vertebrates multiple amino acids in the capsid structure of canine parvovirus coordinately determine the canine host range and specific antigenic and hemagglutination properties detection of canine parvovirus in fecal samples using loop-mediated isothermal amplification real-time pcr assay for rapid detection and quantitation of canine parvovirus type in the feces of dogs canine parvovirus-a review of epidemiological and diagnostic aspects, with emphasis on type c light assisted antibody immobilization for bio-sensing quartz crystal microbalance with dissipation monitoring: enabling real-time characterization of biological materials and their interactions evaluation of a novel diagnostic test for canine parvovirus detection of infectious canine parvovirus type by mrna real-time rt-pcr rapid detection of bacillus anthracis using monoclonal antibody functionalized qcm sensor sensitive detection of canine parvovirus dna by the nested polymerase chain reaction mapping of determinants of the host range for canine cells in the genome of canine parvovirus using canine parvovirus/mink enteritis virus chimeric viruses multivalent anchoring and oriented display of single-domain antibodies on cellulose biosensor as a molecular malaria differential diagnosis comparative study of random and oriented antibody immobilization techniques on the binding capacity of immunosensor an enteric disease of dogs resembling feline panleukopenia detection of h n canine influenza virus using a quartz crystal microbalance antibody arrays prepared by cutinase-mediated immobilization on self-assembled monolayers three-year serologic immunity against canine parvovirus type and canine adenovirus type in dogs vaccinated with a canine combination vaccine pro-teochip: a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-protein interaction studies characterization of a quartz crystal microbalance with simultaneous mass and liquid loading site-directed antibody immobilization techniques for immunosensors fiber-optic biosensors-an overview comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of canine parvovirus dna directly in faecal specimens canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses emergence, natural history, and variation of canine, mink, and parvoviruses host range relationships and the evolution of canine parvovirus temperature-induced apparent mass changes observed during quartz crystal microbalance measurements of atomic layer deposition real-time and sensitive detection of salmonella typhimurium using an automated quartz crystal microbalance (qcm) instrument with nanoparticles amplification construction and characterization of the direct piezoelectric immunosensor for atrazine operating in solution disposable, low cost, silver-coated, piezoelectric quartz crystal biosensor and electrode protection comparison of enzyme-linked immunosorbent assay, dna hybridization, hemagglutination, and electron microscopy for detection of canine parvovirus infections surface plasmon resonance-based immunosensor with oriented immobilized antibody fragments on a mixed self-assembled monolayer for the determination of staphylococcal enterotoxin b behavior of quartz crystal microbalance in nonadsorbed gases at high pressures electrochemically oriented immobilization of antibody on poly-( -cyanoethylpyrrole)-coated gold electrode using a cyclic voltammetry latex agglutination test for detecting feline panleukopenia virus, canine parvovirus, and parvoviruses of fur animals quartz crystal microbalance in elevated temperature viscous liquids: temperature effect compensation and lubricant degradation monitoring immobilization of unraveled immunoglobulin g using well-oriented zz-his protein on functionalized microtiter plate for sensitive immunoassay immobilization of antibodies on the selfassembled monolayer by antigen-binding site protection and immobilization kinetic control we are grateful for the technical assistance provided by proteogen (chuncheon, south korea). this study was supported by a grant (project code number: b-ad - - - ) from the animal and plant quarantine agency (qia), ministry of agriculture, food and rural affairs, republic of korea. key: cord- -lgqup ug authors: ayers, m.; adachi, d.; johnson, g.; andonova, m.; drebot, m.; tellier, r. title: a single tube rt-pcr assay for the detection of mosquito-borne flaviviruses date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: lgqup ug mosquito-borne flaviviruses include several important agents of human disease and have provided striking examples of emerging infections. in this study we present the design and validation of a single tube rt-pcr assay using a pair of consensus primers for the detection of mosquito-borne flaviviruses. sequencing of the amplicons permits the species identification. the assay was validated using rna from the yellow fever virus vaccine strain and from representative strains of dengue viruses , , and , west nile virus, kunjin virus (a clade of west nile virus), and st. louis encephalitis virus. within the family flaviviridae, the genus flavivirus comprises at least species, of which have been associated with human disease (burke and monath, ) . nucleotide sequence analysis has shown that the genus can be divided in three clusters lindenbach and rice, ) , namely the mosquito-borne viruses, the tick-borne viruses and a third cluster for which no vectors have been identified. in turn the mosquitoborne cluster encompasses nine clades, including the clades of the yellow fever virus (yfv), and of dengue fever viruses (the four serotypes of which also form a serocomplex). the japanese encephalitis virus (jev) serocomplex include viruses from three clades; the species in that complex include most notably jev itself, west nile virus (wnv), kunjin encephalitis virus, murray valley encephalitis virus (mve), st. louis encephalitis virus (sle) and usutu virus (burke and monath, ; kuno et al., ; lindenbach and rice, ) . the epidemiology of these viruses is complex and depends on the ecology of their vectors in relations with human habitats. consequently, many human-dependant factors, such as increase in population/density of population, massive population displacement, unplanned urbanization, deforestation, establishment of urban mosquito populations, and deterioration of public health surveillance and infrastructure, can all influence the spread and activity of these viruses, which have frequently provided striking examples of emerging infections in both the developing world and industrialized nations (gubler, ; mackenzie et al., ; tomori, ) . yellow fever virus, the prototype of the genus, originated from africa and spread to south america. at least , cases, causing , deaths, occur every year worldwide; % of cases occur in africa. these numbers are widely acknowledged to be underestimates because of the insensitive surveillance in endemic areas (gubler, ; mackenzie et al., ; tomori, ) . dengue fever viruses have undergone a dramatic increase in geographic spread and frequency of infections over the past years. currently, the incidence is estimated at - million cases per year, causing , deaths. in all tropical areas, more than one serotype circulate and re-introduction of these viruses in the continental usa cannot be ruled out (gubler, ; mackenzie et al., ) . japanese encephalitis virus (jev) is encountered throughout southeast asia and is the most important cause of viral encephalitis in these countries. the current estimate of , - , cases/year is likely an underestimate and jev activity is on the increase (kabilan et al., ; mackenzie et al., ; promed-mail, a,b) . west nile virus (wnv) underwent a dramatic expansion of its ecological niche over the past few years, first noticed with an encephalitis outbreak in new york city in and followed by spread throughout the usa and canada, as well as mexico and the carribean. in and the virus was responsible for two of the largest arboviral epidemics ever observed and has now become endemic in large areas of the western hemisphere (davis et al., ; drebot and artsob, ; hayes, ; mackenzie et al., ) . kunjin virus, recovered from cases of encephalitis in western australia, has been reclassified as a clade of west nile virus (burke and monath, ; mackenzie et al., ) . st. louis encephalitis virus has been recognized for several decades as a cause of viral encephalitis in the usa (burke and monath, ) . recently a large outbreak in cordoba, argentina was documented (promed-mail, a,b) . in the late summer of , a sudden increase in mortality in several species of birds in central europe was demonstrated to be caused by the emergence of the usutu virus, a flavivirus which had previously remained confined to africa. however, the virus has not so far been linked to human disease (weissenbock et al., ) . given the disease burden caused by these viruses and their emerging behavior, laboratory testing will become increasingly important. yet, the laboratory diagnosis of flavivirus infection remains unsatisfactory; isolation of these viruses is time consuming, and for many of them requires a biosafety level installation. serology tests other than neutralization assays frequently display cross reactivity between species. for all these reasons, development of molecular tests for these agents, including rt-pcr, has attracted a lot of efforts and attention. in this study we present a single tube rt-pcr assay for mosquitoborne flaviviruses, using a single pair of consensus primers. we show that the assay can detect several important species of flaviviruses with high sensitivity. species identification is provided by sequencing the amplicons; in the case of dengue viruses this also permits the serotype determination. yellow fever virus vaccine strain was obtained from a vial of yf-vax (aventis pasteur), reconstituted as per the manufacturer's recommendations. the preparation is certified to contain a titer of at least × pfu/ml. west nile virus strain ny, st. louis encephalitis virus, dengue fever serotype , , , and were grown and titrated in a biosafety level facility at the national microbiology laboratory (winnipeg), as described (weingartl et al., ) . kunjin virus was grown in biosafety level conditions, as described (weingartl et al., ) . additional samples tested consisted of mosquito pools that had been found positive for wnv using a real time rt-pcr taqman based assay (lanciotti et al., ) . the samples included two pool homogenates of culex pipiens collected in the province of ontario, canada in , two homogenates of c. tarsalis collected in the province of manitoba in , and two homogenates of c. tarsalis from manitoba, . for specificity studies, several viral samples were used, including clinical samples found to contain cmv and ebv dna by pcr testing, as described (johnson et al., ) ; a clinical isolate of influenza virus from the - season, typed as h by sequencing of the hemagglutinin gene; echovirus from the laboratory collection at the hospital for sick children; hepatitis c virus rna was obtained by in vitro transcription of the infectious clone pcv-h c (yanagi et al., ) (the clone was kindly provided by dr. j. bukh, niaid, national institutes of health) followed by dnase i digestion, rna purification and serial dilution; and human coronavirus oc- (kindly provided by dr. p.j. talbot, inrs, institut armand frappier). rna extraction was done using the qiagen rna easy kit, as per the manufacturer's recommendations, or, for the yellow fever vaccine strain, trizol (life technologies), as per the manufacturer's recommendations. the rna was resuspended in mm dithiothreitol (promega) with % (v/v) rnasin ( - u/l, promega), and -fold serial dilutions were prepared with the same diluent. consensus primers were designed to target segments of the ns coding region conserved across several species of flaviviruses including yfv, the dengue viruses serocomplex and several members of the jev serocomplex, (fig. ) . the sequences of the primers are: flavi- (sense primer): aatgtacgctgatgacac-agctggctgggacac and flavi- (antisense primer): tccagaccttcagcatgtcttctgttgtcatcca . these two primers correspond to the segments [ - ] and [ , ] , respectively, of the west nile virus ny (genbank accession #af ). rt-pcr with these primers would result in amplicons of similar sizes, depending on the virus species: bp for dengue and and dengue , bp for dengue and dengue , and bp for yfv, wnv (including kunjin), jev, mve, sle, and usutu. amplification was carried out using the qiagen one step rt-pcr kit. each reaction was performed in a . ml thin wall tube (stratagene) in a total volume of l overlaid with l of mineral oil. each reaction mix contained l of × qiagen buffer, l of dntp mix (each dntp at mm concentration), l of each primer ( m stock), l of molecular grade double distilled water (ddh o) and l of the one step enzyme mix (qiagen). the master mix was then aliquoted in tubes, to which l of template rna was added. the pcr thermal cycling was done on a stratagene robocycler , with an initial incubation at • c for min, followed by an incubation at • c for min, and cycles consisting of denaturation at • c for min, annealing at • c for min, and elongation at • c for min s. extensive precautions against pcr contamination, as previously described (johnson et al., ) were strictly observed. positive and negative controls, as well as extraction controls and controls for pcr inhibition, were set up essentially as described (johnson et al., ) . a l volume of each reaction was submitted to electrophoresis on . % agarose gels containing ethidium bromide. the gels were visualized on a uv transilluminator and photographed. amplicons were submitted for automated sequencing, for both strands, using the pcr primers as sequencing primers. sequencing was performed by the dna sequencing facility, the centre for applied genomics, hospital for sick children. sequence editing and analysis were done using the program generunner for windows ver. . (hasting software). sequence alignments were calculated using clustalx for windows ver. . (thompson et al., ) , with the default parameters for gap opening and gap extension. a phylogenetic tree was established using treecon for windows ver. . .b ( van de peer and de wachter, ) . in brief, the distance was calculated without correction, with insertions and deletions taken into account. the topology was inferred using the upgma method. bootstrap analysis was done using replicates. based on the sequence alignment of the ns coding region of several flaviviruses (fig. ) the consensus primers flavi- and flavi- were designed. the expected sequence of the amplicons were deduced from the genomic sequences and the primer sequences, and a phylogenetic tree was inferred (fig. ) , which demonstrates that this assay permits reliable identification of the virus species by sequencing the amplicon. amplicons of the expected size were readily and reproducibly obtained from rna extracted from cultures of wnv (ny strain), sle, kunjin, dengue serotypes , , and , and yfv vaccine strain. in each case, the identity of the amplicon was confirmed by sequencing. for the stocks of viruses that had been titrated, serial dilution of rna allowed to estimate the equivalent limit of detection in plaque forming units (pfu), as shown in table . in addition, for wnv the rna serial dilution was used to compare the sensitivity of the assay described here with that of a commercially available quantitative rt-pcr kit (realart wnv lc rt-pcr kit, artus gmbh). the sensitivity was found to be equivalent, with a measurement of approximately genome copies (average of two experiments). the methods of rna extraction used in the clinical laboratory at the hospital for sick children typically extract rna from a volume of - l, although for wnv testing the qiaamp ultrasense kit (qiagen), with a volume of sample up to l, is preferred. it can be readily seen that the sensitivity obtained for all viruses tested is similar to that reported with assays for other flaviviruses (scaramozzino et al., ; busch et al., ; kao et al., ; kuno, ) . samples from six mosquito pools collected in different provinces in canada over the course of years were also tested. amplicons of the expected size were obtained from all samples, and sequencing of the amplicons confirmed the viral species as wnv. nucleic acids extracted from preparation of cytomegalovirus, influenza h n (clinical isolate, season - ) , echovirus or hepatitis c virus (genotype a) did not generate amplicons of - bp expected from flaviviruses. similarly, nucleic acids extracted from human csf or plasma of uninfected patients yielded negative rt-pcr. in this study the use of a single pair of consensus primers for the detection of mosquito-borne flaviviruses was validated. the targeting of segments of the ns coding region to detect several flaviviruses has been reported before; for example, kuno et al. ( ) have established a precise phylogeny of the flaviviruses by sequencing large amplicons in the ns region from cultured viruses. the consensus primers used in this study show some similarity with two of the primers used by kuno et al. ( ) ; however the version of the primers presented here and a careful optimization of the reaction allow for a sensitivity sufficient for direct detection of viruses in clinical samples and not just from cultured viruses. scaramozzino et al. ( ) have described a highly sensitive pcr assay for the detection of several flaviviruses, but their assay used a two step rt-pcr format followed by a second heminested pcr, a procedure less convenient for clinical samples. the analytical sensitivities are shown in table ; taking into account the volume of extraction, as outlined in the section , the sensitivities obtained for the viruses tested compare favorably to that reported with assays for other flaviviruses (scaramozzino et al., ; busch et al., ; kao et al., ; kuno, ) . with the current sensitivity of molecular tests, laboratory diagnosis of acute yellow fever and dengue fever can be readily accomplished. even though the viremia is short lived, it occurs during the symptomatic febrile phase and reaches high viral titers: up to - pfu/ml for yfv (tomori, ) , and up to . × genome copies/ml and . × genome copies/ml in dengue fever and dengue hemorrhagic fever, respectively (wang et al., ) . the use of molecular tests for the diagnosis of other flaviviruses can be more problematic; thus for wnv, not only is the viremia of short duration but the titer is considerably lower, with a peak of no more than . × pfu/ml (hayes, ) . a single negative rt-pcr would therefore not rule out a current infection and consequently, serological testing continues to play an important role in laboratory diagnosis in immunocompetent persons. molecular testing would arguably be of greater usefulness in cases of suspected infection with wnv in immunocompromised patients, where the humoral response may be blunted and the viremia may be prolonged or of higher titer (huang et al., ) . in addition, molecular assays have been used as part of screening for blood and organ donors (cdc, ; health canada, ; kleinman et al., ) . finally, the use of a single polyvalent flavivirus pcr would be useful for the detection of flaviviruses in various vectors and non-human hosts, to aid in the monitoring of viral activity, including virus emergence. the assay presented here requires sequencing of the amplicons for confirmation and identification of the viral species. given the increasing ease and affordability of automated sequencing, this is not a great imposition on clinical laboratories proficient in molecular techniques. primers capable of detecting several viral species permit the use for a positive control of a viral sequence unlikely to be present in the area where the laboratory is located (for example, kunjin virus rna is now used at the hospital for sick children), thus eliminating a possible source of laboratory contamination of the assay. the implementation of this assay in a laboratory that must monitor, say, wnv, also means that an assay for more infrequent viruses is in fact kept ready in the laboratory. for example the diagnosis of acute infection with dengue in a returning traveler could be confirmed in our laboratory with this assay (data not shown). in summary, a single tube rt-pcr using consensus primers was designed and validated. coupled with sequencing, it could detect with great sensitivity and identify several mosquito-borne flaviviruses including wnv, kunjin, sle, yfv and dengue fever viruses. it is expected that this assay will be useful for clinical diagnosis and for the ecological surveillance of flaviviruses. flaviviruses analytical and clinical sensitivity of west nile virus rna screening and supplemental assays available in west nile virus phylogenetic analysis of north american west nile virus isolates west nile virus. update for family physicians the changing epidemiology of yellow fever and dengue epidemiology and transmission dynamics of west nile virus disease health canada guidance document: measures to prevent west nile virus transmission through cells, tissues and organs for transplantation and assisted reproduction first isolation of west nile virus from a patient with encephalitis in the united states comprehensive pcrbased assay for detection and species identification of human herpesviruses japanese encephalitis in india: an overview laboratory diagnosis of dengue virus infection: current and future perspectives in clinical diagnosis and public health update: west nile virus screening of blood donations and transfusion-associated transmission universal diagnostic rt-pcr protocol for arboviruses phylogeny of the genus flavivirus rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a taqman reverse transcriptase-pcr assay flaviviridae: the viruses and their replication emerging flaviviruses: the spread and resurgence of japanese encephalitis, west nile and dengue viruses flavivirus infection, fatal, argentina (cordoba) ( ): st louis encephalitis japanese encephalitis, india (uttar pradesh) ( ) comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the ns gene sequences the clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools yellow fever: the recurring plague treecon for windows: a software package for the construction and drawing of evolutionary trees for the microsoft windows environment high levels of plasma dengue viral load during defervescence in patients with dengue hemorrhagic fever: implications for pathogenesis comparison of assays for the detection of west nile virus antibodies in chicken serum emergence of usutu virus, an african mosquitoborne flavivirus of the japanese encephalitis virus group transcripts from a single full-length cdna clone of hepatitis c virus are infectious when directly transfected into the liver of a chimpanzee this work was supported by the department of paediatric laboratory medicine, hospital for sick children. key: cord- - lswjro authors: fan, jing-hui; zuo, yu-zhu; shen, xiao-qiang; gu, wen-yuan; di, jing-mei title: development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: lswjro the recent dramatic increase in reported cases of porcine epidemic diarrhea (ped) in pig farms is a potential threat to the global swine industry. therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (pedv) infection or vaccination would be essential in helping to control the spread of ped. we developed and validated an indirect enzyme-linked immunosorbent assay (elisa) based on the recombinant membrane (m) protein of pedv. to detect pedv antibodies in eight herds, serum samples were collected from sows that had been immunized with a ped vaccine, and screened using the developed elisa in parallel with a serum neutralization (sn) assay. of the tested samples, were positive for the presence of pedv antibodies according to both assays, while were negative. an excellent agreement between the elisa and the sn assay was observed (kappa = . ; % confidence interval = . – . ; mcnemar's test, p = . ). no cross-reaction was detected for the developed elisa with other coronaviruses or other common pig pathogens. the developed elisa could be used for serological evaluation and indirect diagnosis of ped infection. porcine epidemic diarrhea (ped) is a devastating swine disease and its etiological agent is ped virus (pedv). porcine epidemic diarrhea is characterized by watery diarrhea, dehydration, and a high death rate among suckling pigs (pensaert and debouck, ) . the disease was first documented in the united kingdom in as a swine disease that resembled transmissible gastroenteritis (oldham, ) . outbreaks of ped have since been reported in europe, asia, and north america (nagy et al., ; takahashi et al., ; sun et al., ; fan et al., ; stevenson et al., ) . porcine epidemic diarrhea virus has spread rapidly across the united states, resulting in the high mortality of piglets, and substantial economic losses (cima, ) . its recent emergence in north america suggests that this virus is a threat to the swine industry worldwide. accurate diagnosis and serological detection of specific antibodies in pigs as a result of pedv infection or vaccination are required to control the spread of ped. porcine epidemic diarrhea virus is a coronavirus within the coronaviridae family. the genome of pedv contains genes encoding spike (s), membrane (m), small membrane (sm), open reading frame , and nucleocapsid (n) proteins (park et al., ) . the m protein is a structural membrane glycoprotein, and the most abundant of all the envelope proteins. it has a short amino-terminal domain that exists outside of the virion, with the long carboxyterminal domain of the protein present inside the virion (utiger et al., ) . immune reactions to the m protein of coronaviruses play an important role in the induction of protection, and in mediating the course of the disease (fleming et al., ; vennema et al., ) . the nucleotide sequence of the pedv m gene exhibits low homology (around %) with the m gene of other coronaviruses; however, its sequence is highly conserved among different pedv strains arndt et al., ) . therefore, the m protein could be a suitable candidate for the detection of pedvspecific antibodies and in diagnosing pedv infections. many enzyme-linked immunosorbent assays (elisas) have been developed for the detection of antibodies against pedv. however, the preparation of an appropriate antigen for most of these methods requires cultivation of pedv, which is time consuming and expensive. in the current study, we expressed and purified the m protein of pedv. was used as a coating antigen in an indirect elisa that we developed. we then screened serum samples from pigs to determine the presence of antibodies specific for the pedv m protein. we collected blood samples from healthy unvaccinated pedv-free pigs of various ages. the sera derived from these samples were negative for the presence of antibodies against pedv according to serum neutralization (sn) assays (sn titers < : ). the sera from blood samples, taken from pedv-infected pigs, were used as reference positive sera (sn titers ≥ : ). the pedv infection status of pigs was confirmed by reverse transcription polymerase chain reaction (rt-pcr) assays targeting the pedv m gene, with viral rna extracted from fecal samples. porcine sera containing antibodies against transmissible gastroenteritis virus (tgev; sn titer : ), porcine rotavirus (prv; : ), porcine reproductive and respiratory syndrome virus (prrsv; : ), porcine circovirus (pcv- ; : ), and classical swine fever virus (csfv; : ) were obtained from the hebei center for disease prevention and control (shijiazhuang, people's republic of china). a total of serum samples were collected from eight pig herds, in which the animals had been administered a pedv vaccine. all samples were tested by the developed indirect elisa. porcine epidemic diarrhea virus strain hb/bd (genbank accession no. jf . ) was isolated from the feces of a pig from hebei province, china, and adapted to cell culture for passages in vero cells. viral rna was extracted from pedv using trizol reagent (invitrogen, carlsbad, ca, usa), following the manufacturer's instructions. the sequence corresponding to the m gene was amplified from viral rna by rt-pcr using oligonucleotide primers mp ( -gga tcc atg tct aac ggt- ) and mp ( -aag ctt tct gtt tag act aaa t- ). the resulting pcr products were separated by agarose gel electrophoresis and purified using a gel extraction mini kit (tiangen biotech co. ltd., beijing, china) according to the manufacturer's recommendations. purified amplicons were cloned into the pmd -t vector (takara biotech co. ltd., dalian, china) to yield the recombinant plasmid pmd-m, which was then transformed into competent escherichia coli jm . positive clones were selected according to their lacz phenotype, and verified by restriction enzyme digestion, pcr screening, and dna sequencing. the m gene contained within pmd-m was subcloned into the prokaryotic expression vector pgex- p- (sunbiotech inc., beijing, china) to yield pgex- p-m. the pgex- p- vector also contained a sequence for a gst tag, which was designed so as to be added at the amino terminal of the expressed protein. the pgex- p-m vector was verified by dna sequencing. the recombinant m protein was transformed into e. coli bl (tiangen biotech co. ltd., beijing, china). e. coli bl cells were cultured and transformed with pgex- p-m, in luria-bertani broth supplemented with g/ml ampicillin until the optical density at nm (od ) was . - . , at which point protein expression was induced via the addition of . mm isopropyl-␤-d-thio-galactopyranoside. at h post-induction, bacterial cells were collected by centrifugation. the recombinant protein was purified from the bacterial lysate using a gst fusion protein purification kit (transgen biotech co. ltd., beijing, china), according to the manufacturer's recommendations. fractions of purified recombinant m protein were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), with polyacrylamide gels stained with coomassie brilliant blue r- . expression of the recombinant m protein was confirmed by immunoblotting, using porcine pedv antisera. the prokaryotic expression vector pgex- p- was also transformed into e. coli bl as a negative control. fractions of purified recombinant proteins were separated by sds-page and electrophoretically transferred to nitrocellulose membranes (gibco brl, gaithersburg, md, usa). to prevent nonspecific reactions, membranes were blocked with % (w/v) non-fat milk powder in tris-buffered saline containing . % (v/v) tween (tbst) at • c for h. membranes were incubated with pig pedv antisera (diluted : ) at • c for h. after three washes with tbst, membranes were incubated with an anti-pig igg conjugated to horseradish peroxidase (hrp; sigma-aldrich, st louis, mo, usa) at • c for h. we used , -diaminobenzidine (sigma-aldrich) for the development of color reactions to visualize protein bands. a checkerboard titration involving each combination of antigen (purified recombinant pedv m protein) and sera was used to determine optimal dilutions for use in the indirect elisa we developed. the antigen and serum were diluted from to . g/ml and : to : (two-fold dilution), respectively. ninety six well plates (nunc maxisorp, denmark) were coated with the recombinant m protein, which was diluted in phosphate-buffered saline (pbs), and allowed the plates to incubate at • c overnight. pbs was used as a blank control. wells were blocked with % fetal bovine serum in pbs (ph . ) for h at • c. wells were then washed three times with pbs containing . % tween (pbst) and diluted serum samples were added, in duplicate, to the antigen-coated wells. following incubation at • c for h, plates were washed three times with pbst and l of diluted goat anti-pig igg conjugated to hrp (kpl inc., gaithersburg, md, usa) was added to each well. after incubation at • c for min, wells were washed with pbst, and l of , , , ,-tetramethyl-benzidine (tmb) was added to each well and the plates were incubated for min at room temperature. color development reactions were stopped with l of m h so . the od was determined for each well using a microplate reader. dilutions that resulted in the highest od ratio between the positive and negative serum samples (p/n value), along with an od value for positive serum samples close to . , were considered optimal working conditions. to establish negative and positive cutoff values for this assay, pedv-negative serum samples, as determined by sn assays, were tested in triplicate using the elisa we developed. a cutoff value was defined as the mean absorbance value plus three standard deviations (sd). repeatability assays were conducted by comparing the ratios of od values for triplicate results from each serum sample tested on the same plate (intra-assay repeatability) or on different plates (inter-assay repeatability). the coefficient of variation (cv) was calculated according to the following formula: where x was the average od for the field serum samples. a cv value less than % was considered an acceptable level of variation. to determine the specificity of the developed elisa, crossreactivity was assessed by determining the reactivity of the purified antigen with serum samples containing antibodies against tgev, prv, prrsv, pcv , csfv and pedv. reference serum samples were diluted : , with three replicates of each dilution tested using the indirect elisa. serum samples from pedv-infected pigs (n = ) were selected at random to determine the sensitivity of the indirect elisa. samples were serially diluted two-fold from : to : . eight replicates of each diluted serum sample were tested to determine the titers of antibodies in samples. the titers were also determined by sn. the indirect elisa that we developed was used to evaluate the presence of pedv antibodies in serum samples from sows with varying immune status. these serum samples were also subjected to sn assays. the level of agreement between the sn and elisa assay was determined using a cohen's kappa, and a mcnemar's test was performed to investigate the differences between both assays. a p-value < . was considered significant. all analyses were performed in spss version . . the m gene (approximately bp) of pedv was successfully amplified and cloned it into a ta vector. positive clones were identified by sequencing. sequence analysis indicated that the pedv m gene was bp, and encoded amino acids. the complete nucleotide sequence of the m gene has been deposited in genbank (accession number jf ). recombinant pedv m gene was expressed in e. coli bl . analysis of the bacterial cell lysate by sds-page and western blotting revealed a prominent band of around kda. the recombinant protein we identified was approximately kda heavier than the predicted molecular weight of kda, which was due to the gst tag (fig. ) . the recombinant protein was predominantly in the insoluble fraction of the bacterial cell extract, and purified using a gst fusion protein purification kit. western blotting analysis showed that the purified protein was recognized by pedv antiserum (fig. ) . the optimum antigen concentration and serum sample dilution were determined to be . g/ml and : , respectively. the optimum dilution of the anti-pig igg conjugated to hrp was found to be : . the incubation conditions for primary and secondary antibodies were h at • c and min at • c, respectively. the optimum blocking buffer was found to be % fetal bovine serum in pbs, while pbst was the most appropriate washing buffer. the detection threshold of our indirect elisa was determined using serum samples that were negative for the presence of pedv antibodies. the cutoff point was specific for each individual assay and was based on the od of negative samples included in each assay. the mean od value for the elisa was . , with an sd of . . the cutoff value was determined to be . (mean + sd); therefore the negative-positive threshold was set at . . serum samples returning an elisa od value below table cross-reaction analysis of the m-based i-elisa with anti-sera against other pig viruses: od value (mean ± sd). pedv, tgev, prv, pcv- , csfv, prrsv represent pig sera that were positive for pedv, tgev, prv, pcv- , csfv, prrsv, respectively. 'noninfected' represents the serum from a pig that was negative for pedv. . were regarded as negative; samples that returned an od value higher than this were considered to be seropositive for pedv antibodies. reproducibility within and between elisa plates was evaluated by testing ten sn-negative serum samples and ten sn-positive serum samples in triplicate. the inter-assay cv of the indirect elisa ranged from . % to . % with a median value of . %. the intraassay cv ranged from . % to . %, with a median value of . %. these results indicate that the indirect elisa we developed to detect antibodies against the pedv m protein was repeatable, with low and acceptable levels of variation. the specificity of our elisa was evaluated by testing the reactivity of the pedv m protein antigen with antibodies against pedv, tgev, prv, prrsv, pcv- , and csfv. we found that pedv antisera significantly reacted with the pedv m antigen, as expected. in contrast, the od values for all other serum sample containing antibodies against other porcine viruses were significantly lower than the established negative cutoff value (table ) . our results suggest that little or no cross-reactivity occurs between the pedv m protein and antibodies against other porcine viruses, and that the m protein antigen was specific for antibodies against pedv. we evaluated the sensitivity of our indirect elisa using ten pedv-positive serum samples, and found that we could detect pedv antibodies in a serum sample that had been diluted out to : , while only : . in sn. this result indicated that our indirect elisa was more sensitive than the sn assay for pedv antibody detection (table ) . of the serum samples collected from animals with a known immunization history across eight pig farms, ( . %) were positive for pedv antibodies according to our elisa, while ( . %) were pedv antibody-negative. in comparison, according to the sn assay we used in parallel with the indirect elisa, % ( / ) of samples were pedv antibody-positive and . % ( / ) of samples were antibody-negative (table ) . according to both assays, and of the screened serum samples were pedv antibody-positive and -negative, respectively. three of the serum samples that were positive according to the sn assay, were porcine epidemic diarrhea virus is a member of the coronaviridae, and is known to cause fatal diarrhea in newborn piglets. the virus was first identified in (pensaert and debouck, ; chasey and cartwright, ) and has since been found to be prevalent in many countries. infection with pedv has resulted in significant economic losses, mainly in europe and asia, and recently the usa (fan et al., ; chae et al., ; martelli et al., ; stevenson et al., ) . although there are commercial vaccines available to prevent and control ped in china, the damage caused by pedv is significant and the threat continuous. to effectively control and prevent this disease, detection methods that can assess the current epidemic situation in herds are required. in addition, for subsequent immunoprophylaxis, assays that can monitor serum antibody levels of immunized or infected pigs need to be developed. before local immunity is actively established, the piglet intestine is protected against pedv infection by maternal antibodies. although the presence of antibodies in serum is not directly related to the protection for sows or for piglets, serological examination facilitates the assessment of humoral response to pedv elicited either through vaccination or natural infection. porcine epidemic diarrhea virus was isolated for the first time in using the vero cell line and trypsin-supplemented medium (hofmann and wyler, ) . various diagnostic methods have been described for the detection of pedv antigen and pedv antibodies (carvajal et al., ; knuchel et al., ; kweon et al., ; song et al., ) . at present, serological diagnostic methods for the diagnosis of pedv infection are more common. given that elisas are simple, sensitive, and convenient serological detection methods, a number of commercial and in-house elisas have been developed to detect pedv antigens, or antibodies against pedv (song and park, ) . the majority of in-house assays for the detection of antibodies against pedv are based on using the whole virus as an antigen (carvajal et al., ; oh et al., ) , or preparations of viral antigen (knuchel et al., ) . the cultivation of pedv is laborious and time consuming, especially considering the bio-security measures that must be taken in handling this virus; therefore it is difficult to upscale the production of these serological assays. the use of a recombinant viral protein as an antigen would allow researchers to avoid some of the problems associated with the large-scale preparation of native pedv antigen. recently, an elisa based on a recombinant n protein was validated in china (hou et al., ) . however, these researchers did not assess the cross-reactivity of the elisa with other coronaviruses. the m protein of pedv protrudes from the viral envelope, and is considered a superior diagnostic antigen compared with other pedv proteins (shenyang et al., ) . in the current study, we chose the pedv m protein for use as an elisa antigen because it is highly conserved among pedv strains, and because it can elicit the formation of protective antibodies (zhang et al., ) . we expressed the m protein in e. coli and confirmed its antigenicity using pedv-specific antibodies. our results indicated that the expressed recombinant m protein was indeed antigenic, and could feasibly be used as a coating antigen in an indirect elisa. the indirect elisa that we developed exhibited low levels of variability among replicates, according to intra-and inter-assay tests. these minor variations in results indicated that the indirect elisa was reproducible. furthermore, this elisa was able to detect pedv antibodies in serum samples that had been diluted out to : , and exhibited no cross-reactivity to antibodies against other common pig pathogens. these findings indicated that the developed indirect elisa could be used widely in the future. vaccination is one of the most effective techniques in controlling ped in china. however, since late , ped has been reemerging in immunized swine herds with devastating impact. to confirm the effects of targeted immunoprophylactic measures, we used our indirect elisa to screen serum samples from vaccinated pigs. we found that . % and % of samples were seropositive according to elisa and sn assay, respectively. this indicated that vaccination elicited neutralizing antibodies against pedv in sows to some degree. however, . % and % of samples from vaccinated pigs were seronegative by the elisa and sn assays, respectively. although these antibodies could not be used to assess protection against pedv, these vaccinated, but seronegative sows are not able to vertically transmit effective antibodies to their neonates. it indicating that the vaccine used requires some improvement in antigenicity. a comparison of the elisa and sn assay results revealed eight differences between positive and negative samples. there were five elisa-positive samples that were negative according to the sn assay we used, which could be explained by the higher sensitivity of the elisa. the reason for the elisa-negative results for the three sn-positive serum samples might be due to errors inherent in the tests that we applied. in conclusion, this is the first report of an indirect elisa using the recombinant pedv m protein as a coating antigen for the detection of antibodies against pedv in china. the developed assay is quick, convenient, and not labor-intensive, and could facilitate the development of a reliable tool or kit for the large-scale detection of antibodies against pedv. the authors have no conflicts of interest concerning the work reported in this paper. a conserved domain in the coronavirus membrane protein tail is important for virus assembly evaluation 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antibodies against porcine epidemic diarrhea virus (pedv) based on the specific solubility of viral surface glycoprotein rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy enterotoxigenic escherichia coli, rotavirus, porcine epidemic diarrhoea virus, adenovirus and calici-like virus in porcine postweaning diarrhoea in hungary comparison of an enzyme-linked immunosorbent assay with serum neutralization test for serodiagnosis of porcine epidemic diarrhea virus infection letter to the editor. pig farm molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea a new coronavirus-like particles associated with diarrhea in swine high-level prokaryotic expression of envelope exterior of membrane protein of porcine epidemic diarrhea virus porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines multiplex reverse transcription-pcr for rapid differential detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, and porcine group a rotavirus emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets an outbreak of swine diarrhea of a new-type associated with coronavirus-like particles in japan identification of the membrane protein of porcine epidemic diarrhea virus primary structure of the membrane and nucleocapsid protein genes of feline infectious peritonitis virus and immunogenicity of recombinant vaccinia viruses in kittens identification of a conserved linear b-cell epitope in the m protein of porcine epidemic diarrhea virus this work was supported by the research foundation of the education bureau of hebei province, china (no. ). key: cord- -fkg d ym authors: wang, leyi; eggett, therese e.; lanka, saraswathi; fredrickson, richard l.; li, ganwu; zhang, yan; yoo, dongwan; bowman, andrew s. title: development of a triplex real-time rt-pcr assay for detection and differentiation of three us genotypes of porcine hemagglutinating encephalomyelitis virus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: fkg d ym porcine hemagglutinating encephalomyelitis virus (phev) is a single-stranded, positive-sense rna virus. phev mainly causes two types of clinical manifestations representing vomiting and wasting and encephalomyelitis in piglets. however, our recent findings provide strong evidence that phev can also cause respiratory disease in older pigs. genomic analysis of new phev strains identified in our former study further classifies phev into three genotypes. detection and differentiation of these new mutants are critical in monitoring phev evolution in the field. in the present study, we report the development of a triplex real-time rt-pcr assay for detection and differentiation of three phev genotypes, , , and . three sets of primers and probes were designed; one set of primers and probe targeting the conserved regions of the ′ end nucleocapsid for detection of all three genotypes and another two sets of primers and probes targeting the regions of ns with different patterns of deletions for detection of both genotypes and , or genotype only. genotype was positive when two probe dyes showed signals, genotype was positive when only one probe dye showed a signal, and genotype was positive when all three probes showed signals. the detection limit of the developed triplex real-time rt-pcr was as low as or dna copies for three sets of primers and probes. the specificity test showed no cross reaction with other porcine viruses. positive field-samples were correctly typed by this new assay, which was further confirmed by dna sequencing. the triplex real-time rt-pcr provides a rapid and sensitive method to detect and differentiate all three us genotypes of phev from clinical samples. porcine hemagglutinating encephalomyelitis virus (phev) is one of six known porcine coronaviruses (covs) causing diseases in pigs (gong et al., ; wang and zhang, ) . phev typically affects pigs less than three weeks of age and the clinical syndromes include the vomiting and wasting disease (vwd) and encephalomyelitis (quiroga et al., ) . phev was first isolated from suckling piglets suffering from encephalomyelitis in canada in (greig et al., ) . phev has since been identified in many countries of europe, asia, and america (cartwright et al., ; dong et al., ; forman et al., ; hirahara et al., ; mengeling, ; pensaert and callebaut, ; quiroga et al., ; rho et al., ) . although phev infection has been endemic in different countries for decades and the primary route of phev infection is through upper and lower respiratory tracts, only few reports suggest an association of the virus with respiratory disease in swine (cutlip and mengeling, ) . recently, we have demonstrated that phev caused an influenza-like illness (ili) in affected pigs and provided evidence for the role of phev as a respiratory pathogen (lorbach et al., ) . in that study, a significantly higher positive rate . % ( of pigs) was observed in pigs at the michigan fairs compared to . % ( of pigs) in indiana and ohio, indicating the phev-associated epizootic behavior in the michigan fairs (lorbach et al., ) . phev is a single-stranded positive-sense rna virus and belongs to the betacoronavirus genus of the coronaviridae family. the first two https://doi.org/ . /j.jviromet. . . received february ; received in revised form april ; accepted april thirds of the phev genome contain two large open reading frames (orf a and orf b) encoding the replicases, and the remaining part of the genome encodes six structural proteins (hemagglutinin-esterase protein, he; spike glycoprotein, s; envelope protein, e; membrane protein, m; nucleocapsid protein, n, and n ) and three non-structural (ns) proteins (ns , ns . , and ns . ). previous genomic characterization and analysis reveal that phev is closely related to bovine cov and human cov oc , and have identified truncated ns and ns . genes in the phev-vw strain (vijgen et al., ) . prior to our study (lorbach et al., ) , only one complete phev genome sequence (phev-vw ) was available in genbank. we completed the full genomic sequencing of phev isolates, and the analyses of complete genomic sequences showed that phev formed three distinct genotypes. interestingly, genotypes had deletions in ns and ns . , while genotype showed no deletion and genotype has a large deletion in the ns gene (lorbach et al., ) . the prevalence and distribution of three genotypes remain unknown in pigs in the united states. the traditional real-time rt-pcr (rrt-pcr) test currently used in diagnostic laboratories does not differentiate emerged phev from classical phev strains (lorbach et al., ) . the triplex rrt-pcr developed in the present study will fulfill this purpose and can be used to monitor phev of different genetic genotypes and differentiate between them. complete genomic sequences of all phev strains available in the genbank database were collected and aligned using clustalw of mega . . program. based on the conserved and variable regions of sequences, three sets of primers and probes were designed; the first set targeting the ′ non-coding region and n to detect all three genotypes (n-g ), the second set targeting the ns region to detect genotypes and (ns -g ) that have deletions for genotype , and the third set targeting the regions of ns for genotype (ns -g ) that there are deletions for genotypes and (fig. ) . the probes were labeled with different dyes for multiplexing purpose (table ) . rna was extracted using qiagen one-for-all vet kit (valencia, ca, usa) according to the manufacturer's instructions. the extracted rna was eluted in μl of elution buffer and stored at - ℃ until use. concentrations of rna samples were measured using qubit fluorometer. step rt-pcr kit (valencia, ca, usa) was used for amplification in the abi real-time pcr system (applied biosystems, ca, usa). the assay was performed in a -μl reaction mixture containing μl of × reaction buffer, μl of each primer ( μm), μl of each fluorogenic probe ( μm), μl of dntp ( mmol), μl of enzyme mix, . μl of purified rna, and an appropriate volume of rnase-free distilled water (dh o). the thermocycler amplification conditions were °c for min, °c for min, and cycles of °c for s, °c for s, and °c for s. rna was extracted from positive samples with known genotypes of sw (genotype ), sw (genotype ), and sw (genotype ), and then quantified using qubit fluorometer. the n gene was amplified using rna of sw (genotype ) and the primer set of phev n f pcr and phev ncr r, and the ns gene were amplified using rna of above three samples and the primer set of phev poly-f pcr and phev he-r pcr (table ). the pcr products were cloned into the pcr . vector (invitrogen, carlsbad, ca, usa). the detection limit of the triplex rrt-pcr assay was determined by testing -fold serially diluted positive phev rnas ( sw and sw ) and plasmid dnas (pcr . - sw -n, pcr . - sw -ns , and pcr . - sw -ns ) in duplicate. intra-specificity of the triplex real time rt-pcr assay was determined using a single probe of n-g , ns -g , and ns -g for detection of all three genotypes, both genotype and , and genotype only, respectively. inter-specificity of triplex real time rt-pcr assay was examined using various swine viruses available in our laboratory. these viruses include porcine reproductive and respiratory syndrome virus, swine influenza virus (h n ), transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine deltacoronavirus, porcine circovirus , and senecavirus a. in the assay, . μl of rna or dna samples were used, . μl of genotype strain sw was used as a positive control, and . μl distilled water was used as a negative control. positive controls for each genotype were used in each run of the triplex real-time rt-pcr. nasal swab and nasal wipe samples were collected from exhibition swine at shows during − . seventyfive samples testing positive for phev were selected for this study and processed for rna extraction. rna samples were first tested for phev by a singleplex rrt-pcr as previously reported (lorbach et al., ) . if a test appeared positive, the triplex rrt-pcr was used to differentiate three genotypes of phev and a conventional pcr using a primer set of pehv poly-f pcr pehv he-r pcr (table ) was applied to amplify ns region. the amplicons were subjected to dna sequencing using a sanger method (acgt, inc.) for confirmation. based on the sequence alignment and analyses of all complete genome sequences of different genotypes, a conserved region in the ′ end of n gene was selected for detection of all three genotypes of phev, whereas ns gene with different patterns of deletions was used to design two sets of primers and probes to detect both genotypes and , or genotype only (fig. ). if only one fam signal is detected, it indicates genotype ; if two signals (fam and cy ) are detected, it indicates genotype ; and if all three signals (fam, cy , joe) are detected, it indicates genotype of phev (table ) . the triplex rt-pcr assay specifically detected genotype by the fam probe only, genotype by the fam and cy probes, and genotype by the fam, cy , and joe probes. by contrast, the triplex rt-pcr did not cross-react with any other swine viruses (porcine reproductive and respiratory syndrome virus, swine influenza virus (h n ), transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine deltacoronavirus, porcine circovirus , and senecavirus a) used in the study. the sensitivity of the triplex rrt-pcr was determined through fold serial dilutions of rnas of known phev genotypes ( sw for g and g , and sw for g ) and plasmids pcr . - sw -n for g , pcr . - sw -ns for g , and pcr . - sw -ns for g in duplicate. the detection limit was . ng/μl and dna genome copies for n-g with ct value as a cutoff, and . ng/μl and dna genome copies for ns -g and -g with ct values and as cutoffs, respectively (fig. ). both replicates of n-g , ns -g , and ns -g produced positive results at or dna copies. there are strong linear correlations ( r > . ) between c t values and amount of viral rna and between c t values and corresponding amount of plasmid copy numbers for different targets ( fig. a, b , and c). a total of positive samples were tested by the singleplex rrt-pcr and were run again by the triplex real-time rt-pcr. fifteen samples appeared positive for genotype , samples were positive for genotype , and were positive for genotype . the sanger sequencing using pehv poly-f pcr-pehv he-r pcr primer set confirmed the genotyping results. to be noted, out of genotype strains have a primers and probes used in the study. different deletion pattern from that originally identified (fig. ) . in recent years, with the help of molecular technology, more novel coronaviruses have been identified in pigs. so far, there are six known porcine coronaviruses including transmissible gastroenteritis coronavirus (tgev), porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus (pdcov), porcine respiratory coronavirus (prcv), bat hku -like porcine enteric alphacoronavirus (peav), and phev. these porcine coronaviruses cause neurologic (phev), digestive (tgev, pedv, pdcov, peav, phev), and respiratory diseases (prcv, phev). vwd and encephalomyelitis caused by phev are common and have experimentally been reproduced in pigs (mengeling and cutlip, ) . in contrast, only a few studies including ours report that phev causes respiratory problems in pigs. in our previous study, we reported that influenza-like illness in market-age pigs was strongly linked to phev (lorbach et al., ) . field and experiment infection studies are needed to elucidate the pathogenic mechanism of respiratory diseases caused by phev. in addition to the uncommon clinical presentation of phev, we show that there are three different genotypes cocirculating in the field in us. different deletion patterns (small, large, none) in ns are observed in genotypes , , and , respectively. genotype has a small deletion in ns . . genotype was predominant in pigs with respiratory disease from michigan fairs (lorbach et al., ) . it remains unknown if different genotypes have similar virulence. to further type samples positive for phev, we have developed a triplex rrt-pcr using one fam dye for genotype , the fam and cy dyes for genotype , and the fam, cy , and joe dyes for genotype . our data indicate that the triplex rt-pcr is highly specific with no cross-reaction with other known swine viruses and is sensitive with a detection limit ranging between . − . ng/μl of viral rna and - dna genome copies. furthermore, the developed assay correctly subtyped phev-positive samples, which was confirmed by sanger sequencing. it should be noted that genotype strains previously identified in our previous study have defective ns gene, and out of genotype strains detected in the present study have a larger deletion in ns , resulting in the complete deletion of ns . furthermore, out of genotype strains identified in the study have a different deletion pattern from other strains in genotype previously identified (fig. ) . similar to the finding that genotype was predominant in pigs of the michigan fair, genotype was also predominant ( %) in samples tested in the present study, whereas positive rates for genotype and were % and %, respectively. future studies are needed to address whether the large deletion in ns benefits virus replication or not. in contrast to conventional singleplex real time rt-pcr, the triplex real time rt-pcr that we have developed in this present study can be used to monitor phev genotypes and potentially to identify new variants if test results cannot be explained as expected. few limitations of our study need to be noted. for the specificity test, not all porcine coronaviruses were tested due to unavailability of prcv and peav. since prcv is a spike deletion mutant of tgev and the triplex assay developed did not show the cross reaction with tgev, it should cross react with prcv. for peav, blast search of sequences of primers and probes used in the triplex pcr showed no any hit to genome of peav, indicating that a cross reaction should not occur. the observed low efficiency of the triplex real-time rt-pcrs in the study was due to unknown factors including amount of pcr reagents and design of primers and probes. in summary, we have developed a triplex real time rt-pcr to detect and differentiate all three genotypes of phev. this triplex rt-pcr assay is highly specific and sensitive. the developed assay may be used to monitor different genotypes circulating in the field. vomiting and wasting disease of piglets lesions induced by hemagglutinating encephalomyelitis virus strain n in pigs identification and genetic characterization of porcine hemagglutinating encephalomyelitis virus from domestic piglets in china haemagglutinating encephalomyelitis virus infection of pigs a new bat-hku -like coronavirus in swine a hemagglutinating virus producing encephalomyelitis in baby pigs isolation of hemagglutinating encephalomyelitis virus from respiratory tract of pigs in japan porcine hemagglutinating encephalomyelitis virus and respiratory disease in exhibition swine incidence of antibody for hemagglutinating encephalomyelitis virus in serums from swine in the united states pathogenicity of field isolants of hemagglutinating encephalomyelitis virus for neonatal pigs characteristics of a coronavirus causing vomition and wasting in pigs hemagglutinating encephalomyelitis coronavirus infection in pigs detection and genetic analysis of porcine hemagglutinating encephalomyelitis virus in south korea evolutionary history of the closely related group coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc animal coronaviruses: a brief introduction key: cord- -zg gkf j authors: yi, li; cheng, shipeng; xu, hongli; wang, jianke; cheng, yuening; yang, shen; luo, bin title: development of a combined canine distemper virus specific rt-pcr protocol for the differentiation of infected and vaccinated animals (diva) and genetic characterization of the hemagglutinin gene of seven chinese strains demonstrated in dogs date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zg gkf j a combined reverse-transcription polymerase chain reaction (rt-pcr) method was developed for the detection and differentiation of wild-type and vaccine strains of the canine distemper virus (cdv). a pair of primers (p /p ) was used to detect both cdv wild-type strains and vaccines. another pair (p /p ) was used to detect only cdv wild-type strains. a bp fragment was amplified from the genomic rna of the vaccine and wild-type strains. a bp fragment was amplified specifically from the genomic rna of the wild-type strains. no amplification was achieved for the uninfected cells, cells infected with canine parvovirus, canine coronavirus, or canine adenovirus. the combined rt-pcr method detected effectively and differentiated the cdv wild-type and vaccine strains by two separate rt-pcrs. the method can be used for clinical detection and epidemiological surveillance. the phylogenetic analysis of the hemagglutinin gene of the local wild-type cdv strains revealed that the seven local isolates all belonged to the asia- lineage, and were clustered closely with one another at the same location. these results suggested that the cdv genotype asia- is circulating currently in domestic dogs in china. canine distemper virus (cdv) is a single-stranded negative rna virus belonging to the morbillivirus genus of the paramyxoviridae family (beineke et al., ) . cdv is a highly contagious viral pathogen causing lethal systemic, nervous, and enteritis diseases in dogs and other carnivores. the cdv genome is approximately , nucleotides (nts) long and consists of genes encoding nucleoprotein (np), phosphoprotein, as well as matrix, fusion, hemagglutinin (h), and large proteins. due to prevention by vaccination, the outbreak of canine distemper disease has been controlled. however, the incidence of canine distemper virus (cdv) in canines and other animal populations throughout china seems to have increased in the past years. several cdv outbreaks in vaccinated animals have been reported (ek-kommonen et al., ; perpinan et al., ) . therefore, cdv wild-type and vaccine strains need to be distinguished for diagnosis and epidemiological monitoring. a previous study (calderon et al., ) has shown significant differences between cdv wild-type and vaccine strains in the h protein, which displays a large number of antigenic variations. based on this finding, several cdv differential diagnoses between wild-type and vaccine isolates have been established (martella et al., ; si et al., ) . however, these methods are all based on reverse-transcription and nested polymerase chain reactions (rt-npcrs); they have the disadvantages of being timeconsuming and susceptible to contamination. therefore, a method for the specific and easy detection of wild-type cdv strains is required. in the present report, a combined reverse-transcription polymerase chain reaction (rt-pcr) method was established. the purpose was to detect and differentiate cdv wild-type and vaccine strains by two separate rt-pcrs effectively. the method provided a quick and affordable protocol for the diagnosis of cdv as well as for the differentiation of infected and vaccinated animals. this protocol can be implemented even in small laboratories. consequently, the diagnostic possibilities for the disease are improved, accessible tools are provided to people involved in cdv eradication in endemic areas, and the cdv genetic variability as well as epidemiology can eventually be elucidated. the cdv vaccine strain cdv- (cheng et al., ) and cdv wild-type strain cdv-ps in african green monkey kidney (vero) cell cultures were identified and preserved by the zoonosis lab, institute of special field economic animal and plant science, the chinese academy of agricultural sciences. the two local commercial cdv vaccine products (vacc-a and vacc-b) were purchased at a veterinary retail shop in china and tested during the current study. the vero cells were purchased from the china institute of veterinary drug control. seven cdv-positive samples were identified using rt-pcr assay, which amplified a bp-long fragment of the np gene (wang et al., ) . the background information on the seven cdv-positive samples collected in different locations in china is listed in table . total rna was obtained from mg of tissue homogenates or l of vaccine. a trizol ® (invitrogen) reagent was used according to the manufacturer's instructions. rt was performed in a final volume of l containing l of rna solutions, l of × rt buffer, l of deoxyribonucleotide triphosphate ( mm each), pmol of random -mer primers, u of reverse transcriptase xl (amv) (takara, dalian, china), and u of rnase inhibitor. the mixture was incubated at • c for h, and at • c for min. the cdv cdna products were further simultaneously amplified by a combined two-step rt-pcr assay using two primer sets to distinguish the vaccine and field strains. the specific primer sets, namely, p /p (wang et al., ) and p /p , targeted either the field isolates or the vaccine strains specifically. the primer pair p /p was used to detect only wild-type cdv strains. the other pair, p /p , was used to detect both the wild-type and vaccine strains of cdv (fig. ). all primers were designed based on the genomic sequences of the cdv strains published in genbank using the primer premier . software. the nt positions and sequences of the primers are shown in table . the amplification conditions for the combined two-step rt-pcr assay were • c for min followed by cycles of denaturation at • c for min, annealing from - • c for min, dna extension at • c for min, and a final extension at • c for min. the two separate rt-pcrs of the combined assay were used to detect non-infected cells as well as cells infected with the cdv vaccine strain cdv- , wild-type strain cdv-ps, canine parvovirus (cpv), canine adenovirus (cav), and coronavirus (ccv) to test their specificities. extracted rna from serially diluted ( , , , , , , − , and − % tissue culture infective dose, tcid ) cdv- strains ( . tcid /ml) in vero cell cultures were assayed by rt-pcrs to determine their sensitivities. the combined rt-pcr was also performed to distinguish among the wild-type strains, field samples, vaccine strains, and commercial vaccines to validate the applicability of the test. the pcr products were resolved by . % agarose gel electrophoresis. the cdv cdna products were amplified with primers reported previously by demeter et al. ( ) . the nt positions and sequences of the primer hc- /hc- are shown in table . the pcr reaction was subjected to cycles. each cycle consisted of denaturation at • c . schematic illustration of cdv differential primers strategy by p /p and p /p for detecting cdv wild-type strains. the * display as the identity nucleotide. the bold letters represent terminus mismatches between cdv wild-type strain and vaccine by primers p /p . for s, annealing at • c for min, and dna extension at • c for min. pcr products of the correct size ( bp long) were amplified and cloned into the pmd -t vector (takara, dalian, china). for each cdv strain, - positive recombinant plasmids were sequenced in both directions using primer m . additional primers selected based on the obtained sequences were provided by the shanghai invitrogen biotechnology company (shanghai, china). seven of the cdv field samples were selected for the amplification of the h gene of cdv by rt-pcr (table ). the amplified products were cloned and sequenced. the sequence data of the fulllength cdv h gene were then assembled to a total length of bp using the seqman and editseq functions of the dnastar software package. entire h gene sequences were aligned with the sequences of other cdv h genes collected from different places around the world and available in genbank. subsequently, the consensus tree was edited in mega . (tamura et al., ) . a phylogenetic analysis was performed using the neighbor-joining (nj) method, and setting the p distance algorithm of correction. this algorithm is expected to produce reliable phylogenies when large sets of closely related sequences are analyzed. the robustness of the phylogenetic analysis was assessed by a bootstrap analysis with replicates. after rna extraction and reverse transcription, the primer pairs p /p and p /p were used to amplify the vaccine and wild-type strains, respectively, at different annealing temperatures. only one specific band was observed at the annealing temperature range of - . • c, with the most distinct band appearing at . • c. hence, . • c was chosen for the annealing temperature in the combined rt-pcr. a bp fragment was amplified from both cdv wild-type and vaccine strains by rt-pcr with the primer pair p /p . on the other hand, a bp fragment was detected only from the cdv wild-type strains by rt-pcr with the primer pair p /p ( fig. a and b) . by the combined rt-pcr method, amplifications were not found for the non-cdv viruses (i.e., cpv, cav, and ccv), the uninfected cells, and the negative control of healthy animal tissues. the detection limit of the rt-pcr was tcid for the cdv wild-type strain cdv-ps by p /p , and was tcid for the vaccine strain cdv- by p /p (fig. ) . as illustrated in fig. , all cdv commercial vaccine and field strains were recognized by p /p , and yielded products that were bp long. in contrast, both cdv wild-type strains yielded bp products and all three cdv vaccines did not yield anything when amplified by p /p . after two rounds of rt-pcrs, cdv wild-type and vaccine strains were recognized. as a result, the cdv wild-type strains showed positive results ( and bp) in both rt-pcr rounds. the cdv vaccine strains showed a positive result ( bp) fig. . sensitivity of rt-pcrs to detect cdv vaccine by primers p /p and cdv wildtype strain by primers p /p . lanes - : tcid cdv cell culture; tcid cdv cell culture; tcid cdv cell culture; tcid cdv cell culture; tcid cdv cell culture; tcid cdv cell culture; and − tcid cdv cell culture. in the first round and a negative result ( bp) in the second round. the non-cdv viruses were negative in both rounds. by pcr with the primer set hc- /hc- , sequences of the h gene ( - nts) were identified from seven cdv local strains and one commercial vaccine (cdv ) used very commonly in china. these sequences were submitted to genbank. the nt sequences aligned using clustal w demonstrated that the sequence identities among the local isolates (cdv wild-type strains) ranged from . % to . %. on the other hand, the cdv vaccine strain was lower at . - . % (table ). these findings indicated that the h sequence variations of cdv circulating in china and the currently used commercial vaccine are significant, as previously reported (zhao et al., ) . a phylogenetic tree based on the h genes of various cdv strains was generated using the mega . software (tamura et al., ) and the nj method with bootstraps, as shown in fig. . the strains cdwuhan- , cdwuhan- , cdwuhan- , cdwuhan- , cdwuhan- , raccoon dog ps- , and bj- were classified into the asia- group. in general, strains from the same city or province shared a common clade. this result suggested a potential link with the phylogenetic relationship between the strains and their geographical source. all strains detected in the present study indicated a close relationship among one another as well as with strains from taiwan (strain dogtaiwan) and japan (strain hamamastu). additionally, the strain cdwuhan- was more distant from all other wild-type cdv strains in china, and it may eventually constitute a different genotype close to the cdv isolates in japan (strain yanaka). the primary objective of the present work was to establish an easy and quick rt-pcr method for the detection and differentiation of cdv wild-type strains and vaccines. presently, there are several methods for cdv testing, such as virus isolation, antigen-enzymelinked immunosorbent assay, and inmunofluorescence. however, these methods are laborious and time consuming. recent studies have revealed that the virus isolation of cdv using vero.dogslam cells can be quick and easy (seki et al., ; woma and van vuuren, ; woma et al., ) . nevertheless, these tests cannot differentiate between cdvs of wild-type and vaccine strains. the cdv combined rt-pcr assay consisted of two steps of rt-pcrs. one was the traditional cdv rt-pcr assay, which detected cdv wild-type and vaccine strains. the other was the specific cdv rt-pcr assay, which only detected cdv wild-type strains. this technique, also called allele-specific oligonucleotide pcr, has originally been designed for the detection of known sequence nt polymorphisms (chulakasian et al., ) . amplification discrimination mainly depended on the mismatched nt at the -terminus of the primers. the pcr process only allowed amplification to occur when the most -terminal nt matched its target sequences. comparing the genome sequences of the wild-type cdv and vaccine strains in genbank, amino acid sequence differences were identified. the cdv wild-type strain amino acid sequence was val and lys at positions and in h protein ( and nts), respectively. on the other hand, the cdv vaccine strain contained ile and arg at the same locations. based on the molecular sequence investigation of the wild-type local cdv strains, the h gene sequence alignment showed that the identity of the nucleic acid between local and vaccine strains ranged from . % to . %. the ca and g nts were also highly conserved at positions - and of the h gene in the vaccine strains. ag and a nts were at the same locations in the local wild-type strains (marked with square in fig. ). the cdv field strain cdwuhan- was not mismatched with the -terminus of the p primer at the h gene position, whereas it had the same nt g at position in the h gene as the cdv vaccine strains. however, the reaction still continued to produce the correct fragment product. therefore, the primer p may be more functional than p . additionally, changes in ag/ca and a/g were also observed in other asia- cdvs, asia- cdvs, america- cdvs, europe, and europe wildlife cdvs. in contrast, a change in gg/ca was found in africa cdvs (fig. ) . given the developments on cdv molecular research, an increasing number of cdv sequences are being deposited in genbank. hence, cdv molecular diagnosis methods, such as rt-pcr and realtime rt-pcr, have been significantly developed (martella et al., ; scagliarini et al., ) . recently, several cdv differential table comparison of the nucleotide sequences of field isolates from china with vaccine-like reference strains and commercial cdv vaccine using the cdv hemagglutinin gene (nucleotides - diagnoses between wild-type isolates and vaccines have been established. for example, an rt-pcr lineage-genotyping system based on specific nt polymorphisms scattered over the h gene has been developed. this system is used to characterize major cdv lineages, such as european, asia- , asia- , arctic, and vaccine strains (martella et al., ) . in another multiplex pcr assay, primers target the h gene to distinguish field strains from china and vaccine-like strains, such as onderstepoort (si et al., ) . a multiplex amplification refractory mutation system pcr is also used to differentiate seven cdv field isolates in taiwan (asia- lineage) from vaccine strains based on the untranslated region region between the m and f genes (chulakasian et al., ) . compared with these cdv differentiation assays, the presently proposed method may be conducted more easily and quickly. it making up by two separate steps rt-pcr assays to reduce contamination percentage and time-less in one pcr procedure but not multiplex or nested rt-pcr. cdv field isolates are also more easily distinguished from cdv commercial vaccines used in china. unfortunately, the present method cannot detect mixed populations of cdv wild-type and vaccine strains, although these are rarely seen in animals. however, the method is still a suitable and easy diagnostic tool for detecting cdvs in animals with suspected cdv disease. in the current study, phylogenetic analysis indicated the close relationship of cdv field strains with one another according to geographical source. the results confirmed and extended those previously reported in other global locations bolt et al., ; haas et al., ; iwatsuki et al., ; pardo et al., ; woma et al., ) . all the cdv field strains analyzed in the present study could be grouped into a phylogenetic cluster clearly separate from the cdv vaccine strains, and they were characterized as having asia- genotypes. this genotype appears to be predominant throughout china, and is infecting domestic dogs as well as raccoon dog species. asia- cdv strains have also been detected in japan (mochizuki et al., ) , taiwan (chan et al., ; lee et al., ) , and korea . in summary, the combined rt-pcr test developed in the current study is a suitable diagnostic tool for detecting and differentiating wild-type and vaccine strains of cdv. fig. . sequence alignment of partial hemagglutinin gene. the partial h gene nucleotide sequences of field strains from china and commercial cdv vaccine were analyzed. the numbering starts at the position nucleotide and of the h gene. only amino acids that differ from the majority sequence are shown. identical residues are represented by dots. the substitution of the ag and a present in the field strains, which was used to design the differentiating primers for rt-pcr, is indicated by a square box. phylogenetic characterization of canine distemper virus isolates from naturally infected dogs and a marten in korea pathogenesis and immunopathology of systemic and nervous canine distemper genetic diversity of the attachment (h) protein gene of current field isolates of canine distemper virus detection by rt-pcr and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in argentina identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus biological characteristics identification of attenuated virus strain cdv of canine distemper virus multiplex amplification refractory mutation system polymerase chain reaction (arms-pcr) for diagnosis of natural infection with canine distemper virus genetic diversity of hungarian canine distemper virus strains outbreak off canine distemper in vaccinated dogs in finland analysis of the haemagglutinin gene of current wild-type canine distemper virus isolates from germany molecular and phylogenetic analyses of the haemagglutinin (h) proteins of field isolates of canine distemper virus from naturally infected dogs the identification of frequent variations in the fusion protein of canine distemper virus genotyping canine distemper virus (cdv) by a hemi-nested multiplex pcr provides a rapid approach for investigation of cdv outbreaks genotypes of canine distemper virus determined by analysis of the hemagglutinin genes of recent isolates from dogs in japan phylogenetic characterization of canine distemper viruses detected in naturally infected dogs in north america outbreak of canine distemper in domestic ferrets (mustela putorius furo) taqman based real time pcr for the quantification of canine distemper virus a multiplex reverse transcriptionnested polymerase chain reaction for detection and differentiation of wild-type and vaccine strains of canine distemper virus efficient isolation of wild strains of canine distemper virus in vero cells expressing canine slam (cd ) and their adaptability to marmoset b a cells mega : molecular evolutionary genetics analysis (mega) software version . establishment and application of rt-pcr for detection of canine distemper virus isolation of canine distemper viruses from domestic dogs in south africa using vero.dogslam cells and its application to diagnosis phylogenetic analysis of the haemagglutinin gene of current wild-type canine distemper viruses from south africa: lineage africa phylogenetic analysis of the haemagglutinin gene of canine distemper virus strains detected from breeding foxes, raccoon dogs and minks in china the present study was partly financially supported by the ji lin province technology and development program ( and ); the ji lin province major scientific and technological achievements transformation program (no. zdzh ) and the ministry of agriculture scientific and technological achievements transformation program (no. gb b ). the authors gratefully acknowledge zhi-gang li and xin-bo guo for their help in revising the manuscript. key: cord- - sqovwb authors: li, hao; li, kai; bi, zhen; gu, jun; song, deping; lei, dan; luo, suoxian; huang, dongyan; wu, qiong; ding, zhen; wang, leyi; ye, yu; tang, yuxin title: development of a reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay for the detection of porcine pegivirus date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: sqovwb a simple and accurate reverse transcription-loop-mediated isothermal amplification (rt-lamp) assay was developed and evaluated for the detection of porcine pegivirus (ppgv). the specific rt-lamp primers targeting the conserved regions of ns a genes were designed and used to detect ppgv. the optimal reaction parameter for rt-lamp assay was ℃ for min. the detection limit of the rt-lamp assay was copies of ppgv genome, which was times more sensitive than that of the conventional rt-pcr and comparable to nested rt-pcr and quantitative rt-pcr (qrt-pcr). there was no cross amplification with other related rna viruses. in the clinical evaluation, the rt-lamp assay exhibited a similar sensitivity with nested rt-pcr and qrt-pcr. the results indicated that rt-lamp assay developed in this study could be a highly specific, sensitive, and cost-effective alternative for a rapid detection of ppgv in field settings. porcine pegiviruses (ppgv) are small enveloped viruses with a single-stranded, positive-sense rna genome that have recently been assigned as the pegivirus genus in the flaviviridae family (berg et al., ) . ppgv genome contains a single large open reading frame (orf) encoding a polyprotein, which is cleaved into individual proteins including e , e , x, ns , ns , ns a, ns b, ns a, and ns b. pegiviruses are detected in diverse mammalian hosts including pigs, humans, horses, chimpanzees, bats, monkeys, and rodents (yang et al., ) . all known pegiviruses have been classified into species (a-k), and ppgv is a member of pegivirus k. ppgv was first discovered in germany in (baechlein et al., ) , and then reported in the united states in (yang et al., ) . in china, ppgv was first identified in guangdong province (lei et al., ) , where vesicular diseases broke out in a swine farm in . the serum samples were submitted to the veterinary diagnostic laboratory of jiangxi agriculture university, china for diagnosis. the tested results indicated that foot-and-mouth disease virus (fmdv) and seneca valley virus (svv) were negative but positive for ppgv by a rt-pcr assay. although it remained yet unknown about the association between ppgv infection and the disease, more epidemiological studies are needed to address the clinical effect of ppgv on swine health. currently, there are some methods available for ppgv diagnosis, including conventional rt-pcr, nested rt-pcr, and semi-quantitative rt-pcr (yang et al., ; lei et al., ; chen et al., ) . therefore, a rapid and accurate method to detect ppgv is urgently needed for monitoring its presence. in the past decade, loop-mediated isothermal amplification (lamp) has become an effective technique, which exhibits high sensitivity and specificity for diagnosing important pathogens in medicine and veterinary medicine (notomi et al., ) . lamp is a nucleic acid amplification-based method that generally requires a group of four specific external and internal primers (notomi et al., ) . besides, additional loop primers can be used to further accelerate the reaction (nagamine et al., ) . in this study, a reverse transcription lamp (rt-lamp) assay was established and evaluated for surveying the prevalence of ppgv in porcine serum samples. a set of three specific primers pairs was designed to amplify the ns a gene, which is suitable for rt-lamp and relatively conserved in ppgv genome. the developed rt-lamp assay showed high sensitivity and specificity for detection of the ppgv. the results tested by rt-lamp assay on clinical samples were % correlated to that of nested rt-pcr and qrt-pcr, indicating the assay could be used for the surveillances of ppgv infection. in this study, serum samples (n = ) of -week-old pigs with a vesicular disease from guangdong province, china in were collected. extraction kit (takara, china), and then stored at - ℃ until use. the extracted rna was converted to the first strand of cdna using primescript rt reagent kit (takara, china) following the manufacturer's instructions. for the construction of plasmid standards, an -bp fragment of the ns a gene of ppgv was initially amplified using a pair of primers (table ) with the st strand of cdna as template. afterwards, the purified fragment was cloned into the pmd -t vector (takara, china) according to the manufacturer's protocol. to assess the successful construction of the plasmid, designated as pmd-ppgv, dna sequencing was performed by sangon biotech company (shanghai, china). the concentration of pmd-ppgv plasmid was measured by a nanodrop spectrophotometer (thermo fisher scientific, usa). the copy number of the cloned gene was quantified as follows: [copy/ μl = plasmid concentration (g/μl) / [(plasmid length) × ) × ( . × )] (park et al., ) . serial dilutions of plasmid standards ( × - × copies) were used as templates for the determination of detection limit of pcr, which would be used as a positive control in the rt-lamp assay system. the three pairs of rt-lamp primers targeting the conserved regions of ns a genes were designed based on the sequences of the reference strain (ppgv_gd/ch/ , genbank: mg . ; ppgv_s / /ger/ , genbank: ku . ; ppgv_ /ger/ , genbank: nc_ . ; ppgv_ f/ger/ , genbank: ku . ; ppgv_ / nd/ , genbank: mg ; ppgv_ /mo/ , genbank: mg ; ppgv_ /ia/ , genbank: mg ) using the online software primer explorer v (http://primerexplorer.jp/e/), which included an outer pair (f and b ), an inner pair (fip and bip), and a loop pair (lf and lb). the inner primers fip and bip contained f and b sequences with the complementary sequences of f and b (f c and b c) in their ′-terminal, respectively (fig. ) . a pair of primers (f and r ) was used for amplifying the ns a gene in conventional rt-pcr and meanwhile served as outer primers for the nested rt-pcr (table ). the primer pair of f and b was used as inner primers for nested pcr. a pair of primers (f and r ) was used for qrt-pcr targeting the ns a gene which was designed using the online software primer (http://bioinfo.ut.ee/primer - . . /). the specificity of the primers was then examined by a basic local alignment search tool (blast) search (http://www.ncbi.nlm.nih.gov/blast) against the ncbi database. the results demonstrated that there was no hits, that is, no nonspecific primer binding site was observed. the final volume of μl reaction mixtures for rt-lamp was prepared, which contained μl of bst dna polymerase (neb, usa) ( u/ml), . μl of × isothermal amplification buffer, μl of betaine ( m), μl of mgso ( mm), μl of dntp ( . mm), μl of each inner primers fip and bip ( μmol), . μl of each outer primers f and b ( μmol), . μl of each loop primers lf and lb ( μmol), . μl of amv reverse transcriptase (takara, china) ( u/μl), μl of rna template, and the sterile distilled water was set as a negative control template. to determine the optimal reaction conditions, the rt-lamp reactions were incubated at different temperatures of , , , , , and ℃ for , , , , , and min, respectively. finally, the reaction was terminated by heat inactivation at ℃ for min. the amplified dna products from the rt-lamp were analyzed by . % agarose gel electrophoresis with the addition of . % ethidium bromide using a gel electrophoresis system (liuyi, china). the results were also observed directly by color change from orange to green with staining due to the presence of sybr green Ⅰ (thermo scientific, usa). after the amplification was completed, μl of coloring agent was added to each reaction tube, and the results were then examined visually. in rt-lamp assay with ppgv rna as template, the amplified dna products showed characteristic ladders of multiple bands on an agarose gel, indicating that the final products were a mixture of stem-loop dna with different stem lengths (khan et al., ) . the results of the rt-lamp reaction were also determined by direct visualization of the color change under daylight and uv light as shown in fig. . in contrast, lamp-specific dna bands and color changes were not observed in the negative control. meanwhile, the dna products of rt-lamp showed the highest intensity when the reaction was carried out for min (fig. ) . in regard to temperature, the optimal reaction temperature of rt-lamp was ℃. therefore, the optimized parameter for the established rt-lamp was ℃ for min. to determine the detection limit of the rt-lamp, -fold serially diluted plasmid standards of pmd-ppgv ( × - × copies) were used as templates in μl rt-lamp reaction system under optimized conditions. the sterile distilled water was set as a negative control template. nested rt-pcr was performed based on the protocol established in our laboratory (lei et al., ) . for the pcr step of the nested rt-pcr, the primer sets of f /r and f /b respectively served as outer ( st set) and inner primers ( nd set) ( table ). the reaction system contained μl of plasmid standards of pmd-ppgv ( × - × copies) or μl of the first pcr product, . μl of × pcr buffer (ta-kara, china), . μl of rtaq ( u/μl), μl of mgcl ( mm), μl of dntp ( . mm), and μl of each primer ( μmol). two rounds of nested pcrs were performed under the following conditions: ℃ for min; cycles at ℃ for s, ℃ for min, and ℃ for min; and a final extension of ℃ for min (wang et al., ) . the conventional rt-pcr was carried out under the same reaction condition as above in nested rt-pcr using the primer pair of f and r based on the procedure described previously (lei et al., ) . the products of nested rt-pcr and conventional rt-pcr were analyzed by . % agarose gel electrophoresis with . % ethidium bromide. for qrt-pcr, the primer set of f and r , targeting conserved region of the ns a gene of ppgv, was used ( table ). the qrt-pcr assay was performed by using a commercial qpcr kit (takara, china) based on the manufacturer's procedure. each μl reaction mixture consists of μl of tb green fast qpcr mix, μl of plasmid standards of pmd-ppgv ( × - × copies), . μl of ( μmol) each forward and reverse primers and . μl of rox. the amplification parameters included an initial denaturation at ℃ for min followed by cycles of ℃ for s and ℃ for min. the melting curve analysis was measured using the software supplied with abi fast real-time pcr system (applied biosystems, usa). the results demonstrated that the detection limit of the developed rt-lamp was copies/μl, which was much higher than conventional rt-pcr ( copies/μl), while it was comparable to that of the nested rt-pcr ( copies/μl) and qrt-pcr ( copies/μl), respectively (fig. ) . to assess the specificity of rt-lamp for ppgv, cdnas of seneca valley virus (svv), food and mouth disease virus (fmdv), porcine epidemic diarrhea virus (pedv), porcine deltacoronavirus (pdcov), and swine acute diarrhea syndrome coronavirus (sads-cov) were used, and the plasmid standards of pmd-ppgv was used as the positive control. the amplified products were analyzed by . % agarose gel electrophoresis with . % ethidium bromide and addition of sybr green Ⅰ staining solution. the results indicated that there was no cross amplification of the rt-lamp assay for these control viruses. using ppgv rna as a template, positive dna bands were amplified as expected (fig. ) , indicating that the rt-lamp assay developed was specific for ppgv. to further evaluate the developed rt-lamp assay, serum samples (n = ) from finishing pigs and sows collected in guangdong and jiangxi province in china during october to january were tested (lei et al., ) . total rna was extracted and used as a template for conventional rt-pcr, nested rt-pcr, qrt-pcr, and rt-lamp according to aforementioned methods. as shown in table , the positive sample rates of conventional rt-pcr, nested rt-pcr, qrt-pcr, and rt-lamp were . % ( / ), . % ( / ), . % ( / ), . % ( / ), respectively, suggesting these data were consistent with our previous findings (lei et al., ) . the detailed information is listed in table s . the results showed the rt-lamp had a similar sensitivity to nested rt-pcr and qrt-pcr, which was superior to conventional rt-pcr. pegiviruses have a broad host range and infect diverse animal species. ppgv is recently proposed as a novel species in the pegivirus genus. ppgv epidemics have been confirmed in germany in , the united states in , and china in . although several pigs positive for ppgv exhibited vesicle and lameness (yang et al., ) , there is limited evidence that infections were associated with a clinical disease. therefore, the clinical significance and epidemiological investigation of ppgv infection should be further studied. nevertheless, the establishment of a sensitive, cost-effective, and rapid assay for detecting ppgv is urgently needed. in this study, a rt-lamp assay was developed to monitor ppgv in pig populations. compared to other diagnostic methods, such as conventional rt-pcr, nested rt-pcr, and qrt-pcr (baechlein et al., ) , rt-lamp assay may eliminate a need for complex procedures and expensive instruments, which makes it suitable for poorly equipped laboratories and clinics (lopez-jimena et al., ) . rt-lamp is considered to be an alternative diagnostic tool in terms of its ability to rapidly amplify target nucleotide sequence(s) under isothermal conditions. lamp has been widely used to detect a variety of important swine pathogens, including pdcov (zhang et al., ) and pedv (mai et al., ) . in the present study, the rt-lamp reaction conditions were optimized in terms of reaction time and temperature. the detection limit of rt-lamp assay established is times higher than that of conventional rt-pcr, and had a similar sensitivity to nested rt-pcr and qrt-pcr. rt-lamp developed in the study showed a high specificity as it did not cross react with other related porcine viruses. in the clinical evaluation, the positive rate of rt-lamp were consistent with that of nested rt-pcr and qrt-pcr, indicating the ppgv-specific rt-lamp assay was a valuable tool for the rapid detection in field settings. in summary, we developed a rapid, simple, accurate, and cost-effective rt-lamp assay for detection of ppgv, and the assay could be used as an alternative for the clinical diagnosis of ppgv infection and epidemiological investigation. none. pegivirus infection in domestic pigs discovery of a novel human pegivirus in blood associated with hepatitis c virus co-infection semiquantitative duplex rt-pcr reveals the low occurrence of porcine pegivirus and atypical porcine pestivirus in diagnostic samples from the united states diagnostic accuracy of loop-mediated isothermal amplification (lamp) for detection of leishmania dna in buffy coat from visceral leishmaniasis patients detection and genetic characterization of porcine pegivirus from pigs in china development of a single-tube one-step rt-lamp assay to detect the chikungunya virus genome development of pooled testing system for porcine epidemic diarrhoea using real-time fluorescent reverse-transcription loop-mediated isothermal amplification assay loop-mediated isothermal amplification of dna loop-mediated isothermal amplification (lamp): principle, features, and future prospects loop-mediated isothermal amplification assay for the rapid and visual detection of novel porcine circovirus integrating nested pcr with high-throughput sequencing to characterize mutations of hbv genome in low viral load samples detection and genetic characterization of porcine pegivirus in pigs in the united states a simple and rapid identification method for newly emerged porcine deltacoronavirus with loop-mediated isothermal amplification rapid specific and visible detection of porcine circovirus type using loop-mediated isothermal amplification (lamp) this work was supported by the national key research and development program (grant number yfd ), the natural science foundation of jiangxi province (grant number acb and bab ), the graduate research & innovation projects in jiangxi (grant number yc -b ), and science and technology project of education department of jiangxi province (grant number gjj and gjj ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/ . /j.jviromet. . . . key: cord- -zz mt be authors: hakhverdyan, mikhayil; hägglund, sara; larsen, lars-erik; belák, sándor title: evaluation of a single-tube fluorogenic rt-pcr assay for detection of bovine respiratory syncytial virus in clinical samples date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zz mt be bovine respiratory syncytial virus (brsv) causes severe disease in naïve cattle of all ages and is a common pathogen in the respiratory disease complex of calves. simplified methods for rapid brsv diagnosis would encourage sampling during outbreaks and would consequently lead to an extended understanding of the virus. in this study, a brsv fluorogenic reverse transcription pcr (frt-pcr) assay, based on taqman principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental brsv infections. by using a single-step closed-tube format, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. according to comparative analyses, the detection limit of the frt-pcr was on the same level as that of a nested pcr and the sensitivity relatively higher than that of a conventional pcr, antigen elisa (ag-elisa) and virus isolation (vi). interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. taken together, the data indicated that the frt-pcr assay can be applied to routine virus detection in clinical specimens and provides a rapid and valuable tool in brsv research. bovine respiratory syncytial virus (brsv), a pneumovirus belonging to the paramyxoviridae family, is a respiratory pathogen of cattle that causes economical losses to beef and dairy production. the infection consists as a part of a disease complex in calves (stott et al., ) , but it also occurs as outbreaks in naïve populations and may be fatal in adults (elvander, ; inaba et al., ) . brsv has enveloped virions; it possesses a negative sense, single-stranded rna genome and exists as a single serotype. genomic phylo-genetic studies have divided isolates into six subgroups with the highest average percentage of sequence divergence in the glycoprotein (g) coding region of the genome (valarcher et al., ) . the fusion (f) protein coding region is less variable and is therefore a more suitable target for the design of diagnostic tests (eleraky et al., ) . due to problems of detection by classical methods, the importance of brsv infections in cattle may be underestimated. the period of virus shedding from the point when clinical signs appear is short in infected animals; the virus is labile and therefore difficult to isolate in cell culture (valarcher et al., ; west et al., ) . brsv-specific antibodies in clinical specimens may interfere with virus isolation (vi) and also with direct detection methods based on serological techniques (west et al., ) . furthermore, the usage of indirect detection by serology (brsv-specific igg detection) is hampered by the suppressive effect of maternal antibodies on the humoral response to infection (kimman et al., ) . the introduction of the sensitive conventional pcr assays for virus diagnosis has lead to a significant improvement in the detection of brsv (larsen et al., ; oberst et al., ; valarcher et al., ; vilcek et al., ; west et al., ) . these methods, however, require complex laboratory manipulations and are therefore time-consuming and expensive (belák and thorén, ) . the purpose of the present study was to develop a singlestep, closed-tube format fluorogenic reverse transcription pcr (frt-pcr) assay for the detection of brsv in clinical samples from cattle. the assay had to meet requirements such as high sensitivity, specificity, short detection time and relatively low costs. before application of the frt-pcr assay, lung samples from cattle, submitted for examination to the danish institute for food and veterinary research, were tested for brsv, bovine coronavirus (bcov) and bovine parainfluenza- (piv- ) virus by antigen elisas (ag-elisas) . thirty-four lung samples from calves included in a brsv challenge experiment (tjornehoj et al., ) were tested for brsv by conventional pcr (larsen et al., ) and/or ag-elisa. nasal swabs from animals included in a second brsv challenge experiment were also tested by virus isolation (hägglund et al., in press) (n = ) or nested pcr (vilcek et al., ) (n = ) . all samples were analysed in a blind manner. clinical samples were obtained from outbreaks of respiratory disease in three swedish dairy herds (a, b and c). a majority of the animals in these herds showed clinical signs of respiratory disease, including cough and decreased appetite. in herd a, sera and nasal swabs were obtained from three cows with rectal temperature exceeding • c. in herd b, similar samples were obtained from a calf with cough and normal rectal temperature as well as from a cow with elevated respiratory rate ( min − ), abnormal sounds on lung auscultation, cough, depression and rectal temperature of . • c. a swab was also obtained from apical parts of the right lung of a cow that was found dead the previous day. in herd c, nasal swabs were obtained from seven animals with clinical signs ranging between serous nasal discharge alone to cough, rectal temperature up to . • c, abnormal sounds on lung auscultation and depression. one cow with abdominal, open mouth breathing, subcutaneous emphysema and rectal temperature of . • c was sampled twice days apart (nasal swabs and sera). rna was extracted from l of template (cell culture supernatant, nasal swab transport medium or approximately mg lung tissue homogenated in l pbs) using tri reagent ls (sigma, st. louis, mo, usa) according to the manufacturer's instructions. rna was recovered in l of sterile, nuclease-free dmpc (dimethylpyrocarbonate) water and either used immediately or stored at − • c until further analysis. the oligonucleotide primers and probe against brsv were designed from the published sequence of the f gene of isolate (genbank accession no. af ), collected from genbank using ncbi blast. alignments of different brsv isolates/strains were performed using lasergene software (dnastar inc., version , madison, wi, usa). primers and probes for the frt-pcr were selected using primer express tm software (version . , applied biosystems, foster city, ca, usa). compatible primer sets were selected to ensure efficient amplification and detection of brsv isolates from all subgroups (valarcher et al., ) (for mismatches, see table ). the sequences of the primers were ( - ): brsv-f- f, aagggtcaaacatctgct-taactag ( bp, forward), brsv-f- r, tctgcct-gwgggaaaaaag ( bp, reverse). the primers were ordered and synthesized at cybergene ab (huddinge, sweden). the taqman ® probe, termed brsv-f-taqman- , had the following sequence ( - ): agagcctgcat-trtcacaataccaccca ( bp, complementary). the probe was labelled with -carboxyfluorescein (fam) at the end and with nonfluorescent black hole quencher dye at the end (bhq, biosearch technologies, novato, ca, usa). the reverse primer and the probe were designed so that they could tolerate one mismatch. the predicted product size of the brsv amplicon was bp. the frt-pcr was carried out on an abi prism sequence detection system (applied biosystems, foster city, ca, usa) and optimised by titration series of primers, probe and mn + . the assay was carried out in a total volume of l using a -well optical reaction plate or microamp ® optical tubes (applied biosystems). each l reaction mixture contained l of extracted rna in dmpc water, . m of each brsv primer and probe, . mm x dntps, . mm mn(aoc) , . u/l of rtth dna polymerase (applied biosystems), × ez buffer and mg/ml of bsa. the final volume was adjusted with dmpc water. the thermodynamic profile used was as follows: • c for min (data collected) and • c for min (rt step), and then • c for s (initial denaturation) followed by cycles of amplifi- table nucleotide mismatches at primer and probe sites of brsv, one orsv and one hrsv f-gene sequences (genbank) cation: • c for s, • c for s (data collected), • c for s. a -fold dilution series of a brsv positive rna template (three dilutions, five replicates of each dilution) were used for generation of a standard curve and calculation of the reaction efficiency (e), using formulae e = (− /a) − , where a is the slope value of the standard curve. a cycle threshold (ct) value (the number of cycles for the fluorescence to reach a threshold) of . was chosen as a negative cut-off for the frt-pcr assay. as described previously (reid et al., ) , any sample that did not generate a sigmoid amplification curve was retested. duplicate analyses of lung samples assessed the intra-assay reproducibility. the detection limit of the frt-pcr was determined by testing nine steps of a -fold dilution series of a brsv strain (no. ) (viuff et al., ) with a concentration of . tcid /ml. total rna was extracted from each dilution and was analysed by frt-pcr (two replicates of each dilution, from to ) and in parallel by nested pcr (vilcek et al., ) . the experiment was repeated with both assays. the analytical specificity of the assay was evaluated by testing nine brsv strains originating from the netherlands ( nl, ), hungary ( / h, ) , sweden ( sw, sw, sw, sw, - and denmark ( dk, ; dk, ; dk, , nasal swabs from healthy calves and virus species, genetically or symptomatically related to brsv. the viruses included bovine coronavirus (n = ), parainfluenza- virus (n = ), bovine vi-ral diarrhoea virus, type (bvdv- ; n = ), border disease virus (bdv; n = ), bovine herpesvirus, type (bhv; n = ), bovine reovirus, types and (n = ), bovine adenovirus, types and (bav; n = ) and human rsv, types a and b (hrsv; n = ). as calculated from the slope of the standard curve (a = − . ; fig. ) and the formulae described above, the efficiency of frt-pcr was . or %. this means that, on average, the number of dna copies increased by % in every cycle. during control visualization of frt-pcr products from six positive and one negative sample in agarose gel, the size of the bands from all positive samples agreed to the predicted size of bp and the negative sample did not yield a visible band. moreover, results of the samples that were tested in duplicate were in complete accordance. obtained ct values for positive samples (n = ) differed with . - . cycles (mean variation . ). when testing dilution series of a brsv strain ( . tcid /ml, dilutions - ), amplification curves with increasing ct-values were achieved for each dilution from to (fig. ) . the obtained detection limit was accordingly . tcid /ml. results of the same rna templates tested by brsv nested pcr were identical with those obtained by frt-pcr (fig. ) . the experiment was repeated and results were the same (data not shown). nine steps of a -fold dilution series of a brsv strain, with an initial titre of . tcid /ml, were tested (dilutions - , two replicates of each dilution). the detection limit is times diluted sample. the results of the frt-pcr, ag-elisa, conventional pcr, nested pcr and vi were in accord with of analyses on samples. the results for the rest analyses were as follows: the frt-pcr detected more positive samples than ag-elisa, more than conventional pcr and more than vi. only nested pcr detected more positive sample than frt-pcr ( table ). the relative diagnostic sensitivity and specificity percentages are shown in table . investigations on the analytical specificity comprised two tests. in the first test, nine brsv strains (see above), human rsv (types a and b), as well as positive and negative controls, were included. all brsv strains and the positive fig. . brsv nested pcr results from analyses on the same rna templates as described in fig. . the products of pcr (a) and pcr (b) are shown by gel electrophoresis. predicted product size is bp for pcr and bp for pcr . the detection limit is times diluted sample (number on gel b). numbers - correspond to the dilutions from to ; number is negative control. controls were amplified successfully, but not human rsv or the negative controls (data not shown). in the second test, symptomatically or genetically related virus species (see above), as well as three different brsv positive and two negative controls, were used. only the positive controls yielded positive results (fig. ) . all nasal swabs from dairy cows with acute respiratory infections in herd a were positive for brsv by the frt-pcr assay and identical results were obtained by the nested pcr. in herd b, nasal swabs from both a calf with mild respiratory signs and a cow with pneumonia were tested positive by frt-pcr. in addition, brsv was detected in a swab from the lung of a dead animal. in herd c, nasal swabs from four animals out of seven were tested positive by frt-pcr. the positive samples were derived from two young animals with mild cough and serous nasal discharge, and from two dairy cows with severe pneumonia. three samples, which were negative in frt-pcr, were obtained from cows with serous nasal discharge as the only observed clinical sign of disease. one of these three samples was positive in nested pcr. nasal swabs from a cow with severe pneumonia, obtained days apart, were positive in both pcr assays. acute sera from animals in all three herds were negative for brsv-specific igg antibodies and a seroconversion to brsv was detected in paired sera in all cases. the conventional laboratory diagnosis of brsv is based on virus isolation, antigen/antibody elisa, immunohistochemistry and pcr. at present, the most sensitive diagnostic tool for direct diagnosis is the nested pcr because it includes two rounds of amplification with two discrete primer sets, which increase drastically the limit of detection (belák and thorén, ) . although the nested pcr is undoubtedly effective, the practical application is rather complicated. it takes five laborious steps to achieve the final results: reverse transcription, two rounds of amplification, gel electrophoresis and photography. the second round of amplification not only delays the results, but also increases the risks of crosscontamination. moreover, gel electrophoresis and photography include handling of ethidium bromide, which with its carcinogenic effects hazards the health of laboratory personnel. considering the needs for a sensitive and specific, as well as safer and more rapid method for the diagnosis of brsv infections, we have developed a single-step, closed-tube frt-pcr assay for the detection of this virus in clinical samples. to our knowledge, this is the first report on the development and application of an frt-pcr system for the direct diagnosis of respiratory diseases caused by brsv. although werling et al. ( ) published recently a brsv taqman pcr assay, their method was developed for research purposes, in order to measure viral mrna levels in the context of cytokine responses. our intention was to develop a simple and high throughput frt-pcr assay and to investigate its diagnostic applicability by testing clinical samples from experimentally and naturally infected animals. compared to nested pcr, the single-tube frt-pcr assay has important advantages. by including rtth dna polymerase, which acts both as a thermoactive reverse transcriptase and a thermostable dna polymerase, a separate rt step was avoided and the reactions require less than h to complete (from rt reaction to results), compared to - h for rt and nested pcr. unlike other protocols, neither the addition of a second enzyme nor an alteration in buffer composition is required between the reverse transcription and the pcr. the frt-pcr allows a considerably increased sample throughput, which is a very important requirement in diagnostic laboratories. during simultaneous examination of up to samples, the results of duplicate tests were in complete agreement. negative controls included in each run gave no amplification signals, suggesting that cross-contamination was avoided. with future development of standardized protocols for different respiratory pathogens, the sample capacity of this assay will allow specimens to be screened for several targets in a single run. as another important requirement for a diagnostic assay, the brsv frt-pcr has provided a high analytical specificity. the assay gave negative results when testing samples obtained from healthy animals or heterologous viruses related to brsv symptomatically or genetically. the lack of amplification with human rsv types a and b, together with positive results of all brsv strains, showed that the assay was highly specific. according to the current data, the detection limit of the frt-pcr was on the same level as that of a nested pcr. only one lung sample amongst tested was positive in nested pcr and negative in frt-pcr. in the rest cases, pcr results were in good agreement, whereas the frt-pcr showed higher relative sensitivity compared to that of ag-elisa, conventional pcr and virus isolation on a limited number of samples. the positive frt-pcr results on lung and nasal secretions from experimental animals, with negative results in other assays (table ) , were probably true positive, since all these animals had been inoculated with brsv. divergent results between frt-pcr and vi might be explained by interfering brsv-specific antibodies in clinical specimens. antibodies may block virus and impede isolation attempts (west et al., ) . indeed, the animal from which samples with divergent results were obtained possessed high brsv-specific antibody titres in nasal secretions (hägglund et al., in press ). these samples also generated the highest ctvalues of all positive samples detected by frt-pcr (data not shown), suggesting that antibodies interfered with vi when viral concentrations in samples were low. a benefit of fluorogenic pcr systems for virus research is the possibility for quantification of the nucleic acid molecules in samples. in this study, the increasing ct-values, in agreement with the brsv dilutions, implied that a relative quan-tification was functioning already in the developed brsv frt-pcr system. for a numeric quantitation of viral copies, however, simultaneous detection of a house-keeping gene in rna extractions would be required (gueudin et al., ) . the practical use of the frt-pcr was tested on a carefully selected collection of clinical samples, representing various cases of respiratory infections in cattle. in the field data presented above, animals with acute respiratory signs including high fever or dramatically elevated respiratory rates were tested positive, inferring that this type of clinical cases represent a suitable target group for sampling. it should be noted that calves, but not adults, shed detectable levels of brsv even with mild clinical signs of disease. brsv was detected in samples obtained days apart from one animal, indicating that the period of possible detection by frt-pcr is reasonably long during natural brsv infections in naïve cattle. nevertheless, to be assured of a reliable herd diagnosis, nasal swabs should be obtained from more than one animal during an outbreak. the results suggest that the frt-pcr assay described above allows a rapid, sensitive and specific detection of brsv from clinical samples. the detection of targets without opening the reaction vessel and combination of reverse transcription with pcr in a single closed-tube format greatly reduces both the risk for cross-contamination of specimens and the detection time. the sensitivity of the frt-pcr assay was comparable to that of nested pcr and seemed higher than that of vi or ag-elisa. taking in account that nested pcr is laborious and demands several practical steps to reach the final results, frt-pcr with its single-step performance is a more suitable tool for detection of brsv. another advantage is that multiple results can be obtained with minimal effort. in summary, the results described above indicate that the method is sensitive, simple and can be applied to routine diagnosis. molecular diagnosis of animal diseases: some experiences over the past decade comparison of targeting f and g protein genes to detect bovine and ovine respiratory syncytial viruses severe respiratory disease in dairy cows caused by infection with bovine respiratory syncytial virus quantitation of respiratory syncytial virus rna in nasal aspirates of children by real-time rt-pcr assay bovine respiratory syncytial virus iscoms-protection in the presence of maternal antibodies. vaccine bovine respiratory syncytial virus local and systemic antibody response to bovine respiratory syncytial virus infection and reinfection in calves with and without maternal antibodies diagnosis of enzootic pneumonia in danish cattle: reverse transcription-polymerase chain reaction assay for detection of bovine respiratory syncytial virus in naturally and experimentally infected cattle identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations detection of all seven serotypes of foot-andmouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay a survey of virus infections of the respiratory tract of cattle and their association with disease an experimental infection model for reproduction of calf pneumonia with bovine respiratory syncytial virus (brsv) based on one combined exposure of calves viral aetiology of enzootic pneumonia in danish dairy herds: diagnostic tools and epidemiology evaluation of a nested reverse transcription-pcr assay based on the nucleoprotein gene for diagnosis of spontaneous and experimental bovine respiratory syncytial virus infections evolution of bovine respiratory syncytial virus development of nested pcr assays for detection of bovine respiratory syncytial virus in clinical samples sites of replication of bovine respiratory syncytial virus in naturally infected calves as determined by in situ hybridization cytokine responses of bovine dendritic cells and t cells following exposure to live or inactivated bovine respiratory syncytial virus a comparison of diagnostic methods for the detection of bovine respiratory syncytial virus in experimental clinical specimens key: cord- -nvrl vyi authors: song, zhenhui; dai, xianjin; ye, cuifang; li, yuntian; wang, li; hu, yang title: morphogenesis and proliferative rule of porcine transmissible gastroenteritis virus in porcine intestinal epithelial cells date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: nvrl vyi to gain a better understanding of the replication, proliferation and infection characteristics of porcine transmissible gastroenteritis virus (tgev) in porcine intestinal epithelial cells (iecs), this study established a cell model of iecs infected with the chongqing (cq) strain of tgev. the morphogenesis and proliferative rule of tgev in porcine iecs were investigated using transmission electron microscopy, indirect immunofluorescence assays and real-time fluorescence quantitative pcr. observations under the tem indicated that the enveloped viral particles were roughly spherical, with diameters of between and nm. the virions entered porcine iecs by membrane fusion and the mature viruses in the vacuoles were transported to the cell membrane before release. the results also showed that from to h after tgev infection of porcine iecs, the intracellular viral rna content did not change significantly. logarithmic growth occurred from to h, after which it gradually decreased. moreover, the extracellular rna content began to rise at h after inoculation and then reduced gradually at approximately h. this study provided a theoretical foundation for further study on the infection characteristics of tgev in target cells. transmissible gastroenteritis virus (tgev), a member of the coronaviridae, is the etiological agent of transmissible gastroenteritis (tge), which is characterized by severe diarrhea, vomiting and dehydration in approximately -week-old suckling piglets (goodwin and jennings, ; mullan et al., ) . tge results in serious economic losses in swine-producing areas worldwide. tgev has a large, . kb single-stranded positive sense rna genome, which comprises four structural proteins encoded by the spike (s), membrane (m), envelope (e), and nucleoprotein (n) genes (lai and cavanagh, ; eleouet et al., ) . many cell types are susceptible to tgev, including pig thyroid cells, swine testis cells (st), pig kidney cells (pk- , ibrs- ) and salivary gland cells (nguyen et al., ; haelterman and hutchings, ) . ibrs- , pk- and st cell lines are used commonly to culture tgev in the laboratory (chen et al., ) . in vivo, tgev replicates in intestinal epithelial cells of susceptible animals, causing cellu-lar degeneration, villus shortening, malabsorption and diarrhea (haelterman, ; schwegmaan-wessels and herrler, ) . most studies concerning tgev pathogenesis in swine have been performed in animal infection models. only rarely have in vitro studies been used with cell lines originating from porcine intestines. cell lines ipec- , ipec-j and ipi- i have been used in in vitro model systems (fangning et al., ; valentina et al., ; peter et al., ) . however, data concerning these cell lines, and the morphogenesis and proliferation of the virus in the target cells, are scarce. thus, we developed an in vitro model based on porcine intestinal epithelial cells (iec) infected with tgev, and used transmission electron microscopy (tem), indirect immunofluorescence assay (ifa) and real-time fluorescence quantitative pcr (fq-pcr) to investigate the infection mechanism of tgev. tgev n genes are highly homologous and are essential for replication. the n gene encodes the nucleocapsid protein, which is a highly basic protein with a molecular mass ranging from to kda, depending on the species and strain (escors et al., ) . the n protein's function involves cessation of cell division and extension of the cell cycle to provide more suitable conditions for virus assembly (liu et al., motokawa et al., kapke and brian, ) . this is especially significant when the viral life cycle is longer than that of the host cell cycle (wurm et al., ) . therefore, to anahttp://dx.doi.org/ . /j.jviromet. . . - /© elsevier b.v. all rights reserved. lyze the tgev proliferation in target cells, a one-step growth curve was constructed according to the fq-pcr data for the transcription dynamics of the n gene. porcine intestinal epithelial cells (iecs) were developed in our institute, and were isolated from the jejunum of a newborn piglet (unpublished data). tgev chongqing (cq) strain was isolated from sick piglets with symptoms of diarrhea (song et al., ) . an anti-tgev-np mouse monoclonal antibody was produced in our laboratory. anti-rabbit igg/cy , anti-mouse igg/fitc and , -diamidino- -phenylindole (dapi) were purchased from sigma (usa). porcine iecs were grown in dulbecco's modified eagle's medium (dmem)/f- medium (gibco) with % fetal bovine serum (fbs, gibco brl) and maintained in maintenance medium (dmem/f- supplemented with % fbs) in a % co incubator. porcine iecs were seeded on cover slips and grown to % confluence. tgev ( × tcid ) was then inoculated into the cells. after culture for , , , and h, respectively, cells were fixed with % paraformaldehyde for min, the cover slips were washed three times with phosphate buffered saline (pbs), and then permeabilized using . % triton x- for min at room temperature (rt). the primary antibodies, diluted in % bovine serum albumin, were incubated with the coverslips for h at rt in a humid chamber, followed by washing three times with pbs. the secondary anti- bodies were then incubated with coverslips for min at rt. after the last incubation step, the cover slips were washed three times with pbs and twice with distilled water to remove precipitates. the coverslips were embedded in % polyvinyl-alcohol (mowiol) and stored at • c until examination under a fluorescence microscope. to detect whether tgev propagated in porcine iecs cells could infect st cells, st cells were infected by the supernatant of iecs infected with tgev for h. a monoclonal antibody against the tgev nucleoprotein (np), at a : dilution, was used to detect infected cells. the secondary antibody comprised fitc-labeled antimouse igg at a : dilution. an ifa was then performed using the method mentioned above. to demonstrate apoptosis, iecs were infected with tgev for , , , and h, and an apoptosis was detected using an anticaspase- antibody. the rabbit anti-caspase- antibody was used at a : dilution, followed by incubation with a cy -labeled anti rabbit igg as the secondary antibody at a dilution of : . cell nuclei were stained by incubation with dapi for min • c. an ifa was then performed using the method mentioned above. at , , and h post-infection, infected and mock-infected cells were harvested and washed with pbs ( . mol l − , ph . ) twice, and fixed with g l − glutaraldehyde at • c for h. the cells were scraped from the glass surface and centrifuged at × g for min. the supernatant was then discarded, and the cell pellet was resuspended in g l − low melting agarose at • c and cen-trifuged at × g for min. samples were post-fixed in g l − aqueous oso . after stepwise dehydration in acetone, the samples were embedded in epoxy resin and polymerized at • c for days. the embedded cells were cut into -nm slices and stained with uranyl acetate and lead citrate for subsequent examination using tem (hitachi h ; hitachi, tokyo, japan). a pair of primers to amplify the n gene of tgev was designed according to the published sequences obtained from genbank at the national center for biotechnology information (ncbi) website. the primer sequences were: sense primer: -ttcaaccccataaccctccaacaa- , and antisense primer: -ggcccttcaccatgcgatagc- . sangon biotech (shanghai, china) co., ltd synthesized the primers. total rna was isolated from infected porcine iecs using the tri-zol regent (takara biotechnology (dalian) co., ltd.), according to the manufacturer's protocol. the following reaction mixture was used to generate cdnas: mgcl l, × rt buffer l, rnase free dh o . l, dntp mixture l, rnase inhibitor . l, amv reverse transcriptase . l, random -mer . l, antisense primer . l and rna l. the reaction conditions were: • c for min, • c for min, • c for min, • c for min. the pcr amplification reaction comprised forward primer . l, reverse primer . l, ex taq hs . l, × pcr buffer l, cdna template . l and l of distilled water for a total volume of l. the pcr conditions were: • c for min, followed by cycles of dna amplification ( s at • c, s at • c, and min at • c) and a min incubation at • c. the pcr products were cloned into the pmd -t vector (takara biotechnology) and sequenced using the classical sanger dideoxy sequencing method (takara biotechnology). the concentration of the recombinant plasmid was determined using a micro-spectrophotometer. the copy number of the plasmid was calculated and the plasmid was then subjected to a -fold serial dilution (from − to − copies/l) for use as the standard template in real-time pcr method. the standard samples were amplified using real-time pcr using the following reaction mixture: ddh o l, sybr premix ex taq ii l, specific primers . l and dna l. the reaction conditions were: cycles of • c for s, • c for s and • c s. one high-concentration and one low-concentration plasmid template were selected for three repeats. the average value was then calculated, which permitted the construction of the kinetic curve and the standard curve. porcine iecs were infected with tgev cq strain, and the supernatant and cells were collected separately at different time points ( , , , , , , , , and h) . the total rna in the different samples was extracted and used to produce cdna as a template for quantitative pcr. the pcr reaction comprised: ddh o l, sybr premix ex taq ii l, specific upstream primer . l, specific downstream primer . l and cdna template l. the reaction conditions were: cycles of • c for s, • c for s and • c for s. the reaction for each sample was repeated three times and a negative control was included. the copy numbers of the gene were obtained from the test sample and used to construct a one-step growth curve for tgev. an indirect immunofluorescence technique was used to observe the distribution of tgev in porcine iecs at different time points. cell nuclei stained by dapi appeared blue, and the cytoplasm of the control group did not generate specific fluorescence compared with the treated cells (fig. a) . after h, h and h of infection, some green fluorescence was observed (fig. b, c, d) . at h after treatment, a large amount of green fluorescence was noted close to the dapi-stained cell nuclei (fig. e) . at h, the green fluorescence had decreased, although the nuclear fluorescence remained clearly visible (fig. f) . to determine whether st cells could be infected by tgev propagated in iecs, the growth of tgev in st cells infected by the culture supernatant of iecs was determined. the ifa results showed that st cells were susceptible to infection by tgev propagated in iecs (fig. ) . in addition, the apoptotic effect of tgev from iecs was assessed using an anti-caspase- antibody. as time increased, more caspase- -positive cells were observed (fig. ) , indicating that tgev induced apoptosis in iecs. to confirm tgev infection of the target cells, the morphogenesis of the tgev virions was observed under tem. at h after infection, virions with high electron density were observed attached to the membrane surface (fig. b) ; however, very few viruses appeared in the cytoplasm or vacuoles (fig. c) . at h after inoculation, the cytoplasm was filled with vacuoles containing tgev particles (fig. d, e) . fig. f shows that the viral envelope was obtained by budding into vacuoles. virions in vacuoles were transported to the cell membrane (fig. g) , with which they fused and then released into the extracellular space via exocytosis (fig. h) . at the later stage ( h post-infection), the golgi complex became disaggregated (fig. i) . a tgev cq strain was used to infect porcine iecs, and the supernatant and cells were collected at different time points for fq-pcr. the one-step growth curve of tgev was plotted based on amount of rna for the tgev n gene in different samples. the resulting growth curve (fig. ) showed that the amount of intracellular tgev rna did not change substantially during the first h post-infection. from h- h after infection, intracellular viral rna increased logarithmically, after which its proliferation slowed down. from h- h post-infection, the extracellular tgev rna content did not change significantly. thereafter, the viral rna level exhibited logarithmic growth, reaching a maximum at h. tgev can proliferate in many kinds cultured cells, especially in st cells and pk- cells (ren et al., ) . after infection by tgev for h, st cells showed a typical cytopathic effect, characterized by shrinkage and rounding, and a gradual weakening of their adherence ability: ultimately, the majority of the cells were floating in the medium (yin et al., ) . sirinarumitr et al. ( ) found that tgev could induce apoptosis during infection of st cells. nuclear chromatin condensation and fragmentation, as well as cell shrinkage and cytoplasmic vacuolation, were observed in pk- cells infected by tgev. this study showed that tgev's infection of porcine iecs has a partial cytopathic phase, characterized by cells changing to a fusiform shape, pyknosis, refraction and activation of caspase . however, the cytopathic effect in iecs was not as obvious as that in st or pk cells. tgev could induce porcine iecs to undergo apoptosis, which is consistent with the results of tong et al. ( ) . tgev-induced apoptosis of porcine iecs might function via blocking cell-cycle transfer from g to s phase; however, its specific mechanism requires further exploration. indirect ifa extends the application range of an antibody and further improves the detection sensitivity by labeling the specific secondary antibodies with fluorescein for signal amplification. in this study, both infected and uninfected cell cultures were detected by ifa using tgev antiserum. the results revealed the distribution of tgev in porcine iecs clearly. at h post-infection, many tgev particles bound to goat anti-rabbit igg-fitc showed green fluorescence when exposed to laser light and were located in the cytoplasm. at h, the fluorescence transferred to the perinuclear area, before decreasing gradually at h. porcine aminopeptidase n (papn), a cellular receptor for tgev, is abundantly expressed in the membrane of porcine iecs (data not shown), which suggested that papn plays a role in mediating tgev infection. in addition, we demonstrated that st cells could be infected with the culture supernatant of iecs infected tgev at h. taken together, these data indicated that iecs, which are derived from a target tissue for porcine coronaviruses, could be infected by tgev successfully, and that tgev propagated in iecs remains infectious for st cells. the tgev s protein is involved in the viral invasion process (trincone and schwegmaan-wessels, ) . combined with papn, the s protein mediates the fusion of tgev and the cell membrane, such that nuclear capsid is released into the cell (gelhaus et al., ) . a previous report also suggested that viral genome and structural proteins were assembled into virus particles in the area between the endoplasmic reticulum and the golgi, and that the particles were transported to the extracellular space through budding of vesicles (corse and machamer, ; lili and masters, ) . until now, the morphology of tgev in target cells had not explored in detail. in this study, tgev particles in porcine epithelial cells were observed to have a diameter ranging from to nm, which was consistent with previous reports. the infectious virions entered the target cells by membrane fusion and mature viruses budded into vacuoles, which were gradually transported to the cell membrane before being released. fq-pcr was used to quantify tgev inside porcine iecs in comparison with the sc-tgev virus inside pk- cells (dai et al., ) . the one-step growth curve of tgev in porcine iecs showed that the amount of viral rna did not change significantly from to h post-infection, whereas dai et al. showed that in pk- cells, the rna increased rapidly from to h. the viral rna displayed a logarithmic increase in porcine iecs from to h, while dai et al. showed that the viral rna was maintained at high level and entered into stationary phase gradually. extracellular tgev rna levels were unchanged at to h post-infection of porcine iecs, and increased logarithmically until reaching maximum at h, before decreasing gradually. in pk- cells, dai et al. demonstrated that extracellular tgev rna levels increased logarithmically at - h post-infection, and maintained relatively stable and high level at - h. the differences between the curves indicated that differences in virulence and cell types might lead to changes in virus replication. the onestep growth curve of tgev revealed the rule of virus proliferation, and thus will provide further guidance for clarifying the infection mechanism of tgev. in conclusion, this is the first study to report the proliferative rule and morphogenesis of tgev in porcine iecs. these results lay the foundation for further study of the infection characteristics between tgev and its target cells. genomic organisation of a virulent taiwanese strain of transmissible gastroenteritis virus infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles isolation and identification of porcine transmissible gastroenteritis virus in sichuan province and determination of one-step growth curve complete sequence ( kilobases) of the polyprotein-encoding gene of transmissible gastroenteritis virus organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core porcine small intestinal epithelial cell line (ipec-j ) of rotavirus infection as a new model for the study of innate immune responses to rotaviruses and probiotics palmitoylation of the alphacoronavirus tgev spike protein s is essential for incorporation into virus-like particles but dispensable for s-m interaction a highly infectious gastroenteritis of pig epidemic diarrheal disease of viral origin in newborn swine on the pathogenesis of transmissible gastroenteritis of swine sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene the molecular biology of coronaviruses evolved variants of the membrane protein can partially replace the envelope protein in murine coronavirus assembly dna mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus comparison of the amino acid sequence and phylogenetic analysis of the peplomer, internal membrane and nucleocapsid protein of feline, canine and porcine coronavirus simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia transmissible gastroenteritis (tge) of swine: in vitro virus attachment and effects of polyanions and polycations characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine importance of cholesterol for infection of cells by transmissible gastroenteritis virus sialic acids as receptor determinants for coronaviruses transmissible gastroenteritis virus induced apoptosis in swine testes cell cultures isolation and identification of porcine transmissible gastroenteritis virus from chongqing, southwestern china influence of the transmissible gastroenteritis virus on activities of swine intestinal epithelial cells looking for a needle in a haystack: cellular proteins that may interact with the tyrosine-based sorting signal of the tgev s protein gene expression study of two widely used pig intestinal epithelial cell lines: ipec-j and ipi- i localization to the nucleolus is a common feature of coronavirus nucleoproteins and the protein may disrupt host cell division molecular cloning and phylogenetic analysis of orf region of chinese isolate th- from transmissible gastroenteritis virus this work was supported by the graduate scientific research in chongqing (cys , cys ), and the chongqing basic research program (cstc jcyja , cstc jcya ), and the fundamental research funds for the central universities (xdjk b ). key: cord- - b ocoh authors: zhang, chao-fan; cui, shang-jin; zhu, chao title: loop-mediated isothermal amplification for rapid detection and differentiation of wild-type pseudorabies and gene-deleted virus vaccines date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: b ocoh a loop-mediated isothermal amplification (lamp) assay was developed specifically for detection and differentiation of pseudorabies virus (prv). one group of primers was designed to detect wild-type strains (i.e., strains with the ge gene) and the other group of primers was designed to detect both prv ge-vaccine and wild-type strains (i.e., strains with the gg gene and with or without the ge gene). after amplification by bst enzyme at a constant temperature of °c, a laddering of bright products was visible following electrophoresis on a % agarose gel. lamp was – -fold more sensitive than the standard pcr. the assay was specific in that it did not amplify other porcine viruses including porcine parvovirus, porcine circovirus type , porcine circovirus type , porcine reproductive and respiratory syndrome virus, classical swine fever virus, swine transmissible gastroenteritis coronavirus, and porcine epidemic diarrhea virus. because of its sensitivity, specificity, and simplicity, the lamp assay could be a useful method for early and rapid differentiation of swine vaccinated with prv ge-deleted vaccine from swine infected with wild virus. pseudorabies virus (prv) is a member of the herpesviridae family and causes aujeszky's disease, which is characterized by neurological signs and death in young piglets, respiratory disorders in older pigs, and abortion in pregnant swine. in addition to swine, prv infects many other animals including sheep, cattle, dogs, and rodents. wild-type strains cause a peripheral neuropathy characterized by violent pruritus in dead-end hosts like sheep, cattle, and dogs but not in pigs. prv has caused substantial damage to the swine stockbreeding worldwide (thomsen et al., ; nauwynck, ; rooij et al., ). an improved system for detecting prv is needed (pejsak and truszczyni, ) . traditionally, prv detection has been based on direct virus isolation or detection of antigens by immunohistological methods. however, both methods are time-consuming. pcr facilitates the rapid detection of prv but requires specialized equipment and an elaborate method for detecting the amplified product (osorio, ; balasch et al., ) . although prv vaccine containing attenuated virus prevents the expression of clinical signs, such vaccination does not eliminate wild-type prv in pigs infected previously nor does it prevent sub-sequent infection by wild-type strains. these latent prv infections can be activated and cause the spread of the wild-type virus. therefore, it is very important to develop a method to identify pigs infected with wild-type virus or immunized with the prv gevaccine (prv lacking the ge gene). compared to the standard pcr, loop-mediated isothermal amplification (lamp) is a simple and rapid nucleic acid amplification method. lamp has very high specificity because it uses four to six primers that recognize six to eight regions of the target dna. lamp is used increasingly for clinical diagnosis of infectious diseases including those caused by bacteria, viruses, and parasites. for example, lamp is being used for detecting newcastle disease virus (hang et al., ) , salmonella enterica (kayoko et al., ) , plasmodium spp. (han et al., ) , porcine circovirus , and porcine parvovirus (chen and cui, ) . although a lamp assay for prv has been described previously (en et al., ) , that assay does not allow genetic diva. the use of lamp is described for determining whether swine are infected with wild-type prv or have been vaccinated with prv ge-and remain uninfected by wild-type strains. the prv ge-vaccine (strains prv-bartha-k and prv-plus), st cells, and the following viruses were obtained from the - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.jviromet. . . table primers for prv-ge and prv-gg. type sequence reverse gtgagcccgtgcttcatg harbin veterinary research institute of chinese academy of agricultural sciences: prv wild-type strains (strains prv-jx and prv-hlj), porcine parvovirus (strain ppv-bq), porcine circovirus type (strain pcv -hlj), porcine circovirus type (strain pcv -sh), porcine reproductive and respiratory syndrome virus (strain prrsv-hb), classical swine fever virus (strain csfv-sm), swine transmissible gastroenteritis coronavirus (strain tgev-hlj), and porcine epidemic diarrhea virus (pedv-hlj). samples from the cerebrum were collected from piglets suspected of being infected with prv in hei long jiang and ji lin provinces. these piglets exhibited the clinical manifestations of disease such as high fever, diarrhea, depression, disgorging, loose stools, ataxia, epilepsy, circling, hindquarter paralysis, and opisthotonos. the disease caused rapid death of the piglets. because the piglets showed some clinical signs of prv and csfv, swine fever infection was also suspected, but csfv was not detected by rt-pcr (unpublished data). the infected piglets that were sampled were - days old. although all the sows on these farms had been treated with the prv ge-vaccine about months before the samples were collected, none of the piglets had been treated with prv ge-vaccine. piglets are generally not immunized with the prv ge-vaccine until they are - weeks old. dna and rna were extracted from referenced virus and clinical samples with a tianamp virus genomic dna/rna kit (beijing tiangen biotech co., beijing, china) in accordance with the manufacturer's instructions. the extracted dna was eluted in a total volume of l of rnase-free ddh o and stored at − • c until use. cdna synthesis reaction was performed by the transcript firststrand cdna synthesis supermix (beijing transgen biotech co.) in accordance with the manufacturer's instructions. because ge has been deleted from all vaccines used in china but have gg, these two genes were targeted for lamp detection. the lamp primers were designed using the primer explorer v software based on both the glycoprotein e gene (ge) and the glycoprotein g gene (gg) (genbank accession number nc ) ( table ). pigs that have been vaccinated but that have not been infected with wild-type prv will have virus with the gg gene but not the ge gene whereas pigs that have been infected with wild-type prv will be infected with virus with both genes whether or not they have been vaccinated. the complete coding sequences of the ge and gg genes were inserted into the vector pmd -t (takara biotechnology co., dalian, china). the resulting pmd -t-prv-ge and pmd -t-prv-gg were amplified in e. coli dh ɑ, and the recombinant plasmids were purified using the axypreptm plasmid miniprep kit (axy-gen biotechnology co., hangzhou, china). the products were kept at − • c until used. except for the primers in the thermal water bath, the reaction mixtures for the prv-ge-lamp and the prv-gg-lamp were identical. the -l reaction mixture contained . l of × ther-mopol buffer, u of bst dna polymerase (new england biolabs (beijing) co., ltd.), l of mm mgcl , l each of outer primers ( m), l each of inner primers ( m), l each of loop primers ( m), . l of dntp mixture ( . mm each), . l of betaine ( m), l of extracted template dna or cdna in a . -ml eppendorf tube, and sufficient distilled water to increase the volume to l. in initial optimization tests, the amplification reaction was performed at , , or • c for min and then terminated by heating at • c for min. lamp products were subjected to electrophoresis on a % agarose gel. in subsequent tests, the amplification reaction was performed at • c (see section ). the reaction mixtures for prv-ge-pcr and prv-gg-pcr were identical except for the primers. the prv-pcr reaction mixture contained . l of × gc buffer, l of dntp ( . mm each), . l of each of forward and reverse primer ( m) (table ) , l of extracted template dna, . l of la taq polymerase (takara biotechnology co., dalian, china), and sufficient distilled water to increase the volume to l. the amplification conditions were • c for min; followed by cycles of • c for s, • c for s, and • c s; and a final extension of • c for min. pcr products were subjected to electrophoresis on a % agarose gel. the sensitivity of prv-ge-lamp and prv-gg-lamp was compared with prv-specific real-time pcr by using serially diluted plasmids with × , × , × , × , × , × , × copies/l. the specificity of prv-ge-lamp and prv-gg-lamp was examined using dna of pcv , pcv , and ppv, and cdna of prrsv, csfv, pedv, and tgev. dna of the prv-jx strain was used as the positive control, and dnas of st cells and three samples from each of five uninfected animals were used as the negative control. the prv ge-vaccines (strains prv-bartha-k and prv-plus) and prv wild-type strains (strains prv-jx and prv-hlj) were subjected to the prv-ge-lamp and prv-gg-lamp assay. the results were visualized by electrophoresis on a % agarose gel. the applicability of the prv-ge-lamp and prv-gg-lamp assay for clinical diagnosis of prv was determined in with brain samples collected from piglets in the hei long jiang and ji lin provinces. the sows, the mothers of the infected piglets, had been immunized routinely with prv ge-vaccine. as noted earlier, the piglets had not been vaccinated and exhibited signs of prv infection. lamp results were compared with those of typical prv-ge-pcr and typical prv-gg-pcr, which were run in parallel. prv-ge-lamp and prv-gg-lamp products from the lamp reactions carried out on the clinical material were purified using the axypreptm dna gel extraction kit (axygen biotechnology co., hangzhou, china) and sent to takara company for sequencing. dnastar software and blast searching of genblank were used to determine the homology with known prv-ge and prv-gg gene sequences. the prv-ge-lamp and prv-gg-lamp reactions with l of template dna extracted from prv-jx strain and with or without loop primers were conducted at , , and • c for min in a water bath. the results were optional at • c because at this temperature the prv-lamp reactions produced bright, ladder-like bands (fig. ) . the detection limits were copies per sample for both the prv-ge-lamp and the prv-gg-lamp, copies for the prv-ge-pcr, and , copies for the prv-gg-pcr ( fig. a-d) . table detection of prv in clinical samples by lamp and pcr. primers were specific for the ge gene or the gg gene of prv. pigs vaccinated with the prv ge-deleted vaccine will lack the ge gene. pigs infected with wild-type prv will contain both kinds of genes. positive negative the prv-ge-lamp and the prv-gg-lamp amplified prv-jx but did not amplify seven other porcine viruses, dna from st cells, or samples from uninfected animals (fig. ) . the prv-gg-lamp detected the prv ge-vaccine strains (prv-bartha-k and prv-plus) and the prv wild-type strains (prv-jx and prv-hlj), but the prv-ge-lamp only detected the wild-type strains (fig. ) . the results for prv-lamp and prv-pcr were similar (table ) , although two samples that were negative for prv based on prv- - contained × , × , × , × , × , × , × , and × copies/ml of recombinant plasmid, respectively. lane was the negative control. ge-pcr were positive for prv based on prv-ge-lamp. based on prv-gg-lamp, of the piglets were infected with wild-type prv and may or may not have been vaccinated. based on both the prv-gg-lamp and the prv-ge-lamp, three of the piglets had antibody from the mothers but had not been infected with wild-type pcr. sequencing of ge and gg products from the lamp reactions was carried out on virus that was isolated from the clinical material and then propagated in tissue culture. the sequences showed completely homology with the sequences of a known prv strain (accession number nc ). the prv-lamp assays described above should be useful for the management of infection and study of prv. in viral assays, specificity and sensitivity are essential in cases where low concentrations of virus are expected. prv-lamp is specific analytically (it did not amplify seven other porcine viruses) and is much more sensitive analytically and easier to perform than classical prv-pcr. although typical pcr is inferior to the prv-specific real-time pcr, the sensitivity of the prv-lamp and the prv-specific real-time pcr was compared in another study; the sensitivity of the two assays was similar, i.e., both assays detected copies per sample (unpublished data). although our unpublished results indicate that the prv-lamp is as sensitive as the prv-specific real-time pcr, the prv-lamp is easier and requires less time to perform than the prvspecific real-time pcr. these characteristics should make lamp very useful for field tests and in other situations where a rapid, simple test is required. eradicating latent infections of prv wild-type strains in swine is very difficult, and producers therefore vaccinate routinely their swine two to three times each year with the prv ge-deleted vaccine to reduce economic losses. many different pcr assays can differentiate between the wild-type prv and gene-deleted virus vaccines (liu et al., ) , but lamp is easier to perform and provides more rapid results. en et al. ( ) described a lamp system for prv detection but that lamp system could not differentiate between swine infected with wild-type prv and swine vaccinated with prv ge-deleted. all the vaccines used in china now are ge-deleted and gg-retained. in our experience, the gg primers used in this study are more specific and more sensitive than primers aimed at gb and gd (data not shown), and so the gg gene was used in the lamp assay. the development of a sensitive antigen-detection method that can distinguish between wild-type strains and attenuated vaccine strains will help producers identify and eliminate swine infected by wild-type prv strains. the nature of the problem is exemplified by the data obtained from clinical samples in this study. although these samples were obtained from piglets produced by vaccinated sows, % of the samples were positive for wild-type prv. the identify of the virus in the clinical samples was confirmed by isolation and sequencing and also by injecting the isolated virus into rabbits, which developed subsequently neurological signs of prv including the chewing of the leg at the injection site, ataxia, hindquarter paralysis, and opisthotonos (data not shown). the prv-ge-lamp in particular should help producers eliminate wild-type prv from their herds. aujeszky's disease (pseudorabies) virus detection in cerebrospinal fluid in experimentally infected pigs detection of porcine parvovirus by loop-mediated isothermal amplification rapid detection of porcine circovirus type by loop-mediatedisothermal amplification loop-mediated isothermal amplification establishment for detection of pseudorabies virus loop-mediated isothermal amplification for rapid detection of newcastle disease virus detection of four plasmodium species by genus-and species-specific loop-mediated isothermal amplification for clinical diagnosis detection of salmonella enterica in naturally contaminated liquid eggs by loop-mediated isothermal amplification, and characterization of salmonella isolates microarray-based detection and differentiation of virulent and attenuated pseudorabies virus functional aspects of aujeszky's disease (pseudorabies) viral proteins with relation to invasion diagnosis of pseudorabies (aujeszky's disease) virus infections truszczynsk: aujeszky's disease (pseudorabies) a dna vaccine coding for gb and gd of pseudorabies virus (suid herpes type ) primes the immune system in the presence of maternal immunity more efficiently than conventional vaccines pseudorabies virus as a live virus vector for expression of foreign genes the study was supported in part by funding from the national high-tech r&d program ( program- aa ) and the chinese national key laboratory of veterinary biotechnology fund (nklvbp ). key: cord- -zij wbzs authors: kim, hyun soo; hyun, jeongwon; kim, jae-seok; song, wonkeun; kang, hee jung; lee, kyu man title: evaluation of the sd bioline norovirus rapid immunochromatography test using fecal specimens from korean gastroenteritis patients date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zij wbzs the analytical and clinical performance of a new rapid immunochromatography test, the sd bioline norovirus test, was evaluated for the detection of human norovirus in fecal specimens. the analytical performance studies were performed for detection limit, reproducibility, cross-reactivity, and interference. for comparison, norovirus-positive stool samples and norovirus-negative samples for which the results were confirmed by different real-time pcr kits were used. the rapid immunochromatography test detected the equivalent of . × ( ) copies/ml of the norovirus genome in stool samples. on performing the repeatability/reproducibility test, samples above this concentration all provided positive results ( %) and . % of the samples slightly below this concentration ( . × ( ) copies/ml) provided negative results. no cross-reactivity or interference was detected. positive percent agreement (sensitivity), negative percent agreement (specificity), and overall percent agreement of the rapid immunochromatography test compared with testing by real-time pcr were . %, %, and . %, respectively. in addition, the rapid immunochromatography test was completed within min. the sd bioline norovirus test was, therefore, easier and more rapid to perform and showed excellent reproducibility, no cross-reactivity, no interference, and high agreement compared with real-time pcr. thus, this test is useful for rapid screening to identity norovirus infection. norovirus is the most common causative organism responsible for acute gastroenteritis epidemics in all age groups worldwide (fankhauser et al., ; glass et al., ). in the us, norovirus was detected in of ( %) epidemic acute gastroenteritis cases between and (fankhauser et al., ) . in addition to epidemic infection, sporadic infection is also common, and the prevalence rate varies slightly by area and by time (glass et al., ; fankhauser et al., ; park et al., a,b) . norovirus infection is common in south korea during autumn and winter, and its prevalence is reported to be . - . % (park et al., a,b; park et al., ; chung et al., ) . norovirus is an rna virus of the caliciviridae family and is classified into genogroups (gi-gv) according to the polymerase and capsid protein sequences (glass et al., ) . over genotypes have been reported to date (patel et al., ; glass et al., ) . of these, several strains of gi, gii, and giv have been identified in humans, and human norovirus infection is known to be caused primarily by genotypes of the gi genogroup and of the gii genogroup (patel et al., ; glass et al., ). many attempts have been made to establish diagnostic methods for norovirus infection, and detection of norovirus in stool specimens has been performed using the techniques of electron microscopy, enzyme immunoassay, immunochromatography (icg), western blotting, rt-pcr, and real-time pcr (atmar and estes, ) . among these assays, rt-pcr and real-time pcr are recognized as the most sensitive methods currently available, but the drawbacks are that they require expensive special equipment and skilled technologists. additionally, they are more time consuming and costlier than the rapid icg test. this study was performed to evaluate the analytical and clinical performance of a newly developed rapid icg test (sd bioline norovirus test) for detecting human norovirus genogroups gi and gii in stool specimens. two hundred and eighteen frozen fecal suspensions ( - % fecal suspension diluted with saline) collected and stored at − • c from august to april were tested retrospectively in this study. these comprised norovirus-positive stool samples and norovirus-negative samples, confirmed using different realtime pcr kits (rida gene norovirus v, r-biopharm, darmstadt, germany; and accupower norovirus real-time rt-pcr kit, bioneer, daejeon, korea). of these, samples ( . %) were from male patients and samples ( . %) were from patients less than years old. this study was approved by the institutional review board of hangang sacred heart hospital. real-time pcr assays using the stored frozen fecal suspensions were performed in june , and the sd bioline norovirus test (standard diagnostics, yongin, korea) was performed on the same stored fecal samples during november , that is, months later. according to the sd bioline norovirus reagent package insert, sd bioline norovirus test is a quick, qualitative icg test that detects human norovirus genogroups gi and gii in stool samples. the norovirus in the specimen reacts with colloidal gold-labeled norovirus-specific monoclonal antibodies recognizing both gi and gii strains, thus forming a complex of antigen-antibody colloidal gold conjugates. as this complex migrates along the result window by capillary action, it is captured by norovirus-specific monoclonal antibodies immobilized in the test line positioned across the result window, and a colored line is generated. in the absence of norovirus, a complex is not formed, and no colored line appears. frozen fecal suspensions were warmed to room temperature, and l of fecal suspension was added to a tube containing ml of diluent ( : dilution) and mixed well. four to drops (approximately - l) were added to the sample well of the test device, and results were read after min. in samples with negative icg and positive real-time pcr results, l of fecal suspension was mixed with l diluent ( : dilution) instead of ml diluent, and the test was repeated. rna extracts were prepared from fecal suspensions by using a qiaamp viral rna mini kit (qiagen, hilden, germany) and a qiacube platform (qiagen). two real-time pcr kits (rida gene norovirus v and accupower norovirus real-time rt-pcr kit) were used. for the rida gene norovirus v assay (r-biopharm, darmstadt, germany), l of rna extract was mixed with l of master mix and real-time rt-pcr was performed using a pcr thermocycler (rotor-gene q, qiagen) with the following program: reverse transcription at • c for min; cycles of denaturation at • c for s and annealing/extension at • c for s. for the assay with the accupower norovirus real-time rt-pcr kit (bioneer, daejeon, korea), l of rna extract was mixed with l of master mix, and real-time rt-pcr was performed using a pcr thermocycler (exicycler real-time quantitative thermal block, bioneer) with the following program: reverse transcription at • c for min; pre-denaturation at • c for min; cycles of denaturation at • c for s and annealing/extension at • c for s. all the procedures were performed according to the manufacturer's instructions. to quantify the copy number of norovirus rna, in vitro transcribed rna calibrators were prepared as follows. the amplicon of the target region in the norovirus genome was cloned into the pgem-t easy vector (promega, madison, wi, usa), and hit-dh ␣ competent cells (rbc bioscience, new taipei city, taiwan) were then used for transformation. the cloned plasmid was purified using the accupower plasmid mini kit (bioneer), and in vitro transcription of the target region was conducted by maxiscript ® sp kit (invitrogen, carlsbad, ca, usa) according to the manufacturer's instructions. the mass of in vitro transcribed rna was determined using a nanodrop system (thermo fisher scientific, wilmington, de, usa) and converted to copy number ([x g/l rna/(transcript length in nucleotides × )] × . × = y molecules/l; http://www. qiagen.com/resources/info/guidelines rtpcr/quantifying.aspx). the rna copy number in the samples was calculated on the basis of the ct (threshold cycle, the number of cycles at which the fluorescence exceeds the threshold) value, with the corresponding rna copy number determined from calibration curves prepared using rna calibrators (yang et al., ) . norovirus genotyping was conducted as described previously (kim et al., ) . for -step rt-pcr, the specific primers gi-f m (nt )/gi-r m (nt ) and gii-f m (nt )/gii-r m (nt ) targeting open reading frame (orf )-encoding capsid protein (vp ) were used. nested pcr was also performed using the primers gi-f (nt )/gi-r m (nt ) and gii-f (nt )/gii-r m (nt ). the products from the nested pcr were purified using a mega quick-spin pcr kit (intron biotechnology, seongnam, korea), and then cloned into the pgem t-easy vector and analyzed by dna sequencing. the nucleotide sequences were analyzed using abi prism bigdye terminator version . (applied cosmogenetech, seoul, korea) and an automated norovirus genotype tool was used to determine norovirus genotypes (available at: http://www.rivm.nl/mpf/norovirus/typingtool) (kroneman et al., ) . twofold serial dilutions of pooled positive fecal suspension prepared in saline were used to evaluate the reproducibility and the detection limit for the icg assay. each diluted sample was tested in triplicate, over a period of days, using sd bioline kits from different lots (lot numbers , , and ). the positive rates of repeated tests for each concentration were calculated to determine the reproducibility. the norovirus concentration in each sample was measured by real-time pcr (bioneer), and the number of norovirus rna copies/ml for each sample was recorded. cross-reactivity was examined with multiple strains of viruses, bacteria, and fungi, which have been listed below. for viruses, the virus culture supernatant was used to evaluate cross-reactivity. for bacteria and fungi, the colonies were diluted with saline to prepare a suspension with a density equivalent to . ; http://knrrb.knrrc.or.kr), type (kbpv-vr- ), type (kbpv-vr- ), type (kbpv-vr- ), type (kbpv-vr- ), type (kbpv-vr- ), type (kbpv-vr- ); enterovirus type (atcc vr- ); cytomegalovirus (atcc vr- ); poliovirus type (atcc vr- ); coxsackievirus a type (atcc vr- ); coxsackievirus b type (atcc vr- ); coxsackievirus b type (atcc vr- ); coxsackievirus b type (atcc vr- ); bk virus (atcc vr- ); herpes simplex virus type (atcc-vr- ); respiratory syncytial virus (atcc vr- ); parainfluenza virus type (atcc vr- ); parainfluenza virus type (atcc vr- ); rhinovirus type (atcc vr- ); echovirus type (atcc vr- ); coronavirus (atcc vr- , - ); and mumps virus (atcc vr- ). interference tests were performed with the following substances: human blood from a patient sample, barium sulfate (contrast medium), loperamide (anti-diarrhea drug; janssen, korea), metronidazole (antibiotic; cj pharma, korea), hemoglobin (sigma-aldrich, usa), bilirubin (sigma-aldrich, usa), and triglyceride mix (sigma-aldrich, usa). each substance ( mg) was dissolved in ml of solvent (triglyceride in ether; bilirubin in chloroform; all others in distilled water), and l of solution was mixed with l of negative stool suspension (negative base pool) and l of low-positive stool suspension (low-positive base pool). for substances in liquid form (e.g., blood), l of the substance was mixed with l of the negative and the low positive base pools. the time required to complete the icg test was determined for operators by using fresh fecal samples. each operator performed the icg test times, and the mean and standard deviation value of the procedure time (in minutes) for all operators was calculated. eighty-three of the real-time pcr-positive samples ( . %) were positive by the icg test, and all real-time pcr-negative samples were negative by the icg test (table ). the positive percent agreement (ppa, or sensitivity), negative percent agreement (npa, or specificity), and overall percent agreement of the rapid icg fig. . the reproducibility between replicate samples, days, and lots was % in positive fecal samples diluted : , : , and : , and in all negative fecal samples (table ). however, . % and . % positivity was observed for the positive fecal samples diluted : and : , respectively. lot no. was slightly more sensitive compared to the others, but the inter-lot variability for the detection limit of all lots was less than that for -fold dilution. the icg test could detect norovirus up to a dilution of : , corresponding to a viral load of . × copies/ml by the accupower norovirus real-time rt-pcr kit (table ) . no cross-reactivity was observed for any of the viruses, bacteria, or fungi tested (see section for details). there was no interference by any of following substances: human blood, barium sulfate (contrast medium), loperamide (anti-diarrhea drug), metronidazole (antibiotic), hemoglobin, bilirubin, or triglyceride mix. the mean ± standard deviation value for process times by operators was . ± . min. in this study, the performance of a newly developed norovirus detection kit that uses the icg technique was evaluated. if consistent positive or negative results from different real-time pcr assays are regarded as "true positives" or "true negatives," the sensitivity of the new assay is . % and the specificity is %. commercial norovirus antigen test kits can be broadly classified into types: the icg type and the elisa type. the ridaquick norovirus assay from r-biopharm is the most widely used icg assay, and the quicknavi-noro kit (denka seiken, japan), ip-noro kit (immuno-probe, japan), and ic kit (morinaga milk industry, japan) have also been introduced. for elisa testing, the ridascreen norovirus kit, ideia norovirus kit (oxoid, uk), and denka elisa kit (denka seiken, japan) are currently being used. however, various sensitivities and specificities have been reported for these kits, even for different studies using a single test kit. the sensitivity of . % ( % confidence interval, . - . %) obtained in this study for the sd bioline icg assay is similar to or higher than the reported sensitivities of the ridaquick ( . - %), quicknavi-noro ( . %), ip-noro ( . - . %), ic kit ( . %), ridascreen ( - %), ideia ( - . %), and denka elisa ( - . %) tests (bruggink et al., ; bruin et al., ; bruins et al., ; burton-macleod et al., ; costantini et al., ; derrington et al., ; khamrin et al., khamrin et al., , kim et al., ; kirby et al., ; nguyen et al., ; siqueira et al., ; wilhelmi de cal et al., ) . its specificity of % ( % confidence interval, . - %) is the same as or better than that of existing icg tests ( . - %) and elisa methods ( - %) (bruin et al., ; bruins et al., ; burton-macleod et al., ; castriciano et al., ; costantini et al., ; derrington et al., ; khamrin et al., khamrin et al., , kim et al., ; kirby et al., ; nguyen et al., ; park et al., ; siqueira et al., ; wilhelmi de cal et al., ) . furthermore, there were no cross-reactions with various types of viruses, bacteria, or fungi, and no interference by substances that can appear in the feces, such as blood, hemoglobin, bilirubin, triglyceride, antidiarrhea drug (loperamide), and antibiotics (metronidazole). very few studies have measured the detection limit of norovirus antigen test kits. the detection limit (analytical sensitivity) of the kit used in this study was . × copies/ml, which is similar to the detection limit of another icg method (takanashi et al., ) and the ideia elisa kit (costantini et al., ) , which was determined to be - copies/g of stool. as mentioned previously, different studies using the same test kit have reported differing sensitivities and specificities. therefore, the detection limit of different kits can be accurately compared using serial dilutions of the same clinical specimen, as shown in table . in this study, the icg test had inferior analytical sensitivity (i.e., a higher detection limit) than real time pcr tests. this is supported by the observation that ( ) no specimens tested positive in icg and negative in pcr, whereas specimens tested positive in pcr and negative in icg; and ( ) the ct values of of these specimens were > , indicating low norovirus concentrations. several reasons may contribute to the differences in sensitivity and specificity among the samples analyzed using the same kit. the first reason is the characteristics of the specimen, including whether the specimen is from an adult or a child, whether it is a specimen taken during an outbreak period, whether it has been taken from a subject strongly suspected to be infected with norovirus or from a healthy subject, whether it is a stored or fresh specimen, and the conditions of specimen storage or treatment (if any). the second reason is that the sensitivity and specificity of the test method can vary depending on the sensitivity and specificity of the comparative method, real-time pcr. the third reason is the diversity of genotypes within norovirus-positive specimens or the distribution of norovirus concentrations within samples. as norovirus comprises genogroups i-v and different genogroups or subtypes can be detected with different efficiencies by different kits, the sensitivity or specificity of the kit can differ depending on the range of genotypes in the samples. according to the manufacturers' data, the test kits used in this study could detect the gi. , , and , and gii. , , , , , , , , , and genotypes. another important factor in determining sensitivity is the virus concentration in the positive specimen group. the ct values for real-time pcr for most pcr-positive samples in this study were - , indicating that they contain high concentrations of norovirus. the fact that the specimens used were identified as positive or negative in different pcr tests could increase the sensitivity and specificity of the assays in this study. another factor that influenced the sensitivity and specificity in this study was the use of pre-diluted ( - % fecal suspension; : - : dilution) frozen fecal suspension. according to the manufacturer's instruction, fresh, refrigerated, or frozen fecal specimens were recommended, not pre-diluted frozen fecal suspensions. in this study, therefore, in the case of samples with negative icg and positive real-time pcr results, l of fecal suspension was mixed with l diluent instead of ml diluent (total dilution titer was : - : dilution, which is similar to that of the original procedure of this assay using stool), and the test was repeated. because of the similar dilution titer in the repeated test, this modified procedure involving pre-dilution might not decrease the sensitivity of the assay. however, the protocol in this study could increase the specificity of the assay due to the absence of repeated measurement with pre-diluted samples showing negative results. an important characteristic of this study is that the detection limit, reproducibility, and inter-lot variability were evaluated by using serially diluted fecal samples, although the test kit is designed to provide qualitative and not quantitative results. the reproducibility was nearly % at concentrations above or below the detection cutoff. although lot (lot no. ) exhibited the least sensitivity and lot (lot no. ) showed the greatest sensitivity, the difference in sensitivity between the lots was within -fold dilution. the difference between lots may explain the varying sensitivities that have been reported although the same test kit was used, and continuous improvements in the kits may explain the increased sensitivity in more recent reports. in conclusion, the sd bioline norovirus test showed good analytical and clinical sensitivity, excellent specificity, no interference, no cross-reactivity, and excellent reproducibility. therefore, this test is recommended as a fast and easy-to-use method for detecting and diagnosing norovirus infection. diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses evaluation of the ridaquick immunochromatographic norovirus detection assay using specimens from australian gastroenteritis incidents diagnosis of norovirus outbreaks by commercial elisa or rt-pcr evaluation of a rapid immunochromatographic test for the detection of norovirus in stool samples evaluation and comparison of two commercial enzyme-linked immunosorbent assay kits for detection of antigenically diverse human noroviruses in stool samples comparison of the ridascreen ® norovirus enzyme immunoassay to ideia nlv gi/gii by testing stools also assayed by rt-pcr and electron microscopy detection of gii- / b variant and recombinant noroviruses in children with acute gastroenteritis diagnostic accuracy and analytical sensitivity of ideia norovirus assay for routine screening of human norovirus norovirus ridaquick: a new test for rapid diagnosis of norovirus epidemiologic and molecular trends of norowalk-like viruses associated with outbreaks of gastroenteritis in the united states norovirus gastroenteritis evaluation of rapid immunochromatography test for the detection of norovirus infection: comparison with elisa and real time quantitative reverse transcription pcr assays evaluation of immunochromatography and commercial enzyme-linked immunosorbent assay for rapid detection of norovirus antigen in stool samples immunochromatogaphy test of rapid detection of norovirus in fecal specimens genetic distribution of human noroviruses detected from acute gastroenteritis patients in seoul an evaluation of the ridascreen and ideia enzyme immunoassays and the ridaquick immunochromatographic test for the detection of norovirus in fecal specimens an automated genotyping tool for enteroviruses and noroviruses evaluation of immunochromatography tests for detection of rotavirus and norovirus among vietnamese children with acute gastroenteritis and the emergence of a novel norovirus gii. variant epidemiological analysis of norovirus infection between evaluation of a new immunochromatographic assay kit of the rapid detection of norovirus in fecal specimens genetic analysis of norovirus gii. variants circulating in korea viral agents associated with acute gastroenteritis in seoul noroviruses: a comprehensive review evaluation of third-generation ridascreen enzyme immunoassay for the detection of norovirus antigens in stool samples of development of a rapid immunochromatographic test for noroviruses genogroups i and ii evaluation of two commercial enzyme immunoassays for the detection of norovirus in faecal samples from hospitalised children with sporadic acute gastroenteritis real-time pcr for quantitation of hepatitis c virus rna we thank standard diagnostics for providing the sd bioline norovirus kits, ridascreen norovirus elisa kits, and rida gene norovirus v kits. we also thank bioneer corporation for providing the accupower norovirus real-time rt-pcr kit. key: cord- -q mgue z authors: terlizzi, maria elena; massimiliano, bergallo; francesca, sidoti; sinesi, franca; rosangela, vendrame; stefano, gambarino; costa, cristina; rossana, cavallo title: quantitative rt real time pcr and indirect immunofluorescence for the detection of human parainfluenza virus , , date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: q mgue z human parainfluenza viruses (hpivs) are distributed worldwide and are involved mainly in the pathogenesis of respiratory tract infections. the development and optimization of three quantitative reverse transcription real time polymerase chain reactions (rt real time qt-pcrs) and an indirect immunofluorescence (ifa) for the detection and quantitation of hpiv- , - and - in clinical samples are described. efficiency, sensitivity, specificity, inter- and intra-assay variability and turnaround time of the two methods were compared. these assays have been validated on bronchoalveolar lavage specimens. based on the results obtained, the molecular methods represent a valid and rapid tool for clinical management and should be included in diagnostic panels aimed to evaluate suspected respiratory tract infections. prototype virus strains of hpiv- (strain c- ), hpiv- (strain greer), hpiv- (strain c- ) were obtained from american type culture collection (atcc, manassas, va) (vr- , vr- , vr- , respectively). one-hundred thirty-one bronchoalveolar lavage specimens, thawed and liquefied with : n-acetylcisteine, obtained from patients (male/female, / ; median age . ± . ) from different clinical settings were examined. informed consent was obtained from all the patients or the nearest relatives and the study was approved by the ethics committee of the university hospital san giovanni battista of turin. human laryngeal epidermoid carcinoma (hep- ) cells and african green monkey kidney epithelial (vero) cells were used for primary viral isolation and propagation. cell lines were obtained from the zooprofilattic institute of lombardy and emilia romagna (bs tcl and bs cl , respectively). hep- and vero cells were maintained at • c and % co with supplemented mem (paa laboratories gmbh, pasching, austria) containing % filtered glutamine, . % antibiotic agent (penstrep, sigma-aldrich, saint louis, mo), . % antimycotic agent (fungizone, bristol-myers squibb, sermoneta, italy) , and % fetal calf serum (fcs; paa laboratories gmbh). infection was carried out at - % confluence. each infected flake (t , falcon, becton dickinson, milano, italy) was inoculated with a solution containing l of each viral stock and . ml of mem with % fcs. the medium for hpiv- infection contained no fcs and was supplemented with g/ml of trypsin (gibco, invitrogen, carlsbad, ca). the flakes which were not inoculated with virus were used as controls. cells were maintained for h at • c and % co to assist viral adsorption. subsequently, the inoculum was removed and mem with % fcs was added. the cell monolayers were observed daily for cytopathic effect (cpe). for virus recovering, the cellular media were centrifuged for min at × g and the supernatants were kept on ice. the cell monolayers were scraped and a thermal treatment was performed to extract viral particles from cells consisting in rapid freezing on liquid nitrogen followed by defrosting for m at • c for three times. the recovered viral particles were stored at − • c. ninety six-well plates at - % confluence hep- and vero were inoculated with l of -fold diluted virus or medium (i.e. negative control) for tcid assay. the plates were observed daily for cpe. for evaluating ifa sensitivity, -fold dilutions (ranging from to − tcid / l) of titrated virus were obtained and different variables, such as days of incubation ( - days) and primary monoclonal antibody dilutions (mab; : - : - : in albumin supplemented with pbs %), were examined. two hundred microliters of tcid dilution were inoculated into shell vials at - % confluence. the inoculum adsorption was enhanced by centrifugation at × g for min. one millilitre of supplemented medium was added and the shell vials were kept in a thermostat. after incubation, the cells were fixed with ml of acetone-methanol ( : ) for min. subsequently, the slides were incubated with a generic anti-hpivs mab ( - lot m -z; argene, verniolle, france) for m at • c and then with goat anti-mouse fluorescein-conjugated monoclonal antibody ( - lot n ; argene), as secondary antibody, for m at • c. the slides were washed and read immediately using a fluorescence microscope. the presence of bright green fluores-cence within intact cells was considered positive. the slides with too few intact cells were considered inadequate for examination. optimal ifa conditions were used for reproducibility within a single run (n = ; intra-assay variability) or different run experiments (n = ; inter-assay variability) using three dilutions: the dilution of the sensitivity level and the -fold above and the -fold below ones (table ) . nucleic acid extraction was performed using nuclisens easy-mag (biomeriéux, marcy l'etoile, france). viral cdna was generated, first by incubation of random primers ( ng/l) and dntps ( mm) (invitrogen) with l of rna for min at • c. subsequently, a mix containing . m dtt, buffer × [ mm tris-hcl (ph . ), mm kcl], superscript tm ii rt ( u/l) and rnaseout tm ( u/l) (invitrogen) was added. the total volume ( l) of the reaction mixture was incubated for min at • c, min at • c, min at • c using fast thermal cycler (applied biosystems, monza, italy). the alignment of virus-specific hn gene, based on available sequences in public databases, was performed with the clc free workbench . software (clc bio a/s). consensus primers and probes were designed within the hn region of hpiv- - using the primer express . software (applied biosystems) and oligoperfect tm designer software (invitrogen) ( table ). primer and probe design was made according to the standard requirements: prevention of primer-dimer formation and melting temperatures (tm) of the primers up to • c. in order to confirm rna-dna extraction and to prevent false negative results, the housekeeping gene glycerin-aldehyde- -phosphate-dehydrogenase (gapdh) was co-amplified and used as internal control (vic ® probes, applied biosystems) ( table ) . for the production of plasmid standard, cdna fragment of virusspecific hn sequences was cloned into the ta vector and propagated in competent escherichia coli top cells. plasmids were created using the topo ta pcr cloning kit (invitrogen), according to the manufacturer's specifications. the concentration of the plasmid dna was quantified by using a high-resolution spectrophotometer. for optimizing the three rt real time qt-pcrs, two different concentrations of target primers and probe were evaluated: . mm/ . mm and . mm/ . mm for hpiv- and hpiv- ; mm/ . mm and . mm/ . mm for hpiv- . two microliters of cdna were added to l of the reaction mix, giving a final reaction volume of l. uracil-dna glycosylase was used to eliminate pcr 'carry over' contamination from previous pcrs (quint et al., ; tetzner et al., ) . the amplification profile was optimized on the real time pcr system (applied biosystems) as follows: one cycle of decontamination at • c for min, one cycle of denaturation at • c for min followed by cycles of amplification at • c for s and • c for s. for each run a standard curve was generated in a -log range by -fold serial dilutions of the plasmid standard. the linearity range was evaluated using -fold dilutions (from to copies/reaction) of each plasmid. the intra-and interassay coefficients of variability (cv) were evaluated using different concentrations of plasmid standard (ranging from to copies/reaction) within a single run (n = ) or different run experiments (n = ). different concentrations of tcid (ranged from to − /reaction) were amplified by the rt real time qt-pcrs in order to compare sensitivity and specificity of the two diagnostic approaches. the generic anti-hpivs mab was tested for potential crossreactivity with unrelated viruses and bacteria (influenza virus a h n , atcc vr- ; a h n , atcc vr- ; influenza virus b, atcc vr- ; adenovirus, atcc vr- ; rsv, atcc vr- ; cmv, atcc vr- ; hsv - , atcc vr- ; human rhinoviruses, atcc vr- /vr- /vr- ; human coxsackievirus type b , atcc vr- ; echovirus types , atcc vr- ; , atcc vr- ; enterovirus type , atcc vr- ; human coronavirus types e, atcc vr- and oc , atcc vr- ; streptococcus pneumoniae, atcc ; legionella pneumophila, atcc ; mycoplasma pneumoniae, atcc ; chlamydia pneumoniae, atcc ). similarly, primers and probes were tested using the above strains and human sequences based on the data available at the blast alignment software (http://blast.ncbi.nlm.nih.gov/blast.cgi). no significant homology to any other sequences was found. viral cpe consisted of cell rounding and focal destruction of the monolayer with syncytial formation. vero and hep- cells showed a different susceptibility to hpivs infection: for hpiv- , tcid /ml × on hep- (vero cells were unable to support growth in the presence of trypsin) at day ; for hpiv- , and × on vero and hep- , respectively, on day ; for hpiv- , × and . × on vero and hep- , respectively, at day . hpiv- -ifa sensitivity was − tcid at day (fig. a) . no significant difference was observed on day . single positive cells showing a granular cytoplasmic fluorescence were seen at lower tcid (e.g. − to − ), while at higher tcid (e.g. to ) rare syncytial formations were appreciable. there was no difference between : and : mab dilutions in terms of fluorescence intensity, while a decrement using : dilution was evident (data not shown). on day , hpiv- -ifa sensitivity on vero and hep- cells was − tcid and − tcid , respectively (fig. b) . a granular cytoplasmic fluorescence in infected cellular foci with a "spread" infection pattern at lower tcid (e.g. − to − ) was observed; while at higher tcid (e.g. to ), the fluorescence involved the nucleus. with vero cells, the stain intensity resulted dependent on mab dilution and reduced constantly with the increase of mab dilution from : to : ; while no significant difference was observed on hep- cells. on day , hpiv- -ifa sensitivity on vero and hep- cells was − tcid and tcid , respectively (fig. c) . a lower intensity of fluorescence was seen with high mab dilutions, such as : and : , in comparison to : . since hep- cells had a very low sensitivity on day post-infection, cell culture was prolonged until day ; however, this resulted in only -log increment of sensitivity. a widespread and homogeneous cytoplasmic fluorescence pattern in both cellular models at lower tcid (e.g. − to − ) was observed; while at higher tcid (e.g. to ), syncytial formation was seen. interestingly, hpiv- infection pattern was "all or nothing", as the first dilution higher than that of sensitivity threshold (spread staining) appeared completely negative. in table the reproducibility and sensitivity (i.e. the lowest tcid concentration detectable at a frequency of %) of ifa are reported. for the rt real time qt-pcrs, the optimal parameters in obtaining the lowest detection limit with a high specificity resulted in the following concentrations: both primers . mm and probe . mm for hpiv- and hpiv- ; forward primer mm and reverse . mm, and probe . mm for hpiv- amplification. the dynamic range of the three rt real time qt-pcr assays was assessed by carrying out serial dilutions of the plasmid standard (from to copies/reaction) and was as follows: to copies/reaction ( fig. a) for hpiv- ; to (fig. b ) for hpiv- ; to (fig. c) for hpiv- . the sensitivity of the rt real time qt-pcrs (defined as the lowest concentration of target quantified at a frequency of %) was found to be copy/reaction for hpiv- and copies/reaction for hpiv- and hpiv- . the rt real time qt-pcr sensitivity was found to be − tcid /reaction for hpiv- and hpiv- and − for hpiv- (data not shown). the inclusion of an internal control, gapdh target, did not induce loss of primary target sensitivity during amplification (data not shown). the results of intra-and inter-assay reproducibility are summarized in table . for rt real time qt-pcr quantitation, the following formula was used: lower limit of virus-specific dynamic range × (correction factor derived from analytic procedure). the inferior limit was geq/ml of bronchoalveolar lavage for hpiv- and geq/ml for hpiv- and hpiv- . in the case of bronchoalveolar lavage specimens, / ( . %) were positive by ifa versus / ( . %) by rt real time qt-pcrs; ( . %) for hpiv- , ( . %) for hpiv- , and ( . %) for hpiv- (three specimens with co-infections: one hpiv- + and two hpiv- + ). viral loads were as follows: both specimens < for hpiv- , ranging from < to , geq/ml for hpiv- (mean , ; median ), and from < to , (mean , ; median ) for hpiv- . the development and standardisation of three "in-house" rt real time qt-pcrs and an ifa for the detection of hpiv- , - and - in clinical samples are described. different ifa conditions were evaluated, such as cellular models, days post-infection, and mab concentrations. according to the features of the replication cycle of hpiv (vainionpaa and hyypia, ) , a cytoplasmic staining of infected cells was observed, although it became nuclear (hpiv- infection) or syncytial (hpiv- infection) on day - post-infection. the two cellular models showed different susceptibility to hpivs infection. in the case of hpiv- , hep- had a sensitivity of − tcid on day post-infection using a mab concentration of : (similar to that obtained by others (aguilar et al., ) on nci-h cells), while tcid calculation on vero cells was not attained because of their inefficient growth in the presence of trypsin, necessary for attachment of hpiv- . vero cells were more suitable for detection of hpiv- and - . hpiv- sensitivity was − tcid on day post-infection using : mab dilution versus × − found by aguilar et al. ( ) . the typical "spread" staining reflects hpiv- infection strategy, in which contiguous cells are infected subsequently. hpiv- had a sensitivity of − tcid on vero cells on day post-infection using : mab dilution versus ∼ × found by aguilar et al. ( ) . the pattern of hpiv- infection (i.e. "all or nothing") is likely to reflect the simultaneous infection of both nuclear and cytoplasmic structures. it should be note that, although the primary mab is able to recognize simultaneously hpiv- , - and - without differentiating the virus, but not hpiv- a-b, as indicated by the manufacturer, the fluorescence pattern observed was specific for each virus, thus permitting discrimination by ifa in the absence of co-infections. also the intensity differed in relation to the mab dilution and cellular model employed: staining on vero cells was mab dilution-dependent, with high dilutions ( : - : ) resulting in unfocused and blurred fluorescence; whereas on hep- cells, : or : showed a similar intensity, with a reduction at : dilution. considering the three rt real time qt-pcr assays, the identical thermal profile was used, thus permitting to amplify the targets in the same work session. the assays have been optimized by identifying an improved primer/probe concentration in order to obtain the highest amplification efficiency and by evaluating the dynamic range, sensitivity, and reproducibility of each virus. the sensitivity of the two diagnostic approaches were compared in terms of amplification of fixed tcid concentrations and was as follows: − for hpiv- and - and − for hpiv- by rt real time qt-pcr versus − for hpiv- / and − for hpiv- by ifa. the sensitivity data obtained by the rt real time qt-pcrs were superior to those obtained by other investigators that employed multiplex rt-pcr or rt real time pcr protocols, including hpivs (osiowy, ; aguilar et al., ; templeton et al., ; hamano-hasegawa et al., ; cordey et al., ) . as regards multiplexing, these differences could be due to the decrease of sensitivity attributed to the simultaneous amplification of different targets. as shown by the evaluation of clinical specimens, the sensitivity of the rt real time qt-pcrs, expressed in tcid , was higher than ifa (data not shown); this is likely to be due to the fact that the detection limit of ifa was higher in comparison to that of the molecular methods. another factor to be considered in large volume laboratories and for a prompt clinical decision is the turnaround time. in this study, ifa required from (for hpiv- ) to days (for hpiv- /- ), while the results of the rt real time qt-pcrs were obtained within h, according to previous studies (kuypers et al., ) . these molecular methods should be included in the diagnostic workup able to detect a wide range of respiratory viruses for the clinical management of patients with suspected airway infections (tivjelung-lindell et al., ) . another relevant advantage is the automation of molecular protocols (from nucleic acid extraction to quantification of fluorescence signal), thus limiting the drawbacks derived from a labour intensive, time-consuming, and operatordependent method, such as ifa. in conclusion, molecular methods resulted in a significant improvement over ifa for hpiv- - in terms of sensitivity, applicability, and turnaround time and represent a valid tool for clinical management of patients with suspected respiratory tract infections. detection and identification of human parainfluenza viruses , , and in clinical samples of pediatric patients by multiplex reverse transcription-pcr parainfluenza viruses simultaneous detection of parainfluenza viruses and by real-time reverse transcription-polymerase chain reaction respiratory syncytial virus and parainfluenza virus comprehensive detection of causative pathogens using real-time pcr to diagnose pediatric community-acquired pneumonia parainfluenza viruses comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children clinical virology in real time direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-pcr assay the order mononegavirale-current status reliability of methods for hepatitis b virus dna detection orthomyxoviral and paramyxoviral infections in transplant patients rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses , , , and control of carry-over contamination for pcrbased dna methylation quantification using bisulfite treated dna development and implementation of a molecular diagnostic platform for daily rapid detection of respiratory viruses biology of parainfluenza viruses polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia molecular diagnostics in virology this study was supported by "compagnia di san paolo". we thank paolo solidoro and daniela libertucci, division of pneumology, azienda ospedaliero-universitaria s. giovanni battista of turin for biological samples. key: cord- -gzou b k authors: beier, d.; riebe, r.; blankenstein, p.; starick, e.; bondzio, a.; marquardt, o. title: establishment of a new bovine leukosis virus producing cell line date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: gzou b k due to the prevalence of different bovine leukosis virus (blv) species in the cattle population in europe, problems may arise in the serological diagnosis of blv infections. in addition, earlier investigations demonstrated that contamination of the blv antigen-producing cell culture systems by bovine viral diarrhea virus (bvdv) may give rise to misinterpretation of serological test results after bvdv vaccination of cattle. by co-cultivation of peripheral leukocytes of a blv-infected cow with a permanent sheep kidney cell line, a new blv-producing cell line named po was established. this line carries a blv provirus of the belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. investigations of a panel of well-characterised sera by agar gel immunodiffusion (agid) and capture elisa (celisa) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line flk/blv yielded comparable results. false positive results caused by bvdv cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line. although many attempts have been made either to improve the antigen-producing properties of existing permanent bovine leukosis virus (blv) expressing cell lines or to generate corresponding new lines (graves and ferrer, ; mamoun et al., ; altaner et al., ; altanerova et al., ; wagner et al., ) , the cell line flk/blv still is the one that is used mainly for blv antigen production for commercial diagnostic test kits. this line was established by cocultivation of fetal lamb kidney (flk) cells with lymphocytes of an infected cattle in the usa (van der maaten and miller, ) . analysis by polymerase chain reaction (pcr), restriction fragment length polymorphism analysis (rflpa) and sequencing showed a close relation of the flk/blv provirus to the australian and japanese strains (fechner et al., ; beier et al., ; licursi et al., ) . blv infected cattle in europe, especially in germany, however, mostly harbor proviruses closely related to the belgian provirus line (rice et al., (rice et al., , mamoun et al., ; molteni et al., ; blankenstein et al., ) . this may cause problems with regard to the sensitivity of serological diagnostic assays. furthermore, the cell lines flk/blv and fetal lamb spleen fls/blv, which are both used for antigen production, are known to be contaminated with bovine viral diarrhea virus (bvdv) (bolin et al., ; dees et al., ; own data, unpublished) . this contamination may result in additional diagnostic problems concerning the specificity of the reactions (roberts et al., ) . in , an inactivated bvdv vaccine was first applied in bovine leukosis free herds of cattle. subsequently, an increasing number of positive results - /$ -see front matter © elsevier b.v. all rights reserved. doi: . /j.jviromet. . . was observed in routine diagnostic investigations using a distinct elisa test system for detection of blv antibodies. further analysis of these reactions by means of other tests for blv antibody detection and blv provirus pcr always yielded negative results. therefore, it was assumed that crossreactivity occurred between the bvdv antibodies induced by the vaccine and bvdv in the blv antigen preparation. in the meantime, this assumption could be confirmed in a vaccination study in blv-free cattle (beier and conraths, ) . this prompted us to develop a new blv-producing permanent cell line expressing a european blv isolate of the belgian species without contamination by other viruses. this cell line should be useful for antigen production to improve current tests. peripheral blood mononuclear cells (pbmc) were prepared from edta-blood of a naturally infected cow from the federal research centre for virus diseases of animals (frc, wusterhausen, germany) using the ficoll method according to the manufacturer's recommodation (ficoll-paque, pharmacia). short time cultures were seeded in rpmi medium with % fcs containing - × cells/ml. an established ovine kidney cell line from the collection of cell lines in veterinary medicine (cclv), frc, isle of riems, was used as carrier line for blv uptake and replication. the po cell line (po, cclv rie ) is a fast and stable growing line also suitable for cultivation in roller-bottles. it was shown to be free of bovine, ovine and porcine viruses by immunofluorescence tests (ift). all viruses but bvdv were investigated by direct ift using commercially available fitc labelled antibodies (gamakon, bioveta, nitra), or in-house preparations (entero-, rota-and coronavirus, brsv, bsv, blv). the test for bvdv was carried out indirectly using the pan-pesti specific monoclonal antibody wb / (c.c.pro gmbh, neustadt/germany), : diluted, and anti-mouse igg (sigma/germany), : diluted. for bvdv, bdv, blv and brsv, the results were confirmed by pcr assays. bvdv pcr assay was done as described (vilcek, ) . no virus particles were detectable by electron microscopy (em). tests for mycoplasm using the culture method, dna staining and pcr were repeatedly negative. flk/blv cells (flk/blv, cclv, rie - ) served as reference line for blv antigen production. this line is contaminated with bvdv (data not shown). short time pbmc cultures and po cells were co-cultivated at a ratio of about : . using eagle's mem in earle's bss containing % non-essential amino acids, mg/l napyruvate, % fcs, ph . - . . when the monolayer was confluent, we used a split ratio of : for the first three subcultures. subsequently, cells were subcultivated regularly twice a week at a split ratio of : using the same culture medium. the cells could easily be detached by an edta/trypsin solution. the monolayer was stable for at least days, if the medium was changed every other day after confluence. after cryopreservation of - × cells/ml culture medium containing . % dmso and % fcs, the thawed cells show a viability of more than %. the new cell line was named po (po , cclv, rie ). tests for contamination by foreign viruses and mycoplasms were negative. thereafter, several experiments were carried out to show whether multiple virus harvests from one cell culture were possible without antigen loss. cultures of different passage ranges were included. cell culture supernatant was harvested on day , , and after subcultivation, and antigen titres were determined as described. in initial experiments, blv was pelleted from the cell culture supernatant by ultracentrifugation (rotor tft . (kontron), , rpm/min, h, • c) avoiding any previous freeze-thawing step. the pellet was resuspended in / of the starting volume (v ) in ten-zwittergent buffer ( . m tris-buffer, . m nacl, . m na-edta, . m zwittergent . (calbiochem), ph . ). the remaining antigen in the supernatant was concentrated by ultrafiltration (regenerated cellulose membrane ym , millipore corp., usa) to / v . for further investigations, antigen was prepared by ultrafiltration of the native cell culture supernatant after removal of cell debris by low speed centrifugation. tissue culture flasks were seeded with × cells/ml in leibowitz (l )/dulbecco's modified eagle's medium (dmem) [seromed], corresponding to × cells per cm flask, and were examined for blv proteins for days over passages. samples of cell culture supernatant ( . ml) were collected daily, centrifuged at rpm for min and supernatants were stored at − • c. the presence of blvp was determined as an indicator for viral gene expression using a capture enzyme immunoassay as described by platzer et al. ( ) with some modifications. the amount of antigen (blv gp and p ) in cell culture supernatants was determined using a standard blv preparation. this was adjusted to quantified blv preparations kindly provided by c. platzer. a duplicate set of serial dilutions of this reference was included in each test run and a standard curve of relative antigen concentration versus optical density was developed. the limit detection was about ng/ml. genomic dna was extracted from the cell line po using a commercially available extraction kit (macherey-nagel, düren/germany). the dna was subjected to pcr, followed by a nested pcr (n-pcr) that amplifies the env gene between nucleotides (nt) and (fechner, ; beier et al., ) . primers were designed based on published sequence data (sagata et al., ) . forward primers were: reverse primers were: primers env and env were identical with those previously used for blv provirus detection by pcr (naif et al., , brandon et al., ) . all primers were custom synthesised (mwg biotech, ebersberg/germany). the pcr protocol has been described previously (fechner et al., ) . the primer pair env / env was used for the first round of amplification resulting in a bp fragment. for the n-pcr, l of the product was transferred into a new tube containing fresh pcr mix and primer pair env / env . the n-pcr product consists of bp. pcr assays were carried out using a dna thermal cycler (perkin-elmer cetus inc., weiterstadt/germany). in order to visualise pcr products, l of the total assay volume was run on a . % agarose gel followed by ethidium bromide staining. direct digestion of l env nested pcr product ( bp) with the restriction endonucleases bcli, pvuii and bamh (boehringer, mannheim/germany) was done at • c for h to verify the specificity of the amplicons and to determine the blv subgroup (fechner et al., ) . pcr products were separated from primers and nucleotides using qiaquick columns (qiagen, hilden/germany). sequences of the purified amplicons were determined using the fluorescent dye deoxy-terminator cycle sequencing kit (perkin-elmer cetus inc., weiterstadt/germany) and analysed by an abi prism dna sequencer (applied biosystems, weiterstadt/germany) as previously described (marquardt and haas, ) . the primers were the same as used for nested pcr. the resulting sequences were aligned to published sequences from different blv provirus species over bp. in addition, blv provirus sequences of cattle from germany were included in the phylogenetic analysis. the phylogenetic tree was established using the software package dnasis for windows . (hitachi, japan). standard and reference sera from the national reference laboratory for bovine leukosis, located at the federal research centre for virus diseases of animals (frc), and from an agar gel immunodiffusion (agid) test kit producer were used for comparative examination of the different serological test systems. in addition, sera from naturally infected cattle kept at the federal research centre and field sera with contradictory results in serological detection and pcr as well as in several serological tests were investigated. blood samples for serological tests were obtained together with the blood used for dna preparation. antigen preparations were characterised by agid as described (wittmann, ) . sera from two cattle, which had been investigated clinically, virologically and serologically over several years, served as internal reference for detection of blvp and gp , respectively. serial lg -dilutions of the antigens were prepared with tris-nacl buffer ph . and were tested against the two reference sera in parallel with an internal standard antigen, using l of each. agid plates were read after and h (p test) and and h (gp test). all tests were at least done in duplicate. the titre of the last antigen dilution causing a visible reaction was used as measure for the relative p or gp content in the preparation. standard and field sera were investigated in parallel with a commercially available test kit (riemser rinderleukose test kit, rtam/germany) in order to compare sensitivity and specificity of the antigens from the cell lines po and flk/blv. elisa tests were carried out either by using the chekit-leucotest kit (bommeli ag, bern, switzerland) according to the manufacturer's recommendations, or by a capture elisa (celisa). the celisa was done using two monoclonal antibodies (mab), kindly provided by dr. c. platzer (faculty of medicine, university of jena, germany). mab gp / was directed against epitope b of the blv gp , and mab p / against p antigen. capture elisa was performed as described previously (platzer et al., ) . cell culture supernatant of flk/blv or po , used as antigen, was diluted in phosphate buffer with . % tween and % horse sera. the cut off for sera is twice the od of the negative control. sera with an od between . times and twice of od of neg-ative control are counted as suspicious. the capture elisa was also used for quantitation of blv antigen in cell culture supernatant as described above. cells were lysed with two volumes ripa buffer ( % triton x- , % sodium desoxycholate, . % sds, mm nacl, mm tris-hcl [ph . ], mm pmsf, . % gelatine) by three cycles of freezing and thawing. twenty micrograms whole protein from each sample was separated on a sds- % polyacrylamide gel. the flk/blv control lysate was adjusted with standard antigen preparation and contained about . ng gp and . ng p in g whole protein. western blots were developed with mab gp / and p / (platzer et al., ) and goat anti-mouse alkaline phosphatase. for detection of bound antibodies, nbt/bcip color substrate (promega) was used. during the first co-cultivation, po and pbm cells were observed without any mutual interference. however, already after two subcultures, lymphocytes, macrophages and dendritic cells could no longer be seen microscopically. the newly established cell line po started to show small syncytia typical for blv replication in the th passage. in the th passage, cell culture supernatant was investigated for blv by celisa. the amount of gp and p was about - % compared to flk/blv. higher passages ( th- th passage) showed a higher blv antigen expression, which was nearly comparable with that of flk/blv under the same conditions. as shown in table , the p content in the supernatant was relatively stable for days after passaging for at least subcultivation steps. in addition, western blot analysis of cell lysates from flk/blv and po showed that both cell lines express similar amounts of gp , p and different precursor proteins. cells of the th subculture were shown to express blv by ift and em. particles with typical retrovirus morphology were detectable in ultrathin sections. cells and cell culture supernatant of the rd and th subculture revealed positive pcr results (earlier passages were not tested by pcr assay). the rflpa products from env amplicons showed a high homology with the belgian subtype (fig. ) . fur- fig. . phylogenetic tree based on env fragment ( bp) sequence analysis. po derived blv (accession number ay ) was compared to sequences from blv of different geographical origin (japanese: sagata et al., ; australian: coulston et al., ; belgian: rice et al., ; flk/blv and a cow from germany of belgian provirus type: fechner et al., ). thermore, the sequence of the env pcr product (genbank accession number: ay ) exhibited a . % homology with the belgian subtype over the analysed bp (fig. ) . all tests for foreign viruses and mycoplasma were negative. an important result of the investigations regarding antigen production was the fact that from one culture, up to four virus harvests were possible without loss of antigen content. in addition, the amount of p antigen clearly increased within that time. the content of gp antigen did not increase as strongly as that of p but showed the same tendency. furthermore, it is possible to use a wide range of passages of cell line po for blv antigen production: there were no table analysis of blv antigen prepared from cell line po after the rd and th subculture by means of agid using gp -and p -specific reference sera antigens presented in the table were prepared after the second ( rd subculture) and fourth ( th subculture) harvest of cell culture supernatant, respectively. serial lg -dilutions of the antigens were prepared with tris-nacl buffer (ph . ), and the intensity of the precipitation bands in the agar gel was evaluated using a classification from ++ (strong) to (+) (weak). a does not contain p . significant differences between the antigen yields of the rd and the th subculture (table ) . this is valid for p as well as for gp . comparable results were obtained using maintenance medium with and % fcs, respectively. the cell line is stable and produces blv, so far up to the th subculture. agid antigen prepared from cell line po was compared to antigen from a commercial agid test kit ( table ). the precipitation lines of the po antigens were confluent with those of the control antigen showing homology between both preparations. the lines were long, clear and distinct and, thus, offer the same quality as the commercial antigen. on an average, the final titres were slightly lower, which, however, had no influence on the test results. twenty-seven standard and reference sera were investigated in a first agid test. they included highly positive, weakly positive and negative sera and the eu standard serum "e " ( : ). all highly and weakly positive sera were detected in this test, and no false positive results occurred. in the following, sera from field and laboratory animals infected with blv of different provirus subtypes were tested in parallel with a commercially available test kit and po antigen. the sera had been characterised using up to five commercial elisa systems and full blood was tested by env nested pcr. out of these sera, were positive ( strongly and weakly positive sera), were negative and were doubtful. both agid test systems revealed the same results. furthermore, these serum samples were investigated in gp -and p -celisa tests using cell culture supernatant of flk/blv as well as of po . fig. shows representative results of the gp -celisa. the sera reacted in the same manner when testing by the p -assay. thus, both cell lines are suitable antigen sources for celisa tests. most of the sera reacted similarly with both antigens. in a few cases, differences occurred in the strength of the reactions: some sera reacted better with flk/blv antigen, but others showed a stronger reaction with po antigen. this should not be important for clearly negative or strongly positive sera, but in particular cases (see sample in fig. and table ), it may result in differences in the positive/negative-grading of the sample. this means that weakly positive sera might be detected by only one of the antigens and, thus, might be missed if tested using only one system. different reactivities were also seen between flk/blv and po antigen in p celisa (data not shown). comparative investigations of sera from cattle infected with blv of different provirus subtypes with antigen from both cell lines using the gp celisa. efforts to establish new blv-producing cell lines free of extraneous agents and attempts to isolate additional blv strains in cell culture continue. the new permanent cell line po productively infected with blv was created by cocultivation of po cells with a short time culture of leukocytes from a blv-infected cow. po was of special interest because it is free of other contaminating infectious agents and because it carries a blv provirus which is different from that of flk/blv with an env region closely related to the belgian species. this is the dominant provirus subtype among cattle in germany (fechner, ) . further attempts to isolate blv from cattle with indifferent serological but positive pcr results and from tumour cells of an intensely tumourous cow killed in a moribund state were not successful. the lymphocytes as well as the tumour cells were contaminated with bvdv and/or bovine foamy virus (bfv) which in each case caused a massive infection of the carrier cells. the yield of gp antigen produced by cell line po as analysed by agid test was slightly lower than that of the commercially available antigen. this fact had no consequences for the sensitivity of the test. sensitivity and specificity of po -antigens prepared and used for agid are comparable to those of commercial test kits making the cell line po attractive for production. the amount of p antigen appears to be stable and comparable to that produced by the most commonly used flk/blv. this is an important parameter for the production of elisa antigen. cell culture supernatant of both cell lines was proven to be useful as antigen in celisa tests for serological investigations. most of the sera showed the same reaction with both antigens. only a few sera reacted differently in the two tests demonstrating that there are differences in antigenicity. thus, antigen from both cell culture systems should be used to reliably demonstrate the absence of blv during the last stage of eradication programs and to confirm the blv-free status of a herd. to improve viral yield, further investigations need to be performed to optimise culture conditions as well as subcloning in order to enhance the antigen titres. on the other hand, po cells represent a good production cell system because they are easy to handle and do not require special media conditions. they grow fast and stable also in rollerbottles, and several harvests of supernatants are possible from one cell culture. isolation and characterization of cell clones producing various amounts of bovine leukemia virus infection of rats with bovine leukemia virus: establishment of a virus-producing rat cell line abklärung fraglicher und falsch-positiver ergebnisse in einem testbesteck zum nachweis von antikörpern gegen das bovine leukosevirus (blv) und mögliche zusammenhänge zu bvd/md-impfungen möglichkeiten und grenzen der anwendung der polymerasekettenreaktion (pcr) bei der diagnose der infektion mit dem bovinen leukosevirus (blv) des rindes identification of different blv provirus isolates by pcr, rflpa and dna sequencing pcr-elisa zum nachweis proviraler dna des bovinen leukosevirus (blv). biochemica inf survey of cell lines in the american type culture collection for bvdv early detection of bovine leukosis virus dna in infected sheep using the polymerase chain reaction molecular 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derived from the leukocyte of a leukaemic cow sequence variability of bovine leukemia virus env gene and its relevance to the structure and antigenicity of the glycoproteins vp -coding sequences of recent isolates of foot-and-mouth disease virus types a, o and asia molecular characterization of a variant of proviral bovine leukaemia virus (blv) bovine leukaemia proviral dna detection in cattle using the polymerase chain reaction early detection of bovine leukemia virus by using an enzyme-linked assay for polymerase chain reaction-amplified proviral dna in experimentally infected cattle use of monoclonal antibody against major internal protein p of bovine leukemia virus in capture elisa the nucleotide sequence of the env gene and post-env region of bovine leukemia virus the gag and pol genes of bovine leukemia virus: nucleotide sequence and analysis comparison of the agar gel immunodiffusion test and elisa in the detection of bovine leukosis virus antibody in cattle persistently infected with bovine virus diarrhoea virus complete nucleotide sequence of the genome of bovine leukemia virus: its evolutionary relationship to other retroviruses identification of pestiviruses contaminating cell lines and fetal calf sera replication of bovine leukemia virus in monolayer cell cultures increase of antigen production in blv-infected cell-lines via additional expression of tax leukosen der wiederkäuer we thank h. schirrmeier for investigating the po cell line for the presence of contaminating viruses by means of ift. we also thank u. polster (mycoplasm tests) and h. granzow (em). key: cord- -hkh grys authors: turnage, nicole l.; gibson, kristen e. title: sampling methods for recovery of human enteric viruses from environmental surfaces date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: hkh grys acute gastroenteritis causes the second highest infectious disease burden worldwide. human enteric viruses have been identified as leading causative agents of acute gastroenteritis as well as foodborne illnesses in the u.s. and are generally transmitted by fecal-oral contamination. there is growing evidence of transmission occurring via contaminated fomite including food contact surfaces. additionally, human enteric viruses have been shown to remain infectious on fomites over prolonged periods of time. to better understand viral persistence, there is a need for more studies to investigate this phenomenon. therefore, optimization of surface sampling methods is essential to aid in understanding environmental contamination to ensure proper preventative measures are being applied. in general, surface sampling studies are limited and highly variable among recovery efficiencies and research parameters used (e.g., virus type/density, surface type, elution buffers, tools). this review aims to discuss the various factors impacting surface sampling of viruses from fomites and to explore how researchers could move towards a more sensitive and standard sampling method. acute gastroenteritis causes the second highest infectious disease burden worldwide with an estimated . million deaths per year (ahmed et al., ) . in the united states alone, acute gastroenteritis causes . million illnesses, , hospitalizations, and deaths (scallan et al., ) . there are approximately major pathogenic agents known to cause acute gastroenteritis and/or foodborne illness including human enteric viruses such as astrovirus, rotavirus, hepatitis a virus (hav), and human norovirus (hnov) (scallan et al., ) . the most common enteric viruses that cause foodborne illnesses are hnovs and hav (cliver, ; koopmans and duizer, ) . generally, viral acute gastroenteritis is transmitted through food and water contamination, contaminated environmental surfaces, direct person-to-person contact, and other unknown sources (wikswo et al., ) . furthermore, enteric viruses are spread by fecal-oral contamination, and there is growing evidence of viral transmission occurring through contaminated fomites in a variety of ways and settings including food preparation environments (boone and gerba, ; rzezutka and cook, ) . enteric viruses have been shown to maintain infectivity on fomites over prolonged periods of time (escudero et al., ) . for instance, seminal research by kiseleva ( ) reported on the survival of echovirus, coxsackievirus, and poliovirus on representative surfaces (painted wood, glass, cotton fabric) in households and showed that these viruses maintained infectivity for two to more than days. human norovirus survival for up to days has also been reported on carpets subject to vomiting episodes after an initial outbreak in a hospital ward (cheesbrough et al., ) . there are some studies focusing on the role of fomites and environmental contamination in the transmission of enteric viruses however this specific route of transmission is difficult to determine during outbreaks (rzezutka and cook, ) . to better understand the role of environmental surface transmission during outbreaks due to human enteric viruses, the persistence of viruses on various surface types must be investigated. to do this, a surface sampling method must be applied for recovery of viruses. for instance, understanding the persistence of human enteric viruses on inanimate fomite surfaces in relation to various environmental conditions could provide insight on ways to limit and prevent virus transmission and subsequent outbreaks. however, studies on surface sampling techniques are typically limited to swabs for application in environmental sampling during foodborne outbreaks or for investigation of baseline virus prevalence. as a result, information is lacking on evaluating tools used in laboratory sampling studies for the optimal recovery of viruses. thus, this review aims to: ( ) discuss and compare evaluations of surface sampling methods for optimal recovery of human enteric viruses from inanimate fomite surfaces and ( ) explore how researchers could move towards one standard methodology for surface sampling of human enteric viruses and their surrogates. the most common foodborne viruses are categorized based on the type of disease they cause: ( ) gastroenteritis (e.g. rotavirus, hnov, aichi virus a, coronavirus, and others), ( ) enterically transmitted hepatitis viruses (e.g. hepatitis e and a), and ( ) viruses that replicate in the human gut then migrate to other organs to cause disease (e.g. poliovirus) (koopmans and duizer, ) . enteric viruses are typically spread by vomiting or shedding into the stool and have a greater chance of transmission the longer the virus is able to survive outside the host. this survival is impacted by various environmental conditions such as ph, moisture, and temperature (koopmans and duizer, ; rzezutka and cook, ) . as indicated previously, enteric viruses have been shown to maintain infectivity on surfaces over prolonged periods. human noroviruses have been detected on a variety of surfaces including cellular phones, public phones, televisions, chairs, keyboards, microwave ovens, bathroom light switches, various handles and knobs of kitchen and bathroom items, bed frames, and chairs (boxman et al., ; gallimore et al., gallimore et al., , . boxman et al. ( ) reported year round prevalence of hnovs on environmental surfaces of catering facilities even without a recently reported outbreak of acute gastroenteritis. the authors reported that hnov was recovered from . % of catering settings with recent outbreaks in contrast to only . % of catering settings without a recent outbreak. elderly homes and pension/hotels catering company types had the highest prevalence of positive swab samples for hnovs (boxman et al., ) . moreover, multiple studies have shown institutional settings such as cafeterias and long-term facilities are more likely to have hnovs on surfaces compared to food service settings (boxman et al., ; hall et al., ; verhoef et al., ) . for environmental surface sampling, the international organization of standardization ( ) recommends swabbing with a sterile cotton swab presoaked in pbs followed by rna extraction and reverse transcription, real time pcr (rt-qpcr) analysis for hav and hnov sampling and detection on nonporous fcs. in the u.s., there is not a standardized method available. however, the centers for disease control and prevention (cdc, ) does recommend the use of swabs for obtaining norovirus from environmental surfaces; however, the cdc has also reported that swabbing is highly variable and that the interpretation of results should be conducted with caution. currently, hnovs are most often detected by rt-qpcr due to its high sensitivity and low detection limits using measurements such as pcr amplifiable units (pcru/ml). these pcrus are determined by a standard curve produced from a -fold dilution series of the virus where one pcru corresponds to the highest dilution with a quantifiable rt-qpcr value (or cycle threshold [c t ] value) (knight et al., ; tung et al., ) . however, knight et al. ( ) pointed out that the determination of pcrus in correspondence to specific c t values is dependent on the sample matrix and the standard used. moreover, the cutoff c t values (i.e. endpoint of detection) for hnovs also vary across studies ranging from to (knight et al., ) . the presence of inhibitory components within some sample matrices could impact amplification efficiencies especially in contaminated food and environmental samples that typically have low viral loads (knight et al., ; sair et al., ) . regardless, rt-qpcr is primarily chosen for the analysis of viruses in environmental and food samples to allow for increased sensitivity to detect low viral concentrations that are typically present (knight et al., ) . however, as the authors of the review indicated, this method cannot determine infectivity since it may recognize intact or degraded viral nucleic acid, nonviable viruses, or defective viral particles (knight et al., ) . consequently, the use of surrogates and other infectivity assays remain important in investigating enteric viral viability and infectivity in lab-based studies as further discussed in section . . . virus density, the rate of positive environmental samples of total samples collected, and exposure magnitude provide information about virus contamination on surfaces (julian et al., ) . however, these factors are impacted by the surface sampling method and detection assay selected. subsection . . to . . will examine the variability among the many factors impacting recovery of viruses from surfaces, specifically surface type, virus type/density, drying time, elution buffers, and implement/recovery tool selection. fomites are generally categorized as either nonporous or porous. examples of nonporous surfaces are ceramic, glass, acrylic, and stainless steel, and examples of porous surfaces include carpets, lettuce, deli meats, wood, latex, and fruits. surface type has been shown to have some effect on surface sampling recovery efficiencies (table ) . tung-thompson et al. ( ) swabbed foods (cheese, apple, green pepper, tomato) and hard surfaces (stainless steel and ceramic) with wipes that were inoculated with μl of varying pcr-units (pcru)/ml of hnov gii. . the study obtained a mean range recovery efficiency of % to approximately % for all surfaces except for cheese, which was significantly different from the other surfaces with % to % recovery for high inoculum levels ( to pcru) and no detection at low inoculum levels ( to pcru) (tung-thompson et al., ) . the authors were not able to determine if the lipid content of the cheese contributed to the possible absorption and recovery of the virus samples even though a previous study suggested this possibility for hnovs (fumian et al., ; tung-thompson et al., ) . furthermore, surface properties can also impact recovery efficiencies in a variety of ways. for instance, stainless steel is a hydrophilic (contact angle of . °in water, surface energy of . mj/m ) and negatively charged surface in which microorganisms have been shown to develop irreversible attachment within one minute potentially making surface recovery more difficult (mafu et al., ; mafu et al., ) . the orientation of a surface could interfere with adequate surface sampling and collection as seen in a study involving vertical and horizontal stainless steel surfaces. taku et al. ( ) determined that greater recovery efficiency could be obtained by allowing the elution buffer to sit on the surface for min-something that cannot be performed on a vertical surface. the mean recovery for horizontal surfaces and sinks using the cell scraper-aspiration method ranged from % to % while vertical stainless steel surfaces only obtained a mean recovery of % since the buffer was not in contact with the surface long enough to facilitate virus recovery (taku et al., ) . scherer et al. ( ) suggested physical properties of nonporous and porous could reduce virus recovery via trapping virus particles within the matrix/ crevices or facilitate enhanced virus recovery by smooth/porous surfaces. mattison et al. ( ) suggested the low mean recovery of feline calicivirus (fcv) from strawberries might be due to its surface texture and how the crevices may shield viruses against environmental conditions. furthermore, the authors observed a ph change in the elution buffer from . to . when strawberries were immersed, which could impact virus recovery by either partial viral inactivation or interference with fcv recovery (mattison et al., ) . overall, physical and chemical properties of nonporous and porous food and food contact surfaces could impact recovery efficiencies of enteric viruses. this review will focus on surface sampling techniques for enteric viruses from nonporous, inanimate surfaces. (rönnqvist and maunula ). there has not been an in vitro cell culture system for hnovs available until recently (ettayebi et al., ) , and until reproducible and readily available infectivity assays are developed, surrogates still provide much needed information on infectivity of hnovs. multiple surrogates are important for understanding infectivity due to variations in their genetic relatedness to hnovs and the diversity among hnov genotypes. other cultivable viruses utilized in environmental persistence research include aichi virus a (aiv) and hav-both known human enteric pathogens (cannon et al., ; koopmans and duizer, , yeargin et al., cannon et al., cannon et al., duizer, , yeargin et al., ) . diversity among hnov genotypes could impact the recovery efficiency from surfaces; however, studies focus mainly on hnov gii. (table ). this focus is a result of gii. being the pandemic genotype of hnov and accounting for over % of all hnov outbreaks in the u.s. since (glass et al., ). surrogates provide essential information on hnov infectivity in relation to viral persistence on food contact surfaces (fcs), and numerous studies have shown fcv, mnv, and tuv to remain infectious on multiple surfaces for at least days or more (arthur and gibson ; fallahi and mattison ; mattison et al., ) . some studies have compared the recovery efficiency between different types of enteric viruses. scherer et al. ( ) compared hnov gii. and rotavirus recovery efficiencies using a cotton swab from various porous and nonporous fcs. table shows the recovery varied between virus types for a given surface. for instance, scherer et al. ( ) reported the highest percentage of hnov was recovered on ceramic ( - %) while rotavirus was recovered at a slightly higher percentage ( - %) on the same surface. the authors suggested the varying recovery rates observed between the two enteric viruses may be due to the abilities of the different viruses to adhere to the various surfaces as well as differences in virus properties affecting attachment (scherer et al., ) . a greater variety of surrogates and enteric viruses need to be evaluated for surface sampling to ensure accurate prevention and detection methods are being implemented. virus density could also impact the amount of virus recovered from a given surface. in general, higher starting densities of viruses equal greater recovery efficiencies-primarily due to the limit of detection of the downstream assay. tung-thompson et al. ( ) reported recovery efficiency variability by virus density when using wipes on food and nonporous food contact surfaces. the authors showed that recovery was consistent at high inoculum levels ( - pcru/ml) of gii. while more variability was observed at lower inoculum levels ( − pcru/ml). in contrast, rönnqvist et al. ( ) also reported variability among lower concentrations of gii. with higher mean recoveries for hnov gii. at pcru than pcru when evaluating four different swabs on environmental surfaces. for pcru of gii. , there was no significance difference for recovery efficiency among the swabs evaluated except on latex surfaces with polyester swabs regardless of buffer type. meanwhile, microfiber swabs combined with glycine buffer for elution was found to be a significantly better recovery method for pcru of gii. on all the surfaces (rönnqvist et al., ) . scherer et al. ( ) reported that the mean recovery efficiencies for rotavirus and hnov gii. were higher from various nonporous and porous surfaces using a cotton swab-rinse method at higher inoculum levels ( × pcru for hnov; × pcru for rotavirus) than lower inoculum levels ( × pcru for hnov; × pcru for rotavirus). the authors also mentioned how reverse transcription became less efficient at low inoculum levels resulting in an increase in statistical errors. overall, the higher the inoculum level for all enteric viruses, the higher the mean recovery rate regardless of the variability among methods, pa = plaque assay; pbs = phosphate buffered saline; pbst = pbs + . % tween ; pcru = polymerase chain reaction units; pe = polyethylene; pf = porous formic; pfu = plaque forming units; rh = relative humidity; rb = rubberized surface; rt-qpcr = reverse transcription quantitative pcr; rt = room temperature; ss = stainless steel. virus type, and high standard deviations of the mean recovery rates. additionally, organic matter such as coagulated food and other debris while on environmental surfaces may impact the effect of virus density on recovery efficiency. for instance, fatty foods such as cheese have been known to contribute to absorption and recovery of virus samples for hnovs due to lipid content (fumian et al., ) . furthermore, abad et al. ( ) studied the effect of fecal matter on the persistence of enteric viruses and reported varying results between virus types and fomites. the authors found no effect on the persistence of hav and human rotavirus with the exception of longer persistence of hav on latex surfaces. overall, abad et al. ( ) observed longer persistence for adenovirus and poliovirus on nonporous fomites (china, glazed tile, aluminum, and latex), and a decrease in persistence of adenovirus and poliovirus on porous fomites (cotton cloth and paper). for hnovs, the preparation of stool samples (i.e. because hnov does not have a routine culture method) is not always specifically stated in studies on virus persistence and recovery from surfaces. for example, park et al. ( ) include a clarification step-a brief centrifugation to separate the large particulates from the viruses in % fecal suspensions-while others (de keuckelaere et al., ; ronnqvist et al., ) use hnovs in the original % fecal suspension for their studies. the presence or absence of organic matter can certainly impact both virus persistence and recovery; however, it should also be noted that the presence of organic matter could also impact downstream analysis such as rt-qpcr via inhibition (wilson ) , also indicated in section . . even though virus persistence and recovery from food matrices are not within the scope of this review, enteric virus recovery from nonporous environmental surfaces as a function of particle association (e.g., food and debris) is lacking and does need further study. drying time for enteric virus surface sampling is highly variable and dependent on factors including volume of virus suspension and desiccation (table ) . drying times range from min to overnight at ambient conditions with volumes ranging from μl to μl. drying time impacts the recovery efficiencies of surface sampling methods, and generally, the longer a virus is on a surface, the harder it is to recover the virus from the surface. mattison et al. ( ) tested recovery of fcv from stainless steel surfaces using vortexing at min post inoculation versus immediate recovery after inoculation of . × fcv in μl. the difference in recovery between elution immediately following and after min of drying was and %, respectively-a three-fold difference. while this review is focused on fcs and not food, the authors did note that the difference between viral recovery from lettuce and stainless steel may be due to viruses being more influenced by the effects of air drying when on a flat nonporous surface. park et al. ( ) observed a reduction in the recovery efficiency of hnov gii. from stainless steel and toilet representative surfaces as a function of drying time. on stainless steel surfaces using macrofoam swabs, the recovery efficiency was . % ± . % without drying, . % ± . % at h, . % to . % ≤ h, and . % ± . % after h (park et al., ) . based on the evidence presented above, there is a need for uniformity among studies and standardization in drying time and inoculum amount in order to properly evaluate virus recovery and surface sampling methods. the recovery efficiencies for the numerous eluent-tool combinations are variable and often impacted by both intrinsic factors related to the actual tool and eluent types as well as the extrinsic factors already introduced (sections . . - . . ). the differences in eluent formulations such as ph, salinity, and use of a surfactant can impact the recovery efficiency of viruses from surfaces. ionic strength and ph of eluents have been known to impact the net charge of viral particles (gerba, ) . rönnqvist et al. ( ) obtained slightly higher recovery efficiencies using an alkaline glycine buffer (ph . ) than eluting with pbs (ph . ). conversely, taku et al. ( ) recovered more fcv from stainless steel surfaces using a slightly acidic glycine buffer (ph . ) with a mean recovery of % compared to and % recovery using glycine buffer (ph . ) or culture medium (ph . ), respectively. surfactants are another common component added to elution buffers. these are known to increase the water content of the surface, assist in solubilization of proteins and cells from the surface, and can disrupt hydrophobic interactions between charged viruses and surfaces thus enhancing virus recovery (farrah ; lukasik et al., ; moore and griffith ) . park et al. ( ) suggested that adding a surfactant ( . % tween ) to the pbs elution buffer of a swab rinse protocol enhanced viral recovery efficiency of hnov gii. even though no significance was observed. meanwhile, another study found higher recovery of hnov gii. and mengovirus from laminated wooden surfaces when using lysis buffer compared to mm tris-hcl − mm glycine − . % beef extract (tgbe, ph . ); however, again no significance difference was observed (ibfelt et al., ) . for ms recovery, two separate studies found the eluent type to not be significantly different (casanova et al., ; julian et al., ) . furthermore, eluent type for ms recovery was suggested to be selected based on experimental design such as considering eluents compatible with nucleic acid extraction for molecular detection-based sampling studies or with tissue culture for infectivity-based studies (julian et al., ) . moreover, rönnqvist et al. ( ) suggested an elution buffer be selected based on the specific situation with the consideration of factors such as the time elapsed between swabbing and sample analysis. overall, eluent type can impact viral recovery, and thus eluent-tool combinations must be chosen with consideration of surface, virus, and eluent interactions for efficient surface sampling and recovery. therefore, a matrix of elution buffers and when to apply given a certain situation or parameters would be a valuable resource. the majority of tools used in laboratory-based studies for evaluation of surface sampling methods have focused on various types of swabs (table ). this finding comes as no surprise since swabbing is known as the gold standard for hnov sampling and detection on fcs (iso, ). evaluation of swabs has shown varying recovery rates for enteric viruses; however, while the swab itself may be the primary driver in recovery, numerous other factors can play a role as indicated previously. more specifically, the material and properties of the recovery tool can impact recovery efficiencies. for example, the dying process of microfiber cloths can change its net surface charge, which could impact viral attachment and detachment from surfaces (rönnqvist et al., ) . taku et al. ( ) suggested the selection of swabs are due to the ease of operation over small surface areas even though swabs yield consistently poor results in comparison to other methods evaluated, possibly due to surface area of the swab head and smearing virus over surfaces. macrofoam, polyester-tipped, and/or cotton swabs have been shown to be more efficient among swabs tested in viral recovery from fomites depending on a given study's conditions and parameters (ibfelt et al., ; julian et al., ; scherer et al., ) . for instance, julian et al. ( ) reported that polyester-tipped swabs recovered a greater amount of infectious ms than antistatic cloths. however, as indicated in section . . , the elution buffer and tool combination complicates matters. for instance, rönnqvist et al. ( ) reported that elution buffer type only impacted the recovery efficiency of microfiber cloths composed of polyester and polyamide materials where mm glycine buffer (ph . ) performed better than pbs. additionally, the authors reported better recovery of low inoculum hnov gii. on latex surfaces when using polyester swabs, though it is unclear why. unfortunately, it is difficult to compare swab types across studies due to differences among surface types, virus types, virus volume, and virus concentrations used for the evaluations of the swab sampling protocols. as evidenced by table , surface sampling methods used in the recovery of enteric viruses are highly variable and diverse. a majority of studies focus on swabbing for a variety of reasons. in fact, the international organization of standardization ( ) recommends hnov sampling and detection on nonporous fcs to be collected with a cotton swab moistened with pbs followed by rna extraction and reverse transcriptionquantitative pcr (rt-qpcr) analysis. other tools and methods such as repeated pipetting, cell scraping, and sonication/stomaching have been used for viral persistence and disinfection studies (arthur and gibson ; mattison, ; yeargin et al., ) . studies involving environmental surface sampling for applications in detecting viruses during outbreaks can be used as a baseline for standard surface sampling techniques for enteric viruses. swabbing is the technique typically used for enteric virus studies involving applications in detection of viruses during outbreaks. thus, studies have focused on evaluating swab protocols on surfaces associated with outbreaks such as on cruise ships and fcs (table ) . rönnqvist et al. ( ) evaluated four swab types (e.g. flocked nylon, cotton wool, microfiber, and polyester) in either pbs or glycine buffer at ph . for collecting hnov gii. from stainless steel and plastic surfaces. park et al. ( ) evaluated five swab types (e.g. cotton, rayon, polyester, antistatic cloth, and macrofoam) using hnov gii. from stainless steel and toilet representative surfaces with macrofoam swabs producing the highest recovery efficiencies. during comparison of these two studies, microfiber performed better than macrofoam swabs with . % ± . % and . % ± . % recovery efficiency, respectively, when elution buffer (glycine buffer) and surface type (stainless steel) were the same. however, the amount and concentration of hnov gii. varies between the two studies, and this could also impact recovery efficiencies as reviewed in section . . . rönnqvist et al. ( ) also provides information on using swabs on plastic surfaces. overall, there is a need for more studies involving more viruses and nonporous surfaces to properly determine a standardized approach for surface sampling of enteric viruses during outbreaks. several different methods have been used to optimize recovery of enteric viruses from inanimate fomites in laboratory-based persistence studies. furthermore, differences among the studies include virus types, volume and concentration of virus as well as tools, fcs, and type of analysis. in this subsection, we will further examine these differences and how they could contribute to the varying results of surface sampling method evaluation studies. summaries of these studies are available in table . as stated in section . , swabbing has traditionally been the focus in studies on virus detection and persistence (table ) . a few studies focused on evaluating one swab implement for use in recovering enteric viruses from a variety of surface types and virus inoculum levels. scherer et al. ( ) evaluated a cotton swab with pbs (ph . ) elution buffer for collecting hnov gii. and rotavirus from different fcs (i.e. stainless steel, ceramic, high-density polyethylene, and wooden chopping board) with recovery efficiencies ranging from . ± . % (wood, pcru) to . ± . % (ceramic, pcru) for gii. and . ± . % (wood, tcid ) to . ± . % (ceramic, t-cid ) for rotavirus. the authors found recoveries for both hnov and rotavirus to be higher from fcs than food surfaces at both inoculum concentrations (scherer et al., ). additionally, ganime et al., ( ) evaluated the recovery rates of mnv- and bacteriophage pp from porous formic, non-porous formic, and rubberized surfaces using a rayon swab with culture media with recovery efficiencies ranging from . to . % (pp ) and . - . % (mnv- ). while these two studies evaluate how one particular swab performs, other studies expand their evaluations to provide a better comparison of different swabs and tools and their recovery of particular enteric viruses. for example, ibfelt et al. ( ) evaluated three different swabs (i.e. cotton, foamed cotton, and polyester) and two elution buffers (i.e. direct lysis or alkaline tgbe − ph . ) for recovery of hnov gii. and mengovirus from cm laminated wooden surfaces. the authors found a significantly better virus recovery using polyester swabs with the direct lysis in comparison to other combinations tested; however, recovery efficiencies were ≤ % for all combinations. ibfelt and others ( ) suggested their low recovery rates may be due to the size of the surface or differences in experimental design in comparison to other swab studies. furthermore, julian et al. ( ) also recommended the use of polyester swabs pre-moistened in either ringer's or . % saline solution for ms recovery from plastic and stainless steel surfaces following evaluation of three tools (cotton swab, polyester swab, and antistatic cloth) and four elution buffers (saline, ringer's solution, viral transport media, and acid/base). based on a meta-analysis of ms surface sampling, the authors noted that polyester swabs obtained significantly higher positive ms rates in comparison to rayon and cotton (julian et al., ). conversely, de keuckelaere et al. ( found cotton and polyester swabs to not be significantly different in their recovery efficiencies of hnovs gi. and gii. from nitrile gloves, polyethylene, or neoprene rubber surfaces. park et al. ( ) reported a similar result when evaluating the recovery efficiencies of four swab types (macrofoam, rayon, cotton, and polyester). the authors applied the different swabs for recovery of hnov gii. from stainless steel and toilet representative surfaces and found that rayon, cotton, and polyester were not significantly different. however, macrofoam swabs obtained significantly higher recovery efficiencies of hnov gii. in comparison to the other three swabs after h of drying on a given surface (park et al., ) . additionally, some studies found other tools and methods such as biowipes and cell scraper-aspiration methods to be potentially more efficient for enteric virus recovery from surfaces in comparison to cotton and/or polyester swabs. these studies are further examined in sections . . and . . (de keuckelaere et al., ; taku et al., ) . cloths and wipes have also been introduced as possible alternatives to swabbing methods for obtaining higher recovery efficiencies of enteric viruses from surfaces. de keuckelaere et al. ( ) evaluated two swabs (cotton and polyester) along with biowipes (biomérieux, lyon, france) composed of a mixture of fibers and microfibers (cotton, polyester, and polyamide fibers) moistened in pbs (ph . ) by recovering gi. and gii. hnovs from fcs (high-density polyethylene, nitrile gloves, and neoprene rubber). there was no significant difference among any of the three tools evaluated based on recovery efficiency from polyethylene surfaces and nitrile gloves for hnov gi. . meanwhile, the authors found significantly higher recovery efficiencies using biowipes ( . ± . %) compared to cotton swabs ( . ± . %) on the coarser rubber surface (de keuckelaere et al., ) . the authors also found that the mean recovery efficiency of biowipes for gi. from rubber surfaces was higher than using polyester swabs even though no significant difference was observed. for hnov gii. , there was no significant difference in recovery observed between all three tools tested on polyethylene surfaces and nitrile gloves even though the biowipes had significantly higher recovery efficiency ( . ± . %) on rubber surfaces compared with both polyester ( . ± . %) and cotton ( . ± . %) swabs (de keuckelaere et al., ). another study further confirmed the effectiveness of these biowipes in collecting hnov gii. at various inoculum concentrations ( to pcru) from stainless steel and ceramic fcs (tung-thompson et al., ) . the authors reported a range of mean recovery efficiencies of gii. using biowipes (biomerieux sa, grenoble, france): . - . % (stainless steel) and . - . % (ceramic). it should be noted that recovery efficiencies reported by tung-thompson et al. ( ) were generally much higher than other studies included in table . however, a few studies showed certain swabs to be more efficient for recovery of enteric viruses than cloths. for example, macrofoam swabs had a higher recovery efficiency of hnov gii. ( . ± . %) from large ( . cm ) stainless steel surfaces than antistatic cloths ( . ± . %) (park et al., ) . additionally, julian et al. ( ) determined that polyester swabs obtained higher recoveries of infectious ms than antistatic cloths as well. overall, cloths and wipes may be a valuable tool for collecting enteric viruses from fcs, and there is a need for further studies using cloths and wipes involving a greater variety of virus types, cloth types, surface types, and infectivity analyses. other surface sampling methods such as vortexing, repeated pipetting, stomaching/sonication, and cell scraping have been used for baseline information for viral persistence studies and disinfection studies (table ). the studies summarized in table use different surrogates, initial drying times, and elution buffers making it difficult to adequately compare the studies. fallahi and mattison ( ) recovered % of mnv- from stainless steel after a min drying time using a repeated pipetting method with ebss eluent. mattison et al. ( ) recovered % of fcv from stainless steel after a min drying time by vortexing for s in ebss eluent. arthur and gibson ( ) obtained recovery efficiencies of % and % for tuv from acrylic and stainless steel surfaces, respectively, after a drying time of h using a cell scraping techniques. the cell scraping technique was confirmed as possible with tuv and has also been evaluated using fcv previously (taku et al., ) . taku et al. ( ) found consistently better mean virus efficiencies for fcv using mm glycine (ph . ) from stainless steel surfaces in comparison to mm glycine (ph . ) and modified eagle's medium (ph . ) using the scraping-aspiration method. the mean fcv recovery efficiencies for the scraping-aspiration method from stainless steel were reported to be % (glycine ph . ), % (glycine ph . ), and % (modified eagle's medium). the authors suggested the modified eagle's medium complex composition may have played a role in being less efficient than the glycine buffers (taku et al., ) . taku et al. ( ) added cell scraping to the aspiration method for better recovery efficiencies speculating that cell scraping may facilitate release of virus from surface. in addition, yeargin et al. ( ) recovered a range of . % (cotton) to . % (glass) for fcv and . % (cotton) to . % (glass) for mnv- from three surface types (i.e. polyester, cotton, and glass) using a stomaching/sonication method. the authors also found the recovery efficiencies to be highest for glass and lowest for polyester and cotton for both virus types. the recovery efficiencies were also reported to be significantly different among all surface types for the same virus type while only cotton swab recoveries showed a significant difference between mnv- and fcv (yeargin et al., ) . similar to other techniques, more studies with inclusion of more virus types and standardized drying times are needed to provide information on using these alternative techniques for future persistence and environmental sampling studies. surface sampling of enteric viruses varies across studies throughout the literature. this variability in results may exist due to varying human behavior, the tool used, and/or the elution buffer type used to recover the virus from the surface as well as numerous other factors outlined in the present review. most surface sampling evaluations have focused on various swab types while there are limited studies focused on evaluation of other possible tools and techniques such as repeated pipetting and cell scraper application, historically used in a laboratory setting. as a result, food and environmental virology researchers may have difficulty in selecting the most appropriate surface sampling method for a particular study. additionally, we found that no single standard approach to recover enteric viruses from fcs exists. the following suggestions are based on our review to assist researchers in moving towards one standard methodology for optimizing the recovery of enteric viruses from fomite surfaces: • eluent buffer used to recover sample needs to be standardized. • concentrations and volumes of virus need to be more consistent and include standard low and high inoculum levels. • the impact of organic materials on enteric virus recovery from surfaces needs further investigation. • infectivity assays such as plaque assays are highly recommended for the analysis of surface sampling optimization in order to distinguish infectious particles from non-infectious viral particles. however, this is currently only possible with cultivable viruses and hnov surrogates. • results need to be reported in one standard form of measurement. • more techniques and tools need to be evaluated along with the swab protocols and these evaluations should include of a variety of human enteric viruses and their surrogates. survival of enteric viruses on environmental fomites global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis environmental persistence of tulane virus: a surrogate for human norovirus significance of fomites in the spread of respiratory and enteric viral disease year-round prevalence of norovirus in the environment of catering companies without a recently reported outbreak of gastroenteritis norovirus: specimen collection surrogates for the study of norovirus stability and inactivation in the environment: a comparison of murine norovirus and feline calicivirus methods for the recovery of a model virus from healthcare personal protective equipment possible prolonged environmental survival of small round structured viruses virus transmission via food semi-direct lysis of swabs and evaluation of their efficiencies to recover human noroviruses gi and gii from surfaces persistence and transferability of noroviruses on and between common surfaces and foods replication of human noroviruses in n stem cell-derived human enteroids evaluation of murine norovirus persistence in environments relevant to food production and processing chemical factors influencing adsorption of bacteriophage ms to membrane filters a rapid procedure for detecting noroviruses from cheese and fresh lettuce environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season applied and theoretical aspects of virus adsorption to surfaces norovirus gastroenteritis. new eng evaluation of the swab sampling method to recover viruses from fomites division of viral diseases, national center for immunization and respiratory diseases, c.d.c., . vital signs: foodborne norovirus outbreaks -united states microbiology of food and animal feed-horizontal method for determination of hepatitis a virus and norovirus in food using real-time rt-pcr-part : method for quantification test and validation of methods to sample and detect human virus from environmental surfaces using norovirus as a model virus comparison of surface sampling methods for virus recovery from fomites survival of enteric viruses in water and foodstuffs and on various surfaces a critical review of methods for detecting human noroviruses and predicting their infectivity foodborne viruses: an emerging problem influence of salts on virus adsorption to microporous filters attachment of listeria monocytogenes to stainless steel, glass, polypropylene, and rubber surfaces after short contact times characterization of physicochemical forces involved in adhesion of listeria monocytogenes to surfaces survival of calicivirus in foods and on surfaces: experiments with feline calicivirus as a surrogate for norovirus problems associated with traditional hygiene swabbing: the need for in-house standardization evaluation of a new environmental sampling protocol for detection of human norovirus on inanimate surfaces noroviruses on surfaces: detection, persistence, disinfection and role in environmental transmission swabs as a tool for monitoring the presence of norovirus on environmental surfaces in the food industry survival of human enteric viruses in the environment and food improved detection of human enteric viruses in foods by rt-pcr foodborne illness acquired in the united states-major pathogens application of a swab sampling method for the detection of norovirus and rotavirus on artificially contaminated food and environmental surfaces concentration and detection of caliciviruses from food contact surfaces efficacy of commonly used disinfectants for inactivation of human noroviruses and their surrogates evaluation of a surface sampling method for recovery of human noroviruses prior to detection using reverse transcription quantitative pcr reported behavior, knowledge and awareness toward the potential for norovirus transmission by food handlers in dutch catering companies and institutional settings in relation to the prevalence of norovirus outbreaks of acute gastroenteritis transmitted by person-to-person contact, environmental contamination, and unknown modes of transmission-united states inhibition and facilitation of nucleic acid amplification recovery and disinfection of two human norovirus surrogates, feline calicivirus and murine norovirus, from hard nonporous and soft porous surfaces this review is based upon work that is supported by the arkansas biosciences institute with the grant received by author gibson key: cord- - zyy y authors: lorusso, alessio; faaberg, kay s.; killian, mary lea; koster, leo; vincent, amy l. title: one-step real-time rt-pcr for pandemic influenza a virus (h n ) matrix gene detection in swine samples date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: zyy y in the spring of , a novel (h n ) influenza a virus began to spread among humans worldwide. although the h n is related genetically to swine influenza viruses, human infection has not been connected to pig exposure. because the virus is now circulating widely in the human population, swine herds are at increased risk of becoming infected. in order to investigate potential outbreaks of the pandemic virus in pigs, a quantitative real-time reverse transcriptase-polymerase chain reaction (qrt-pcr) for the detection of the (h n ) rna in clinical specimens was developed. to evaluate the applicability of the test as a diagnostic tool in the screening of field specimens from swine, field isolates of north american swine, equine and avian influenza viruses collected during diagnostic investigations were analyzed retrospectively as well as samples collected during an experimental in vivo infection with two novel h n isolates, a/california/ / (h n )v virus and a/mexico/ / (h n )v. the sensitivity of the qrt-pcr was shown to be higher with respect to standard techniques such as virus isolation and the reproducibility was satisfactory. the present unique and highly sensitive assay is able to detect as little as × ( ) copies of rna per μl of template and it represents a rapid and useful approach for the screening and quantitation of (h n ) rna in porcine specimens. swine influenza is an acute respiratory disease caused by influenza a viruses that belong to orthomyxoviridae, a family of enveloped negative-sense, segmented, single stranded rna viruses. based upon the major differences within the hemagglutinin (ha) and neuraminidase (na) proteins, ha and na subtypes have been identified thus far (rohm et al., ; webster et al., ; fouchier et al., ) . it is recognized that influenza viruses evolve by reassortment and/or point mutation, thus giving rise to new viral subtypes with different host tropism. in april , a novel swine-lineage influenza virus capable of rapid human transmission was reported, although infection with (h n ) was not connected to pig exposure or to a contemporary infection in the swine population (dawood et al., ). this novel pandemic h n possessed a unique genome arrangement. six genes, including pb , pb , pa, ha, np and ns, cluster together with those belonging to the viruses identified as triple-reassortant swine influenza viruses of the north american lineage, whereas the m and na genes are derived from eurasian lineage swine influenza viruses (dawood et al., ) . other than sporadic transmission to humans (myers et al., ) , classical swine influenza a viruses of the h n subtype were historically distinct from avian and other mammalian influenza viruses based on host specificity, serotype, and/or genotype (vincent et al., ) . swine influenza virus was first recognized as an agent of respiratory disease in pigs in (shope, ) , and the north american swine influenza virus-lineage genes of the pandemic virus have its genetic origins with this ancestral h n . three predominant swine influenza virus subtypes are currently circulating in us swine following the emergence of the triple reassortant h n in : reassortant h n (rh n ), h n , and h n and their drift mutant derivatives, all containing the triple reassortant internal gene cassette (trig) (for review see vincent et al., ) . the novel (h n ) is not known to be circulating widely among swine. pigs have been shown to be susceptible to the human pandemic (h n ) infection (lange et al., ; vincent, unpublished data) . the chance of cross-species transmission may lead to serious consequences in terms of human risk of infection by increasing the reservoir of the virus in addition to dramatic costs for the pork industry. swine have been shown to possess receptors for avian and human influenza viruses in the tracheal epithelium, leading to the suggestion that the pig is a mixing vessel for the emergence of new subtypes with human pandemic potential (ito et al., ; scholtissek et al., ) . in order to recognize promptly the novel pandemic (h n ) in swine, reducing the potential serious economic damage as well as exposure of humans to the virus, the development of a rapid and sensitive test capable of identifying and differentiating the pandemic strain from type a influenza viruses circulating in pigs is necessary. in this manuscript the development of a quantitative real-time reverse transcriptase-polymerase chain reaction (qrt-pcr) using taqman technology for the rapid and sensitive detection of pandemic (h n ) matrix gene and quantification of viral nucleic acid in diagnostic samples, is reported. for this purpose, field isolates of north american swine, equine and avian influenza viruses were analyzed retrospectively as well as samples (swabs and lavage fluid) collected during an experimental in vivo infection with a/california/ / (h n )v and a/mexico/ / (h n )v isolates. data obtained by the qrt-pcr analysis were compared with those achieved from virus isolation of the clinical samples collected during the in vivo study. the matrix (m) gene sequences of endemic swine influenza virus isolates, novel pandemic (h n ) , and sequences from a panel of human and avian type a influenza virus strains, including type a human seasonal strains, were retrieved from the genbank database (http://www.ncbi.nlm.nih.gov/genbank/index.html) and aligned using the dnastar software package (dnastar inc., madison, wi, usa). the primers were designed using the geneious software (biomatters, ltd.) to amplify a conserved bp within the aligned m genes. however, the probe was purposely designed using the sequence of a/california/ / (h n ) (fj ), in a conserved, yet lineage-specific region shared by all pandemic (h n ) isolates sequenced thus far. the primers and probe were synthesized by idt (coralville, ia, usa). the taqman probe was dual-labeled with -carboxyfluorescein (fam) at the end and with tetramethylrhodamine (tamra) at the end. the position and sequence of primers and probe used for the assay are reported in table . to obtain a standard for the taqman assay, a -bp rt-pcr product containing the full-length m gene of a/california/ / (h n ) virus was amplified using primer pair m+ and m- (hoffmann et al., ) , and the rt-pcr product was cloned into pgem ® -t easy vector system (promega, madison, wi, usa), then linearized and transcribed with ribomax tm large scale rna production system-t (promega, madison, wi, usa), from the t promoter, according to the manufacturer's guidelines. after dnase treatment to remove residues of plasmid dna, the transcripts were purified using a commercial column (rneasy kit, qiagen s.p.a., germantown, md, usa) and quantified by spectrophotometric analysis. tenfold dilutions of the rna transcript, representing to copies rna l − of template, were prepared in sterile water, and aliquots of each dilution were frozen at − • c. each aliquot was used only once. to evaluate the applicability of the test as a diagnostic tool for the screening of field specimens, field isolates of north american swine, equine and avian influenza viruses, collected during diagnostic investigations and samples collected during an experimental in vivo study were examined. the in vivo study was conducted in two separate groups of -week-old pigs inoculated with two pandemic (h n ) isolates, a/california/ / (h n )v (pigs - ) and a/mexico/ / (h n )v (pigs - ), respectively, kindly provided by the centers for disease control and prevention (cdc). all pigs came from a herd free of swine influenza virus and porcine reproductive and respiratory syndrome virus (prrsv). they were treated with ceftiofur crystalline free acid (pfizer, new york, ny, usa) to reduce bacterial contaminants preceding the start of the study. the two groups were housed in individual isolation rooms at a-bsl and cared for in compliance with the institutional animal care and use committee of the national animal disease center. pigs were humanely euthanized with a lethal dose of pentobarbital (sleepaway, fort dodge animal health, fort dodge, ia, usa) at the appropriate time during the course of the study. thirty pigs, per group, were inoculated intratracheally with × tcid of a/california/ / (h n )v and × tcid of a/mexico/ / (h n )v, both isolated and prepared on mdck cells. five pigs remained nonchallenged as negative controls. the pigs were anesthetized by intramuscular injection of a cocktail of ketamine ( mg/kg), xylazine ( mg/kg) and telazol ( mg/kg, fort dodge animal health, fort dodge, ia, usa) followed by virus inoculation. pigs were observed daily for clinical signs and sample collection. nasal swabs were taken and placed in ml minimal essential medium (mem) on , , , and dpi to evaluate nasal virus shedding and stored at − • c until the end of the study. five inoculated pigs per group were euthanized on , , and dpi and five control pigs were euthanized on dpi. after euthanasia, each lung was lavaged with ml of mem to obtain bronchioalveolar lavage fluid (bal fluid). each nasal swab sample was subsequently thawed and vortexed for s, centrifuged for min at × g and the supernatant passed through . m filter. subsequently, l of the nasal swab sample was then placed on confluent mdck cells in -well plates to incubate for h. after h of incubation the sample was removed and l mem w/tpck trypsin was added. the plate was checked at and h for cytopathic effects. after h, l of cell culture supernatant from each well of the -well plate was subsequently passed onto a confluent -well plate after a freeze and thaw cycle. after h evidence of cytopathic effects was evaluated and presence of virus antigen confirmed by immuno-cytochemical staining with an anti-influenza a nucleoprotein monoclonal antibody as described previously (kitikoon et al., ) . tenfold serial dilutions in serum-free mem supplemented with tpck trypsin and antibi-otics were made with each bal fluid sample. each dilution was plated in triplicate in l volumes onto pbs-washed confluent mdck cells in -well plates. plates were evaluated for cpe between and h post-infection. at h, plates were fixed with % phosphate-buffered formalin and stained using immunocytochemistry as above. rna isolation was carried out from cell culture-grown virus isolates, nasal swab filtrates in mem, and bal fluid with the magmax tm - total rna isolation kit (ambion, austin, tx, usa) in accordance with the manufacturer's protocol. template rna was eluted in l of buffer and stored at − • c prior to use. duplicates of each rna sample and standard were amplified by the qrt-pcr assay performed on a real-time pcr system (applied biosystem) with the agpath-id tm one-step rt-pcr kit (ambion, austin, tx, usa) with rox added as passive reference dye. the l reaction volume for each sample contained l of extracted rna, . l of agpath kit × buffer, l of agpath × enzyme mix, nm of primers m( )-for and m( )-rev, nm of m( )-probe, . l of agpath detection enhancer and . l of ultrapure dnase-rnase-free distilled water. the thermal profile consisted of a single cycle of reverse transcription for min at • c and min at • c for reverse transcriptase inactivation and dna polymerase activation. the amplification of cdna was performed by cycles including denaturation at • c for s, annealing for min at • c and extension at • c for s. the increase in fluorescent signal was registered during the annealing step of the reaction and the data were analyzed with sequence detector software ( system software v. . . , applied). data reported represent the average of the duplicates for each sample and standard. in order to exclude cross-reactivity between pandemic (h n ) and other viruses responsible for respiratory diseases of pigs, (h n ) qrt-pcr test specificity was evaluated by analysis of the following: endemic swine influenza virus, avian and equine influenza viruses, coronaviruses (porcine respiratory coronavirus (prcov), transmissible gastroenteritis virus (tgev)), prrsv, porcine circovirus type (pcv ), porcine adenovirus, porcine parvovirus, blue-eye paramyxovirus and pseudorabies virus. nasal swab and bal fluid samples collected from five uninfected pigs as well as sterile water were also included in the analysis as negative controls and no-template controls, respectively. to determine the detection limit of the (h n ) qrt-pcr assay, -fold dilutions of a bal fluid sample containing × copies of a/mexico/ / (h n )v rna l − were made and subsequently analyzed. serial -fold dilutions of standard rna which contained from to copies of rna transcript and the corresponding c t values were used to plot the standard curve for the pandemic (h n ) rna absolute quantification. reproducibility of the assay was evaluated by testing several clinical samples containing a/california/ / (h n )v rna quantities that included the full range covered by the qrt-pcr. the intra-assay reproducibility was measured by testing the same samples times in the same experiment, whereas the inter-assay reproducibility was confirmed by testing the same samples in independent experiments. coefficients of variation (cvs) were calculated by dividing the standard deviation of each tested sample by its mean and multiplying that result by (decaro et al., (decaro et al., , . swine and equine influenza virus isolates and the clinical samples from pigs infected experimentally with (h n )v were subjected to the usda-validated qrt-pcr procedure for the general detection of type a influenza virus rna (matrix screening assay), following procedures described previously (spackman and suarez, ) . the no-template controls and (h n ) negative specimens did not produce detectable fluorescence signal. the detection limit of the assay was assessed as × rna copies l − , whereas, in general, gel-based rt-pcr is limited generally to detect to × copies l − of template. tenfold dilutions of standard rna were used to create a standard curve representing to copies of viral rna standards and linearity was observed over the entire quantitation range (slope = − . ). the coefficient of linear regression (r ) was . . in order to verify the reproducibility of the assay, intra-assay and inter-assay cvs were calculated and satisfactory results were obtained. intra-assay cvs ranged from % (samples containing × rna copies) to % (samples containing × rna copies), whereas the inter-assay cvs ranged from % ( × rna copies) to % ( × rna copies). north american swine influenza virus isolates as well as equine influenza virus isolates were successfully detected by the usda-validated qrt-pcr (data not shown). all endemic north american swine influenza virus isolates were negative for (h n ) specific matrix gene rna using the present qrt-pcr assay, whereas the (h n ) strains used as positive control were positive. cross-reactivity with other extant swine viral pathogens was not detected. amplification of equine and avian influenza virus for (h n ) remained below detection threshold, although weak cross-reactivity was observed at later cycle numbers. a single avian influenza isolate (a/mynah/mass/ h n ) was detected by (h n ) qrt-pcr. in the group of pigs infected with a/california/ / (h n )v, / nasal swabs (table ) and / bal fluids (table ) were positive for isolation on cell culture. one nasal swab sample as well as all bal fluid samples at dpi were negative by virus isolation. in the group infected with a/mexico/ / (h n )v virus, virus was isolated from / nasal swabs and from / bal fluid samples. seven nasal swabs collected at dpi, collected at dpi, and all bal fluids collected at dpi were negative for virus isolation. the qrt-pcr results from the clinical specimens from the in vivo study are summarized in tables and qrt-pcr and negative by using vi whereas only samples were positive by vi and negative by (h n ) qrt-pcr. the samples analyzed by the (h n ) qrt-pcr contained a wide range of (h n ) rna copies per l of template, from . × to . × (bal fluid), and from × to . × (nasal swabs). the (h n ) is of significant human and swine health concern and the future role of pigs in the ecology of this newly emerged virus remains unknown. there is an immediate and critical need for a rapid differential diagnostic method for pandemic (h n ) virus detection in swine. pandemic (h n ) isolated from humans has had limited detection in the swine population so far (http://www.oie.int/eng/en index.htm). however, pigs are susceptible to the infection, as demonstrated by the clinical signs and viral loads that found in nasal swabs and bal fluids at day , , and dpi. similar results have been described by others (lange et al., ) . it is likely that the virus will continue to jump from humans to naïve pigs and may become established as an endemic infection in the swine population. in that case, two consequences will be obvious: first, a reservoir of h n virus in the swine population poses an elevated risk for human infection via aerosol transmission from clinically ill pigs, and second, dramatic economic losses for the pork industry due to direct disease related costs as well as indirect market losses. the long-term consequence is the increased chance for novel reassortment between endemic swine influenza viruses and the novel h n in the swine host, posing further human and animal health risks. it is apparent that pigs may be infected at least transiently with wholly avian and/or human viruses, allowing reassortment with swine viruses to acquire avian and/or human virus gene segments (karasin et al., ; ma et al., ) . the (h n ) underscores the potential risk to the human population of other influenza virus subtypes and genotypes with the swine influenza virus trig backbone. increased surveillance and monitoring for the (h n ) as well as other swine influenza virus in both the swine and human populations are critical to under-stand the dynamic ecology of influenza a viruses in susceptible host populations. in the event of the pandemic virus spread in the swine population, the assessment of a specific innovative diagnostic tool that permits a rapid identification of the pandemic (h n ) in pigs is an absolute necessity. in fact, a sensitive and specific diagnostic test is critical for the implementation of response measures to outbreaks in swine to reduce human risk of infection. the qrt-pcr assay described is able to detect the new pandemic (h n ) viral rna with the ability to differentiate the new lineage from the extant swine influenza viruses circulating in the north american swine population. the assay was shown to be reproducible and linear over a range of orders of magnitude, from to rna copies, thus ensuring an accurate measurement of (h n ) viral loads in clinical samples. if compared with the classical gelbased rt-pcr protocol, the processing time required by taqman rt-pcr is shorter, the contamination risks are lower because of the lack of post-amplification steps, and the specificity is enhanced by the probe hybridization. the specificity of the (h n ) qrt-pcr was assessed against a set of viruses associated with respiratory disease in swine, including endemic north american swine influenza virus isolates, prcov, tgev, prrsv, porcine circovirus type , swine adenovirus, porcine parvovirus, blue-eye paramyxovirus and pseudorabies virus. cross-reaction was not identified in swine specimens, thus providing evidence for high fidelity of the assay for the exclusive detection of (h n ) in clinical samples from swine. there is a potential to detect matrix genes not of the classical swine lineage, especially those of avian lineage including the avian-like eurasian lineage viruses. since no eurasian avianlike matrix genes have been reported in the us, any identification of avian-like or equine-like matrix genes would be a novel finding and should be investigated by further molecular diagnostics such as sequencing. importantly, no endemic us swine influenza viruses tested here were shown to have the eurasian swine influenza viruslineage matrix gene, indicating the (h n ) virus was not circulating in the us prior to based on current knowledge. further testing of swine influenza virus repositories at veterinary diagnostic laboratories is warranted to rule out the existence of this lineage of viruses in north america prior to . the (h n ) qrt-pcr was shown to be more sensitive with respect to vi in pigs inoculated experimentally over the duration of the shedding period. indeed, samples were demonstrated to be negative by vi but positive by qrt-pcr. however, nasal swabs positive by vi were negative when tested by both qrt-pcr assays. the qrt-pcr may not be more sensitive than vi early in the course of infection when viral titers are extremely low, but it is more rapid and more specific and was more sensitive later in the course of infection. additional testing in the diagnostic laboratory setting is necessary to compare further the (h n ) qrt-pcr with vi. indeed, the titers of the three qrt-pcr-negative samples were very low, . , . and . tcid /ml. vi is recognized as the gold standard for the detection of influenza viruses but is time-consuming and labor intensive, and lacks specificity; thus the qrt-pcr assay described here can be useful in (h n ) outbreaks, experimental challenge studies, and vaccine trials as well. although a large collection of (h n ) virus isolates were not available in our laboratories for testing, analysis of published m-gene sequences of strains from worldwide geographical areas allowed us to pinpoint a novel lineage-specific nucleotide sequence for diagnostic development. the sensitivity of the (h n ) qrt-pcr is comparable to the canonical usda-validated type a influenza virus assay reported by spackman and suarez ( ) , thus encouraging the use of both assays, first for influenza a screening, followed by differentiation and quantification of pandemic (h n ) rna in clinical samples as described here. in fact, while the type a influenza virus real-time rt-pcr matrix screening assay is able to detect all viral isolates tested in this study, the (h n ) qrt-pcr selectively detects only the novel pandemic (h n ) viral rna. this assay can be a powerful tool in the diagnostic laboratory setting for specific simultaneous analysis of up to samples on the same plate in minimal time. quantitation of canine coronavirus rna in the faeces of dogs by taqman rt-pcr genotype-specific fluorogenic rt-pcr assays for the detection and quantitation of canine coronavirus type i and type ii rna in faecal samples of dogs characterization of a novel influenza a virus hemagglutinin subtype (h ) obtained from black-headed gulls universal primer set for the full-length amplification of all influenza a viruses molecular basis for the generation in pigs of influenza a viruses with pandemic potential genetic characterization of h n influenza viruses isolated from pigs in north america, - : evidence for wholly human and reassortant virus genotypes the immune response and maternal antibody interference to a heterologous h n swine influenza virus infection following vaccination pathogenesis and transmission of the novel swine origin influenza virus a/h n after experimental infection of pigs identification of h n influenza a viruses from swine in the united states cases of swine influenza in humans: a review of the literature emergence of a novel swine-origin influenza a (h n ) virus in humans characterization of novel influenza hemagglutinin, h : criteria for determination of influenza a subtypes analysis of influenza a virus nucleoproteins for the assessment of molecular genetic mechanisms leading to new phylogenetic virus lineages swine influenza iii. filtration experiment and etiology type a influenza virus detection and quantitation by real-time rt-pcr swine influenza viruses a north american perspective evolution and ecology of influenza a viruses the authors thank michelle harland and david michael for technical assistance and dr. becky jepsen, brian pottebaum and jason huegel for assistance with animal studies. isolates of pandemic h n were generously provided by dr. alexander klimov (cdc) and endemic swine influenza virus isolates provided by dr. marie gramer (university of minnesota). mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. funding was provided by usda-ars and dhhs-cdc. key: cord- -k engcx authors: naguib, mahmoud m.; el-kady, magdy f.; lüschow, dörte; hassan, kareem e.; arafa, abdel-satar; el-zanaty, ali; hassan, mohamed k.; hafez, hafez m.; grund, christian; harder, timm c. title: new real time and conventional rt-pcrs for updated molecular diagnosis of infectious bronchitis virus infection (ibv) in chickens in egypt associated with frequent co-infections with avian influenza and newcastle disease viruses date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: k engcx in egypt, currently two geographically restricted genotypes of the infectious bronchitis coronavirus (ibv) are circulating with detrimental effects for poultry industry. a sensitive real-time rt-pcr assay targeting the ibv nucleocapsid gene (n) was developed to screen clinical samples for presence of ibv. conventional rt-pcrs amplifying hypervariable regions (hvrs – and ) of the ibv s gene were developed and amplificates used for nucleotide sequence-based typing of ibv field strains in egyptian chickens directly from clinical samples. in total, fifty samples from poultry comprising swabs, tissues, and allantoic fluid were examined. twenty eight samples from chickens showed ibv-positive results. genetic analysis of the hvrs - of seven samples revealed closest amino acid homology of . - . % in four viruses and . - . % in the others to the previously described egyptian variant ii (eg/ b/ ), while all seven samples shared > . % amino acid homology at the hvr locus with that genotype. in addition, in most of samples a high degree of co-infections with highly pathogenic aiv h n , low pathogenic h n , and newcastle disease was found. mixed infections in this study were detected in out of ibv positive samples. this indicates an intricate situation in egyptian poultry populations with unknown putative synergistic effects on pathogenicity and spread of these pathogens. effective control measures including vaccination may be severely compromised. avian infectious bronchitis virus (ibv) is a member of the genus gammacoronavirus in the coronaviridae family (king et al., ) . ibv induces an acute, highly contagious infectious disease of chicken. ibv is globally distributed and responsible for huge economic losses in the poultry industry. ibv was first reported in north dakota, usa, as a novel respiratory disease affecting chickens (schalk and hawn, ) . ibv infects initially the respiratory tract; for some ibv strains further virus spread may involve kidneys and oviduct causing reduction of growth rate, decreased performance and reduction of egg quality and quantity (cavanagh, ) . also, some strains showing a shift of tissue tropism may cause proventriculitis (yu et al., ) .the infection is spread by aerosols, direct contact and indirectly through contaminated fomites (ignjatovic and sapats, ) . ibv harbors an unsegmented rna genome of positive polarity which is approximately . kb in size and codes for four structural proteins: the spike (s) glycoprotein, the membrane (m) glycoprotein, the nucleocapsid (n) phosphoprotein, and the envelope (e) protein (spaan et al., ) . the n protein is a major structural protein, and highly conserved among different ibv serotypes the spike (s) glycoprotein, an integral membrane protein, is another major structural protein; it is cleaved post translationally into the s (n terminal part) and s fragments. mature s proteins trimerize and form the globular head (s ) and the stalk domain (s ) of the viral peplomer spikes (belouzard et al., ; cavanagh, ) . the s protein carries the receptor binding site and thus plays an important role in both tissue tropism and induction of protective immunity (belouzard et al., ; wickramasinghe et al., ) . numerous distinct serotypes have been described which differ by - % and sometimes up to % in the s protein sequence (adzhar et al., ) . in particular, three hypervariable regions (hvrs; amino acid residues - , - and - ) along the s gene are affected that elicit neutralizing and serotype-specific antibodies koch et al., ; moore et al., ) . variation in these epitopes has been implicated in escape from vaccine-induced immunity (belouzard et al., ) .genotypes of ibv are classified based on the genetic variation of the s gene encoding the spike protein, in particular its s fragment (belouzard et al., ; cavanagh, ; valastro et al., ) . consequently, cross protection between these different serotypes is limited (cavanagh, ; jackwood, ; kuo et al., ; wickramasinghe et al., ) . the s gene therefore is used for ibv strain differentiation (gough et al., ; kingham et al., ) . new s genotypes of ibv that often show antigenic variation and, hence, define new serotypes, appear to emerge frequently in different parts of the world (jackwood, ) . a number of mutation processes account for the emergence and evolution of multiple serotypes (cavanagh et al., ; jackwood, ) including point mutations, insertions, deletions, and also recombination between strains (adzhar et al., ; hewson et al., ) . in egypt, highly pathogenic avian influenza viruses (hpaiv) of subtype h n and co-circulating low pathogenic aiv h n have established endemic status (naguib et al., ) . various chicken flocks are suffering from respiratory disease caused, at least in part, by infection with ibv and ndv (abd el rahman et al., ) . over the last decades, different genotypes of ibv have been recognized in egypt which were related to the massachusetts, d , d , d- and / genotypes (abdel-moneim et al., ; jackwood, ; sheble et al., ) . in , a unique egyptian variant (type isolate: egypt/beni-suef/ ), closely related to an israeli variant strain ii, was identified in different chicken farms and classified as egyptian variant i (abdel-moneim et al., ) . in , yet another genotype, egyptian variant ii (ck/eg/bsu- , / ), was isolated, and representatives of these genotypes have been co-circulating since with the previously detected classical (vaccine like) and variant ibv genotypes as mentioned above (abdel-moneim et al., ) . this study is aimed at providing novel molecular diagnostic tools that can be used to detect and characterize ibv genotypes circulating on chicken farms in egypt. an n gene specific real time rt-pcr (rt-qpcr) specifically tailored to detect ibv circulating in egypt complements with previously published rt-qpcrs targeting another ibv orf e.g., (callison et al., ) . in addition, conventional rt-pcrs for nucleotide sequence analysis of the s gene hvrs have been developed and validated. fifty field samples including tracheal swabs, allantoic fluid (from isolation attempts), and tissues (kidney and trachea) were obtained in the frame of routine veterinary measures from commercial poultry farms showing severe respiratory problems and/or performance losses in egypt between and (table s ). detailed information on species, type, age and mortality is presented in table s . the locations of the farms are depicted in table s at the gover-norate level. swab samples from alive birds were jointly obtained according to standard procedures by authorized veterinarians of the national laboratory for veterinary quality control on poultry production (nlqp, ministry of agriculture, giza) and the faculty of veterinary medicine, beni-suef university (bsu), egypt. tissues were collected from birds that had died spontaneously. following initial examination at these institutions samples were submitted to the friedrich-loeffler-institut, germany. viral rna was extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions. rna was eluted in l nuclease-free water, aliquoted at l and stored at − • c until use. by using a newly developed rt-qpcr (see below), presence of n-specific ibv rna was examined and positive samples were further subjected to two conventional rt-pcrs specific for hypervariable regions (hvrs) of the s gene of ibv (see below). in addition, all samples were screened for presence of the influenza a virus matrix (m) gene (fereidouni et al., ) , and positive samples were further subtyped using rt-qpcrs specific for subtypes h and h (monne et al., ) . also, samples were examined for ndv rna using an rt-qpcr specific for the avian paramyxovirus- matrix gene (wise et al., ) . pcr reactions for aiv and ndv were performed in l volumes using superscript ® iii one-step rt-pcr kit with platinum ® taq dna polymerase (invitrogen) on a cfx thermocycler machine (bio-rad). pathotyping of h -and ndv-positive samples was achieved by sequencing of the ha (naguib et al., ) or f gene cleavage sites, respectively (aldous et al., ) . primers were selected based on alignments of the n and s genes of a selection of avian gamma-coronavirus sequences available from genbank (ncbi). pre-selected primers were then screened in silico for their binding properties to ibv and shannon entropy plots using entropy one software (http://www.hiv.lanl.gov/content/ sequence/entropy/entropy one.html) were produced to confirm the specificity of the primers and probes for different ibv genotypes ( fig. ) . shannon entropy analysis for ibv-specific primers and probes targeting a fragment of the nucleocapsid (n) orf was carried out using an alignment of sequences of the n orf of representative ib viruses from different lineages. entropy values of primers used for amplification of the hvr of the s gene was performed on alignments of sequences representing various genotypes including egyptian variants i and ii, different israeli variants, qx viruses as well as different vaccine strains. the oligonucleotides finally designed are shown in table . furthermore, the specificity of the newly developed rt-qpcr was evaluated using different ib reference viruses: m , ma , h , h , beaudette, qx, qx-like, cr - , / , d , d , and the egyptian variants i (eg/ibv ) and ii (ibv-eg/ b- ). turkey coronavirus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, equine torovirus, bovine and canine coronaviruses, in addition to aiv subtypes h n , h n and ndv were used to further define specificity. the optimized thermal cycling conditions for the newly developed rt-qpcr specific for the ibv n gene fragment using the agpath-id one-step rt-pcr (thermofisher) kit were as follows: a reverse transcription step was carried out at • c for min, followed by an initial denaturation step at • c for min, and cycles of pcr amplification at • c for s, • c for s, and • c for s in a l reaction mixture using . pmol of each forward, pmol of the reverse primer and pmol probe per reaction. fig. . analysis of the detection limit of the ibv n-specific rt-qpcr based on serial ten-fold dilutions of transcribed viral rna ranging from to copies/reaction (a). linear regression analysis was constructed revealing the correlation coefficients (r ) and the slope value (b). this new diagnostic tool was compared to a previously published rt-qpcr targeting ibv orf a; thermal cycling conditions of this method were described before (callison et al., ) . for the conventional rt-pcrs targeting the s hvrs - primer concentrations of . pmol each were used per reaction. the superscript ® iii one- step rt-pcr kit with platinum-taq was used with the following cycling condition: • c for min, followed by an initial denaturation step at • c for min followed by pcr amplification cycles were run at • c for s, • c for s, and • c for s with a final extension step of • c for min. an amplificate was produced from ibv strain m using the n-specific rt-qpcr primer set and cloned into the topo ta cloning dual promoter kit (invitrogen) containing t and sp promoter sequences. rna was transcribed with t rna polymerase (promega) from bamhi linearized plasmids according to the manufacturer's instructions. transcribed rna was further purified and quantified. the detection limit of the rt-qpcr assays was determined by testing in triplicate serial ten-fold dilutions of the in-vitro transcribed viral rna ranging from to rna copies/l/reaction, and standard curves were produced. a cq value < was considered positive. rna copy number was calculated based on the dna/rna copy number calculator online tool http:// endmemo.com/bio/dnacopynum.php for amplification of the hvr and of the s gene of ibv, forward and reverse primers were designed in the frame of this study, while those for the hvr were modified from adzhar et al. ( ) (table ) . amplificates were size-separated by agarose electrophoresis, excised and purified from gels using the qiaquick gel extraction kit (qiagen, hilden, germany). purified pcr products were used directly for cycle sequencing reactions (bigdye terminator v . cycle sequencing kit, applied biosystems). the reaction products were purified using nucleoseq columns (macherey-nagel gmbh ® genetic analyzer (life technologies). the obtained s hvr sequences were assembled and edited using geneious software, version . . (kearse et al., ) . alignment and identity matrix analyses were performed using mafft (katoh and standley, ) and bioedit (hall, ) . sequences generated in this study were deposited in the genbank database, and assigned accession numbers are shown in table s . sequences of other viruses required for further analyses were extracted from public databases. phylogenetic analyses were based on manually edited alignments of the full-length open reading frames. for maximum likelihood analysis of phylogenetic relationship, a best fit model was chosen first on which further calculations and an ultrafast bootstrap equivalent analysis was based. the iq-tree software version . . was used for all operations (minh et al., ; nguyen et al., ) . trees were finally viewed and edited using figtree v . . software (http://tree.bio.ed.ac.uk/software/figtree/) and inkscape . . . . performance characteristics of quantitative (n-specific) and conventional (s -specific) rt-pcrs for ibv detection shannon entropy plots, used to evaluate the specificity of the designed primers, predicted a broad reactivity against different table comparison of the analytical specificity of two real time rt-qpcrs targeting utr (callison et al., ) or n (this study) using a panel of ibv and other coronaviruses as well as other avian viral pathogens. rt-qpcr n rt-qpcr orf a -a positive, cq values obtained with the two rt-qpcrs did not differ by more than - values indicating similar analytical sensitivity. b negative; cq values > . ibv genotypes circulating in the middle east (fig. s ). considerable sequence variability was evident despite selecting regions with pronounced overall conservation. thus, primers were degenerated at key positions as shown in fig. s to allow broader, yet specific, target hybridization. the specificity of rt-qpcr primer set was evaluated by examining different avian respiratory viruses circulating in poultry in egypt including aiv h n , h n and ndv as well as different coronaviruses including a panel of ibv reference strains as listed in the materials section. this comprises all ibv genotypes reported to be circulating in egypt. no amplification was detected for h n -, h n -and ndv-specific rna ( table ) . as predicted by shannon entropy plots, a distinctly specific amplification of ibv rna but not of other coronaviruses, with the exception of turkey coronavirus, was observed with the n-specific rt-qpcr primer set. genomic rna from ibv isolate (m ) was used as template for initial validation experiments. analytical specificity and sensitivity of the newly developed rt-qpcr compared favorably with the utr −specific rt-qpcr of callison et al. ( ) (table ) which also does not differentiate between ibv and turkey coronaviruses. run-off rna transcripts of the ibv n gene fragment were used to evaluate analytical sensitivity of the ibv-n-specific rt-qpcr. investigations using copy-based rna run-off transcripts showed a detection limit of that pcr close to copies per assay. the cq values corresponding to rna copies were ± . . the standard curves as shown in fig. depict a dynamic linear range across at least log units of rna copies. linear regression analysis revealed excellent reproducibility characteristics (fig. ) . based on these results the n-specific rt-qpcr was deemed fit as a screening tool for ibv infections in clinical poultry samples from egypt. the two primer sets designed or modified to amplify ibv s hvr - and produced a band of -bp covering hvr and hvr (set ), or a -bp band covering hvr (set ) as predicted from an alignment of all different serotypes and variants detected in egypt so far (fig. s ) . the specificity of the two hvr primer sets was further confirmed using reference strains compromising different genotypes circulating in egypt as mentioned in the material section. an amplificate of bp was also generated using the forward primer for hvr - and the reverse primer of hvr ; this amplificate covers all three hvrs of the s gene (fig. s ) . a trade-off had to be observed between a shorter pcr product which is less vulnerable to be affected by target rna degradation in clinical samples and a longer one yielding more sequence information (moss and thein, ) . therefore, a decision was taken to generally amplify the three s hvrs in two separate pcrs when examining clinical samples. generally, a sequenceable product was generated with these s rt-pcrs from clinical samples if the cq value obtained by use of the n-specific screening rt-qpcr was lower than (equivalent to approximately rna target copies). thus, this method gives maximum flexibility that can be adjusted according to the sample quality (virus isolate versus clinical sample). a total of field samples were examined by ibv n-specific rt-qpcr. samples yielded positive signals for presence of ib viruses (table ). in the same sample set other avian respiratory viruses were detected by further rt-qpcrs specific for the hemagglutinin gene of aiv subtypes h (n = ), and h aiv (n = ), or specific for the m gene of ndv (n = ). molecular pathotyping for the h viruses revealed presence of highly pathogenic strains of clade . . as genetically characterized in a previous study (see also (naguib et al., ) ). for nd, three velogenic viruses all clustering with genotype vii were detected while the remaining eight were lentogenic vaccine strains. in two ibv-positive samples also h hpaiv was found; sixteen ibv positive samples also harbored h aiv, and seven ibv positive samples also contained ndv. only nine samples showed solely ibv infection without aiv or ndv. furthermore, aiv and/or ndv were detected in several ibv negative samples. details of the various co-infections are shown in table . sequenceable hvr fragments were generated from seven ibv positive samples (shown in bold-face in table s ). no rt-pcr amplicates were obtained from two further samples that showed cq values < in ibv rt-qpcr; failure to amplify ibv s -specific rna from these samples may be related to advanced rna degradation. the sequences obtained were added to an alignment that had also been used for the in silico analysis of the hvr primers: the sequences derived from three samples (ar , ar and ar ) revealed a high similarity of about . - . % at the amino acid (aa) level with egyptian ibv variant ii (e.g., strain eg/ b/ , genbank accession number: kc . ) but only . - . % to egyptian variant i (eg/beni-suef/ , genbank accession number: af ) within their hvrs and . the remaining four viruses (ar , ar , ar and ar ) showed . - . % aa identity in those regions with egyptian variant ii viruses (table a ). this indicates that two groups of viruses can be distinguished within egyptian variant ii with respect to hvr , sequences. all seven samples revealed . - % aa identity with egyptian variant ii for the hvr and . - . % identity compared to egyptian variant i (eg/beni-suef/ genbank accession number: jx ). all sequences obtained from seven clinical samples are remarkably distinct (more than % aa difference) to classical vaccine strains (ma , m and h ) that are used in egypt. the seven field-type sequences were also grossly distinct from other vaccine strains currently or previously used in egypt ( / , cr or d ) (table b) . phylogenetic analysis confirmed the clustering of the seven ibv positive samples with egyptian variant ii sequences (fig. ) . within hvr , sequences, however, the existence of two distinct table rt-qpcrs reveal frequent co-infections in egyptian poultry samples with avian influenza (aiv), newcastle disease (ndv) and infectious bronchitis viruses (ibv). ibv aiv m aiv-h aiv-h ndv collective results eg/ar - / , ----ib eg/ar - / ------ eg/ar - / , ----ib eg/ar - / ------ eg/ar - / - , groups was confirmed ( fig. a) . interestingly, one group of hvr , sequences was closely related to vaccine strain ( fig. a , black dot) which, in turn, belonged to egyptian variant i in terms of hvr sequences. the other group was closely related to vaccine strain d whose hvr was closely related to mass-like vaccine strains. controlling ib in egypt remains a challenging task due to the wide circulation of at least two different genotypes and emerging of novel strains which may pose risks of ibv vaccination failure. there also is a lack of consistent biosecurity levels in some intense production regions which opens the door to the possibility of mixed infection with other respiratory viruses as well as bacterial pathogens like mycoplasma spp. and e. coli. therefore, rapid molecular diagnostic tools covering the broad spectrum of ibv circulating in egypt are required as well as subsequent genotype identification. the ibv n-specific rt-qpcr and hvr - -specific rt-pcr assays developed and adapted in this study are shown to provide a reliable sensitive and specific approach for screening of suspect samples as well as for downstream genetic characterization of viruses. the hvr-specific pcrs allowed for genotyping directly from clinical samples omitting the need to isolate virus, provided a well preserved rna sample quality and sufficient viral loads. partial s gene analysis of the three hvrs of seven ib viruses detected and characterized in this study showed that they were closely related genetically and phylogenetically to the currently circulating egyptian variant ii. in particular, hvr of these viruses formed a monophyletic cluster (fig. b) . analyses of hvrs and , however, revealed two distinct phylogenetic groups ( fig. a) . thus, there seem to exist two populations of egyp- table comparison of nucleotide and deduced amino acid sequences of the s hvr - (a) and hvr (b) of ibv from egyptian field samples with reference and vaccine strains of different ibv serotypes. tian variant ii viruses which can be distinguished in their hvr , loci. the molecular mechanisms having caused this split remain to be elucidated. to confirm or rule out recombination full length s sequences will be required of a larger panel of egyptian ib viruses. in addition, it remains to be clarified whether antigenic differences exist between the two clusters within egyptian variant ii. in this set of samples, no ibv of egyptian variant i was detected; this seems to confirm previously published data from genbank indicating a dominance of egyptian variant ii. infectious bronchitis, avian influenza and newcastle disease are the three major causes of economic losses in the poultry industry; they are able to induce disease independently or in association with each other. avian influenza h n and h n subtypes continue to circulate in egypt since and , respectively, causing many outbreaks in poultry farms (naguib et al., ) . velogenic ndv circulating among chickens in egypt resembles genotype viid (chicken/china/sdwf / ) and is associated with outbreaks in commercial poultry farms despite adherence to strict vaccination regimes (radwan et al., ) . similar co-infections of avian influenza and velogenic ndv were observed also in neighboring countries such as jordan and libya (kammon et al., ; roussan et al., ) . in addition, a recent similar single case was reported in egypt (hussein et al., ). an at least partial but not sterilizing, possibly vaccine-induced, specific immunity against these pathogens is likely to be at the basis of these observations. continuing clinically disguised virus circulation in the presence of specific immunity not only fosters spread of these agents but also drives the development of viral escape mutants. it should be noted that the mortality rates described for the holdings do not correlate with the presence of ibv, hpaiv h n or velogenic ndv (table , e.g., holding ). this investigation focused on ibv, aiv and ndv and did not consider further avian viral or bacterial pathogens. it is highly likely that also various bacterial co-pathogens contribute to and complicate the overall clinical picture. the current situation of ib infections in egypt seems to be the result of a continuing evolution starting with infections caused by egyptian variant i since , egyptian variant ii since , and mass-like strains since (abdel-moneim et al., ; selim et al., ) . tracheal ciliostasis is one of the early and characteristic pathogenetic sequelae of ibv infection (cook et al., ) . cileostatic respiratory epithelium is known to be more vulnerable for infections with further viral and bacterial co-pathogens and may dispose for infection with aiv and ndv even in the presence of (suboptimal) vaccine-induced immunity. in conclusion, the continuous circulation of the egyptian variant ii ibv and co-infections with aiv and/or ndv severely complicate the epidemiology of viral respiratory infections of chicken in egypt. intensive surveillance is required for a better understanding of this situation. moreover, molecular identification of circulating viruses and experimental vaccination-challenge studies are required to provide data for eventual updating of vaccine strains and to strategically strengthen application programs. isolation and characterization of new variant strains of infectious bronchitis virus in northern egypt isolation and identification of egypt/beni-suef/ a novel genotype of infectious bronchitis virus s gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in egypt emergence of a novel genotype of avian infectious bronchitis virus in egypt molecular analysis of the /b serotype of infectious bronchitis virus in great britain rapid pathotyping of newcastle disease virus (ndv) using fluorogenic probes in a pcr assay mechanisms of coronavirus cell entry mediated by the viral spike 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recombination in the nucleocapsid gene ultrafast approximation for phylogenetic bootstrap development and validation of a one-step real-time pcr assay for simultaneous detection of subtype h , h , and h avian influenza viruses identification of amino acids involved in a serotype and neutralization specific epitope within the s subunit of avian infectious bronchitis virus sequencing of pcr products evolutionary trajectories and diagnostic challenges of potentially zoonotic avian influenza viruses h n and h n co-circulating in egypt iq-tree: a fast and effective stochastic algorithm for estimating maximum likelihood phylogenies isolation and molecular characterization of newcastle disease virus genotypes ii and viid in egypt between molecular survey of avian respiratory pathogens in commercial broiler chicken flocks with respiratory diseases in jordan an apparently new respiratory disease of baby chicks molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in egypt during present status of infectious bronchitis in egypt coronaviruses: structure and genome expression s gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity the avian coronavirus spike protein development of a real-time reverse-transcription pcr for detection of newcastle disease virus rna in clinical samples characterization of three infectious bronchitis virus isolates from china associated with proventriculus in vaccinated chickens the authors thank diana wessler, cornelia illing, aline maksimov and gabriele adam, fli, germany, for excellent technical support. thanks are due to günther keil for the m reference strain. we are grateful to colleagues and co-workers at nlqp, cairo, egypt. m. naguib is recipient of a doctoral scholarship from the german academic exchange service (daad). all authors declare that they have no conflict of interest. m. m. naguib is funded by a grant from the german academic exchange service (daad grant number a/ / ). sample collection by veterinarians was achieved from poultry kept as commercial livestock in farms in egypt. obtaining swab samples from the trachea of poultry is minimal invasive and does not require any anesthesia of the animal; minimal restrainment is used for a very short time. sampling, analysis and shipment of samples from egypt to germany was under the legal auspices of the national animal health and research institute, giza, egypt. three co-authors of the manuscript are employees of this governmental institution. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.jviromet. . . . key: cord- -waxtb authors: pratelli, annamaria; tinelli, antonella; decaro, nicola; camero, michele; elia, gabriella; gentile, arturo; buonavoglia, canio title: pcr assay for the detection and the identification of atypical canine coronavirus in dogs date: - - journal: j virol methods doi: . /s - ( ) - sha: doc_id: cord_uid: waxtb comparative sequence analysis of the pcr products of the m gene and fragments of the pol a and pol b genes of canine coronavirus (ccov) have demonstrated that two separate clusters of ccov are present in dogs. this note describes a pcr assay to identify atypical ccov strains with nucleotide substitutions in the m gene. a total of faecal samples from dogs ccov positive previously with the pcr assay were analysed. sixty-two of the samples were amplified with the pcr described in the present study and were thus considered atypical ccovs. the specificity of the pcr typing assay was confirmed by sequence analysis of the pcr products. the coronaviridae, a family of viruses belonging to the order of the nidovirales (de vries et al., ; siddell, ; enjuanes et al., ) , can be grouped into three clusters on the basis of genetic comparison. canine coronavirus (ccov) is included in group i together with transmissible gastroenteritis virus (tgev) of swine, porcine epidemic diarrhoea virus (pedv), feline coronaviruses (fcovs) and human coronavirus e (hcov e). ccov is an enveloped ('/) rna virus that is Á/ kb in length (siddell, ) and causes moderate or severe enteritis in pups. the ? two-thirds of the genome is occupied by open reading frames (orfs) a and b, the expression of which yields large polyproteins. downstream to orf b, there are smaller orfs which encode for the structural proteins s, e, m, for the nucleocapsid (n) protein and for a number of presumptive non-structural proteins (luytjes, ) . virus isolation onto cell cultures and electron micro-scopic examinations appear to be useful tools, for the diagnosis of ccov infection. therefore, ccov is difficult to isolate in vitro and the common presence of coronavirus-like particles in faeces requires confirmation by other diagnostic methods. recently, a nested-pcr assay on a fragment of the gene encoding for the m protein was developed for the diagnosis of ccov infection. the test proved to have a high sensitivity and specificity (pratelli et al., ) . the results obtained in another investigation also suggested that the role of ccov as enteric pathogen has been underestimated and that the virus is largely widespread in the dog population (pratelli et al., ) . a subsequent study revealed nucleotide variability in the sequence of the gene encoding for the transmembrane protein m of ccov (pratelli et al., a) . by comparative sequence analysis of the m gene and fragments of the pol a and pol b genes, it has been observed that ccovs segregated in two separate clusters. the first cluster was intermingled with reference strains of ccov genotype and therefore could be assigned to this genotype. the second cluster segregated separately from ccov and fcov genotypes and therefore these isolates may represent genetic outliers (pratelli et al., submitted for publication) . recently, severe enteritis has been described in two different pups which had been caused by ccov, as confirmed by pcr. the viruses, unfortunately, were not isolated in cell culture. the sequence analysis of the pcr products of the m and s genes carried out on the faecal samples from the two pups confirmed that the fcov-like ccov had caused the disease (personal observations). it has been demonstrated that ccov is particularly difficult to isolate in cell cultures (tennant et al., ; pratelli et al., ) ; this both hampers the acquisition of fundamental information on the pathogenetic role of ccov in dogs and means that fcov-like ccov can be identified only after sequence analysis. on the basis of these preliminary results, a pcr assay was developed to detect and identify the fcov-like ccov strains from faecal samples of infected dogs. a total of faecal samples from dogs, about Á/ months old, with enteritis and positive for ccov by standard pcr (pratelli et al., ) were used to develop a pcr assay for detecting fcovlike ccov. the samples were collected over a period of years from either housed dogs ( specimens) or dogs living in kennels ( specimens) where periodical outbreaks of ccov enteritis had been described. the pups showed moderate to severe enteritis and their faecal samples were negative for canine parvovirus (cpv ) by the haemoagglutination test. the samples were stored immediately at (/ c until tested. three additional coronavirus strains were also examined: feline infectious peritonitis virus (fipv), isolated from a cat with clinical signs of disease (buonavoglia et al., ) , fcov type ii ( - strain) and tgev (purdue strain). viral rna was extracted from clinical specimens using the rneasy kit (qiagen gmbh, germany). the target sequence is a fragment of the gene encoding for the membrane protein m of ccov, as reported previously (pratelli et al., ) . the primer pair ccov /ccov amplified a bp fragment. for the n-pcr, the first amplicon was subjected to a second round of amplification using the ccov and ccov primers and the same pcr cycling procedure. in a previous study, similar nucleotide substitutions in the binding site of the internal primer ccov used for the n-pcr were demonstrated in the sequence analysis of the pcr products from five faecal samples of pups with diarrhoea. these variations were assumed to affect partially the efficiency of the n-pcr amplification, with the production of an undetectable amount of c-dna. moreover, these nucleotide substitutions occurred consistently in all five samples examined (pratelli et al., a) and the sequence and phylogenetic analyses performed on the entire gene identified the viruses as fcov-like ccov. based on these preliminary results an internal primer flanking the variable region (ccov a) was chosen on the basis of the mismatch between the typical ccov and fcovlike ccov strains (fig. ) . pcr was then performed with the primer pair ccov a/ccov , where the sense primer ccov a was able to detect these atypical ccovs. while the primer pair ccov /ccov amplifies a bp sequence of the m gene, a bp band is obtained with the primer pair ccov a/ccov if the target sequence of fcov-like ccov is present in the sample. the sequence of the primers and their positions in the m gene segment are displayed in table . the reverse transcription was carried out in a total reaction volume of ml containing pcr buffer )/ (kcl mm, tris Á/hcl mm, ph . ), mgcl mm, mm of each deoxynucleotide (datp, dctp, dgtp, dttp), rnase u, mulv reverse transcriptase . u, random hexamers . u. synthesis of c-dna was carried out at c for min, followed by a denaturation step at c for min. the mixture was brought up to a total volume of ml, containing pcr buffer )/ , mgcl mm, amplitaq gold dna polymerase . u and pmol of each primers ccov a (sense primer) and ccov (antisense primer). amplification was carried out under the following pcr conditions: cycles of denaturing at c for min, annealing at c for min and extension at c for min. the final products were detected by gel electrophoresis, ethidium bromide staining and uv light transillumination. one hundred and seventy-seven faecal samples of dogs with diarrhoea were examined. of these, samples that had showed the conventional fragment ( bp) of the typical ccov strain, were not amplified with the primer pair ccov a/ ccov . instead, fcov-like ccovs were found in faecal samples, generating a bp fragment. all specimens had been collected from dogs in two separate kennels, where periodical outbreaks of ccov enteritis had been observed. the specificity of the pcr typing assay was confirmed by sequence analysis of the pcr products. as expected, the fipv, fcov type ii - and tgev purdue strains were amplified by the primer pair ccov /ccov , but they were not amplified by the pcr typing test with the primer pair ccov a/ccov . ccov is an enteric pathogen of dogs responsible, in young pups, for mild or severe symptoms characterized by diarrhoea, vomiting, dehydration, loss of appetite and occasionally death. ccov shedding in faeces occurs over a range of Á/ days post-infection (keenan et al., ; tennant et al., ) , although virus faecal shedding in infected pups has been detected by n-pcr for periods of up to days (pratelli et al., b) . the detection limits of virus isolation have been overcome by the development of pcr and n-pcr for the diagnosis of ccov (bandai et al., ; pratelli et al., ; naylor et al., ) . furthermore, sequence and phylogenetic analyses of the m gene clearly confirm that two different clusters of ccov circulate in the dog population (pratelli et al., submitted for publication) . to date, the sequence analysis is the only available method to identify the two genotypes of ccov. to overcome this diagnostic limit, a pcr assay has been developed for the rapid identification of fcovlike ccov from faecal samples of naturally infected pups. this test may be instrumental in furthering epidemiological studies on the occurrence and distribution of ccov strains in dog population. feline coronavirus strains (fcov - and fipv) and the tgev purdue strain were not amplified by this pcr typing assay, because, as observed in the sequence and comparative analysis of the amplicons from typical ccov, fcov-like ccov, fcov and tgev strains, several nucleotide substitutions are present in the binding site of pratelli et al. ( ) . the sense primer ccov a, thus affecting the efficiency of the pcr amplification (fig. ) . the pcr assay described in the present study for the identification of fcov-like ccov strains also shows a very high specificity. in conclusion, a pcr typing test has been described for the detection and identification of fcov-like ccovs confirming that this new genotype may cause occasional enteritis in pups and is particularly common in kennel populations thereby causing epizootic infections. canine coronavirus infections in japan: virological and epidemiological aspects isolamento e caratterizzazione di uno stipite di virus della peritonite infettiva felina the genome organization of the nidovirales : similarities and differences between arteri-, toro-, and coronaviruses nidoviridales. in: regenmortel intestinal infection of neonatal dogs with canine coronavirus Á/ : studies by virologic, histologic, histochemical and immunofluorescent techniques coronavirus gene expression: genome organization and protein expression identification of canine coronavirus strains from feces by s gene nested pcr and molecular characterization of a new australian isolate development of a nested pcr for the detection of canine coronavirus diagnosis of canine coronavirus infection using nested-pcr variation of the sequence in the gene encoding for severe enteric disease in an animal shelter associated with dual infections by canine adenovirus type and canine coronavirus the coronaviridae: an introduction canine coronavirus infection in the dog following oronasal inoculation studies on the survival of canine coronavirus under different environmental conditions this study was supported by grants from cegba (centro di eccellenza di genomica in campo biomedico e agrario) and from ministry of university, italy (project: enteriti virali del cane). we thank dr. athina papa for revising the english of the manuscript. key: cord- -crtpzad authors: neill, john d.; bayles, darrell o.; ridpath, julia f. title: simultaneous rapid sequencing of multiple rna virus genomes date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: crtpzad comparing sequences of archived viruses collected over many years to the present allows the study of viral evolution and contributes to the design of new vaccines. however, the difficulty, time and expense of generating full-length sequences individually from each archived sample have hampered these studies. next generation sequencing technologies have been utilized for analysis of clinical and environmental samples to identify viral pathogens that may be present. this has led to the discovery of many new, uncharacterized viruses from a number of viral families. use of these sequencing technologies would be advantageous in examining viral evolution. in this study, a sequencing procedure was used to sequence simultaneously and rapidly multiple archived samples using a single standard protocol. this procedure utilized primers composed of bases of known sequence with random bases at the ′-end that also served as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. this conferred sequence independence by random priming both first and second strand cdna synthesis. viral stocks were treated with a nuclease cocktail to reduce the presence of host nucleic acids. viral rna was extracted, followed by single tube random-primed double-stranded cdna synthesis. the resultant cdnas were amplified by primer-specific pcr, pooled, size fractionated and sequenced on the ion torrent pgm platform. the individual virus genomes were readily assembled by both de novo and template-assisted assembly methods. this procedure consistently resulted in near full length, if not full-length, genomic sequences and was used to sequence multiple bovine pestivirus and coronavirus isolates simultaneously. next generation sequencing technologies are used to screen environmental and clinical samples for the presence of viral pathogens. this has resulted in discovery of new, previously unknown viruses that are often uncultivable (victoria et al., ; li et al., ; phan et al., ; reuter et al., ) . these technologies have allowed screening of samples collected from domestic animals (shan et al., ; phan et al., ; reuter et al., ) and from animals in the wild to determine if they are carriers of potentially zoonotic viral pathogens (donaldson et al., ; li et al., ; phan et al., ; wu et al., ) . additionally, deep sequence analysis was applied to screening of commercially available vaccines for the presence of contaminants and variants . methods to sequence complete viral genomes have been developed. these include methodologies based on pcr amplification of viral sequences, both in fragments (rao et al., ) or fulllength genome amplification (christenbury et al., ) . these have primarily focused on specific, closely related viruses. the sequence-independent, single primer amplification (sispa) procedure was first used to amplify dna populations through the use of random priming and ligation of adaptors for pcr amplification (reyes and kim, ) . this was modified for amplification of viral sequences from serum to include a step where dnase i was used to first degrade host dna (allander et al., ) . this protocol has since been used in the identification of a novel pestivirus (kirkland et al., ) , bocaviruses (cheng et al., ) and identification of viruses associated with cytopathic effect in cell cultures of unknown origin (abed and boivin, ) . many virological laboratories, especially those associated with diagnostic laboratories, have freezers that contain large numbers of virus isolates collected over varying time spans. often these isolates have clinical histories associated with them that detail where and when the isolations were made and descriptions of the disease state of the host. there is a wealth of information in these isolates, but up till now, it has been time consuming and expensive to sequence these viral genomes, often requiring sets of strain-specific primers for pcr amplification and sequencing. the protocol outlined in this study provides the means to rapidly sequence archival collections of viruses to study the evolution of specific viruses over time. this information is important in determining the relevance of virus strains used in currently marketed vaccines. viral isolates passaged in cell culture and stored frozen at − • c were used in this study. the two virus groups sequenced were bovine viral diarrhea virus (bvdv) isolates and bovine coronavirus (bocv) isolates. after isolation/propagation of the viruses on appropriate host cells, the medium was frozen/thawed twice, cell debris removed by centrifugation, and aliquots frozen at − • c. routinely, the libraries were constructed from groups of viruses. the virus stock was thawed at room temperature and l was mixed with l of × dnase i buffer ( mm tris-hcl (ph . ), mm mgcl , mm cacl ). a cocktail of nucleases was added to degrade host nucleic acids as previously described (victoria et al., ) . the cocktail consisted of (per virus sample) units dnase i (life technologies, grand island, ny, usa), units turbo-dnase (life technologies), units base-line zero dnase (epicenter, madison, wi, usa), units benzonase (emd millipore, billerica, ma, usa), g rnase a/ units rnase t (thermo scientific, waltham, ma, usa), and units rnase one (promega, madison, wi, usa). the virus stock/nuclease mixture was incubated at • c for at least min. the viral rna was purified using the minelute virus spin filter kit (qiagen, valencia, ca, usa) according to manufacturer's specifications. the rna was eluted in l of ultra-pure, rnasefree h o (final yield ∼ l) and immediately placed on ice. twenty microliters of rna were mixed with pmol of a mer primer consisting of nucleotides of a known sequence followed by random nucleotides as previously described . table shows the sequence of the primers used in this study. these primers were developed so that the base known sequence was used for pcr amplification of the library as well as served as a barcode for identifying each viral library following pooling and sequencing. the rna/primer mix was heated at • c for min and immediately placed on ice. first strand synthesis was done in a reaction mix consisting of . mm dntps, × first strand buffer, units rnasin (promega) and units superscript ii reverse transcriptase (life technologies). the first strand reactions were incubated at • c for h, heated at • c for min and immediately quenched on ice. the second strand reaction was carried out using sequenase . (affymetrix, santa clara, ca, usa) and the nucleotides and primers still present following the first strand reaction (yozwiak et al., ). an equal volume of × sequenase buffer with units of sequenase per reaction was added on ice. the reaction tubes were placed in a thermocycler that was prechilled to • c and were ramped from • c to • c at . • c/s. the reaction was continued at • c for min. the tubes were heated to • c for min and placed immediately on ice. a second aliquot of sequenase was added ( units/reaction) and the ramping and incubation repeated with the exception that the • c reaction was done for min. the double stranded cdna was purified using minelute pcr spin columns (qiagen) and eluted with l of ultra-pure h o. the cdna was amplified by pcr using primers consisting of the first known bases of the mer used in the cdna synthesis . the pcr reactions consisted of l of double-stranded cdna, × pfx buffer, m dntps, pm of appropriate mer primer and units of pfx polymerase (life technologies). the reactions were cycled at • c for min and cycles of • c for s, • c for s and • c for min. the number of cycles was empirically determined so as not to over amplify the cdna, where cycles worked well. the dna was again purified and eluted in l ultra-pure h o, and l subjected to agilent bioanalyzer analysis (agilent technologies, santa clara, ca, usa). the percentage of the dna that was - bp in length was determined for each reaction as was the total dna concentration in each sample using picogreen (life technologies). all ten reactions were pooled so that the - bp cdnas from each were present in equimolar amounts. the pooled dna was placed in an end-repair reaction (ion plus fragment library kit, life technologies) followed by ligation of ion torrent sequencing adaptors. the dna was size fractionated using agencourt ampure beads (beckman coulter, indianapolis, in, usa) using . volumes of the ampure beads and was done according to manufacturer specifications. this step removed dna of bp and less which eliminated adaptor dimers and unligated adaptors. at this point, two -genome libraries, for a total of genomes, were pooled for each sequencing run. the - bp fraction was isolated from the dna pool using the pippin prep (sage science, beverly, ma, usa). the - bp dna was subjected to sequence analysis with the ion torrent pgm and standard chemistries (life technologies) using the sequencing chip. the raw data files that contained the sequence data from each sequencing run of the pgm were demultiplexed using the base barcode sequence. the raw sequencing reads obtained from the ion torrent sequencer were exported as sff files. barcode sequences unique to this study were used to populate a custom configuration file that was used in conjunction with the roche "sfffile" tool to separate the reads into individual barcode-specific sff files. all the sequences in each barcode-specific sff file was extracted to a fastq file by applying the "sff extract" utility (http://bioinf. comav.upv.es/sff extract/index.html) and specifying the "-clip" option to remove the sequencing tags, barcode sequences, and low quality sequences. the "fastx trimmer" program (part of the fastx toolkit; http://hannonlab.cshl.edu/fastx toolkit/index.html) was used to remove the random primer ( -mer) by trimming an additional eight nucleotides from the -end of each read. as a final post-trimming step, a custom script was used to remove all reads that were less than nucleotides in length. the resulting sequence files were used to assemble full length genomic sequences using seqmanngen and were edited with the seqman software of the lasergene package (dnastar, inc., madison, wi, usa) using related viral sequences obtained from genbank as the assembly reference. assembled genomic sequences were further edited using aligner (codoncode, inc., centerville, ma, usa). the sequences from each library were submitted to the ncbi sequence read archive with the bioproject number prjna , biosamples samn , and samn through samn . the ion torrent raw sequence data was trimmed of sequencing adaptors, barcodes, the base random primer sequence, low quality sequences and sequences less than bases. after preassembly processing of the reads, an average of % of initial reads remained for downstream analysis. the bvdv and bocv sequencing runs produced , and , , individual sequences, respectively. the percentage of viral sequences in each barcoded library varied but was as high as % ( table ). the ratio of virus:host sequences was highly dependent on the titer of virus in the stock sample. the number of sequences for each barcoded sample in the library varied, even following pooling at concentrations that should have resulted in equimolar amounts of each sample in the library. to test the accuracy of the ion torrent sequencing, a virus previously sequenced by pcr amplification was included to compare sequences generated by this method and by sequencing pcr amplicons. this virus, a bvdv b strain isolated from alpaca (genbank accession jx . ; table , library , barcode ), was assembled from ion torrent data and was found to have only base difference from the sequence determined earlier (data not shown). this base was ambiguous in the amplicon sequenced by sanger chemistry (equal peak height of a and g) but was clearly a g by ion torrent sequencing. the sequences from two libraries were analyzed to determine source of any non-viral sequences. the remaining non-viral sequences were assembled into contigs and were used in blast searches of genbank. this revealed that the non-viral sequences were derived from host nucleic acids, both dna and rna. the rna sequences were primarily ribosomal rnas but some were from more abundant mrna transcripts. assembly of genomic sequences was done using templateassisted assembly. bvdv and bocv genomic sequences used as templates for assembly were obtained from genbank (accession numbers jn . and ef . , respectively). all of the individual virus library sequences resulted in assembled sequences that spanned at least % of the genome from which they were derived (table ). in libraries and , bvdv genomic sequences were determined for virus isolates. the percentage of viral sequences for each barcoded library ranged from . to . %. bvdv assemblies contained no internal gaps but the extreme and termini were not present, with the exception of sequences of four viruses ( table ). the number of nucleotides missing from the termini varied but ranged from to bases. one virus, library , barcode , had only viral sequence reads but . % of the genome was assembled. this genomic assembly had internal gaps, ranging from to nucleotides. the other viruses had no assembly gaps and genomic coverages of . - . %, depending on variable coverage of the genome termini. the low number of sequences for library , barcode was believed to be a result of a pipetting error during the quantitation or pooling of the genomic libraries. the results of sequencing of bocv isolates are listed in table , under libraries and . the coronavirus genomic rna is . times longer than that of bvdv, roughly , nucleotides versus , . thus, it required more sequences overall to assemble the complete genome to the same coverage depth. additionally, because bocv typically grows to a lower titer, there were lower ratios of virus:host sequences, ranging from . to . % of the total sequences. a major difference in genome assembly between bvdv and bocv was that, with only a few exceptions, both the and terminal sequences were determined for each bocv isolate. also, due to the longer length of the bocv genome, more assembly gaps were observed. however, in all cases, assembled sequences spanned more than % of the viral genome. more assembly gaps were observed in individual virus libraries having lower total sequences. the bvdv isolate sequenced in library , barcode gave results similar to that observed with bvdv isolates sequenced in libraries and . the advent of new dna sequencing technologies has led to the development of improved sequencing protocols for detection and characterization of viruses in environmental and diagnostic samples. many of these protocols use deep sequencing techniques to detect and identify novel, often uncultivable, viruses. this sequencing protocol was designed to sequence viruses rapidly that were isolated from clinical specimens and archived. often, a clinical report and history is available for isolates making it possible to begin association of genotype with specific phenotypes and time of isolation. particularly valuable is that this protocol confers the ability to sequence archival collections of specific viruses rapidly in order to study the evolution of the pathogen over time and determine the genotype of the viruses currently in circulation. this information is useful in determining relevance of vaccine strains based on sequences of currently circulating viruses. the viruses used for sequence analysis were all isolated and passaged in cell culture. thus, the abundance of host nucleic acids posed a problem in obtaining sufficient numbers of viral sequences to assemble the viral genomes. it was necessary to pre-digest virus samples with high levels of nucleases before purification of viral rna. the viral rna was protected within the intact virions while the host rna and dna were degraded. the rna purification procedure included a protease digestion step aiding in the removal of the nucleases before the viral rna was released from the virion. the nuclease digestion step aided in reaching as much as % of all sequences in a barcoded library being of viral origin. again, the titer of the virus in the original sample was also important in the number of viral sequences obtained. viruses that grow routinely to low titers require less than libraries in the sequencing run to ensure sufficient reads in each barcoded library to assemble the genomes. an important aspect of this procedure was sequence independence, requiring no previous knowledge of sequence or pathogens present in a sample. the use of base barcode sequence with base random nucleotides (victoria et al., ) served both to prime cdna synthesis and to differentiate each library following sequencing. the base known sequence was also used for pcr amplification of the individual libraries. the ion torrent platform was chosen because it allowed variation in the depth of sequencing based on the size of the chip that was used in the sequencing run. for the most part, the chip worked well, routinely giving sufficient sequencing depth to assemble the genomes of viruses in each sequencing run. the ion torrent is known to have indels in the sequence data, especially associated with homopolymeric runs of bases (bragg et al., ) . use of template assisted assembly of the genomes greatly assisted in producing accurate genome assemblies. limited numbers of de novo assemblies were done and low numbers of errors were found that were attributed to indels. the accuracy of sequences generated by this method was demonstrated by the comparing sequences to those previously generated by sanger pcr amplicon sequencing. only nucleotide difference was detected in sequences generated using the two different methods demonstrating the accuracy of the sequencing using the protocol described in this paper. the genomic sequences of the bvdv isolates were readily assembled from the sequence data, partially owing to the relatively small size of the genomic rna. however, the extreme and terminal nucleotides were difficult to obtain, most likely due to the high degree of secondary structure known to be present in these sequences. this may have made priming of first strand cdna synthesis near the termini more difficult. with the exception of one virus, all bvdv genomes were assembled with no internal gaps in the sequence. the bvdv genome that was not fully assembled had only total viral sequences, yet this was sufficient to assemble greater than % of the genome. the low number of sequences was most like due to a pipetting error during the pooling of the individual genomic libraries. this illustrates the importance of accurate quantitation and pipetting of the individual libraries in making the dna pool used in the template preparation for sequencing. the bocv assemblies tended to have more assembly gaps, due in part to the longer genomic rna of these viruses. conversely, the genome termini of the bocv were determined more often then the bvdv, the reasons for which are unclear. the assemblies containing the most gaps were those that had lower numbers of total sequences. still, the sequences spanned greater than % of the total genome of all bocv isolates examined in this study. these gaps were easily filled by either resequencing the library or by pcr amplicon sequencing of selected regions. routinely, there was sufficient double-stranded dna to be included in a second pooled library if additional sequence data was necessary. in addition to sequencing archived viruses, this protocol can readily be applied to diagnostic applications. with modifications of the sample preparation, most clinical samples submitted to diagnostic laboratories can be used for analysis. one difference in a diagnostic application is that complete genome assembly would not be necessary but detection of specific viral sequences would be indicative of the presence of the virus. this would require, at least initially, de novo assembly and blast analysis to determine if viral sequences were present. preliminary studies showed that viruses can be successfully detected, and in some cases, near full-length genomic sequences assembled, from serum and fecal samples (data not shown). additionally, with minor modification of the initial double-stranded cdna synthesis step, both dsrna and dna viruses can be sequenced. a deep sequencing method for rapid determination of genomic sequences of multiple viruses simultaneously is described. the practical application of this protocol was demonstrated by the generation of full-length or near full length genomic sequences of archived pestivirus and coronavirus isolates. with minor modifications, this protocol could be used to sequence multiple dsrna and dna viruses. this procedure is sequence independent, requiring no previous knowledge of sequence or pathogens present in a sample. additionally, this shows promise for the rapid identification of multiple viral pathogens in diagnostic settings and comparison of viruses currently in circulation with those in vaccines. molecular characterization of viruses from clinical respiratory samples producing unidentified cytopathic effects in cell culture a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species shining a light on dark sequencing: characterising errors in ion torrent pgm data identification and nearly full-length genome characterization of novel porcine bocaviruses a method for full genome sequencing of all four serotypes of the dengue virus metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat identification of a novel virus in pigs-bungowannah virus: a possible new species of pestivirus genomic characterization of novel human parechovirus type bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses the fecal viral flora of wild rodents acute diarrhea in west-african children: diverse enteric viruses and a novel parvovirus genus deep sequencing as a method of typing bluetongue virus isolates identification of a novel astrovirus in domestic sheep in hungary sequence-independent, single-primer amplification (sispa) of complex dna populations the fecal virome of pigs on a high-density farm rapid identification of known and new rna viruses from animal tissues metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis viral nucleic acids in live-attenuated vaccines: detection of minority variants and an adventitious virus virome analysis for identification of novel mammalian viruses in bat species from chinese provinces virus identification in unknown tropical febrile illness cases using deep sequencing the authors would like to thank kathryn mcmullen, renae lesan and patricia federico for excellent technical assistance. additional thanks go to kerrie franzen and mary lea killian of the national veterinary services lab, aphis, usda for their help in sequencing of the libraries. key: cord- - rsgeag authors: han, xueqing; bartlam, mark; jin, ying-hua; liu, xiangtao; he, xiaojing; cai, xuepeng; xie, qingqe; rao, zihe title: the expression of sars–cov m gene in p. pastoris and the diagnostic utility of the expression product date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: rsgeag high-level protein expression is an important means of obtaining large amounts of viral proteins to investigate further their biological properties. to express the membrane (m) protein of sars–cov at high-level in vitro, the m gene fragment was amplified and cloned it into the pichia pastoris expression vector ppiczαa. sds–page and western blotting analysis of the induced products of recombinant yeast transformant indicated that successful high-level expression of m protein was achieved, and that the expression product was similar antigenically to the natural protein. purified recombinant m protein was used subsequently as an elisa antigen for detection of eight serum samples screened previously by whole virus elisa and immunofluorescence assay, and consistent results were obtained. these findings suggest that the recombinant m protein may be useful as a diagnostic reagent. severe acute respiratory syndrome (sars), also called atypical pneumonia, is a human severe respiratory infectious disease that emerged recently in asia, north america and europe. the transmission of sars occurs mainly by direct contact, and its incubation period is - days. infection is usually characterized by high fever, which is followed a few days, later by a dry non-productive cough and shortness of breath, and may progress to generalized interstitial infiltrates in the lung. death from progressive respiratory failure occurs in between - % of cases . at present, no effective therapy or vaccine is available. a novel coronavirus, named as sars-cov, has been identified as the main causative agent of sars. the virions are - nm in diameter, with - nm complex surface projections surrounding the periphery. hemagglutinin esterase type glycoprotein projections were not seen (ksiazek et al., ) . the genome of sars-cov is a single-stranded, positive-sense polyadenylated rna molecule of approximately , nucleotides. the genomic organization is typical of coronaviruses, having the characteristic gene order [ -replicase (rep) , spike (s), envelope (e), membrane (m), and nucleocapsid (n)- ] and short untranslated regions at both termini. the sars-cov rep gene is predicted to encode two polyproteins that undergo co-translational proteolytic processing. there are four open reading frames (orf) downstream of rep that are predicted to encode the four structural proteins s, e, m and n, which are common to all known coronavirus. additionally, sars-cov also encodes several non-structural proteins . the clinical case definition of sars is essentially one of fever and pneumonia, with or without a contact history. there are many causes of pneumonia and in the absence of a definite history of contact with other patients with sars, therefore, it is difficult to differentiate sars from other causes of pneumonia in clinic diagnosis. laboratory tests that can confirm a diagnosis of sars-cov infection early in the course of the illness are, therefore, a critical clinical need (riley et al., ) . since the outbreak of sars in , several laboratory diagnostic methods have been established, including real-time rt-pcr assay, whole-virus-based immunofluorescence assay (ifa), recombinant protein-based enzyme-linked immunosorbent assay (elisa) and immunochromatographic tests, antigencapturing enzyme-linked immunosorbent assay, and western blot (wb) assay. these methods were critically important for controlling the transmission of sars; furthermore, many new laboratory diagnostic methods are being developed (poon et al., (poon et al., , shi et al., ; guan et al., ; he et al., ) . the m protein is an important structural protein of the coronavirus. in the cases of other known coronaviruses, it is the most abundant among the structural proteins and can induce antibody-dependent complement-mediated virus neutralization (woods et al., ) . in addition, the m protein is also involved in the assembly and budding of virions together with the e protein. (rottier, ; de haan et al., ; molekamp and spaan, ; narayanan and makino, ; corse and machamaer, ; vennema et al., ) . the m protein contains highly conserved glycosylated sequences, and its glycosylation may be related to the interaction between virus and host (de haan et al., (de haan et al., , . the important role of the m protein in the life cycle of coronaviruses and inducing immunity make it an attractive target for anti-sars drug research, vaccine development and the establishment of a serological detection assay. here, we report the expression of recombinant m protein in p. pastoris and its antigenicity. the genomic cdna of sars-cov strain bj was provided by huada gene company. the p. pastoris expression vector ppicz␣a and yeast host strain gs were all purchased from invitrogen company. sera from healthy people and sars patients were collected from peking union medical college hospital between april and june . sample # - were from healthy people, sample # - were from four sars patients (age - ; two males, two females) during the acute phase of infection, which were collected at day , , , post sars onset, respectively. all four sars patients had a history of contact with other patients infected with sars, and exhibited symptoms including persistent fever (> . • c), cough and shortness of breath for several days before they were hospitalized. patients were subsequently confirmed to be infected with sars by clinical diagnosis combined with laboratory diagnostic methods. according to the published genomic sequence of sars-cov strain tor (poutanen et al., ) , a pair of primers were designed and synthesized. the sequence of the primers were -gagccgcggccgctcaatgt-ggtcattc- (forward primer) and -cgttctaga-tgtactagcaaagcaata- (reverse primer), which carried a sacii and xbai restriction site, respectively. the primers were used to amplify the m gene fragment without its transmembrane domain from the genomic cdna of sars-cov strain bj- . the pcr reaction contained l of cdna, pmol of each of two primers, l of buffer concentrate, l of dntp and . l of ex taq enzyme (takara, japan), and sterile distilled water up to l. pcr was performed with the following settings: • c for min, followed by cycles at • c for s, • c for s and • c for min, and ending with • c for min. the amplified products were then sent to the takara biotechnology (dalian) co. ltd. for sequencing. the determined sequence was analyzed further by dnaman biological software. the m gene fragment and expression vector ppicz␣a were digested by sacii and xbai, respectively. the larger fragments were purified by qiaquick gel extraction kit (qiagen, germany). the concentrations of purified products were measured by beckman dv- (beckman, u.s.a). after mixing in a : molecular ratio, the purified products were ligated by t dna ligase at • c overnight and then transformed into escherichia coli top competent cells. transformants selected on low-salt lb plates containing zeocin ( g/ml) were screened by direct colony pcr, and by restriction digestion of purified plasmids. the sequence of the inserts were verified. the sequence data obtained were compared with the sequence of the m gene of sars-cov strain bj (accession number ay ). the transformation of yeast cells and screening of transformants, as well as expression in p. pastoris, were performed as described previously (david and james, ) . the recombinant plasmid was linearized by the restriction enzyme saci, the purified products were transformed into yeast cells gs and transformants were selected on low-salt md plates containing zeocin ( g/ml). the colonies of recombinant yeast cells with high copies of the m gene were screened on lowsalt md plates containing different concentrations of zeocin ( g/ml, and mg/ml). a screened colony of yeast cells was selected and inoculated into ml of bmmy in the presence of zeocin ( g/ml). the culture was grown at - • c until the optical density at nm (od ) reached . . the culture was then centrifuged at • c at × g for min, the cells were re-suspended in / prime volume of mmy, and continued to grow at - • c until the optical density at nm (od ) reached - . methanol was added every h to a final concentration of . % to induce the expression of recombinant m protein, and the incubation was continued for further - days. control cultures were processed in parallel. the recombinant m protein was secreted into the culture supernatant as soluble form. the supernatant was harvested by centrifugation and used for analysing the expression level of recombinant m protein by sds-page. the highly expressing pichia colony was selected for inoculation into ml of bmmy, and grown at - • c until the culture reached an od = . . the culture was centrifuged at • c at × g for min, the cells were resuspended in / prime volume of mmy, and continued to grow at - • c until the optical density at nm (od ) reached - . glycerol and methanol were added every h to final concentrations of . and . %, respectively, to induce the expression of recombinant m protein, and the incubation was continued for a further - days. the supernatant of culture was harvested by centrifugation at × g for min at • c, then ammonium sulphate was added to a final saturation of % to precipitate proteins. the pellet was collected by centrifugation at × g for h at • c and dialyzed to ml of pbs overnight. the dialysis buffer was replaced every h until it was confirmed that the ammonium sulphate was completely removed. the prepared sample was loaded onto an nta column equilibrated with niso . this process was repeated three times. the loaded column was first washed with a -fold column volume of mcac ( mm tris, ph . ; mm nacl) to remove the contaminant proteins, and then eluted with ml of mm imidazole in mcac . the different elution parts were analyzed by sds-page, and the part containing recombinant m protein was concentrated by superfiltration. two microgram of the purified recombinant m protein was separated by sds-page and transferred to polyvinylidene difluoride membrane (pvdf) by standard methods. after blocking with pbst ( mmol/l nacl, . mmol/l kcl, mmol/l nahpo , . mmol/l kh po and . % tween- ) containing % non-fat milk for h at • c, the membrane was incubated for h at room temperature with sars-covpositive human serum ( : dilution). after washing three times in an appropriate volume of pbst, the membrane was incubated for h with peroxidase labeled goat antihuman igg ( : , no: a- , sigma). the membrane was washed three times with pbs-t and once with distilled water, for min each time. finally, the washed membrane was transferred into an appropriate volume of ecl solution (amersham) and reacted at room temperature for about min, then put onto a sensitization film for - min to develop and fix. the purified recombinant m protein was diluted with mmol/l carbonate buffer ( mmol/l naco , mmol/l nahco , [ph . ]), and then used to coat -well plates (costar inc.) ( g/well) overnight at • c. after washing with pbs-t (pbs containing . % tween- , ph . ) for three times, each well of the plate was incubated with blocking solution ( % bsa in carbonate buffer) for h at • c. the wells were washed three times with phosphatebuffered saline (pbs) containing . % tween- (pbs-t). hundred microlitres of the primary antibody (sars patient sera), diluted : in pbs-t, was added in duplicate and the plates were incubated for h at • c. after four washes with pbs-t, plates were then incubated with goat anti-human igg antibody labeled with peroxidase ( : , , no.: a- , sigma) for h at • c. tmb [ , , , -tetramethylbenzidine] substrate ( l/well) was added after seven washes with pbs-t and the wells were incubated for min at • c. fifty microlitres of stop buffer ( mol/l h so ) was added to each well and the optical density (od) was read at nm in bio-rad . the cut-off value was defined as the mean od plus three standard deviations calculated from the four negative samples used as control. the m gene fragment without transmembrane domain was amplified from the genomic cdna of sars-cov strain bj . sequence analysis indicated that the nucleotide sequence of that amplified fragment is completely identical to that of sars-cov strain bj (accession number ay ). the deduced amino acid sequence of the m protein of sars-cov strain bj has identities of . % or less with the m proteins of other human or animal coronaviruses (fig. ) . the amplified product of bp was cloned in the ppicz␣a expression vector, and the sequences of inserts were verified by sequencing (data not shown). the nucleotide sequence alignment performed by dnaman software re- vealed % nucleotide identity between the inserted sequence and that of sars-cov bj . the m gene fragment was cloned in ppicz␣a expression vector and transformed into yeast host strain gs . the colonies of recombinant yeast cells with high copies of m gene were screened. after inducing with methanol, the supernatant of culture was harvested by centrifugation. the recombinant protein with a polyhistidine tag at the c-terminus was produced in soluble form in the culture supernatant (fig. ) . twenty microlitres of the supernatant ( g of protein) was subjected to sds-page and coomassie blue staining, and a protein band corresponding to the expected molecular mass of kda was revealed in supernatant, which was not present either in the culture before the induction or in the control culture before and after the induction. the recombinant m protein was purified by nickel affinity chromatography and the purity was confirmed by single banding in sds-page (fig. ) . upon elution from the nta column, the protein was > % pure as estimated from in order to test the reactivity of the recombinant m protein, western blotting was carried out using sars-cov-positive human serum. the protein band of kda showed a strong and specific reaction with the sars-cov-positive serum, whereas the supernatant of the induced culture of recombinant yeast without the insert of the m gene fragment did not react with the sars-cov-positive serum (fig. ) . these results confirmed the authenticity of the recombinant m protein. to test whether the recombinant m protein is effective as an elisa antigen for detecting sars-cov patient serum, the sera from four healthy people and four sars patients were used. the mean and the standard deviation obtained using four sars-cov-negative sera were . and . , respectively. therefore, the cut-off value of ods was determined as . . sera from four sars patients were determined to be positive using only the m protein as an elisa antigen. the mean and standard deviation were . and . , respectively (see fig. for detailed data). the same sera from four sars patients were also positive by whole sars-cov elisa in parallel, using a diagnostic fig. . detection results of eight human sera by elisa using purified recombinant m protein as antigen.# - : sera from four healthy people, respectively, # - : sera from four sars patients, respectively. kit for antibodies to coronavirus (elisa) obtained from beijing bgi-gbi biotech co., ltd., china (unpublished results). coronaviruses are important pathogens in human and animals, where they cause mostly respiratory and enteric diseases. sars-cov is a newly identified human coronavirus that differs from all previously known human coronaviruses (for example, hcov- e and hcov-oc ) in both the speed of transmission and the severity of disease. both the lack of understanding and effective methods to control the virus means that the threat of sars still exists, and therefore, there is an urgent need to develop effective vaccines and anti-viral drugs. the key role of the m protein in coronavirus assembly, budding and inducing virus neutralization make it an attractive target for the development of vaccines and drugs, as well as for effective diagnostic reagents. sars-cov differs from other coronaviruses in nucleotide sequence, and the exact function of its m protein is unknown. since sars-cov is a pathogen with high infectivity and mortality, special facilities are required when the live virus is used to prepare viral proteins given the high risk of infection. therefore, it is important to obtain large amounts of m protein by expression in vitro. the m protein is a structural glycoprotein, and the glycosylation modification is important for its activity. a previous report indicated that the recombinant m protein, when expressed in inclusion body form in e. coli, reacted weakly with human sars sera . p. pastoris is a eukaryotic expression system with high efficiency, and proteins expressed in this system can get appropriate post-translational modifications, including glycosylation. therefore, the p. pastoris system is more suitable for expression of the m protein in vitro than e. coli (grinner and tschopp, ; kukuruzinska et al., ; romanos et al., ) . however, we cannot rule out the possibility that the glycosylation sites of the sars-cov m gene in p. pastoris, while suitable for detection of antibodies, may be quite different from those present in the virus. we expressed successfully the m protein of sars-cov strain bj in p. pastoris, and the expressed m protein was similar to the native protein in its immune response. this point can be demonstrated by the reaction of recombinant m protein with sars-positive sera in western blotting and elisa. the availability of large amounts of recombinant m protein offers a better chance to study its biological characteristics and its role in sars immunity during infection. due to the lack of an effective vaccine or special therapeutic drugs, the control of sars infection relies mainly on early detection of the virus or its antibodies, and rigid quarantine measures. at present, well-established laboratory diagnostic methods for sars include real-time pcr, wholevirus-based indirect immunofluorescence assay and elisa. in these assays, however, the viral nucleic acid and anti-gens needed were commonly prepared from live pathogen, so there is a strong associated risk of individual exposure to this pathogen. compared with these methods, preparing viral antigens by expression in vitro is both easier and safer. among the proteins encoded by the sars coronavirus, the m protein can induce an immunological response and thus may be used for detection of sars-cov antibodies. elisa with the expressed recombinant m protein of canine coronavirus has been reported, and was demonstrated to be a valid and effective detection method (elia et al., ) . in order to evaluate whether our expressed recombinant m protein could be applied as a diagnostic antigen for sars, we used it as an elisa antigen to detect eight collected sars-positive and sars-negative human sera. the results were in complete accordance with those of other assays, thus indicating that the recombinant m protein may be useful as an elisa antigen for detecting specific antibodies to sars-cov in human sera. furthermore, the recombinant m protein offers several advantages over the cell-prepared sars-cov antigen. in summary, the p. pichia system is a viable tool to express the m protein at high-levels in vitro. the availability of large amounts of sars-cov recombinant m protein offers an opportunity to better investigate the biological properties of the m protein during sars-cov infection as well as its immunological role, and thus provide a basis for development of sars-cov vaccine and therapeutic drugs. the recombinant m protein expressed in p. pastoris may have better antigenicity than that expressed in e. coli, and is more suitable for use as a diagnostic antigen. infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles pichia protocols coronavirus particle assembly: primary structure requirements of the membrane protein assembly of the coronavirus envelope: homotypic interactions between the m proteins o-glycosylation of the mouse hepatitis coronavirus membrane protein recombinant m protein-based elisa test for detection of antibodies to canine coronavirus size distribution and general structural features of n-linked oligosaccharides from the methyltrophic yeast picha pastoris recombinant protein-based enzyme-linked imunosorbent assay and immunochromatographic tests for detection of immunoglobulin g antibodies to severe acute respiratory syndrome (sars) coronavirus in sars patients development of a western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome protein glycosylation in yeast identification of a specific interaction between the mouse hepatitis virus a nucleocapsid protein and packaging signal cooperation of an rna packaging signal and a viral envelope protein in coronavirus rna packaging early diagnosis of sars coronavirus infection by real time rt-pcr detection of sars coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-pcr assays identification of severe acute respiratory syndrome in canada transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions foreign gene expression in yeast: a review the coronavirus membrane protein diagnosis of severe acute respiratory syndrome (sars) by detection of sars coronavirus nucleocapsid antibodies in an antigencapturing enzyme-linked immunosorbent assay nucleocapsidindependent assembly of coronavirus-like particles by coexpression of viral envelope protein genes complement-dependent neutralization of transmissible gastroenteritis virus by monoclonal antibodies over-expression in escherichia coli and purification of nucleocapsid and membrane protein of sars coronavirus we are grateful to hai pang from tsinghua university for technical assistance, and to gewei lian and hongkui deng from peking university for providing us with sera and for help with elisa experiments. we also thank hui wang from oxford university for comments and critical reading. this work was supported by project & of the ministry of science and technology of china (grant no. gz ( / ): "structural proteomics of sars coronavirus"). key: cord- -adul nzf authors: wu, qingfa; xu, zuyuan; wei, tian; zeng, haipang; li, jingxiang; gang, haixue; sun, min; jiang, fangbo; wang, xiang; dong, wei; yang, ling; wang, jian title: development of taqman rt-nested pcr system for clinical sars-cov detection date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: adul nzf severe acute respiratory syndrome (sars) is an acute newly emerged infectious respiratory illness. the etiologic agent of sars was named ‘sars-associated coronavirus’ (sars-cov) that can be detected with reverse transcription-polymerase chain reaction (rt-pcr) assays. in this study, sets of nested primers covering the sars-cov genome have been screened and showed sufficient sensitivity to detect sars-cov in rna isolated from virus cultured in vero cells. to optimize further the reaction condition of those nested primers sets, seven sets of nested primers have been chosen to compare their reverse transcribed efficiency with specific and random primers, which is useful to combine rt with the first round of pcr into a one-step rt-pcr. based on the sensitivity and simplicity of results, the no. primer set was chosen as the candidate primer set for clinical diagnoses. to specify the amplicon to minimize false positive results, a taqman rt-nested pcr system of no. nested primer set was developed. through investigations on a test panel of whole blood obtained from sars patients and control persons, the specificity and sensitivity of the taqman rt-nested pcr system was found to be and %, respectively, which suggests that the method is a promising one to diagnose sars in early stages. an outbreak of a newly emergent infectious disease referred to as severe acute respiratory syndrome (sars), first identified in guangdong province, china in november , infected as many as individuals and resulted in deaths around the world by july (http://www.who.int/csr/sars/en/). the etiologic agent of sars has been identified as a new coronavirus and named as the sars-associated coronavirus (sars-cov) ksiazek et al., ; drosten et al., ) . sars-cov is an enveloped, positive-strand rna virus. genomes of more than strains of sars-cov have been sequenced (marra et al., ; rota et al., ; ruan et al., ; qin et al., ) . the size of genome is about - kb, which is the longest rna positive strand virus to date. based on the genetic analysis of the genome sequence, the sars-cov has been classified as a distinct group from any previously known coronavirus . the symptoms of sars resemble those of other forms of 'atypical pneumonia' that are usually caused by mycoplasma, chlamydia species, and others. in the absence of effective drugs and vaccines for sars, rapid identification of this disease is of importance for controlling spread. at present, virus isolation by cell culture, elisa, ifa and reverse transcription-polymerase chain reaction (rt-pcr) are the major methods to diagnose sars. elisa and ifa methods depend on the detection of igg and/or igm against sars-cov virus that emerged in the blood during the later phase of infection drosten et al., ) . there is a critical need for a test that can detect sars at early stages of illness (days - ) since persons infected with the sars-cov can infect a large number of individuals easily (rosling and rosling, ) . the detection of viral rna with rt-pcr assays is the most widely used approach for early detection of pathogens poon et al., ) . based on the genomic sequence of bj (accession number: ay ), sets of nested primers covering sars-cov have been synthesized. in this study a methodology for selecting the no. nested primers as the most promising primers for clinical purposes and a taqman rt-nested pcr system using the no. nested primer set are described, and the system was evaluated on a test panel. the primers were designed with the genome of bj strain of sars-cov (ay ) as the target. each set of primers was designed with primer software freely available through internet (http://www.basic.northwestern.edu/bio tools/primer .html). the size of outer amplicon and inner amplicon are - and - bp, respectively. the annealing temperature was restricted at the range of - • c. the primer sequences are summarized in table . these primers were synthesized by a dna biotechnology company (shanghai). the fluoregenic probe for no. nested primer sets -fam-aag gtg aca ctc gct gat gct g-tamra- was provided by sangon biotechology company (shanghai). fam represents the fluorescent reporter, and the tamra quencher is linked with the last nucleotide g. the rna of viruses cultured in vero cell was extracted with the qiaamp viral rna mini kit (qiagen). whole blood samples of patients diagnosed clinically with sars were used in the panel. all patients (female/male = / , age = . ± . ) had high fever, and the day with the highest temperature was regarded as day . in the meantime, whole blood samples of nine control persons were also included in the test panel. the qiaamp rna blood mini kit (qiagen) was used to prepare the whole blood samples of the test panel. according to the manufacture's instructions, rna was eluted in a final volume of l of elution buffer. all the above procedures were performed in biosafety level laboratory. the reverse transcription reaction was carried out separately with a random primer and a specific primer. each reaction mixture includes ng random primers or l of m specific primer, l of total rna and l dntp ( mm). the mixture was heated at • c for min and then placed immediately on ice. the following reagent was then added to the mixture: l of first strand-synthesis buffer ( ×, promega), l of . m dtt (promega), l rnase inhittor ( u/l, promega) and l of superscript ii rnase h-rt ( u/l, invitrogen). the rt reaction was carried out at • c for min, followed by incubation at • c for min, and then finally inactivation of the enzyme at • c for min. the first round pcr was carried out in a l volume containing l of rt product, . m of each primer, unit of taq dna polymerase (promega), . mm dntps, mm tris-hcl (ph . ), mm kcl, . mm mgcl . thermocycling was performed on a thermocyler of mj-research ptc- (mj-research inc.), with initial denaturation for min at • c, followed by cycles of ( • c for s, optimal tm for s, • c for s). the final extension reaction was performed for min at • c to complete all the pcr reactions. the second round of pcr was on undertaken on icycler (bio-rad). to detect the pcr product, sybr green i or taqman probe in the second round reaction was used in the mixture. the reaction volume for the second round of pcr was also l, containing l product of first round pcr reaction, . m of each primer, unit of taq dna polymerase (promega), . mm dntps, mm tris-hcl (ph . ), mm kcl, . mm mgcl and . l of sybr green i or . l of taqman probe ( m). for using the taqman probe in mixture, thermocycling was carried out with initial denaturation for min at • c, followed by cycles of ( • c for s, • c for min). for detection with sybr green i, thermocycling was carried out with initial denaturation for min at • c, followed by cycles of ( • c for s, optimal tm for s, • c for s). a melting curve was completed from to • c, increasing by . • c per cycle, to investigate the amplicons. the sets of nested primers used in the study distributed unevenly in the sars genome, but they spanned all known genes and the two predicted genes identified in the sars-cov genome (fig. ) . to compare the sensitivities of these sets of nested primers, serial -fold di-lution genome cdna of bj that reverse transcribed with random primer was used as the template to carry out the nested pcr. the initial concentration of total rna from virus and vero cells was ng/l, which corresponds to < molecules of sars-cov per microliter. all nested primers sets could detect up to − diluted rna, which corresponds to - copies of sars genome per microliter. for nos. , , and nested primers sets, several copies of sars genome could be detected. although few sporadic signals occurred for diluted templates from − to − , they represent random, unreliable results (fig. ) . to compare the reverse transcribed efficiency of random primer versus specific primer, seven sets of nested primers covering each known gene were chosen for testing. with the -fold diluted bj genome rna series, the reverse transcriptions were carried out separately with a random primer and a specific primer. then, amplification with outer primer on the cdna was performed. the products were monitored with sybr green i that binds with double dna. the results showed that three out of seven sets of nested primers have better efficiency on templates reverse transcribed with the specific primer, and three other sets of primers showed that the reverse transcription with the random primer has higher efficiency than with the specific primer. it is worthy to note that the four sets of nested primers located on the terminus of the genome have higher efficiency when transcribed with the random primer. in contrast, the other three sets of primers targeting the initial region of genome have higher efficiency when transcribed with the specific primer. the outer reverse primer pairs of no. are the most sensitive on cdna reverse transcribed with specific primer. meanwhile, the no. primers can attain the same sensitivity on the cdna reverse transcribed with the random primers (fig. ) . as the no. nested primers set were found to be the most promising primers for clinical diagnosis, a taqman probe targeting the internal region of the no. inner amplicon has been designed to minimize false positive results. the taqman probe and no. nested primers pairs together constitute a taqman rt-nested pcr system. to test the specificity of the taqman rt-nested pcr system, a panel consisting of sets of rna from sars patients' whole blood and sets of rna from control person's whole blood was used. these rna samples were reverse transcribed with the no. outer reverse primer. after the first round of pcr, a second round of pcr was performed and monitored with real time pcr. none of the control samples produced a signal, whereas, of the samples from sars patients produced a detectable signal. the results showed the sensitivity and the lower margin of each bar shows the level of sensitivity of these outer primers. the nos. , , and have higher reverse transcription efficiency when transcribed with specific primer, whereas those primers of nos. , , , and have higher reverse transcription efficiency when transcribed with the random primer. the values on vertical axis represent the diluted concentration of rna compared with that of initial rna ( ng/l). the specificity of the taqman rt-nested pcr system to be and %, respectively (table ) . the most striking features of the sars-associated virus are its spikes on the sphere-like virion, which can be clearly observed under the electron microscope and are the basis of the name 'coronavira'. the spike (s) protein, together with the e and m proteins, constitute the components of the surface of coronaviruses. the replicase (r) protein, which occurs in limited amounts, including a and b is the defined unique non-structural protein responsible for rna replication. according to the northern blot, the subgenomic regions for s, m, and e are transcribed in larger quantities . with the -fold diluted rna as template, the sensitivity of primers targeting the s, m, e, and n proteins is higher than - -fold than those primers targeting the r protein, which suggests that the sensitivity of detection is determined mostly by the quantity of transcripts. nucleic acid amplification techniques combining reverse transcription and the polymerase chain reaction have been applied to the detection of sars. the primer is the primary component necessary for developing rt-nested pcr. the size of outer amplicon was set range from to bp, which were usually sufficiently long to design inner primers with spacing intervals of ∼ bp. compared with longer pcr products, this range and flexibility enables many assays to be produced successfully. for these primers, the reverse transcriptions with random primer and specific primer have different effects. according to the sensitivity and simplicity of our test result, the no. primer appears to be the most promising primers set for clinical diagnosis, which is very useful to simplify the detection process through combining rt and first round of pcr reactions to one-step rt-pcr reaction. because the nested pcr can detect low amounts of virus, it is necessary and important to adopt strict criteria for confirmation of positive results. the product can be confirmed as sars-cov by a number of other methods including taqman, dna chip, pcr-elisa, and sequencing. in this study, the taqman probe was applied to confirm the pcr product. the taqman system was developed by abi and employs a fluoregenic probe-based , exonuclease technology that enables amplification and detection to be carried out simultaneously, eliminating the need for post-pcr analysis (heid et al., ; fortin et al., ) . use of the fluorescent probe in real time pcr provides an additional level of assay specificity. although fluorescence increases in direct proportion to the amount of specific amplicons, the taqman probe was used to specify the product in this study because the fluorescence increases non-linearly in the second round of amplification compared with the original virus rna concentration. to confirm positive amplicon, the pcr procedure should include appropriate negative and positive controls in each run, including negative and positive controls for the extraction procedure and the pcr run. sars-cov has been detected in multiple specimens including extracts of lung, sputum, upper respiratory tract swabs, aspirate, stool, and blood samples via pcr or viral isolation drosten et al., ; poon et al., ) . high concentration of viral rna of up to million molecules per milliliter has been detected in sputum. viral rna has also been detected at extremely low concentrations in plasma during the acute illness phase and in feces during the late convalescent phase, suggesting that the sars-cov may be shed in feces for prolonged periods of time . compared with sputum and feces, blood is used more frequently in clinical practice because it is relatively easy to obtain. in this study, whole blood was therefore used as test sample in order to develop a clinically useful detection method for the early stage of infection. according to the protocol developed in the study, the sensitivity and specificity of the taqman rt-nested pcr system are and %, respectively. negative results cannot exclude the existence of sars-cov in tested samples, which may be attributed to low amounts of rna in the samples. the sensitivity of rt-nested pcr tests for sars depends on the specimen and the time of testing during the course of the illness. rapid diagnosis not only offers considerable benefits when a positive case is quickly identified, but is also equally informative when rapid negative result are obtained for optimal patient management and appropriate therapy. the taqman rt-nested pcr system described here provides a rapid, sensitive, and cost effective approach for the diagnosis of sars-cov infection. identification of a novel coronavirus in patients with severe acute respiratory syndrome use of real-time polymerase chain reaction and molecular beacons for the detection of escherichia coli o :h real time quantitative pcr a novel coronavirus associated with severe acute respiratory syndrome rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) coronavirus as a possible cause of severe acute respiratory syndrome pneumonia causes panic in guangdong province characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection the authors would like to acknowledge the specific grant for sars research from the ministry of science and technology of china. key: cord- - uqlimov authors: bolotin, s.; robertson, a.v.; eshaghi, a.; de lima, c.; lombos, e.; chong-king, e.; burton, l.; mazzulli, t.; drews, s.j. title: development of a novel real-time reverse-transcriptase pcr method for the detection of h y positive influenza a h n isolates date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: uqlimov during the – influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza a h n isolates. although oseltamivir resistance rates vary from region to region, % of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue of the neuraminidase gene of influenza a. in order to implement effective resistance testing locally a novel real-time reverse-transcriptase pcr (rt-pcr) assay was developed for the detection of the h y mutation. to evaluate this method, oseltamivir resistant and oseltamivir sensitive h n influenza isolates were tested using sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel h y rt-pcr assay. in comparison to sanger sequencing, the sensitivity and specificity of the h y rt-pcr assay were % ( / ) and % ( / ) respectively, while the sensitivity and specificity of pyrosequencing were % ( / ) and . % ( / ) respectively. although all three methods were effective in detecting the h y mutation associated with oseltamivir resistance, the h y rt-pcr assay was the most rapid and could easily be incorporated into an influenza subtyping protocol. during the - influenza season global strain surveillance for antiviral resistance revealed the sudden emergence of oseltamivir resistance in influenza a h n isolates. although oseltamivir resistance rates vary from region to region, % of isolates tested globally were found to be oseltamivir resistant by a histidine to tyrosine mutation of residue of the neuraminidase gene of influenza a. in order to implement effective resistance testing locally a novel real-time reverse-transcriptase pcr (rt-pcr) assay was developed for the detection of the h y mutation. to evaluate this method, oseltamivir resistant and oseltamivir sensitive h n influenza isolates were tested using sanger sequencing, which is the reference method for detection of resistance, pyrosequencing and the novel h y rt-pcr assay. in comparison to sanger sequencing, the sensitivity and specificity of the h y rt-pcr assay were % ( / ) and % ( / ) respectively, while the sensitivity and specificity of pyrosequencing were % ( / ) and . % ( / ) respectively. although all three methods were effective in detecting the h y mutation associated with oseltamivir resistance, the h y rt-pcr assay was the most rapid and could easily be incorporated into an influenza subtyping protocol. © elsevier b.v. all rights reserved. the beginning of the - influenza season marked a significant and sudden increase in resistance of influenza a h n to the neuraminidase (na) inhibitor oseltamivir through a histidine to tyrosine mutation at residue of the na gene (sheu et al., ) (residue in the n gene). by the end of the - influenza season the resistance rate to oseltamivir was % globally. countries with little or no previous oseltamivir resistance, such as the united states, canada and norway reported resistance rates of %, % and % respectively (world health organization, ) . early data from the southern hemisphere for the influenza season suggested oseltamivir resistance rates among h n isolates of as high as % (besselaar et al., ) . in addition to reports of resistance in seasonal h n isolates, reports of oseltamivir resistance have also emerged in patients infected with h n avian influenza (de jong et al., ; kimm-breschkin et al., ; le et al., ) , bringing into question the use of this antiviral for pandemic influenza treat-ment and prophylaxis (moscona, ; world health organization, ) . this emerging oseltamivir resistance has prompted laboratorians to look at time effective means to identify h n influenza with the h y mutation. currently, the gold standard for antiviral resistance screening is phenotypic methods (meijer et al., ) . however, genotypic methods such as sanger dideoxy sequence analysis of the na gene can also be performed to detect mutations such as h y that are associated strongly with oseltamivir resistance. while both phenotypic and sequencing methods are effective in detecting resistance, they can be labour intensive, have a long turn-around-time and be quite expensive, suggesting the need for a rapid, high-throughput approach to influenza drug resistance testing. in this study we introduce the utilization of a novel real-time reverse-transcriptase pcr (rt-pcr) assay for detection of the histidine to tyrosine mutation at residue . this assay is designed to produce a sigmoidal curve when an isolate is oseltamivir susceptible and no curve when an isolate is oseltamivir resistant, thus serving as a quick screening tool that can be incorporated into the influenza subtyping protocol. current taqman technology utilizes a minor groove binder (mgb) that forms a stable complex with the minor groove of singlestranded dna, increasing stability and specificity (afonina et al., ; uchiyama et al., ) . due to these qualities mgb probes aaatgcacccaat reisdorf et al. ( ) are characterized by a shorter length and a higher melting temperature (t m ). mgb-based probes are therefore highly applicable for detecting the presence of polymorphic sequences (uchiyama et al., ) ; however their increased stability may result in false positives. probe cross reactivity is therefore one reason that mgb chemistry is not generally used to detect point mutations. to avoid incorrect assignment of the h y point mutation an effort was made to reduce binding to oseltamivir-resistant isolates which have a c→t point mutation by designing the mgb probe to detect wildtype sequence only. to limit cross reactivity of similar sequences, the mgb probe was designed so that the mgb was linked to the mutable base pair. this was accomplished by designing the probe so that the c→t mutation was bp from the of the probe, which is the site of mgb linkage. this study compares the utility of the h y rt-pcr assay to sanger sequencing, which is the current reference method for h y detection, as well as pyrosequencing, an emerging method for the detection of the h y point mutation (duwe and schweiger, ) . specimens used in this study were from a population that was moderately resistant to oseltamivir, with resistance rates of % in ontario isolates in - . one hundred and one patient isolates were used in this study. these were influenza a h n positive isolates most similar genetically to the a/brisbane/ / -like lineage collected during the - influenza season in ontario, canada. upon arrival in the laboratory, neuraminidase (n ) was detected from specimens using the quantitect probe rt-pcr kit (qiagen) and published primers and probes (schweiger et al., ) (table ) using an mxpro real-time pcr thermocycler (stratagene). this n subtyping assay has been validated previously as a sensitive and specific method for the detection of n in clinical specimens (schweiger et al., ) . it has also been used extensively in external quality assurance panels and classic phenotypic assays. mutations at residue of the n gene were detected using sanger dideoxy sequencing. forty specimens with a wild-type histidine at residue and specimens with a tyrosine at residue were chosen randomly for analysis. patient specimens were grown in rmk cells and nucleic acid was extracted from viral culture isolates using the nuclisens ® easymag tm system (biomérieux) according to the manufacturer's instructions. rna was extracted from l of eagle's minimum essential media (emem) (biowhittaker) with an elution volume of l. to control for extraction all specimens were tested for human target gapdh by using the gapdh taqman control reagents (applied biosystems). to perform sanger sequencing, reverse transcription and amplification of the n gene was performed using the onestep rt-pcr kit (qiagen). three l of rna were combined with l × onestep rt-pcr buffer, l ( m) of dntps, l of onestep rt-pcr enzyme mix, . l ( u) of rnase inhibitor, l of each primers an b- forward (takao et al., ) and n - reverse (yuen et al., ) at a final concentration of . m (table ) and . l of water. reverse transcription and amplification was performed using an icycler (biorad) conventional pcr thermocycler at the following conditions: cycle at • c for min and cycle at • c for min, followed by cycles of amplification at • c for s, • c for s and • c for min, and a final extension cycle of • c for min. following visualization of the pcr products using gel electrophoresis, labelling of the pcr products was performed using the big dye terminator cycle sequencing kit v . (applied biosystems). one l of dna was combined with l of bigdye, l of × bigdye buffer, l of m an b- forward primer and l of water. an icycler (biorad) was utilized for labelling using cycle at • c for min and cycles of • c for s, • c for s, • c for min. labelled pcr products were purified using the dyeex column kit (qiagen) according to the manufacturer's instructions. nucleotide sequences were determined using the abi genetic analyzer (applied biosystems) and analyzed using finchtv (geospiza) and chromas (technelysium) dna sequencing software. for pyrosequencing, reverse transcription and amplification of the na gene from the extracted rna of influenza a h n isolates was performed using the onestep rt-pcr kit (qiagen). three l of rna was combined with l of onestep rt-pcr buffer, l ( m) of dntps, l of onestep rt-pcr enzyme mix, . l ( u) of rnase inhibitor, l of each primers na-forward and na-reverse (reisdorf et al., ) at a final concentration of . m ( table ) . reverse transcription and amplification was performed using an icycler (biorad) conventional pcr thermocycler at the following conditions: cycle of • c for min, • c for s, • c for min, • c for min and • c for min, followed by cycles of amplification at • c for s, • c for s and • c for min, and one extension cycle of • c for min. after visualization on an agarose gel, l of pcr product was combined with l of binding mix composed of l of binding buffer (biotage), l of water and l of streptavidin-sepharose hp beads (amersham biosciences). samples were shaken using a plate shaker (labnet) at rpm for min at room temperature and then combined with l of annealing mix composed of l of annealing buffer (biotage) and . l of m of sequencing primer na-forward sequencing (table ) . pyrosequencing was carried out using a pyromark tm id (biotage) according to the manufacturer's instructions. to design a novel h y rt-pcr assay, reference sequences were aligned using clustal w (larkin et al., ) . primers and probe were designed by eye using the consensus sequence. the assay design was evaluated using primer express (applied biosystems) in order to test for t m and secondary structure. the sequences of the primers and probe for the h y assay are listed in table . rt-pcr was carried out using l of rna was combined with l of × quan-titect probe rt-pcr master mix, . l of quantitect rt mix, l of × h y primers and probes and . l of water. reverse transcription and rt-pcr was carried out on the stratagene mxpro using cycle of • c for min, cycle of • c for min, and cycles of • c for s and • c for min. to ensure that h y probe failure was due to mutation at residue and not sample degradation or inhibition, the n subtyping rt-pcr was run in parallel on the same plate. to determine the limit of detection (lod) of the h y and n control rt-pcr assay, serial ten-fold dilutions of nucleic acid from an oseltamivir-sensitive clinical influenza a/brisbane/ / h n isolate were tested. the starting concentration of virus for this assay was tcid /ml. this dilution series was tested by both methods in triplicate to determine reproducibility as well as in singles to simulate clinical testing. the lod for the h y and n assays were determined by probit regression ( % confidence interval) using spss version . to assess the cross-reactivity of the h y rt-pcr assay with non-target respiratory pathogens, a panel of nucleic acid from respiratory pathogens was tested using the h y assay. nucleic acid used in this panel was derived either from clinical specimens or from atcc strains. extraction and inhibition controls for the clinical specimens were performed upon initial specimen testing using the seeplex rv detection kit (seegene) (drews et al., ; roh et al., ) . the lod of the h y rt-pcr assay and the n control assay were determined using serial ten-fold dilutions of nucleic acid from a sequence-confirmed oseltamivir-sensitive clinical influenza h n solomon islands specimen ( fig. a and b) . the lod was calculated using probit regression ( % confidence interval) and found to be . tcid /ml for the h y assay and . tcid /ml for the n control assay. inter-assay and intra-assay variability experi- fig. . determination of the lod of the h y and n assays: nucleic acid extracted from an influenza a h n oseltamivir-sensitive isolate was used to determine the lod of the h y and n assays. the starting concentration of virus was tcid /ml. amplification curves for (a) h y and (b) n are shown. determination of oseltamivir resistance in influenza a h n isolates using the h y rt-pcr assay. representative amplification curves are shown of both oseltamivir susceptible and resistant clinical isolates. isolates which were oseltamivir susceptible produced a sigmoidal curve (green) while oseltamivir-resistant isolates produced no curve (blue). approximately % of resistant isolates tested produced a low-fluorescence, non-sigmoidal curve (red). ments indicated that the assays were reproducible. the intra-assay coefficient of variation (%cv) was found to be no higher than . % for the h y assay and . % for the n assay. the inter-assay %cv was found to be no higher than . % for the h y assay and . % for the n assay. the cross reactivity of the h y rt-pcr assay was assessed using a panel of nucleic acid from respiratory pathogens. viral nucleic acid was derived from clinical specimens while bacterial nucleic acid was extracted from clinical specimens or atcc strains. ) were only obtained with oseltamivir-sensitive influenza a h n nucleic acid, which was used as a positive control. specificity of the n probe has been published previously (schweiger et al., ) and was not repeated in this study. the sensitivity and specificity of the h y rt-pcr assay were evaluated using influenza a h n isolates, including h ypositive and h -positive isolates which were tested previously for the h y mutation using sanger sequencing (the reference method) and pyrosequencing. all isolates were collected from ontario patients during the - influenza season and were most similar genetically to a/brisbane/ / -like lineage. the sensitivity and specificity of the h y rt-pcr assay were found to be % ( / ) and % ( / ) respectively (table ) . this compared favourably to the sensitivity and specificity of pyrosequencing, which were % ( / ) and . % ( / ) respectively. in contrast to h -positive isolates, approximately % of h ypositive isolates produced no cycle threshold (ct) value, while approximately % produced a non-sigmoidal, low fluorescence amplification curves when tested using the h y rt-pcr assay (fig. ) . interpretation of these curves was not problematic as the difference between amplification curves of oseltamivir-sensitive isolates and the non-sigmoidal flat curves of a subset of the resistant isolates was apparent. the tat of the h y rt-pcr assay was hrs after nucleic acid extraction. this study was initiated during the - influenza season, the first season during which significant oseltamivir resistance was observed globally in influenza a h n strains at residue (sheu et al., ; world health organization, ) . as a result of this sudden increase in global resistance to the drug it was felt that an in addition to national and global surveillance programs, an oseltamivir resistance detection protocol should be implemented in ontario clinical laboratories to assess resistance locally. this study evaluated the use of a novel h y rt-pcr assay for detection of h y-positive isolates in comparison to sanger sequencing and pyrosequencing, and found that while all three methods may have a role in influenza diagnostic testing, the h y rt-pcr assay is a rapid and effective test for the detection of oseltamivir resistance through mutation at residue . sanger sequencing is the most established method for detection of point mutations and was therefore used as the reference method for this study. although it was accurate in determining mutations at residue , it was quite expensive and not time-efficient to sequence every h n isolate in a moderately resistant population like ontario, where only % of isolates were oseltamivir resistant. in addition to time restrictions, sequence alignment analyses for sanger-sequenced isolates must be done using a separate program that generally requires some expertise for operation. when compared to sanger sequencing, pyrosequencing was also quite effective in determining h y mutations, with a sensitivity and specificity of % and . % respectively. pyrosequencing was less time consuming and provided unambiguous sequence from the primer binding site, in contrast with sanger sequencing, which does not provide sequence for the first - bp of sequence (pourmand et al., ) . unlike sanger sequencing instruments, the biotage pyrosequencer has built-in alignment capabilities making sequence analysis less difficult. while in this study pyrosequencing was found to be highly sensitive, the success of pyrosequencing is dependent on the sequence to be analyzed since homopolymeric sequences (repetition of the same nucleotide three or more times) cannot always be discerned (parameswaran et al., ) . both sanger sequencing and pyrosequencing are hampered by the need to pcr amplify the na gene of every isolate before sequencing. this lengthens the turn-around-time (tat) for oseltamivir-resistance testing and adds an extra step in the influenza testing algorithm. the h y rt-pcr assay was found to be as sensitive and specific as sanger sequencing while decreasing the tat. the tat for the h y rt-pcr assay was only h after nucleic acid extraction, compared to h for sanger sequencing and . h for pyrosequencing. designing the mgb probe to detect wild type and not mutant sequence allowed for confirmatory sequencing of a minority of resistant isolates as opposed to a majority of wild type isolates, which would not have been time or cost efficient. implementation of the rt-pcr assay during the - influenza season would have reduced time and cost of isolate sequencing by %, since only isolates which did not produce a sigmoidal curve would have to be sequenced using sanger or pyrosequencing technologies to confirm the presence of a mutation at h y. although this assay was validated using virus from cultured cells and carried out separately from the influenza a subtyping assay, it has also been performed successfully using nucleic acid extracted directly from specimens. this assay can therefore be incorporated easily into the current subtyping protocol, thus decreasing the tat even more. in addition to detecting oseltamivir resistance in seasonal influenza, the h y rt-pcr assay can also be utilized during a pandemic influenza outbreak, since it is thought that oseltamivir resistance, which has already been reported in h n avian influenzas strains (de jong et al., ; kimm-breschkin et al., ; le et al., ) will have a significant impact if an influenza pandemic occurs (lipsitch et al., ) . currently, oseltamivir is the recommended treatment and prophylaxis for avian influenza (schunemann et al., ) , and has been stockpiled by most countries. the h y rt-pcr assay will serve as a screening tool for resistance and could be performed rapidly enough to affect treatment decisions. the short tat and high-throughput nature of this assay will help to conserve resources during a pandemic. the global emergence of h y-mediated oseltamivir resistance in influenza a h n specimens has resulted in the recent development of several molecular methods for resistance detection in addition to sanger sequencing and pyrosequencing. for example, an allelic-discrimination (snp) assay to detect the h y mutation has been published by carr et al. ( ) . the novel h y rt-pcr assay reported here compares favourably with the carr assay, exhibiting parallel test characteristics, no cross reactivity and a similar tat. however, since the h y rt-pcr assay requires only one probe while snp assays by nature utilize two probes, the h rt-pcr assay has a lower cost per test and can be multiplexed easily into influenza detection algorithms that are real-time pcr based. a drawback of the h y rt-pcr assay is that oseltamivir resistance conferred by mutations in residues other than will not be detected. in addition, compensatory mutations in other genes, such as the hemagglutinin gene (abed et al., ) or in conserved genes would not be recognized using this method. however, since oseltamivir resistance is mediated currently by the h y mutation globally, with emerging resistance in many jurisdictions (world health organization, ), a rapid and cost efficient screen targeted at h y is the only method for high-throughput resistance testing of clinical specimens. in conclusion, the emergence of h y mediated oseltamivir resistance globally in influenza a h n isolates has indicated the need for a rapid molecular test for the detection of this mutation. the rapid screening approach presented in this study may allow for high-throughput testing for oseltamivir resistance in a timely fashion. the ability to incorporate this method into realtime pcr-based influenza detection testing algorithms and the potential of this assay for use in an influenza pandemic make this test a valuable tool for use in the clinical microbiology laboratory. characterization of influenza a(h n ) clinical isolates with reduced susceptibility to neuraminidase inhibitors due to mutations in the hemagglutinin gene sequencespecific arrest of primer extension on single-stranded dna by an oligonucleotide-minor groove binder conjugate widespread oseltamivir resistance in influenza a viruses (h n ) rapid molecular detection of the h y oseltamivir resistance gene mutation in circulating influenza a (h n ) viruses oseltamivir resistance during treatment of influenza a (h n ) infection use of the seeplex rv detection kit for surveillance of respiratory viral outbreaks in a new and rapid genotypic assay for the detection of neuraminidase inhibitor resistant influenza a viruses of subtype h n , h n , and h n reduced sensitivity of influenza a (h n ) to oseltamivir clustal w and clustal x version . avian flu: isolation of drug-resistant h n virus antiviral resistance and the control of pandemic influenza influenza antiviral susceptibility monitoring activities in relation to national antiviral stockpiles in europe during the winter oseltamivir resistance-disabling our influenza defenses a pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing rapid and highly informative diagnostic assay for h n influenza viruses comparison of the seeplex reverse transcription pcr assay with the r-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses who rapid advice guidelines for pharmacological management of sporadic human infection with avian influenza a (h n ) virus application of a fluorogenic pcr assay for typing and subtyping of influenza viruses in respiratory samples surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide in neuraminidase subtyping of human influenza a viruses by rt-pcr and its application to clinical isolates short consensus probes with [prime]-minor groove binder of the immunoglobulin heavy-chain gene for real-time quantitative pcr in b-cell non-hodgkin lymphomas who intercountry consultation. influenza a/h n in humans in asia influenza a(h n ) virus resistance to oseltamivir - influenza season clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus key: cord- -p dg jw authors: bigault, lionel; brown, paul; bernard, cécilia; blanchard, yannick; grasland, béatrice title: porcine epidemic diarrhea virus: viral rna detection and quantification using a validated one-step real time rt-pcr date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: p dg jw since , porcine epidemic diarrhea virus (pedv) has reemerged in europe. rt-pcr methods have been described for the detection of pedv, but none have been validated according to a norm. in this study we described the development and validation of a sybr™ green one-step rt-qpcr according to the french norm nf u - , for the detection and quantification of pedv viral rna. the method was validated from sample preparation (feces or jejunum) through to nucleic acid extraction and rt-qpcr detection. specificity and sensitivity, limit of detection (lod), limit of quantification (lq), linearity, intra and inter assay variability were evaluated using transcribed rna and fecal and jejunum matrices spiked with virus. the analytical and diagnostic specificities and sensitivities of this rt-qpcr were % in this study. a lod of genome copies/ µl of extract from fecal matrices spiked with virus or rna transcript and genome copies/ µl of extract from jejunum matrices spiked with virus were obtained. the lower lq (llq) was genome copies/ µl and the upper lq (ulq) ( ) copies/ µl. this method is the first, validated according a norm for pedv and may serve as a global reference method to harmonize detection and quantification of pedv viral rna in both field and experimental settings. porcine epidemic diarrhea (ped) was first described in europe in . it is characterized by watery diarrhea, vomiting, dehydration, and is most notable in young piglets. the etiologic agent, porcine epidemic diarrhea virus (pedv) which was first identified by electron microscopy (em) in (chasey and cartwright, ; debouck and pensaert, ) is now characterized as an enveloped virus with a single stranded positive sense rna genome, member of the order nidovirales, suborder cornidovirinae, family coronaviridae, subfamily orthocoronavirinae, genus alphacoronavirus, subgenus pedacovirus (walker et al., ) . in the 's, pedv was detected for the first time in asia whilst in europe it was endemic. during the 's only few sporadic cases were reported in europe and most of these were reported in italy were it remains endemic (martelli et al., ) . during the last two decades new pedv strains have appeared in china and some of these strains have caused extremely severe outbreaks characterized by a morbidity of % and a mortality of - % on suckling piglets (sun et al., ) . this has led to the naming of pedv as either s-non-indel or s-indel genotypes. in general the more virulent viruses belong to the s-non-indel group. in the last decade both s-non-indel and s-indel viruses have emerged in the usa with serious consequences for the industry. throughout europe, the predominant types are now closely related to the viruses circulating in asia and north and central america (boniotti et al., ) . furthermore, all viruses reported in europe since belong to the s-indel group (grasland et al., ; stadler et al., ; steinrigl et al., ; theuns et al., ) except for one in the ukraine (dastjerdi et al., ) . this data highlights the importance of pedv diversity across several continents. in france, since , ped caused by s-non-indel is a notifiable disease. for territory monitoring purpose, all pedv suspicions have to be notified to french ministry of agriculture and the pedv genotype has to be confirmed by the national reference laboratory at the french agency for food, environmental and occupational health safety (anses). until today, no official method has been validated for the detection and quantification of the pedv viral rna. since the s, real-time pcr emerged as a tool of choice for the detection and quantification of viral rna and has multiple benefits: i) these tests are highly specific ii) are easily standardized compared to "classical" virology procedures, iii) are much less time consuming, and iv) are highly reproducible. several rt-pcrs have been described for the detection of pedv rna (kim et al., ; miller et al., ) . for a rapid, accurate and reliable diagnosis of ped in the veterinary laboratory, a method for the detection of pedv viral rna has been developed and more importantly validated according to the "association francaise de normalisation" (afnor) french nf u - norm entitled "requirement and recommendation for the implementation, development and validation of pcr in animal health" (afnor, a; afnor, b) . this validated sybr tm green one-step rt-qpcr was based on a previously published taqman® probe real time rt-qpcr (kim et al., ) and targeted the same zones of sequence in the conserved n open reading frame (orf) as this had previously allowed for broad range detection and the capability to differentiate between the closely related virus transmissible gastro-enteric virus (tgev). the method developed in the current study under nf u - , unlike other molecular tests developed for pedv, evaluates the whole process from sample preparation through to the detection and quantification by rt-qpcr. this method should help harmonize detection and quantification of viral rna from pedv belonging to both s-non-indel and s-indel strains in both field and experimental settings. all commercial methods were performed according to the manufacturers' recommendations unless otherwise stated. an alignment of pedv n orf sequences that were available on the data base at the time of the study ( ) was made using mafft (katoh and standley, ) and the probabilities of the nucleotides at the priming zones defined by kim et al. ( ) (pednf : '-cgcaaagactgaacccactaattt- ', and pednr : '-ttgcctctgttgttactt-ggagat- ') were calculated using r (wagih, ) (fig. ). based on these probabilities forward primer mpednf ( '-cgcaaagactgaacccactaa- ') and reverse primer pednr were chosen (fig. ) . these primers were subsequently checked against n orfs of the s-indel and s-non-indel pedv strains circulating in europe (dastjerdi et al., ; grasland et al., ; hanke et al., ; martelli et al., ; stadler et al., ; steinrigl et al., ; theuns et al., ) . original cv , the pedv reference strain isolated in , was collected from perfused jejunum performed in and kept at - °c. this stock was named wtcv . wtcv was propagated in cell culture as previously described (hofmann and wyler, ) and was named cccv . a stock of cccv was produced as follows: x mm confluent monolayer of vero cells (atcc® ccl- ) were infected each with µl of . x tcid of cccv in infection media; emem (thermofisher scientific, france) supplemented with . % tryptone phosphate broth, . % yeast extract, % penicillin/streptomycin and µg/ml trypsin. after hours of infection, cells were subjected to three freeze thaw cycles and the culture medium was clarified by centrifugation at g for minutes. a total volume of l of supernatant was then centrifuged for four hours at g to pellet the virus. the pellet was then resuspended in ml of pbs. the infectious viral titer of cccv was determined by immuneperoxidase monolayer assay according kärber's method (kärber, ) . the virus stock solution was titrated by immuno-peroxidase monolayer assay to . x tcid /ml. four other pedv strains were used: three french field strains (pedv/fr/ / genbank accession number (gb acc) kr , pedv/fr/ / and pedv/fr/ / gb acc mn ), and one american strain (pedv/usa/ /iowa gb acc mf , kindly provided by dr p.gauger from iowa state university). nine other 'non-pedv' rna viruses were also used: one pig alpha-coronavirus (porcine respiratory coronavirus, prcv), and two gamma-coronaviruses (infectious bronchitis virus (ibv) gb acc fj ), turkey coronavirus (tcov) gb acc kr ) as well as other pig viruses: a pig artevirus (porcine reproductive and respiratory syndrome virus (prrsv), gb acc ky ), a pestivirus (classical swine fever virus (csfv)), three pig ortomyxoviruses (swine influenza viruses h ni, h n , h n ), and two swine dna virus, one circovirus (porcine circovirus type (pcv ) gb acc af ), and an asfavirus (african swine fever virus (asfv) bankit anses-mada ). jejunum and fecal samples were collected from both specific pathogen free (spf) pigs confirmed negative for coronavirus rna by deep sequencing and from pedv infected pigs positive for pedv rna. the pedv positive samples had been collected during previous experimental studies (gallien et al., a; gallien et al., b; gallien et al., ) . spf samples were used as negative controls or were spiked with pedv produced in vitro as described in section . . spiked spf samples were used for the validation of the method and are later referred to as 'infectious reference materials'. for each jejunum sample, mg were homogenized in ml of phosphate buffered saline (pbs) (merck, france) with mm stainless steel beads in a tissuelyserii (qiagen, france). samples were then clarified by centrifugation at g for minutes. for each fecal sample, ml was diluted in ml of pbs and vortexed for minutes before clarification by centrifugation as describe above. to determine the limit of quantification (lq) of the pcr and produce standard for quantification, a rna transcript was produced by in vitro transcription of the pedv wtcv n orf sequence. wtcv rna was extracted using trizol (thermofisher scientific, france). viral rna extract was subjected to reverse transcription using hexanucleotide primers and superscript iii reverse transcriptase (thermofisher scientific, france). reverse transcription was performed at °c for hour followed by enzyme inactivation at °c for minutes. to amplify the n orf, µl of rt were subjected to pcr amplification in µl reaction containing nm of primers ogvb -f (gtcggatccactttatggcttct) and ogvb -r (gtcctcgagatt gtttaatttccterror! reference source not found.), . units of platinum taq hifi (invitrogen, france), µl of x high fidelity pcr buffer, and mgso at a final concentration of mm. the pcr was performed as follows: °c for minutes for initial denaturation, cycles of °c for seconds, seconds at °c decreasing by . °c per cycle and then °c for minutes, follow by cycles of °c for seconds, °c for seconds and °c for minutes. amplified pedv n cdna was separated on % agarose gel and extracted using montage gel extraction kit (millipore, france). ng of extracted product were cloned in pcr -topo vector (invitrogen, france). plasmid dna was prepared using nucleospin® plasmid kit (macherey nagel, france). in vitro transcription was performed with maxiscript tm t transcription kit (thermofisher scientific, france) using µg of precipitated spei linearized n orfs plasmid. rna was purified with agencourt® rnaclean xp kit (beckmancoulter, france), and quantified using qubit® fluorometer (life technology, france, saint aubin). stock of in vitro transcribed rna was stored at - °c. number of molecular copies was calculated according the following formula: ) × . × rna transcript was diluted to molecules/ µl, aliquoted in µl, supplemented with µl of rnastable® (m, france) and dried in speedvac® vacuum concentrator (thermoelectron, france). the standard transcript was resuspended in ml in deionized nuclease-free water and then log serially diluted from to copies/ µl and stored at - °c. all rna extractions were performed using rneasy® mini kit (qiagen, france) with the following modifications. µl of sample mixture containing µl of sample, µl of an external exogenous control (eec) and µl of proteinase k were used as opposed to µl of sample alone as recommended by the kit. rna was eluted with µl of nuclease-free water and stored at - °c until use. eec used in this study was viral rna genome (mengovirus). reactions were carried out in an applied biosystems real-time pcr system, with power sybr tm green rna-to-ct tm -step kit (applied biosystems, saint aubin, france). the final pcr mix volume was composed of . µl of master mix ( x), . µl of enzyme mix, µl of rna template, primers mpednf and pednr at nm or nm, h o to final volume of µl. rt-pcr cycles were as follows: reverse transcription at °c for minutes, followed by °c for minutes, then cycles of °c for seconds, °c for minute, and a final melting curve analysis step as defined by the applied software v . . all sample amplifications with a melting temperature corresponding to the standard with a viral rna concentration equal to, or above to the limit of detection (lod) were considered positive. all of the following tests were performed using primers at nm. j o u r n a l p r e -p r o o f the analytical sensitivity and specificity were determined as described in the nf u - norm. all nucleic acid extractions from viruses listed in . were tested. five strains of pedv were tested for inclusivity, and eleven other virus for exclusivity, among which, four coronaviruses, five other rna viruses, and two dna virus, all known as pathogens in pigs. the diagnostic sensitivity and specificity were determined as described in the nf u - according to nf u - , lod is the last dilution of reference material that allows a detection of the target with a confidence level of %. n rna transcript dilutions were tested for the lod of the pcr. six points of a two-fold dilution series ranging from to . genome copies/ µl were analyzed in eight replicates. three independent assays were performed for rna transcripts (lodpcr). to determine the lod of the method, spf jejunum and fecal samples spiked with cccv from to - tcid /ml, were tested in two independent assays on a hundredfold serial dilution ranging from to and n transcripts equivalent/ µl, as infectious reference materials (lodjejunum or lodfeces). lod's were determined by probit calculation (finney and stevens, ) . according to nf u - , lq is defined as the lowest (lower lq, llq) and highest level (upper lq, ulq) between which, for each dilution, the statistical bias is under or equal to . log . the bias is the difference between the measured value and the theoretical value calculated by linear regression on all dilutions. uncertainty is calculated as the variance of calculated point plus the medium bias value. the statistical bias is defined as the medium of uncertainty. for the lq, seven points of a ten-fold serial dilution of n rna transcript were tested ( to ). ten independent assays were performed on four independent serial dilutions. the lq for organic matrices were calculated on results obtained for the lod assessment (hundredfold dilution from to ). pcr efficiency was evaluated by plotting the ct against an expected rna copy number in respect to the tcid /ml (data not shown) for infectious reference material or by qubit j o u r n a l p r e -p r o o f quantification for rna transcript. in agreement with the nf u - norm, an efficiency of - % was accepted. the forward primer of kim et al. (kim et al., ) (pednf) had perfect base pairing with of the ( . %) n orfs sequences. the forward primer designed in the current study (mpednf) which did not contain the last three bases of kim et al. ( ) had perfect base pairing with of ( , %) and of those that did not match at % only one had a mismatch at the last ' position ( fig. a) . sequence of the reverse primer (pednr) had perfect base pairing with of sequence ( . %) and those that did not match at % did not have any mismatches in the last three nucleotides of the ' end ( fig. b) . concerning the alignments with the european strains available after may , pednf had perfect base pairing with of n orfs sequences (fig. c) . mpednf had perfect base pairing with of sequences, those sequences that did not match at % only contained one mismatch and these were localized close to the ' end ( fig. c) . pednr had perfect base pairing with of sequences and only one single miss-match with the remaining sequence at the ' end. amongst the different viruses strains listed in . , only the pedv strains (cv , american field strain, and three french field strains) were positive. wtcv (ct = ), cccv (ct = ), all with a tm of . ± . °c which is the expected tm for the pedv sequence amplicon according to the in vitro transcription control. all the other viruses were negative. the analytical specificity and sensibility were both %. efficiency of the method, calculated by linear regression, was . % ± . ( . ) for rna transcripts, . % ± . ( . ) for spiked jejunum and . % ± . ( . ) for spiked feces. different concentrations of primers had no effect on the efficiency of the method (data not shown), however melting curve analysis showed the presence of primer dimers at nm and not at nm (figure ). the lod was determined at copies/ µl for the rna transcript, copies/ µl ( . x . tcid /ml for the spiked feces and copies/ µl ( . tcid /ml) for spiked jejunum (table ) . for every selected rna dilution tested, from to copies/ µl, bias enlarged of uncertainty were included in the norm limits (- . to . ) and statistical bias (mean of uncertainty) were < . log ( table ). the ulqs and llq were and copies/ µl respectively for all matrices. calculations were done when a minimum of out of results were positive for the lod and for all replicates for lq. all coefficients of variation (cv) were below the . limit given by the norm nf u - with . - . , . - . , . - . , for rna transcript, jejunum and feces intra-assay cvs respectively and . - . , . - . , . - . for rna transcript, jejunum and feces inter-assay cvs respectively (table ) . the diagnostic sensitivity was % at two and fourteen dpi, pedv viral rna were detected in all true positive pigs. the diagnostic specificity was % as all non pedv infected pigs were found negative all along all experiments. pedv is of global importance to the pig industry with many different strains and genotypes existing in different continents. after and the introduction of both s-indel and s-non-indel strains to north america and the resulting huge economic losses, the french ministry for agriculture classified ped caused by the s-non-indel virulent strains as a notifiable disease. thus there was a need for a reliable method for rapid, accurate and specific detection and quantification of a broad range of pedv strains and one that was completely validated according to french norm nf u - . many methods have been developed and used for pedv detection and quantification as previously reviewed (diel et al., ) such as direct viral isolation, but it is laborious, time consuming, and requires a reliable model for all possible strains. furthermore, many pedv strains cannot be isolated in vitro. many immuno-assay tests have been developed to detect viral proteins (ifa, blotting, elisa) but all these methods are time consuming, have a low sensitivity and reaction, and are subject to cross reactivity decreasing the specificity. for these reasons the current study focused on developing and validating a specific and rapid diagnostic test for the detection of pedv viral rna. basing this test on a taqman® multiplex rt-qpcr, published by kim et al. ( ) , we developed and validated a sybr tm green one-step rt-qpcr method. the development and validation of the complete method, including the steps of sample preparation, rna extraction, and rt-qpcr, were done according to the french standard nf u - . this norm is an adaptation to the french context of the manual of diagnostic tests and vaccines for terrestrial animals (international office of epizootics, ) and respects the criteria stated by the world organization for animal health (oie). these standards describe the validation criteria for a pcr method in animal health and allows the characteristics not only of rt-qpcr to be determined, but also of the complete method, including sample preparation and extraction. for this, fecal and jejunum samples were used as this material has previously been described as the best matrices for detection of pedv rna in animals (gallien et al., a) . validating the complete method in this way means that the method is applicable for both experimental and diagnostic purposes. in the current study the primers used by kim et al. in were refined by in silico analysis. n orf alignments of the priming site showed that the pednf forward primer of kim et al. ( ) had mismatches with several different pedv n orfs and that the last three nucleotides at the ' end only matched with . % of the sequences. removing these three nucleotides in primer mpednf allowed a % match with . % of international sequences and with . % of european strains. the method using the new coupled primers demonstrated sufficient sensitivity to detect all tested pedv strains (historical, s-indel and s-non-indel strains). although sybr tm green pcrs are characteristically less specific than probe based pcrs, the specificity of the method was % against all viral types tested. primer dimer formation, which are problematic for fluorescent dye based methods as they interfere dramatically with quantification, were eliminated by optimizing the primer concentration to nm. during validation, the sample preparation and rna extraction step were optimized by the addition of a proteinase k treatment step which allowed the statistical bias to be maintained in acceptable limits (< . log ). the statistical bias obtain with the proteinase k treatment confirms a correct reproducibility at all quantification points, and guarantees a near or equivalent lod ( and copies/ µl for feces and jejunum) for the different matrices than for the transcribed rna ( copies/ µl). in addition, the detection limit determined in this study ( . tcid /ml) is very similar to other rt-qpcrs ( . tcid /ml) (miller et al., ) . in conclusion, many pcrs have been developed to detect and monitor the presence of pedv, but, as yet to the authors' knowledge none have been developed with a complete validation according to a norm such as the french nf u - . this fully validated method is the first of its kind for pedv and should help harmonize detection and quantification of pedv viral rna in both field and experimental settings. nucleotide probabilities at each position are shown as coloured text above the alignments. red text in the alignment sequences represent a mismatch. sequences of primers are shown above the alignment (pednf, mpednf or pednr). pednr is shown as reverse complement. each line represents a hybridization sequence, the number of strains presenting this sequence is indicated to the left of the sequence. j o u r n a l p r e -p r o o f afnor nf u - - méthodes d'analyse en santé animale -pcr (réaction de polymérisation en chaîne) -partie : exigences et recommandations pour la mise en oeuvre de la pcr en santé animale afnor nf u - - méthodes d'analyse en santé animale -pcr (réaction de polymérisation en chaîne) -partie : exigences et recommandations pour le développement et la validation de la pcr en santé animale porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus virus-like particles associated with porcine epidemic diarrhoea porcine epidemic diarrhea virus among farmed pigs experimental infection of pigs with a new porcine enteric coronavirus, cv porcine epidemic diarrhea virus: an overview of current virological and serological diagnostic methods a table for the calculation of working probits and weights in probit analysis better horizontal transmission of a us non-indel strain compared with a french indel strain of porcine epidemic diarrhoea virus evidence of porcine epidemic diarrhea virus (pedv) shedding in semen from infected specific pathogen-free boars limited shedding of an s-indel strain of porcine epidemic diarrhea virus (pedv) in semen and questions regarding the infectivity of the detected virus complete genome sequence of a porcine epidemic diarrhea s gene indel strain isolated in france porcine epidemic diarrhea in europe: in-detail analyses of disease dynamics and molecular epidemiology propagation of the virus of porcine epidemic diarrhea in cell culture manual of diagnostic tests and vaccines for terrestrial animals : (mammals, birds and bees) beitrag zur kollektiven behandlung pharmakologisher reihenversuche mafft multiple sequence alignment software version : improvements in performance and usability multiplex real-time rt-pcr for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in italy evaluation of two real-time polymerase chain reaction assays for porcine epidemic diarrhea virus (pedv) to assess pedv transmission in growing pigs first detection, clinical presentation and phylogenetic characterization of porcine epidemic diarrhea virus in austria outbreak of porcine epidemic diarrhea in suckling piglets complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium ggseqlogo: a versatile r package for drawing sequence logos changes to virus taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses the authors wish to thanks ms. cherbonnel-pansart for her help with afnor validation methodology, phd. le guyader for the furniture of the mengovirus and phd p.gauger for the s-indel strain furniture. this work was partially funded by "direction générale de l'alimentation" of the french ministry of agriculture (project n° - ). key: cord- -jaue mv authors: simons, fermin a.; vennema, harry; rofina, jaime e.; pol, jan m.; horzinek, marian c.; rottier, peter j.m.; egberink, herman f. title: a mrna pcr for the diagnosis of feline infectious peritonitis date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: jaue mv a reverse transcriptase polymerase chain reaction (rt-pcr) for the detection of feline coronavirus (fcov) messenger rna in peripheral blood mononuclear cells (pbmcs) is described. the assay is evaluated as a diagnostic test for feline infectious peritonitis (fip). it is based on a well-documented key event in the development of fip: the replication of virulent fcov mutants in monocytes/macrophages. to detect most feline coronavirus field strains, the test was designed to amplify subgenomic mrna of the highly conserved m gene. the test was applied to feline blood samples ( from healthy, from sick cats suspected of fip) and returned % of the diseased cats as positive for feline coronavirus mrna in their peripheral blood cells; of the healthy cats, % tested positive. of a group of animals in which fip had been confirmed by post-mortem examination, ( %) tested positive, whereas cats with different pathologies (non-fip cases) all tested negative. in view of the low rate of false-positive results (high specificity) the mrna rt-pcr may be a valuable addition to the diagnostic arsenal for fip. coronaviruses are enveloped, positive-stranded ssrna viruses, a genus in the family coronaviridae, order nidovirales. they are ubiquitous in cat populations, with particularly high prevalence in catteries and multiple-cat households. feline coronaviruses (fcovs) show a bimodal pathogenicity distribution, with subclinical or mild enteric infections in young kittens at one extreme and the deadly feline infectious peritonitis (fip) at the other. the low virulence strains are referred to as feline enteric coronaviruses (fecv), the highly virulent ones as fip viruses (fipv) . though occurring only sporadically (i.e. not causing epidemics), fip is an important disease: it is mostly fatal, its biology is still poorly understood and prevention is difficult, to say the least. fip is an immune mediated, progressive polyserositis and pyogranulomatosis. it occurs worldwide, affecting both domestic and wild felids (holzworth, ; horzinek and osterhaus, ) . antibodies against fcovs are found in - % of the animals living in catteries or multiple-cat households and in up to % of solitary cats; however, only some - % of the seropositive cats eventually come down with fip. the reason for this discrepancy became clear when the biological and genetic properties of fecv and fipv isolates had been studied (addie and jarrett, ; hohdatsu et al., ; horzinek and osterhaus, ) : the avirulent fcov strains causing inconspicuous infections are responsible for the high sero- prevalence; in cats experiencing some immunosuppressive event, expansion of the quasispecies cloud and mutations in the fecv genome lead to virulent variants that induce fip (vennema et al., ) . at present, there are no routine serological and virological assays available for an aetiological diagnosis of fip, and to distinguish avirulent from virulent fcovs. although serology is still used in the diagnosis of fip, it is of very limited value. results can only be interpreted in correlation with clinical symptoms. currently the presumptive diagnosis of fip is based on clinical data and characteristic changes in some blood parameters (cammarata parodi et al., ; gouffeux et al., ) . a definite diagnosis can only be made on the basis of histological examination of biopsy material or postmortem (sparkes et al., (sparkes et al., , . our pcr technique using primers targeted to conserved regions of the viral genome, the -utr (lai and cavanagh, ) , and its modifications (using the s gene (gamble et al., ) ) became a valuable tool for the detection of fcov in body fluids and tissue samples. unfortunately, the technique detects also avirulent fcovs in healthy cats. although the percentage of pcr-positive healthy animals is much lower when compared to fip cats, a positive pcr result alone does not allow a definite diagnosis gunn-moore et al., ). an important event in fip pathogenesis is the infection of monocytes and macrophages (stoddart and scott, ). originally, it was thought that the avirulent fecvs would remain confined to the digestive tract and not spread beyond the intestinal epithelium and regional lymph nodes. virulent fipvs, on the other hand, would leave the gut, enter the bloodstream, generalize and reach different organ parenchymas via infected monocytes. not unexpectedly, however, fcov were detected in blood samples of healthy cats that never developed fip, and also after experimental fcov infection (gamble et al., ; gunn-moore et al., ; herrewegh et al., ; kipar et al., ) . there may be a difference between the sheer presence of fcov in peripheral blood mononuclear cells (pbmcs) and their replication in pbmcs, and we hypothesized that the latter may be a correlate of virulence. a rt-pcr that detects messenger rna of the highly conserved m gene of the fcov genome in peripheral blood cell samples (lai and cavanagh, ; zhang et al., ) would detect the macrophage-tropic variants and bypass non-virulent fcov strains in blood. the present study presents the results of this approach. the fcov reference strains and their sources are listed in table . strains fipv - , fecv - , and fipv ucd were grown in felis catus whole fetus (fcwf) cells. fipv ucd was obtained from tissue cell culture from in- kapke and brian ( ) a assignment according to pedersen et al., a. b tentative assignment (hohdatsu et al., ) . fected fcwf cells (pedersen et al., b) . fecv ucd was acquired from feline faeces as described by pedersen ( ) and grown to low titers in fcwf cells. fipv dahlberg was obtained from brain of a mouse inoculated with homogenate as described by osterhaus et al. ( ) . fipv wellcome was derived from feline embryonic lung (fel) culture cells as described by o'reilly et al. ( ) . blood samples were collected from diseased cats suspected of having fip based on clinical symptoms (n = ) as well as from healthy cats (n = ). the healthy cats were mainly animals living in the same household or cattery as the cats suspected of having fip. these samples were obtained from different veterinary clinics in the netherlands. blood: a maximum of ml of non-coagulated edta blood was centrifuged for min at rpm. plasma was separated from the cell pellet and stored at − • c. one volume of pbs was added to the blood cells and total rna was isolated following the total quick rna blood kit protocol (talent). the oligonucleotide primers were chosen from the highly conserved m gene sequence (primer ) of the fcov genome combined with a primer aiming at the leader sequence of the fcov-genome (primer ). primer sequences are shown in table . as a control to check the efficiency of the rna isolation from all the blood samples and the subsequent reverse transcriptase reaction, a glyceraldehyde- -phosphate dehydrogenase (gapdh) rt-pcr was performed for every clinical sample (primers and ). for the reverse transcriptase (rt) reactions, l of the rna solution and l of reverse primer or (each samples were stored at − • c before using it in the mrna rt-pcr assay. following reverse transcription, l of the rt reaction mixture was added to l of the pcr reaction mixture. the pcr mix consisted of l pcr buffer ( ×; perkin elmer usa, × mm tris-hcl, ph . , mm kcl), . l magnesium chloride ( mm; gibcobrl life technologies), l dntps ( mm each dntp; gibcobrl life technologies), l primer ( mm), and l primer ( mm) (both invitrogen), . l taq polymerase ( u/ l; gibcobrl life technologies). for the gapdh rt-pcr reaction the same pcr mix was used but with different primers: l primers and (each mm) (both invitrogen). the reaction mixture was placed in a thermal cycler (biozym). the temperature cycling protocol consisted of min incubation at • c followed by cycles of min denaturation at • c, min primer annealing at • c and min primer extension at • c. the cycles were followed by min at • c and finally the reaction mixture was cooled to • c. twenty microliters of each pcr sample was analysed by electrophoresis using a . % tae agarose gel (gibcobrl life technologies) for min at v. a bp molecular weight marker (invitrogen) was used to control the size of the amplified pcr product. amplification products were visualised using ethidium bromide staining and uv radiation. samples revealing a bp fragment for the primers and and another fragment of bp for the primers and were considered positive for coronavirus. amplification products were photographed using the biorad geldoc . twenty-three of the obtained pcr products were sequenced to confirm the rt-pcr product. in order to avoid contamination due to carry-over of amplification products several precautions were taken including physical separation of the pre-and post-pcr procedures, the use of aerosol-resistant filter tips (biozym), and during each step from rna isolation to reverse transcriptase and amplification, negative controls of rnase free water were included to try to rule out any false positives. if possible, necropsy of the cat was performed to confirm or rule out a clinical diagnosis of fip. a total of cats were subjected to post-mortem examination. when macroscopic observations were inconclusive, sections of different organs like liver, kidney, spleen etc. were prepared and examined histopathologically. to determine if a rt-pcr for m gene mrna detects different coronavirus isolates, several laboratory isolates were subjected to this assay (table ) . rna from fipv serotype i (strains ucd , ucd ), and serotype ii (strains - , nor , wellcome), fecv serotype i (ucd, rm), fecv serotype ii ( - ), fipv wellcome, ccv-k and tgev purdue could all be detected in cell culture and faeces material or tissue homogenates. after amplification, fragments of the expected size of bp were obtained with all isolates, as shown in fig. . in all samples tested, gapdh amplicons were demonstrated. the gapdh gene, which is constitutively expressed at high levels in most tissues, was used for reference as a positive result in the gapdh rt-pcr will rules out any failure of sample rna isolation or reverse transcription. an example of a positive mrna rt-pcr assay and gapdh control is shown in fig. . blood samples from healthy cats were assessed for the presence of fcov in peripheral blood cells. these animals had been living in catteries or multiple cat households, where other cats with fip-related clinical signs were living. twentythree cats out of the cats ( %) indeed tested positive for fcov in the mrna rt-pcr test. two cats from the pcrpositive animals became sick within months, both showing different clinical symptoms, one of them indicative of fip. unfortunately, the cause of death could not be assessed by necropsy. veterinary practitioners had submitted samples from cats they suspected to suffer from fip. the animals had shown one or several of the following symptoms: fever, anorexia, weight loss, diarrhea, poor growth, enlarged abdomen, presence of ascitic or thoracic fluid, uveitis and neurological signs. of these, samples ( %) were positive for fcov degrees of freedom: ; chi-square = . ; p is less than or equal to . ; the distribution is significant. mrna in blood cells. a summary of the pcr results is shown in table . microscopy was performed on cats tested for fcov mrna in the blood. in cases fip was confirmed. of these, cats ( %) were found to have fcov mrna in their peripheral blood cells (table ). in none of animals that were shown to have suffered from other diseases than fip, fcov mrna was detected in peripheral blood cells (e.g. heart failure, neoplastia, and bacterial infections. the obtained results were statistically significant when controlled by a chi-square (table ) . this report describes an rt-pcr assay to detect fcov mrna in blood samples of cats. our approach was based on the assumption that during the pathogenesis of fip, the mutant virus would replicate in monocytes and macrophages. we postulated that detection of fcov mrna in blood samples would correlate with the development of fip. there are several observations that led to this assumption. infection of monocytes and macrophages is considered as the most important pathogenetic event in fip. the mutant virus has acquired a new tropism and replicates to high titers in monocytes and macrophages (stoddart and scott, ; kipar et al., ) . in vitro, the virulence of fcov strains correlated with their ability to infect macrophages: avirulent fcovs infected fewer cells and produced lower titers than virulent fcovs. the avirulent fcovs were also inferior in sustaining viral replication and spreading to other macrophages (haijema, personal communication) . in coronavirus-infected cells a nested set of subgenomic mrnas is synthesized, each molecule possessing a "leader sequence". this stretch of - nucleotides (coronavirus species dependent) has been derived from the -end of the genome through discontinuous transcription and is not translated. making use of primers specific for the m-gene mrna leader sequence (lai and cavanagh, ) and a conserved part of the m-gene, a molecule of bp will be amplified. using the mrna rt-pcr assay, type ii fcov genomes like fipv - , fipv wellcome, fipv nor and fecv - could be detected in cell culture. type i fcovs like fipv ucd , fipv ucd , fecv ucd, and fipv dahlberg were detected as well. in view of the fact that also canine (ccv k ) and porcine (tgev purdue) coronaviruses tested positive the assay should detect most, if not all, fcov variants. the high detection rate of fcov from cats suspected of suffering from fip in the field supports this assumption. from its design, the mrna assay would appear to be more specific (only replicating virus detected) and more sensitive (only nucleated blood cells employed) for the diagnosis of fip than previous rt-pcr assays focused on genomic rna in body fluids, feces, and tissues (gamble et al., ; gunn-moore et al., ; herrewegh et al., ) . using this assay, we detected mrna in about % of edta blood samples from confirmed fip cases. in the genomic rna pcr, - % of fip cats were found to test positive (gamble et al., ; gunn-moore et al., ; herrewegh et al., ) . more importantly, of the healthy cats living in catteries or multiple cat households with a notoriously large virus burden, only % tested positive for fcov. the presence of fcov rna in blood monocytes in healthy cats infected with fcov is an indication that the development of fip is not associated with the capability of an fcov to cause viraemia and systemic infection (meli et al., ) previous studies quote figures between and % (gamble et al., ; gunn-moore et al., ; herrewegh et al., ) , which can be expected in view of the high sensitivity of the pcr. the specificity of our test format would therefore appear as a significant improvement over previously published methods. the question remains if the mrna positive, healthy cats harbour virulent mutants in an early stage of fip pathogenesis. quantitative analyses of fcov mrna levels would be needed to identify potential differences between healthy and diseased cats. a study of naturally occurring feline coronavirus infections in kittens using direct immunofluorescence to detect coronavirus in peritoneal and pleural effusions sequence analysis of the -end of the feline coronavirus fipv - genome: comparison with the genome of porcine coronavirus tgev reveals large insertions fip, easy to diagnose? characterization of a feline infectious peritonitis virus isolate development of a nested pcr assay for detection of feline infectious peritonitis virus in clinical specimens feline infectious peritonitis. proteins of plasma and ascitic fluid detection of feline coronaviruses by culture and reverse transcriptase-polymerase chain reaction of blood samples from healthy cats and cats with clinical feline infectious peritonitis detection of feline coronavirus rna in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase pcr elimination of feline coronavirus infection from a large experimental specific pathogen-free catbreeding colony by serologic testing and isolation the prevalence of type i and ii feline coronavirus infections in cats some important disorders of cats the virology and pathogenesis of felineinfectious peritonitis sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene histopathological alterations of lymphatic tissues in cats without feline infectious peritonitis after long-term exposure to fip virus the molecular biology of coronaviruses isolation of feline coronaviruses from two cats with divers disease manifestations high viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (fcov) type i and in naturally fcov-infected cats feline infectious peritonitis: isolation of a coronavirus feline infectious peritonitis (fip) virus; propagation in suckling rat and hamster brain virologic and immunologic aspects of feline infectious peritonitis virus infection infection studies in kittens, using feline infectious peritonitis virus propagated in cell culture an enteric coronavirus infection of cats and its relationship to feline infectious peritonitis experimental studies with three new strains of feline infectious peritonitis virus: fipv-ucd , fipv-ucd , and fipv-ucd . th annual symposium of viral diseases of feline infectious peritonitis: a review of clinicopathological changes in cases, and a critical assessment of their diagnostic value coronavirus serology in healthy pedigree cats intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence genomic organization and expression of the end of the canine and feline enteric coronaviruses feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and cis the authors would like to thank the veterinary practitioners and referring cat owners; without their help this study could not have been completed. this study was supported by a research grant from id-lelystad, the netherlands. key: cord- -z jvq authors: li, yan; wu, tao; qi, xian; ge, yiyue; guo, xiling; wu, bin; yu, huiyan; zhu, yefei; shi, zhiyang; wang, hua; cui, lunbiao; zhou, minghao title: simultaneous detection of hemagglutinin and neuraminidase genes of novel influenza a (h n ) by duplex real-time reverse transcription polymerase chain reaction date: - - journal: j virol methods doi: . /j.jviromet. . . sha: doc_id: cord_uid: z jvq a novel reassortant influenza a (h n ) virus emerged recently in china. in this study, a duplex real-time reverse transcription polymerase chain reaction (rrt-pcr) assay was developed for the simultaneous detection of hemagglutinin (ha) and neuraminidase (na) genes of h n influenza viruses. the sensitivity of the assay was determined to be rna copies per reaction for both ha and na genes. no cross-reactivity was observed with other influenza virus subtypes or respiratory tract viruses. one hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. the assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility. in this study, a duplex real-time reverse transcription polymerase chain reaction (rrt-pcr) assay was developed for the simultaneous detection of hemagglutinin (ha) and neuraminidase (na) genes of h n influenza viruses. the sensitivity of the assay was determined to be rna copies per reaction for both ha and na genes. no crossreactivity was observed with other influenza virus subtypes or respiratory tract viruses. one hundred and forty-six clinical and environmental specimens were tested and compared with reference methods and were found to be consistent. the assay is suitable for large-scale screening due to short turnaround times and high specificity, sensitivity, and reproducibility. © elsevier b.v. all rights reserved. since february , a novel reassortant influenza a (h n ) virus has emerged in china , which has become a global public health concern (horby, ; uyeki and cox, ; wu et al., ) . human infections with h influenza viruses (h n , h n , and h n ) have been reported (hirst et al., ; nguyen-van-tam et al., ) . unlike h n viruses, infection with h n , h n , and h n often cause mild to moderate symptoms in humans, and most of those infections are associated with virus outbreaks in poultry (belser et al., ) . a total of human infections including fatal cases have been reported as of july , (who, a). the novel h n virus has acquired some characteristic mutations that facilitate viral recognition of human-type receptors and efficient replication in mammals (kageyama et al., ) . the early and rapid detection of the virus in patients or in environmental samples is crucial for preventing virus transmission, and for initiating treatment as soon as possible. detection methods for this novel virus are urgently needed in a large number of public health laboratories. real-time reverse transcription polymerase chain reaction (rrt-pcr) is a powerful tool for the sensitive and specific detection of viruses. an rrt-pcr protocol (who taqman assay) for the detection of h n viruses was provided by the who collaborating center for reference and research on influenza at the chinese national influenza center on april , (who, b). another rrt-pcr assay was also developed for the specific detection of h n (corman et al., ) . both of these assays are single rrt-pcr methods that detect hemagglutinin (ha) and neuraminidase (na) genes, respectively. in this study, a duplex taqman rrt-pcr assay was developed which can detect the ha and na genes of the novel h n virus simultaneously. the forward primer and the probe for the detection of the ha gene are the same as those described in the who protocol. one degenerate base was introduced into the reverse primer to minimize nucleotide mismatches. in order to design primers and probes for the detection of na genes, available n gene sequences from genbank and the global initiative on sharing avian influenza data (gisaid) were aligned using the clustalx multiple alignment program (thompson et al., ) . primer and probe sequences were designed and analyzed for secondary structure formation, g + c content, primer dimer formation, hairpin formation, and their compatibility with multiplex pcr using the software primer express . (applied biosystems). primers and probes were synthesized by sangon (shanghai, china) (table ) . rna was extracted using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions. the duplex taqman rrt-pcr assay was conducted using the superscript iii - /$ -see front matter © elsevier b.v. all rights reserved. http://dx.doi.org/ . /j.jviromet. . . table primers and probes used in the duplex rrt-pcr assay for the detection of the novel h n virus. sequence ( the specimen was considered positive if the c t value was ≤ . . the analytic sensitivity of the duplex taqman rrt-pcr assay was compared with the who taqman assay and a commercial single h n rrt-pcr kit (bioperfectus technologies, taizhou, china) with a -fold dilution series of a nasopharyngeal aspirate (npa) from a patient infected with the h n virus (approximately . × copies of the viral genome/ml). each dilution series was tested in the duplex rrt-pcr assay in triplicate. the analytic sensitivity of the duplex taqman rrt-pcr assay for the detection of both h and n genes was a − dilution ( . × copies/ml), which was comparable to that of the commercial h n rrt-pcr kit for detecting both h and n genes ( . × copies/ml), and that of the who taqman assay for detecting the h gene ( . × copies/ml). however, it is more sensitive than the who taqman assay for detecting the n gene ( . × copies/ml). to determine the actual detection limit (number of copies per reaction) of the duplex taqman rrt-pcr assay, in vitro rna transcripts of ha and na genes from the h n virus were prepared with t rna polymerase (takara biotechnology co. ltd., dalian, china) according to the manufacturer's instructions using influenza a/nanjing/ / (h n ) rna as a template. the synthetic rna transcripts were then purified, quantified, and mixed in equimolar amounts, and diluted ten-fold from to rna copies/reaction. rna templates were then tested in the duplex taqman rrt-pcr assay. each concentration was tested in triplicate. the actual detection limit of the assay was found to be approximately copies of synthetic rna per reaction for both h and n genes (fig. ) . the specificity of the duplex rrt-pcr assay was determined with a panel of respiratory tract viruses including influenza virus a (seasonal h n , h n , pandemic h n , h n , novel h n , and h n subtypes), influenza virus b, human rhinovirus, human enterovirus, human respiratory syncytial virus, human parainfluenza virus (types , and ), human metapneumovirus, human coronavirus ( e, nl , hku , and oc ), and human adenovirus. all tested samples were negative except for the novel h n virus. intra-assay variation was assessed by testing three separate dilution series ( - copies/reaction) of rna standards within a single run. inter-assay variation was determined by testing each dilution of the rna standard in triplicate on three different days. since only one test for the h gene obtained a c t value ( . ) at the highest dilution ( rna copies/reaction), we recorded the c t values that were positive for rna ranging from to copies per reaction and calculated the coefficients of variation (cv) and confidence intervals (ci) separately for each rna dilution. the mean cvs of intra-assay variability for h and n genes were . % ( % ci, . - . %) and . % ( % ci, . - . %), respectively. the mean cvs of inter-assay variability for h and n genes were . % ( % ci, . - . %), and . % ( % ci, . - . %), respectively. these results suggest that the duplex taqman rrt-pcr assay was sufficiently reproducible. to evaluate the performance of the duplex rrt-pcr assay, a reference standard was established that combined the results from the viral culture, who taqman assay and the commercial h n rrt-pcr kit. a sample was determined to be positive for h n virus when at least two of the three reference methods were positive. for the who taqman assay and commercial h n rrt-pcr kit, a sample was determined to be positive for h n virus when both h and n genes were detected. a total of specimens including clinical specimens from suspected patients ( tracheal swabs or wash and nasopharyngeal swabs) in hospitals and environmental specimens in living animal markets ( poultry cage swabs, poultry water, and poultry feces) were tested using the duplex rrt-pcr assay and reference methods (table ) . when the results of the duplex rrt-pcr were compared with the combined results of the viral culture, who taqman assay and commercial h n rrt-pcr kit, they were found to be identical. both methods identified specimens that were positive for the h n virus. two clinical specimens and one environmental specimen which tested fig. . sensitivity and dynamic range of the duplex rrt-pcr assay for detection of h n virus. serial -fold dilutions of viral rna standard (from to copies) were plotted against the threshold cycle (ct). the specimen was considered positive if the ct value was ≤ . . the minimum detection limit of the assay was found to be approximately copies per reaction for both h and n genes. the coefficient of determination (r ) and the equation of the regression curve (y) were calculated. , h gene; , n gene. negative using the who taqman assay were determined to be positive by both the duplex rrt-pcr assay and the commercial h n rrt-pcr kit. this novel h n lineage has become a major public health concern. although sustained person-to-person transmission of the virus has not been reported, there is a risk that mutations in the virus could facilitate this. in fact, the asp asn substitution in the pb gene associated with mammalian adaptation is present in human h n isolates . molecular diagnostic assays that can specifically detect h n viral rna are powerful tools to monitor h n virus infections, investigate risk factors, and can provide important information such as the duration of viral shedding, the infectious period, genetic variability, potential virulence, and the spectrum of clinical illness (uyeki and cox, ) . two currently available rrt-pcr assays are not duplexed and must amplify two to three gene segments separately to identify h n viruses (corman et al., ; who, b) . moreover, the who taqman assay was less sensitive for the detection of n genes compared with h genes, which makes it difficult to interpret the results when detecting low copies of viral rna. although no other h viruses are known to circulate in human populations, we could not exclude the possibility of new reassortant viruses emerging in the future, as co-infection with two viruses has been observed recently . therefore, samples were only scored as positive for h n virus when both h and n genes were detected. the high sensitivity, specificity, and reproducibility of the duplex rrt-pcr assay described in this study was found to be similar to the single rrt-pcr assay, indicating that the duplex rt-pcr assay can serve as a substitute method for the current single rrt-pcr method. in this study, both h and n genes were detected simultaneously in one tube which provides a cost-and time-saving method for the detection of novel h n viruses and is suitable for large-scale screening purposes. it should be noted that this assay is optimized for human samples, and therefore care must be taken in the interpretation of the results, since h or n avian influenza viruses from other lineages can also be detected by this assay, especially when applied to environmental specimens. if only h genes were detected while n genes was not, the result could be explained by the presence of other h aiv lineages in the samples, such as h n and h n . likewise, if only n genes was detected while h genes were not, the result could be explained by the presence of other n viruses in the samples, such as h n and h n . past, present, and possible future human infection with influenza virus a subtype h human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome specific detection by real-time reverse-transcription pcr assays of a novel avian influenza a(h n ) strain associated with human spillover infections in china novel avian influenza h n strain outbreak genetic analysis of novel avian a(h n ) influenza viruses isolated from patients in china the clustal x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools global concerns regarding novel influenza a (h n ) virus infections real-time rt pcr (rrt-pcr) protocol for the detection of a(h n ) avian influenza virus emerging risk of h n influenza in china human co-infection with novel avian influenza a h n and influenza a h n viruses in jiangsu province the study was supported in part by the jiangsu province health development project with science and education (zx ), the jiangsu province key medical talent foundation (rc , rc , and jkrc ), the science and technology pillar program of jiangsu province (be ), and the projects of jiangsu province. key: cord- -kftffes authors: mohon, abu naser; oberding, lisa; hundt, jana; van marle, guido; pabbaraju, kanti; berenger, byron; lisboa, luiz; griener, thomas; czub, markus; doolan, cody; servelitta, venice; chiu, charles; greninger, alexander; jerome, keith; pillai, dylan r. title: optimization and clinical validation of dual-target rt-lamp for sars-cov- date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: kftffes a novel reverse-transcriptase loop mediated amplification (rt-lamp) method targeting genes encoding the spike (s) protein and rna-dependent rna polymerase (rdrp) of sars-cov- has been developed. the lamp assay achieves a comparable limit of detection ( copies per reaction) as commonly used rt-pcr protocols using clinical samples quantified by digital droplet pcr. precision, cross-reactivity, inclusivity, and limit of detection studies were performed according to regulatory standards. clinical validation of dual-target rt-lamp (s and rdrp gene) achieved a ppa of . % ( % ci . % to . %) and npa . % ( % ci . % to . %) based on the e gene and n gene reference rt-pcr methods. the method has implications for development of point of care technology using isothermal amplification. over the last several decades, we have witnessed the rise of both known and novel viruses, areas. in december and early january , a cluster of pneumonia cases from a novel coronavirus, sars-cov- , was reported in wuhan, china ( ) ( ) ( ) . sars-cov- has now resulted in a global pandemic with the epicentre at the time of writing in europe and north america ( ) . a common theme in the public health response to covid and similar threats is the lack of rapidly deployable testing in the field to screen large numbers of individuals in exposed areas, international ports of entry, and testing in quarantine locations such as the home residences, as well as low-resourced areas ( ) . this hampers case finding and increases the number of individuals at risk of exposure and infection. with the ease of travel across continents, delayed testing and lack of screening programs in the field, global human-tohuman transmission will continue at high rates. these factors make a pandemic very difficult to contain. early identification of the virus and rapid deployment of a targeted point of care test (poct) can stem the spread through immediate quarantine of infected persons ( ) . we used existing viral genome sequences to develop a sars-cov- loop mediated amplification (lamp) assay for clinical use ( , ) . lamp relies on an alternate set of reagent chemistry that does not depend on or hinder critical elements of the rt-pcr supply chain which is now under duress ( ) . our group has previously demonstrated the utility of lamp for other infectious agents like malaria and dengue ( ) ( ) ( ) . clinical samples used in this study were standard archived nasopharyngeal (np) in viral transport medium (vtm) stored at - o c at alberta precision laboratories (calgary, canada), j o u r n a l p r e -p r o o f university of washington, and university of california, san francisco. ethical approval for use of the archived samples was obtained from the conjoint health research ethics board (chreb) of the university of calgary (reb - ). this study was approved by the institutional review board (irb) at university of california, san francisco (ucsf irb # - ) as a no subject contact study with waiver of consent. remnant np swab after clinical testing and pcr cycle threshold results were collected for analysis. the use of de-identified specimens were deemed non-human subject work by the university of washington institutional review board (irb). genomic sequences (cdna) of the sars-cov- were retrieved from the genbank database (https://www.ncbi.nlm.nih.gov/genbank/sars-cov- -seqs/) and multiple sequence alignment analysis (https://www.ebi.ac.uk/tools/msa/clustalo/) was conducted with other related viruses. from the multiple sequence alignment, several regions unique to the sars cov- were identified. the primers were designed using the primer explorer v software (http://primerexplorer.jp/lampv e/) by uploading the sequences of the s and rdrp gene. initially, primers were designed with the default settings of the software and then screened based on the optimal self-dimer, ' end, and 'end g values. additionally, the intervening sequence between f /f and b /b primer binding sites was manually set at a minimum of nucleotides. other lamp primer design software (eg. http://www.optigene.co.uk/lampdesigner/) are available commercially but were not used in this study. initial screening experiments were performed on several potential primer sets and two chosen based on amplification efficiency (data not shown). lamp primer sets were designed targeting unique regions of the spike (s) protein gene and rna-dependent rna polymerase gene (rdrp) were ultimately used (table ) . for the external lamp amplification control, primers were used against bacteriophage ms as previously described ( ). in silico analysis of primer combinations to determine cross-reactivity and inclusivity j o u r n a l p r e -p r o o f a blast search alignment (https://blast.ncbi.nlm.nih.gov/blast.cgi) for primers in set (spike gene) and set (rdrp gene) were performed against a critical list of infectious agents that cause upper respiratory tract infections. a nucleotide local alignment using blastn with the default parameters was performed against the national center of biotechnology information (ncbi) nucleotide database (see supplementary data). the twelve rt-lamp primers were aligned against , sars-cov- (taxid: ) viral sequences in the ncbi nucleotide database available on august th, . the output of the "discontiguous megablast" algorithm showed no divergence available between the primers and the sars-cov- database and all of the query sequences showed a % identity against the expected sequences from the sars-cov- virus ( genomes). four fragments of specific sars-co-v regions (orf ab (nsp , - ), rdrp (nsp ), and spike (s)) were synthesized by sgi-dna inc. (san diego, ca). fragments were ligated to make one large concatenated dna template using the bioxp (sgi-dna, san diego, ca) automated gibson assembly system. the final template was base pairs long containing concatenated single artificial construct together with flanking plasmid sequence in that order ( figure ). to create a recombinant virus expressing the relevant rna, the template containing the targeted sequences of interest was cloned into sindbis virus (sv) viral vector system (sinrep ) containing green fluorescent protein (egfp) and then transfected into bhk cell lines ( , ) . the number of rna genome copies was based on the number of fluorescent focus forming units generated by the recombinant sv vector. the dual-target lamp reaction was conducted using a combination of warmstart rtx reverse limit of detection of the lamp assay was evaluated by using a nasopharyngeal (np) swab sample infected with sars-cov- for which the viral load was quantified using digital droplet pcr (see supplementary methods). the np sample was serially diluted to achieve the described copies of virus per lamp reaction. calculation of viral copy number using digital droplet pcr is j o u r n a l p r e -p r o o f the workflow used to conduct rt-lamp is depicted in figure . the primer sequences used to target the spike gene (set s ) and rdrp gene (set ) are listed in table . the limit of detection was evaluated using a patient sample (np swab in vtm viral load confirmed by digital droplet pcr). the quantified sample was serially diluted to achieve a range from to . copies per reaction. the limit of detection was confirmed at copies per reaction when using mm guanidine hydrochloride (ph . ) in the reaction mix ( table ). twenty four replicates from a serial dilution containing - copies of sars-cov- which equates to x lod (patient sample np swab in vtm viral load confirmed by digital droplet pcr) per reaction were tested using dual-target rt-lamp (table ) . twenty-three of samples ( . %) were positive. in silico analysis confirmed that no significant cross-reactivity that affects lamp reactions that rely on six primers per reaction were present (supplementary table ) . finally, further studies were performed for precision on a daily basis ( replicates for days twice day and replicates on instruments daily for days) that demonstrated % concordance (supplementary table and ). a clinical validation sample set of nasopharyngeal swabs were used in the analysis. given no gold standard exists, percent positive agreement (ppa) and negative percent agreement (npa) were calculated. reference methods included rt-pcr (e gene and n gene) methods employed by reference laboratories. dual-target rt-lamp dual-target rt-lamp (s and rdrp gene) achieved a ppa of . % ( % ci . % to . %) and npa . % ( % ci . % to . %) based on the e gene and n gene reference rt-pcr methods (table ) . one false negative sample by rt-lamp was positive by both e gene and n gene. one sample out of j o u r n a l p r e -p r o o f was strongly positive by the n gene and negative by e gene and considered a true positive. no cross-reactivity was observed with known circulating respiratory viruses, namely (hcov) oc , e, nl , and hku or influenza virus a (h n ) pdm (data not shown). the global pandemic with sars-cov- has resulted in the need for diagnostic test development at a scale never seen before. rapid deployment of validated laboratory-developed diagnostic tests or commercial tests is essential to the containment of the virus as it allows for selfquarantine measures to be imposed in a strategic fashion before widespread community transmission occurs ( , ) . diagnostic tests have to be analytically sensitive in order to not to miss any cases in the acute phase of viremia ( ) . as such, nucleic acid amplification tests serve this purpose. in particular, rt-pcr has been employed as the primary diagnostic countermeasure ( ) . however, reagent supply chains for key items are under immense pressure. local solutions to reagent sources have become paramount because barriers to trade of these selected items have been a concern. the dual-target rt-lamp test for sars-cov- developed in this study has comparable analytical sensitivity and specificity, limit of detection, precision, and achieved excellent agreement compared to the reference rt-pcr methods used internationally. the addition of guanidine hydrochloride (ph . ) at mm in the lamp reaction improves the limit of detection ( copies per reaction) to a level comparable to rt-pcr methods. lamp does not rely on the same reagents as rt-pcr and thus alleviates pressure on key supply chain items. the lamp method is amenable to high throughput testing in either -well or -well. other groups have j o u r n a l p r e -p r o o f presented rt-lamp solutions in the literature ( ) ( ) ( ) ( ) ( ) ( ) . the lamp solutions differ in several ways: first, the target genes of choice vary between studies as do the specific primer sequences chosen; second, the limits of detection reported vary in terms of sars-cov- copies detected per reaction; third, the detection systems vary from thermocycler based detection for laboratory developed test (ldt) solutions to near-patient solutions based on visual detection of dyes or fluorophores; fourth, the extent which data reflect requirements for clinical validation. the rt-lamp assay described is unique in that it offers the most thorough clinical validation to date meeting regulatory standards which include precision studies on several instruments, reproducibility studies over days, a robust clinical validation sample set, and a limit of detection equal or superior to other lamp studies ( copies per reaction). these data should enable a clinical laboratory to perform this assay as a ldt. additionally, the lamp assay chemistry presented in this work is able to detect sars-cov- in vtm without the need for a kit-based rna extraction method using lyophilized reagents and visual detection (manuscript in preparation). this format may be of particular interest to resource-limited settings. limitations of the study include not testing other sample types such as alternate swabs, nasal washes, saliva, sputum, or stool. this work is ongoing with a special emphasis on swab-free testing and direct visualization. lamp presents a much needed alternative approach to sars-cov- diagnostic testing that is available for deployment immediately in a laboratory-developed test format as it relies on other key reagents that do not cannibalize rt-pcr reagents. in the future, the lamp chemistry has potential to be adapted to a microfluidic device poct to be deployed in the community, either at ports of entry, homes, pharmacies, or workplaces. cd is an employee of illucidx inc. (a start-up company of the university of calgary) which retains patents related to lamp technology. drp is a scientific advisor to illucidx inc. nasopharyngeal and oropharyngeal swabs at hampshire hospitals nhs foundation trust. preprint, infectious diseases (except hiv/aids). figure : map of the gene fragments from sars-co-v (genbank id mt . ) that were used for synthesizing the genetic construct template. four fragments of specific sars-co-v regions (orf ab (nsp , - ), rdrp (nsp ), and spike (s)) were concatenated into a single artificial construct base pairs long. j o u r n a l p r e -p r o o f figure : workflow used to analyze samples in this study. images were obtained from the centers for disease control (www.cdc.gov) and bio-rad laboratories (www.bio-rad.com). abu naser mohon: conceptualization; data curation; formal analysis; methodology data curation; resources; writing -review & editing data curation; resources; writing -review & editing writing -review & editing funding acquisition; investigation; supervision; writing -review & editing funding acquisition; investigation; supervision; writing -review & editing funding acquisition; investigation; supervision; writing -review & editing pillai: conceptualization; data curation; formal analysis; funding acquisition; investigation roles/writing -original draft avian flu, sars, mers, ebola, zika… what next? feng z. . early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a new coronavirus associated with human respiratory disease in china severe outcomes among patients with coronavirus disease (covid- ) -united states diagnostic testing for severe acute respiratory syndrome-related coronavirus- : a narrative review novel coronavirus disease (covid- ): paving the road for rapid detection and point-of-care diagnostics phylogenetic network analysis of sars-cov- genomes genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding predicting the impacts of epidemic outbreaks on global supply chains: a simulation-based analysis on the coronavirus outbreak (covid- /sars-cov- ) case risk prediction for severe disease and better diagnostic accuracy in early dengue infection; the colombo dengue study ultrasensitive loop mediated isothermal amplification (us-lamp) to detect malaria for elimination prevalence and epidemiological characteristics of asymptomatic malaria based on ultrasensitive diagnostics: a cross-sectional study molecular diagnostic field test for point-of-care detection of ebola virus directly from blood detection of novel coronavirus ( -ncov) by realtime rt-pcr comparative performance of sars-cov- detection assays using seven different primer/probe sets and one assay kit rt-lamp for rapid diagnosis of coronavirus sars-cov- a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov- development of reverse transcription loop-mediated isothermal amplification (rt-lamp) assays targeting sars-cov- rapid detection of novel coronavirus/severe acute respiratory syndrome coronavirus (sars-cov- ) by reverse transcription-loop-mediated isothermal amplification a reverse-transcription loop-mediated isothermal amplification dr. ranmalee amarasekara, dr. tara winstone, and barbara chow for expert technical assistance, daniel castaneda mogollon for performing bioinformatics analysis, and omar abdullah and noah toppings for research analytical support. key: cord- -qbm tr authors: krüttgen, alexander; cornelissen, christian g.; dreher, michael; hornef, mathias w.; imöhl, matthias; kleines, michael title: determination of sars-cov- antibodies with assays from diasorin, roche and idvet date: - - journal: j virol methods doi: . /j.jviromet. . sha: doc_id: cord_uid: qbm tr there is an ongoing need for highly reliable serological assays to detect individuals with past sars-cov- infection. using sera from patients tested positive or negative by sars-cov- pcr, we investigated the sensitivity and specificity of the liaison sars-cov- s /s igg assay (diasorin), the elecsys anti-sars-cov- assay (roche), and the id screen sars-cov- -n igg indirect kit (idvet). we determined a sensitivity of . %, . %, and % and a specificity of . %, . %, and , % for the diasorin assay, the roche assay, and the idvet assay, respectively. we conclude that serologic assays combining very high sensitivity and specificity are still not commercially available for sars-cov- . for maximizing sensitivity and specificity of sars-cov- serological diagnostics, the combination of two assays may be helpful. the new coronavirus sars-cov- emerged in (zhou et al., ; zhu et al., ) causing an ongoing pandemic. diagnostic laboratories are facing the challenge of correctly identifying acute and past infections with sars-cov- . whereas nucleic acid amplification assays are the gold standard for detection of acute infections (eis-hübinger et al., ) , sensitive and specific serologic assays are needed for detection of past infections. developing accurate serological assays for coronaviruses is considered a technical challenge (meyer, drosten, and müller, ) . we and others have published results of the assessment of the first commercially available serological assays, such as the anti sars-cov- elisa (igg) from euroimmun (krüttgen et al., ; okba et al., ) . most recently, highly anticipated chemoluminescence immunoassays (clia) for widely-implemented automated high-throughput platforms (cobas, liaison) and further elisas became available. to allow the comparison of performance indicators, these assays have to be compared using identical collections of serum samples. we therefore compared these three new assays with respect to their sensitivity and specificity to detect sars-cov- specific antibodies using a collection of serum samples employed previously for the analysis of four other assays. these values were compared to four assays previously evaluated in our laboratory (krüttgen et al., ) : the anti sars-cov- elisa (igg) (euroimmun, germany), the edi novel coronavirus covid- igg elisa, (epitope diagnostics (edi), usa), the recomwell sars-cov- igg elisa (mikrogen, germany), and the sars-cov- virachip igg (viramed, germany). thus we aimed at directly comparing the performance of seven commercially available serological assays (two clias, four elisas and one immunoblot) in terms of sensitivity and specificity. we used previously characterized sera of different patients hospitalized in the university hospital rwth aachen, germany; approval of medical ethics committee ek / ). in brief, sera were collected from patients with a negative sars-cov- pcr result in respiratory specimens and sera were collected from patients with a positive sars-cov- pcr result in respiratory specimens. the sera were stored for about four weeks before use with the assays described in this manuscript. one freeze thaw cycle was applied. of the sera were defined sars-cov- igg positive and sera were defined sars-cov- igg negative. in analogy to a previous study (de ory et al., ) the sars-cov- igg status of the sera was defined as follows: a serum was regarded as sars-cov- igg negative if at least three of the four assays compared here had a negative test result applying the manufacturer's interpretation criteria. on the other hand, a serum was regarded as sars-cov- igg positive if at least two of the four assays had a positive test result. borderline samples were handled as not positive for determination of the sensitivity and not negative for determination of the specificity. three semiquantitative enzyme immune assays, the liaison sars-cov- s /s igg assay (diasorin, italy), the elecsys anti-sars-cov- assay (roche, usa), and the id screen sars-cov- -n igg indirect kit (idvet, france) were compared for their ability to detect sars-cov- -specific antibodies. to allow comparison of semiquantitative values between assays, the values were divided by the assayspecific cut off value for normalization. normalized values of >= thereby represented a positive test result. we included previously characterized sera in this study (krüttgen et al., ) , of which were collected from patients with a negative sars-cov- pcr result and from patients with a positive sars-cov- pcr result in respiratory specimens. the sera of patients with negative sars-cov- pcr result were obtained at the same day as the pcr analysis of the respiratory specimen. the sera of the patients with positive sars-cov- pcr were collected . days (± . days) post onset of symptoms. each serum was tested in parallel for the presence of sars-cov- igg antibodies as recommended by the manufacturers. using the diasorin assay, sera were classified as sars-cov- antibody positive, were classified as negative. using the roche assay sera had a positive result and a negative result. the idvet assay yielded positive test results as well as negative results. the rate of correct positive and the rate of correct negative test results of the assays is displayed in table a . they resulted in a sensitivity of . %, . %, and % for j o u r n a l p r e -p r o o f the diasorin assay, the roche assay, and the idvet assay, respectively. the corresponding results for the specificity were . %, . %, and , %. these values were comparable to four assays previously evaluated in our laboratory (krüttgen et al., ; from euroimmun, epitope diagnostics (edi), mikrogen, and viramed) which exhibited a sensitivity between . % and % and a specificity between , % and % (table b) . thus, none of the seven assays combined a very high sensitivity with a very high specificity. two assays reached a sensitivity of % (idvet and edi) whereas two other assay had a specificity of % (mikrogen and viramed). the roche assay offered the best balance between high sensitivity and high specificity ( , % and , %). using the assays of roche, diasorin and idvet we also compared the kinetic of antibody titers for two patients of whom consecutive sera were available. we found that all three assay delivered positive results - days after onset of symptoms for both patients (figure ). in comparison with the three previously characterized semiquantitative assays (euroimmun, mikrogen and edi), the two new assays confirmed the previously defined time span of - days after onset of clinical symptoms to obtain a positive serological test result. the assays target two different antigens: the nucleocapsid protein and the spike protein. however, neither an individual assays nor a group of assays using the same target protein is positive first or shows the strongest quantitative increase for both patients. this is also true for other patients with consecutive sera available (data not shown). reliable serological assays are urgently needed to supplement the diagnostic repertoire and identify patients with past sars-cov- infection. this would allow the detection of patients presenting during later stage of the disease when direct pathogen detection has turned negative due to viral clearance (vogel, ) . during the initial stage of the outbreak, the elisa of euroimmun was a commonly used commercial elisa and first evaluated by okba and coworkers (okba et al., ) . in the meantime, several additional assay using different methodologies (clia, elisa, immunoblot) targeting different virus antigens (such as the s-and n-antigen) have become available, increasing the commercially available diagnostic tools of clinical laboratories. we found that the highly anticipated clias for the widely-used automatons from diasorin and roche both offer a high sensitivity of > %. using our collection of samples, the roche assay offers a higher specificity ( , % roche vs , % diasorin). this difference might be related to the different antigens targeted by the assay (the nucleocapsid protein by roche, idvet, edi and microgen; the spike protein by diasorin and euroimmun; the viramed assay targets both the spike protein and the nucleocapsid protein), however, based on our data there is no general trend indicating a higher sensitivity of assays targeting the nucleocapsid protein. the higher sensitivity of the roche assay may be based on the determination of total antibody (igg and igm). the diasorin assay is targeting igg antibodies only. although somewhat less specific, the diasorin assay offers the attractive possibility of measuring neutralizing antibodies targeting the viral spike protein (although no serological assay for sars-cov- has so far been j o u r n a l p r e -p r o o f demonstrated to measure neutralizing antibodies). compared to the assays of roche and diasorin, the idvet elisa had the highest sensitivity and an intermediary specificity. our comparative approach to test in total seven different sars-cov- antibody assays with an identical collection of serum samples allowed for the first time the direct comparison of performance indicators of such a large number of automated assays. a perfect serological test would offer > % sensitivity and > % specificity (mallapaty, ) . we found that two assay reached a sensitivity of % (idvet and edi) whereas two other assay offered a specificity of % (microgen and viramed). the requested very high sensitivity in combination with very high specificity was not observed in any of the seven assays. thus, the combination of two different assays (one test with % sensitivity and another test with % specificity) might provide a maximum diagnostic value. however, if a laboratory decided to implement a single assay approach, the roche assay offers the best balance of high sensitivity and high specificity ( , and , %). a general advantage in terms of sensitivity or specificity by the use of either the nucleocapsid protein or the spike protein as a target could not be observed. the interpretation of this study is limited by a lack of neutralization information for the samples included. further, the use of different targets from two different viral proteins affects the assay's results in an unknown way. taken together, our study will assist diagnostic laboratories to select the appropriate assays or assay combinations according to their sample load, equipment, and diagnostic question. comparison of commercial methods of immunoblot, elisa, and chemiluminescent immunoassay for detecting type-specific herpes simplex viruses- and - igg ad hoc laboratory-based surveillance of sars-cov- by real-time rt-pcr using minipools of rna prepared from routine respiratory samples comparison of four new commercial serologic assays for determination of sars-cov- igg will antibody tests for the coronavirus really change everything? serological assays for emerging coronaviruses: challenges and pitfalls severe acute respiratory syndrome coronavirus -specific antibody first antibody surveys draw fire for quality, bias a novel coronavirus from patients with pneumonia in china