Rapid in vitro assays for screening neutralizing antibodies and antivirals against SARS-CoV-2.
Abstract Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus (SARS-CoV), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals lice...
Source: Journal of Virological Methods - October 14, 2020 Category: Virology Authors: Park JG, Oladunni FS, Chiem K, Ye C, Pipenbrink M, Moran T, Walter MR, Kobie J, Martinez-Sobrido L Tags: J Virol Methods Source Type: research

Facile method of curing toxicity in large viral genomes by high-throughput identification and removal of cryptic promoters.
Abstract Infectious plant virus clones are challenging to construct and manipulate due to the presence of cryptic promoter sequences that induce toxicity in bacteria. Common methods to overcome toxicity include intron insertion to interrupt toxic open reading frames and the use of Rhizobium or yeast species that do not recognize the same cryptic promoters. Unfortunately, intron insertion must be attempted on a trial and error basis within full-length clones and may change the infection characteristics of the virus. We have developed a facile method that can detect multiple cryptic bacterial promoters within large ...
Source: Journal of Virological Methods - October 14, 2020 Category: Virology Authors: Klenov A, Hudak KA Tags: J Virol Methods Source Type: research

Cell culture demonstrates superior sensitivity over one step real time RT PCR and nested VP1 amplification for Enteroviruses.
This study evaluated and compared the sensitivity profile of routine cell culture, nested VP1 amplification and one step real time RT PCR for Enteroviruses. Serially diluted spiked samples of four model viruses (EV71, CVA16, CVB5 and PV1) and 32 true positive samples including Poliovirus (PV1 & PV3), Coxsackie virus (CVB5, CVB3, CVB1 & CVA4, 10, 16), Echovirus (Echo 6, 7, 11, 13, 18, 25 & 30) and Enterovirus 71 (E71), and 32 true negative stool samples were subjected to cell culture, nested RT PCR and one step real time RT PCR. The result of sensitivity test indicated superior sensitivity with one step real tim...
Source: Journal of Virological Methods - October 14, 2020 Category: Virology Authors: Kabuga AI, Nejati A, Soheili P, Yousefipoor S, Yousefi M, Mollaiee Y, Shahmahmoodi S Tags: J Virol Methods Source Type: research

Novel degenerate primer sets for the detection and identification of emaraviruses reveal new chrysanthemum species.
This study used sequence data for emaraviruses to design new degenerate primer sets that identify an extensive range of known and unknown emaraviruses. Sequence alignment of the amino acid and nucleotide sequences of RNA-dependent RNA polymerases for 11 accessions among nine emaraviruses confirmed the presence of seven conserved motifs (Pre-A, F, A, B, C, D, and E). Subsequently, new degenerate primers were designed based on motifs F, A, and B, which were the most conserved among the seven motifs. Reverse transcription-polymerase chain reaction using these primers detected known emaraviruses more efficiently than previousl...
Source: Journal of Virological Methods - October 9, 2020 Category: Virology Authors: Kubota K, Chiaki Y, Yanagisawa H, Yamasaki J, Horikawa H, Tsunekawa K, Morita Y Tags: J Virol Methods Source Type: research

A combined approach of MALDI-TOF Mass Spectrometry and multivariate analysis as a potential tool for the detection of SARS-CoV-2 virus in nasopharyngeal swabs.
Abstract Coronavirus disease 2019, known as COVID-19, is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The early, sensitive and specific detection of SARS-CoV-2 virus is widely recognized as the critical point in responding to the ongoing outbreak. Currently, the diagnosis is based on molecular real time RT-PCR techniques, although their implementation is being threatened due to the extraordinary demand for supplies worldwide. That is why the development of alternative and / or complementary tests becomes so relevant. Here, we exploit the potential of mass spectrometry technology comb...
Source: Journal of Virological Methods - October 9, 2020 Category: Virology Authors: Rocca MF, Zintgraff JC, Dattero ME, Santos LS, Ledesma M, Vay C, Prieto M, Benedetti E, Avaro M, Russo M, Nachtigall FM, Baumeister E Tags: J Virol Methods Source Type: research

Environmental and air sampling are efficient methods for the detection and quantification of foot-and-mouth disease virus.
The objective of this study was to assess the efficiency of sampling methods for the recovery and quantification of FMDV from a range of environmental surfaces and in aerosols. Selected surfaces, based on those likely to be found on farms, were spiked with a range of concentrations of FMDV, left to dry and then the surface was swabbed with an electrostatic dust cloth. For aerosol sampling FMDV was nebulised at different concentrations and distances from the sampler. Recovery of viral RNA and infectious virus was measured by RT-qPCR and virus isolation, respectively. FMDV RNA was detected from all surfaces at all concentrat...
Source: Journal of Virological Methods - October 7, 2020 Category: Virology Authors: Brown E, Nelson N, Gubbins S, Colenutt C Tags: J Virol Methods Source Type: research

Establishment and characterization of a continuous cell line from heart of Nile tilapia Oreochromis niloticus and its susceptibility to Tilapia lake virus.
Abstract In the present study, we have developed a continuous cell line from the heart tissue of the Oreochromis niloticus and used for studying susceptibility to Tilapia Lake Virus (TiLV). The cell line, designated as OnH, has been subcultured for more than 82 passages. The optimal growth of OnH cells was observed at 28- 32 °C L-15 medium supplemented with 20% fetal bovine serum. Karyotype analysis revealed that the modal chromosome number of OnH cells was 44. Partial amplification and sequencing of 16S rRNA gene confirmed the origin of OnH cell line from O. niloticus. Immunophenotyping revealed...
Source: Journal of Virological Methods - October 6, 2020 Category: Virology Authors: Yadav MK, Rastogi A, Criollo Jaoquin MP, Verma DK, Rathore G, Swaminathan TR, Paria A, Pradhan PK, Sood N Tags: J Virol Methods Source Type: research

Development and evaluation of a method for concentration and detection of salmonid alphavirus from seawater.
Abstract Waterborne viral infections represent a major threat to fish health. For many viruses, understanding the interplay between pathogens, host and environment presents a major hurdle for transmission. Salmonid alphavirus (SAV) can infect and cause pancreas disease (PD) in farmed salmonids in seawater. During infection, SAV is excreted from infected fish to the seawater. We evaluated two types of filters and four different eluents, for concentration of SAV3. One L of seawater was spiked with SAV3, followed by filtration and virus elution from membrane filters. For the negatively charged MF hydrophilic membrane...
Source: Journal of Virological Methods - October 6, 2020 Category: Virology Authors: Weli SC, Bernhardt LV, Qviller L, Myrmel M, Lillehaug A, Kjellin ML Tags: J Virol Methods Source Type: research

The use of functionally deficient viral vectors as visualization tools to reveal complementation patterns between plant viruses and the silencing suppressor p19.
Abstract Plant virus transport complementation is classically observed as a helper virus allowing another virus to regain cell-to-cell or systemic movement through a restrictive host plant (Malyshenko et al., 1989). The complementation effect is usually studied by observing virus infection after co-infection or super-inoculation of the helper virus. We herein demonstrate the utility of functionally deficient viral vectors as tools to determine the contribution of individual viral genes to plant viral transport complementation. Two functionally deficient viral vectors were engineered that derive from foxtail mosaic...
Source: Journal of Virological Methods - September 29, 2020 Category: Virology Authors: Zhou Y, Ghidey MR, Pruett G, Kearney CM Tags: J Virol Methods Source Type: research

Concentration of infectious SARS-CoV-2 by polyethylene glycol precipitation.
Abstract The development of medical countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires robust viral assays. Here we have adapted a protocol for polyethylene glycol (PEG)-mediated precipitation of SARS-CoV-2 stocks without the need for ultracentrifugation. Virus precipitation resulted in a ∼1.5 log10 increase in SARS-CoV-2 titres of virus prepared in VeroE6 cells and enabled the infection of several immortalized human cell lines (Caco-2 and Calu-3) at a high multiplicity of infection not practically achievable without virus concentration. This protocol underscores the ...
Source: Journal of Virological Methods - September 22, 2020 Category: Virology Authors: Alexander MR, Rootes CL, van Vuren PJ, Stewart CR Tags: J Virol Methods Source Type: research

High-throughput viral microneutralization method for feline coronavirus using image cytometry.
Abstract 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 recommend...
Source: Journal of Virological Methods - September 22, 2020 Category: Virology Authors: Pearson M, LaVoy A, Chan LL, Dean GA Tags: J Virol Methods Source Type: research

Determination of SARS-CoV-2 antibodies with assays from Diasorin, Roche and IDvet.
eines M Abstract There is an ongoing need for highly reliable serological assays to detect individuals with past SARS-CoV-2 infection. Using 75 sera from patients tested positive or negative by SARS-CoV-2 PCR, we investigated the sensitivity and specificity of the Liaison SARS-CoV-2 S1/S2 IgG assay (DiaSorin), the Elecsys Anti-SARS-CoV-2 assay (Roche), and the ID Screen SARS-CoV-2-N IgG indirect kit (IDVet). We determined a sensitivity of 95.5%, 95.5%, and 100% and a specificity of 90.5%, 96.2%, and 92,5% for the DiaSorin assay, the Roche assay, and the IDVet assay, respectively. We conclude that serologic assays ...
Source: Journal of Virological Methods - September 22, 2020 Category: Virology Authors: Krüttgen A, Cornelissen CG, Dreher M, Hornef MW, Imöhl M, Kleines M Tags: J Virol Methods Source Type: research

Evaluation of immunoglobulin M-specific capture enzyme-linked immunosorbent assays and commercial tests for flaviviruses diagnosis by a National Reference Laboratory.
es SG Abstract Zika and Dengue viruses present considerable immunological cross-reactivity, resulting in a troublesome serodiagnosis due to occurrence of false positive results. Due to Brazil's wide variety of circulating flaviviruses we aimed to access the use of in house serological tests adapted by National Reference Laboratory for Arboviruses in Brazil and evaluate commercial tests available. We evaluated in house IgM ELISAs for the individual detection of anti-ZIKV, -DENV, and -YFV IgM, against a panel of samples positive for dengue, zika, yellow fever, Rocio, Ilheus, Saint Louis encephalitis, West Nile and c...
Source: Journal of Virological Methods - September 20, 2020 Category: Virology Authors: Henriques DF, Nunes JAL, Anjos MV, Melo JM, Rosário WO, Azevedo RSS, Chiang JO, Martins LC, Dos Santos FB, Casseb LMN, Vasconcelos PFC, Rodrigues SG Tags: J Virol Methods Source Type: research

Evaluation of the diagnostic accuracy of an affordable rapid diagnostic test for African Swine Fever antigen detection in Lao People's Democratic Republic.
In this study, an ASF antigen detection RDT from Shenzhen Lvshiyuan Biotechnology Co. Ltd was evaluated using clinical field samples submitted to the National Animal Health Laboratory (NAHL) from ASF suspect cases between June and December 2019 in Lao PDR. Positive (n = 57) and negative (n = 50) samples of whole blood, serum and haemolysed serum were assessed by RDT and PCR, with the latter used as the gold standard reference comparator. Overall the RDT had a diagnostic sensitivity (DSe) of 65%, 95% CI [51 - 77] and diagnostic specificity (DSp) of 76%, 95% CI [62 - 87]. The RDT demonstrated impr...
Source: Journal of Virological Methods - September 17, 2020 Category: Virology Authors: Matsumoto N, Siengsanan-Lamont J, Gleeson LJ, Douangngeun B, Theppangna W, Khounsy S, Phommachanh P, Halasa T, Bush RD, Blacksell SD Tags: J Virol Methods Source Type: research

Value of swab types and collection time on SARS-COV-2 detection using RT-PCR assay.
CONCLUSIONS: In summary, our research discovers that nasopharyngeal or nasal swab collected before washing in the morning might be more suitable for detecting of large-scale specimens from patients infected with low SARS-CoV-2 load during infection late stage. Those results could facilitate other laboratories in collecting appropriate specimens for improving detection of SARS-CoV-2 from patients during infection late stage as well as initially screening. PMID: 32949663 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - September 15, 2020 Category: Virology Authors: Liu M, Li Q, Zhou J, Ai W, Zheng X, Zeng J, Liu Y, Xiang X, Guo R, Li X, Wu X, Xu H, Jiang L, Zhang H, Chen J, Tian L, Luo J, Luo C Tags: J Virol Methods Source Type: research

Optimization and clinical validation of dual-target RT-LAMP for SARS-CoV-2.
Abstract 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-2 has been developed. The LAMP assay achieves a comparable limit of detection (25 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 98.48% (95% CI 91.84% to 99.96%) and NPA ...
Source: Journal of Virological Methods - September 13, 2020 Category: Virology Authors: Mohon AN, Oberding L, Hundt J, van Marle G, Pabbaraju K, Berenger B, Lisboa L, Griener T, Czub M, Doolan C, Servelitta V, Chiu C, Greninger A, Jerome K, Pillai DR Tags: J Virol Methods Source Type: research

Development of an improved dual-promoter-based reverse genetics system for emerging Senecavirus A: Running title: A reverse genetics system for Senecavirus A.
Abstract Senecavirus A (SVA), a recently emerging picornavirus, poses a great threat to the swine industry because it causes swine idiopathic vesicular disease and epidemic transient neonatal losses. Thus far, the progress in SVA viral pathogenesis studies and vaccine development remains sluggish, and an available and convenient reverse genetics system would undoubtedly promote relevant research. Herein, we established an improved universal dual-promoter reverse genetics system with an SVA-specific hammerhead ribozyme and hepatitis delta virus ribozyme at both terminals of the viral genome; this system could be ap...
Source: Journal of Virological Methods - September 13, 2020 Category: Virology Authors: Zhang X, Lu J, Deng T, Zhao P, Peng Z, Chen L, Qian M, Guo Y, Qiao H, Wang Y, Xia Y, Bian C, Wang Z Tags: J Virol Methods Source Type: research

Development of a differential multiplex real-time PCR assay for porcine circovirus type 2 (PCV2) genotypes PCV2a, PCV2b and PCV2d.
Abstract A multiplex quantitative real-time polymerase chain reaction (mqPCR) assay was developed and validated for detection and differentiation of porcine circovirus type 2 (PCV2) genotypes, PCV2a, PCV2b and PCV2d. Single nucleotide polymorphism in primers or probes was deployed for different genotype detections, while conserved sequence in the 3 end of a primer and in the middle of a probe was used for the targeted genotype. In silico analysis of 2601 PCV2 ORF2 sequences showed that the predicted strain coverage of the assay was 93.4 % (409/438) for PCV2a, 95.1 % (1161/1221) for PCV2b and 93.6 % (882/942) for P...
Source: Journal of Virological Methods - September 10, 2020 Category: Virology Authors: Wang Y, Noll L, Porter E, Stoy C, Dong J, Anderson J, Fu J, Pogranichniy R, Woodworth J, Peddireddi L, Bai J Tags: J Virol Methods Source Type: research

Comparison of Commercial Assays and Laboratory Developed Tests for Detection of SARS-CoV-2.
Abstract The global COVID-19 pandemic has led to the rapid development of tests for detection of SARS-CoV-2. Studies are required to assess the relative performance of different assays. Here, we compared the performance of two commercial assays, the cobas® SARS-CoV-2 (Roche Diagnostics) and Xpert® Xpress SARS-CoV-2 (Cepheid®) tests, and a laboratory developed RT-PCR test adapted for use on the Hologic® Panther Fusion® (Hologic®) instrument as well as Bio-Rad and QIAGEN real-time PCR detection systems. Performance characteristics for each test were determined by testing clinical specimens an...
Source: Journal of Virological Methods - September 9, 2020 Category: Virology Authors: Dust K, Hedley A, Nichol K, Stein D, Adam H, Karlowsky JA, Bullard J, Van Caeseele P, Alexander DC Tags: J Virol Methods Source Type: research

Accuracy of a RT-qPCR SARS-CoV-2 detection assay without prior RNA extraction.
Barriga GP Abstract The current COVID-19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected individuals. we sought to evaluate different RT-qPCR kits and protocols to evaluate the best approach to be used omitting an RNA extraction step. We have investigated the sensitivity and performance of different commercially available RT-qPCR kits in detecting SARS-CoV-2 using 80 extracted RNA and NSS from COVID-19 diagnosed patients. We evalua...
Source: Journal of Virological Methods - September 8, 2020 Category: Virology Authors: Beltrán-Pavez C, Alonso-Palomares LA, Valiente-Echeverría F, Gaggero A, Soto-Rifo R, Barriga GP Tags: J Virol Methods Source Type: research

Development and validation of a quantitative pcr assay for detection of terrapene herpesvirus 2 in eastern box turtles (terrapene carolina carolina).
DEVELOPMENT AND VALIDATION OF A QUANTITATIVE PCR ASSAY FOR DETECTION OF TERRAPENE HERPESVIRUS 2 IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA). J Virol Methods. 2020 Sep 07;:113968 Authors: Engel AI, Adamovicz L, Wellehan JFX, Allender MC Abstract Herpesviruses are associated with disease in many chelonian species, resulting in clinical signs such as respiratory tract infections, stomatitis, conjunctivitis, hepatitis, and papillomatosis. Herpesvirus-associated fibropapillomas cause significant morbidity and mortality in marine turtles, and have been identified in an eastern box turtle (Terrapene...
Source: Journal of Virological Methods - September 6, 2020 Category: Virology Authors: Engel AI, Adamovicz L, Wellehan JFX, Allender MC Tags: J Virol Methods Source Type: research

Rabies VLPs adjuvanted with saponin-based liposomes induce enhanced immunogenicity mediated by neutralizing antibodies in cattle, dogs and cats.
Abstract We carried out an investigation on rabies virus-like particles (RV-VLPs) expressed in HEK293 cells using serum free medium. These RV-VLPs were formulated with two different adjuvants in order to analyse the enhancement of the triggered immune response and its stability. In experiments in mice, RV-VLPs showed an enhanced humoral immune response when injected with adjuvant, in contrast to the obtained for the RV-VLPs without adjuvant addition. Besides, higher titers of neutralizing antibodies were induced when RV-VLPs were formulated with LipoSap® in comparison with the obtained with Alhydrogel®. At...
Source: Journal of Virological Methods - September 5, 2020 Category: Virology Authors: Diego F, Federico M, Marina E, Ricardo K, Claudio P Tags: J Virol Methods Source Type: research

Development of two rapid lateral flow test strips for detection of foot-and-mouth disease virus SAT 1 and SAT 3.
Abstract Foot-and-mouth disease (FMD) is a highly contagious disease that affects cattle, sheep, goats, pigs, and over 70 species of wildlife. FMD continues to be a major economic concern for livestock productivity in many countries. FMDV has seven serotypes O, A, Asia 1, C, and Southern Africa Territories (SAT) 1, 2, and 3. Although SAT 1, and SAT 3 outbreaks are not as common as serotypes O, A, Asia 1, and SAT 2, outbreaks have also been reported. The recent outbreaks of SAT 1 occurred in Cameroon, Zimbabwe, South Africa, and Uganda, while most recent SAT 3 occurred in Namibia in 2019. The development of rapid a...
Source: Journal of Virological Methods - September 4, 2020 Category: Virology Authors: Yang M, Mudabuka B, Dueck C, Xu W, Masisi K, Fana EM, Mpofu C, Nfon C Tags: J Virol Methods Source Type: research

Rapid, convenient and efficient kit-independent detection of SARS-CoV-2 RNA.
er A, Otto M Abstract Pandemic SARS-CoV-2 infection has rapidly developed into a socioeconomic and humanitarian catastrophe. Basic principles to prevent SARS-CoV-2 transmission are social distancing, face masks, contact tracing and early detection of SARS-CoV-2. To meet these requirements, virtually unlimited test capacities delivering results in a rapid and reliable manner are a prerequisite. Here, we provide and validate such a rapid, convenient and efficient kit-independent detection of SARS-CoV-2 RNA, termed COVID-quick-DET. This straightforward method operates with simple proteinase K treatment and repetitive...
Source: Journal of Virological Methods - September 2, 2020 Category: Virology Authors: Michel D, Danzer KM, Groß R, Conzelmann C, Müller JA, Freischmidt A, Weishaupt JH, Heller S, Münch J, Michel M, Stamminger T, Kleger A, Otto M Tags: J Virol Methods Source Type: research

A novel real-time PCR to detect Cetacean morbillivirus in Atlantic cetaceans.
This study aimed to develop a molecular technique to detect the most common CeMV strains known to circulate in the Atlantic Ocean: GDMV, Dolphin morbillivirus (DMV) and Pilot-whale morbillivirus (PWMV). A sensible real-time reverse transcription polymerase chain reaction (RT-qPCR) method based on intercalating dye, targeting the P gene was described. This assay successfully detected GDMV, PWMV and DMV from field samples. Its performance was compared to a RT-qPCR method that specifically detects GDMV. Both assays had high sensibility and excellent intra- and inter-assay reproducibility. A total of 109 field samples from 32 ...
Source: Journal of Virological Methods - August 31, 2020 Category: Virology Authors: Groch KR, Taniwaki SA, Favero CM, Brandão PE, Díaz-Delgado J, Fernández A, Catão-Dias JL, Sierra E Tags: J Virol Methods Source Type: research

Establishment of an SYBR Green-based real-time PCR assay for porcine circovirus type 4 detection.
In conclusion, the established SYBR Green I-based real-time PCR may be a cost-effective and rapid method for PCV4 clinical diagnosis. PMID: 32882322 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - August 30, 2020 Category: Virology Authors: Zhang D, Bai C, Ge K, Li Y, Gao W, Jiang S, Wang Y Tags: J Virol Methods Source Type: research

Evaluation of the analytical performance of six rapid diagnostic tests for the detection of viral hepatitis B and C in Lubumbashi, Democratic Republic of Congo.
This study aimed at assessing the effectiveness of RDTs for the detection of HBsAg and anti-HCV antibodies in order to identify infected blood donors in Lubumbashi. A total of 300 serum samples (100 HBsAg positive samples; 100 anti-HCV positive samples and 100 HBsAg and anti-HCV negative samples) were tested simultaneously using the 6 locally used RDTs and as gold standard the chemiluminescent assays for HBsAg and the RT-TMA for HCV.detection. The six evaluated RDTs demonstrated a sensitivity and a negative predictive value (NPV) of 100% whereas the specificity and positive predictive value (PPV) varied from 46% to 98.1%. ...
Source: Journal of Virological Methods - August 25, 2020 Category: Virology Authors: Kabamba AT, Mwamba CM, Dessilly G, Dufrasne F, Kabamba BM, Longanga AO Tags: J Virol Methods Source Type: research

Head-to-head comparison of Enzyme Linked Immunosorbent Assay (ELISA) and Enhanced Chemiluminescence immunoassay (ECLIA) for the detection of Transfusion Transmitted Disease (TTD) Markers; HIV, HCV and HBV in blood donors, in India.
According to this study, it can be concluded both the testing techniques; ELISA and ECLIA have 100% sensitivity for the detection for HBV, HCV and HIV in blood donors and therefore, either can be used for TTD screening in blood banks in India. PMID: 32860798 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - August 25, 2020 Category: Virology Authors: Tiwari AK, Upadhyay AP, Arora D, Wadhwa T, Aggarwal G, Pabbi S, Luthra AS, Rawat SS Tags: J Virol Methods Source Type: research

A simple method for SARS-CoV-2 detection by rRT-PCR without the use of a commercial RNA extraction kit.
Abstract The World Health Organization (WHO) has declared a pandemic caused by a new coronavirus named SARS-CoV-2. The growing demand for commercial kits used for automated extraction of SARS-CoV-2 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-2 from specimens collected in diffe...
Source: Journal of Virological Methods - August 20, 2020 Category: Virology Authors: Ulloa S, Bravo C, Parra B, Ramirez E, Acevedo A, Fasce R, Fernandez J Tags: J Virol Methods Source Type: research

Evaluation of un-methylated DNA enrichment in sequencing of African swine fever virus complete genome.
In this study, the efficiency of the un-methylated DNA enrichment protocol was evaluated for pig and tick samples infected by ASFV. As expected, fewer reads corresponding to ASFV were found in the methylated fraction compared to the un-methylated fraction. However, the sequencing coverage of the un-methylated fraction was not improved compared to the untreated DNA. In our hands, the ASFV DNA enrichment was inefficient for tick samples and very limited for pig samples. This enrichment process represents extra work and cost without a significant improvement of ASFV genome coverage. The efficiency of this enrichment approach ...
Source: Journal of Virological Methods - August 19, 2020 Category: Virology Authors: De Oliveira RP, Lucas P, Chastagner A, De Boisseson C, Vial L, Le Potier MF, Blanchard Y Tags: J Virol Methods Source Type: research

Screening of PRRSV- and ASFV-encoded proteins involved in the inflammatory response using a porcine iGLuc reporter.
Abstract Inflammasome plays a major role in innate immune responses by activating caspase-1, resulting in secretion of interleukin-1β (IL-1β) and inflammatory pathologic responses. IL-1β release is widely used as an indirect readout to study inflammasome activation. Here we report an iGLuc reporter (pro-IL-1β-Gluc) of pig origin to monitor cytosolic pro-IL-1β cleavage and mature IL-1β release. Based on the iGLuc reporter, we reconstructed the inflammasome system in vitro and screened PRRSV- and ASFV-encoded proteins involved in regulating inflammasome activation. We found that three n...
Source: Journal of Virological Methods - August 18, 2020 Category: Virology Authors: Song J, Li K, Li T, Zhao G, Zhou S, Li H, Li J, Weng C Tags: J Virol Methods Source Type: research

Rapid detection of H146-like goose calicivirus using real-time RT-PCR with a Taqman minor groove binder probe.
In this study, a pair of specific primers and a TaqMan minor groove binder (MGB) probe were designed based on a conserved region in the nonstructural (NS) gene sequence. The TaqMan-MGB probe-based one-step qRT-PCR assay was capable of detecting quite low number of targeting nucleic acid as low as 5.07 copies/μL and had excellent intra-assay and inter-assay repeatability with the coefficient of variation (CV) value from 0.558% to 1.293%. The assay was highly specific for H146-like GCV, without cross-reactions with other non-targeted goose-origin viruses, and 62 suspicious tissue samples infected with H146-like GCV from d...
Source: Journal of Virological Methods - August 15, 2020 Category: Virology Authors: Zheng M, Lin S, Zhang S, Chen X, Jiang D, Chen S, Wang S, Chen S Tags: J Virol Methods Source Type: research

Validation of Dried Tube Sample Format Quality Controls for the Monitoring of Viral Load and Blood Screening Assays.
Abstract HIV viral load (VL) and donor screening assays experience variation and require external quality assurance (EQA) programs. NRL sought to confirm a dried tube sample format (HIVDTS) EQA sample type for use in quality control (QC) programs for HIV molecular testing. 50 µL of HIV supernatant at 1 × 10 (Walker et al., 2009) copies per millilitre (copies/mL)) was dried for 48 hours at room temperature. Post-production and shipped integrity studies were undertaken. Dried HIVDTS was reconstituted in PBS buffer and tested in HIV VL (six participants) or blood screening assa...
Source: Journal of Virological Methods - August 13, 2020 Category: Virology Authors: Dimech W, Vincini G, Davies K, Karakaltsas M, van Cauwalaert ND, Guichet E, Koppelman M, Cabuang L Tags: J Virol Methods Source Type: research

Exploring alternative swabs for use in SARS-CoV-2 detection from the oropharynx and anterior nares.
This study evaluated a combined oropharynx/nares (OP/Na) sample collection using two readily-available non-flocked swabs, transported in phosphate-buffered saline, and demonstrates equivalent performance in SARS-CoV-2 detection compared to a previously-validated OP/Na collection kit. PMID: 32783913 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - August 8, 2020 Category: Virology Authors: Patriquin G, Davis I, Heinstein C, MacDonald J, Hatchette TF, LeBlanc JJ Tags: J Virol Methods Source Type: research

Detection of anti-SFTSV nuclear protein antibody in the acute phase sera of patients using double-antigen ELISA and immunochromatography.
Abstract Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening febrile illness that is caused by the SFTS virus (SFTSV). The diagnosis of SFTS is usually performed by detecting viral RNA. However, it has been reported that viral RNA is no longer detectable at 6-12 days after the onset of disease. In the current study, we have constructed a plasmid to express the recombinant nuclear protein (NP) based on the Japanese strain of SFTSV (J1). We developed a double-antigen enzyme-linked immunosorbent assay (ELISA) and immunochromatography (IC) assay using recombinant NP to detect antibody against SFTS...
Source: Journal of Virological Methods - August 7, 2020 Category: Virology Authors: Umeki K, Yasuda A, Umekita K, Megumi R, Nomura H, Kawaguchi T, Matsuda M, Takajo I, Shimojima M, Okayama A Tags: J Virol Methods Source Type: research

Comparison analysis of different swabs and transport mediums suitable for SARS-CoV-2 testing following shortages.
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 6" cotton tipped applicators, and HOLOGIC Aptima) for their efficacy in testing for SARS-CoV-2. Separately, the molecular detection of SARS-CoV-2 was completed from different transport mediums (DMEM, PBS, 100 % ethanol, 0.9 % 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 ...
Source: Journal of Virological Methods - August 7, 2020 Category: Virology Authors: Garnett L, Bello A, Tran KN, Audet J, Leung A, Schiffman Z, Griffin BD, Tailor N, Kobasa D, Strong JE Tags: J Virol Methods Source Type: research

Long amplicon sequencing for improved genetic characterization of African Swine Fever Virus.
In this study, we developed a method for rapid and efficient next generation sequencing of approximately 40% of the ASFV genome using long PCR amplification of six different genomic regions. The amplified regions contain all segments currently used for genotyping and additional genes predicted to contribute to ASFV diversity. The primers used for amplification are broadly compatible with published ASFV genomes, permitting their use on relevant ASFV isolates. This methodology provides the enhanced depth of coverage of amplicon-based sequencing while mitigating complications associated with ASFV whole-genome sequencing. Impl...
Source: Journal of Virological Methods - August 2, 2020 Category: Virology Authors: Meekins DA, Trujillo JD, Gaudreault NN, Morozov I, Pérez-Núñez D, Revilla Y, Richt JA Tags: J Virol Methods Source Type: research

Hepatitis B core antigen-based vaccine demonstrates cross-neutralization against heterologous North American Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-2) strains.
Abstract The U.S. swine industry have been bearing the financial impact of Porcine Reproductive and Respiratory Syndrome (PRRS) for decades. Absent of a safe and efficacious vaccine to combat PRRS virus's genetic heterogeneity, it remains a costly disease on pig farms across the country. We have developed virus-like-particle (VLP) based vaccines that incorporate 4 PRRSV epitopes in the hepatitis B core antigen (HBcAg) backbone. Administration of the vaccines in female BALB/C mice resulted in extremely significant PRRSV epitope specific antibody response. One vaccine candidate GP3-4 was able to mount a significant ...
Source: Journal of Virological Methods - July 27, 2020 Category: Virology Authors: Lu Y, Gillam F, Cao QM, Rizzo A, Meng XJ, Zhang C Tags: J Virol Methods Source Type: research

Development of an SYBR Green I-based real-time PCR assay for the rapid detection of canine kobuvirus.
In this study, an SYBR Green I-based quantitative real-time PCR assay was established for CaKoV detection. Specific primers targeting a highly conserved region of the CaKoV 3D gene were developed. After optimization, the method detected a minimum of 1 × 101 copies/µL with high specificity, stability, and repeatability. Moreover, the entire process only required approximately 1.5 h for completion. Our results were supported by those obtained for clinical samples, in which our developed method was successfully applied. The newly established real-time PCR is a rapid, sensitive, specific, and ...
Source: Journal of Virological Methods - July 26, 2020 Category: Virology Authors: Wang Y, Cui Y, Li Y, Zhang D, Sun J, Guo X, Liu G, Jiang S, Li Y Tags: J Virol Methods Source Type: research

Comparison of Zaire ebolavirus realtime RT-PCRs targeting the nucleoprotein gene.
Vapalahti O Abstract In last five years, the Africa has faced two outbreaks of Zaire ebolavirus. These outbreaks have been the largest so far, and latest outbreak is still ongoing and affecting the Democratic Republic of the Congo. We tested in parallel three different Zaire ebolavirus (EBOV) realtime RT-PCRs targeting the nucleoprotein gene (EBOV NP-RT-qPCRs) described by Trombley et al. (2010), Huang et al. (2012) and Weidmann et al. (2004). These assays are used regularly in diagnostic laboratories. The limit of detection (LOD), intra-assay repeatability using different matrixes, sensitivity and specificity wer...
Source: Journal of Virological Methods - July 20, 2020 Category: Virology Authors: Jääskeläinen AJ, Sironen T, Kaloinen M, Kakkola L, Julkunen I, Hewson R, Weidmann MW, Mirazimi A, Watson R, Vapalahti O Tags: J Virol Methods Source Type: research

Madin-Darby bovine kidney (MDBK) cells are a suitable cell line for the propagation and study of the bovine poxvirus lumpy skin disease virus.
Abstract Lumpy skin disease virus (LSDV) is a poxvirus that causes systemic disease in cattle, resulting in substantial economic loss to affected communities. LSDV is a rapidly emerging pathogen of growing global concern that recently spread from Africa and the Middle East into Europe and Asia, impacting the cattle population in these regions. An increase in research efforts into LSDV is required to address key knowledge gaps, however this is hampered by lack of suitable cell lines on which to propagate and study the virus. In this work we describe the replication and spread of LSDV on Madin-Darby bovine kidney (M...
Source: Journal of Virological Methods - July 20, 2020 Category: Virology Authors: Fay P, Cook C, Wijesiriwardana N, Tore G, Comtet L, Carpentier A, Shih B, Freimanis G, Haga IR, Beard PM Tags: J Virol Methods Source Type: research

A Closed-Tube, Single-Step, Real Time, Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Infectious Bronchitis Virus Detection in Chickens.
Abstract Infectious bronchitis (IB) is a viral infection of the chicken respiratory tract that causes substantial economic burden on the industry. Simple, specific and rapid diagnosis of this disease is critical for the initiation of appropriate control measures. Conventional molecular diagnostic methods require a relatively sophisticated equipment and skilled staff. Here we describe a rapid, simple, semi-quantative, closed-tube, single-step, real-time- reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for IB and compare our assay with quantative, reverse transcription- polymerase chain ...
Source: Journal of Virological Methods - July 16, 2020 Category: Virology Authors: El-Tholoth M, Mauk MG, Anis E, Bau HH Tags: J Virol Methods Source Type: research

Evaluation of three different filters and two methods for recovering viruses from drinking water.
Abstract Among the enteric viruses implicated in waterborne outbreaks, human norovirus and hepatitis A virus (HAV) are a serious public health issue. Most foodborne viruses are difficult or currently unlikely to cultivate. Because of the lack of a cell culture method, real-time reverse transcriptase PCR is commonly used for the detection of norovirus in foodstuffs and environmental samples. Due to low infectious doses in humans and low virus concentration in water sample, filter adsorption methods were used for concentrating viruses from water. The ISO (Anonymous, ISO 15216-1, 2017) describes standardized molecula...
Source: Journal of Virological Methods - July 12, 2020 Category: Virology Authors: Hennechart-Collette C, Dehan O, Fraisse A, Martin-Latil S, Perelle S Tags: J Virol Methods Source Type: research

Immunization of turkeys with a DNA vaccine expressing the haemagglutinin gene of low pathogenic avian influenza virus subtype H9N2.
In conclusion, DNA vaccination with pVAX1-H9 and pCR-H9 could protect turkey from the H9N2 virus, but vaccination regimes need to be improved. PMID: 32663531 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - July 10, 2020 Category: Virology Authors: Shehata AA, Basiouni S, Ali A, Fawzy M, Hafez HM, Ulbert S, Vahlenkamp T Tags: J Virol Methods Source Type: research

Comparison of a Direct Immunofluorescence Assay (Oxoid IMAGEN ®) and a Multiplex RT-PCR DNA Microarray Assay (CLART® PneumoVir) for the Detection of Respiratory Viruses in Hospitalized Children.
The objective of this study was to compare the positive detection rates obtained using the Oxoid IMAGEN® direct immunofluorescence assay (designated as IF) with those obtained using the CLART® PneumoVir multiplex RT-PCR DNA microarray assay (designated as RT-PCR) in the diagnosis of respiratory viruses in hospitalized children. This was a retrospective study of 62 individuals
Source: Journal of Virological Methods - July 10, 2020 Category: Virology Authors: Ferreira ICS, Alegretti AP, De Paris F, Paiva RM, Chakr VCBG Tags: J Virol Methods Source Type: research

A rapid and sensitive lateral flow immunoassay (LFIA) test for the on-site detection of banana bract mosaic virus in banana plants.
This study reports the development of a practical, rapid, sensitive, specific and user-friendly lateral flow immunoassay (LFIA) test for the on-site detection of BBrMV. The BBrMV coat protein (CP) was expressed in Escherichia coli and purified and used to immunize rabbits to produce a polyclonal antiserum (anti-BBrMVCP). The test was based on a double-antibody sandwich format. Protein-A affinity column-purified anti-BBrMVCP Immunoglobulins (IgG) (16 µg/ml), conjugated to ∼30 nm gold nanoparticles, was applied onto the conjugate pad. The anti-BBrMVCP IgG and goat anti-rabbit IgG were printed on the s...
Source: Journal of Virological Methods - July 10, 2020 Category: Virology Authors: Ramasamy S, Selvam KP, Velusamy B, Sundaram SS Tags: J Virol Methods Source Type: research

Brief validation of the novel GeneXpert Xpress SARS-CoV-2 PCR assay.
gli A Abstract The clinical and epidemiologic management of the SARS-CoV-2 pandemic is critically dependent on molecular assays with short turn-around time. We validated the novel Xpert Xpress SARS-CoV-2 assay using a commercial nucleic acid testing (Roche Cobas 6800). We found an excellent concordance over a range of SARS-CoV-2 loads and across established human coronaviruses. PMID: 32659240 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - July 9, 2020 Category: Virology Authors: Goldenberger D, Leuzinger K, Sogaard KK, Gosert R, Roloff T, Naegele K, Cuénod A, Mari A, Seth-Smith H, Rentsch K, Hinić V, Hirsch HH, Egli A Tags: J Virol Methods Source Type: research

Capillary Electrophoresis of PCR fragments with 5 ´-labelled primers for testing the SARS-Cov-2.
In conclusion, we describe and alternative method to identify the SARS-Cov-2 genome that could be scaled to the analysis of approximately 100 samples in less than 5 hours. By combining a standard PCR with capillary electrophoresis our approach would overcome the limits imposed to many labs by the qtPCR and increase the testing capacity. PMID: 32659241 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - July 9, 2020 Category: Virology Authors: Gómez J, Melón S, Boga JA, Alvarez-Argüelles ME, Rojo-Alba S, Leal-Negredo A, Castello-Abietar C, Alvarez V, Cuesta-Llavona E, Coto E Tags: J Virol Methods Source Type: research

Double-quencher probes improve detection sensitivity toward Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a reverse-transcription polymerase chain reaction (RT-PCR) assay.
CONCLUSION: Double-quencher probes decreased the background fluorescence and improved the detection sensitivity of RT-PCR for SARS-CoV-2. PMID: 32650037 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - July 6, 2020 Category: Virology Authors: Hirotsu Y, Mochizuki H, Omata M Tags: J Virol Methods Source Type: research

Replacing the decoy epitope of PCV2 capsid protein with epitopes of GP3 and/or GP5 of PRRSV enhances the immunogenicity of bivalent vaccines in mice.
Abstract Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure and causes economic losses in the domestic swine industry. The decoy epitope (169-180 amino acid (aa)) of the PCV2 capsid (Cap) protein is an immunodominant epitope and diverts the immune response away from protective epitopes. The mixed infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most common co-infections in the pig industry and shows more severe clinical symptoms. Line...
Source: Journal of Virological Methods - July 6, 2020 Category: Virology Authors: Jung BK, Kim HR, Jang H, Chang KS Tags: J Virol Methods Source Type: research