Generation of PK-15 cell lines highly permissive to porcine circovirus 2 infection by transposon-mediated interferon-gamma gene transfer.
Abstract Porcine circovirus 2 (PCV2)-associated diseases affect the swine industry worldwide. Vaccination is the major tool for the disease control, but the vaccine production is hindered by lower propagation rate of PCV2 in vitro. Previous studies showed that interferons (IFNs) can increase PCV2 yield in PK-15 cells. In the present study, we constructed a Sleepy Beauty (SB) transposon vector expressing porcine IFNg gene fused with the coding sequence for immunoglobulin G Fc domain. After dilution cloning, the transposon and transposase vectors were co-transfected into PK-15 cell clones with higher permissivity to...
Source: Journal of Virological Methods - June 16, 2019 Category: Virology Authors: Li Y, Wang Y, Zhou X, Zhang X, Zhang X, Xia X, Sun H Tags: J Virol Methods Source Type: research

Development of a recombinase polymerase amplification assay with lateral flow dipstick for rapid detection of feline parvovirus virus.
In this study, we report a novel FPV lateral flow dipstick RPA (LFD-RPA) instant detection method capable of detecting a range of different FPV strains. The LFD-RPA assay consists of specific primers, probe, and nucleic acid strip. It is capable of detecting 102 copies of target nucleic acid per reaction, which is one order of magnitude higher than the sensitivity of traditional PCR. The most suitable reaction conditions for this assay are at 38 ℃ for 15 min. This paper develops an efficient visual detection system that can eliminate the need for professional staff and expensive and sophisticated equipment for fie...
Source: Journal of Virological Methods - June 16, 2019 Category: Virology Authors: Wang ZH, Wang XJ, Hou SH Tags: J Virol Methods Source Type: research

Development of an enzyme-linked immunosorbent assay for Getah virus infection in horses using recombinant E2 protein as an antigen.
In conclusion, we established rE2-ELISA that could detect horse antibodies against Getah virus after experimental and natural infections; this should be useful in the diagnosis and surveillance of Getah virus infection. PMID: 31207276 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - June 14, 2019 Category: Virology Authors: Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kokado H Tags: J Virol Methods Source Type: research

Development of a Reverse Transcription-loop-mediated isothermal amplification (LAMP) assay for the rapid detection of onion yellow dwarf virus.
Abstract Onion yellow dwarf virus (OYDV) is one of the most important viral pathogens of onion. In particular, on 'Rossa di Tropea' onion, granted with Protected Geographical Indication (PGI) trademarks, this pathogen represents the most limiting biotic stress in terms of spread, severity of symptoms and damage, and its detection is necessary to preserve high quality standards and avoid yield losses. A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed for detection of OYDV. The specificity, sensitivity, repeatability and reproducibility of the assay were validated according...
Source: Journal of Virological Methods - June 13, 2019 Category: Virology Authors: Tiberini A, Tomlinson J, Micali G, Fontana A, Albanese G, Tomassoli L Tags: J Virol Methods Source Type: research

Novel pan-serotype control RNA for dengue virus typing through real-time reverse transcription-polymerase chain reaction.
In this study, we describe the in silico design of the qDENV-Control plasmid with the target sequences to oligonucleotides and probes widely used for DENV serotyping, and the subsequent production of qDENV Control RNA by T7-driven run-off in vitro transcription. The qDENV Control RNA was successfully used to validate the positive and negative DENV serotyping results, allowing its incorporation in routine in-house protocols for virologic surveillance. This Control RNA allowed the absolute quantification of viral RNA copies from unknown samples as required in several fundamental studies. PMID: 31195032 [PubMed - as supp...
Source: Journal of Virological Methods - June 10, 2019 Category: Virology Authors: Álvarez-Díaz DA, Quintero PA, Peláez-Carvajal D, Ajami NJ, Usme-Ciro JA Tags: J Virol Methods Source Type: research

Duplex Real-Time RT-PCR Assay for Detection and Subgroup-Specific Identification of Human Respiratory Syncytial Virus.
t TCT Abstract Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Reliable detection and identification of HRSV subgroup A and B infections are essential for accurate disease burden estimates in anticipation of licensure of novel HRSV vaccines and immunotherapies. To ensure continued reliability, molecular assays must remain current with evolving virus strains. We have developed a HRSV subgroup-specific real-time RT-PCR (rRT-PCR) assay for detection and subgroup identification using primers and subgroup-specific probes targeting a conserved region ...
Source: Journal of Virological Methods - June 7, 2019 Category: Virology Authors: Wang L, Piedra P, Avadhanula V, Durigon EL, Machablishvili A, López MR, Thornburg N, Peret TCT Tags: J Virol Methods Source Type: research

Performance of InBios ZIKV DetectTM 2.0 IgM Capture ELISA in two reference laboratories compared to the original ZIKV DetectTM IgM Capture ELISA.
PMID: 31181219 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - June 7, 2019 Category: Virology Authors: Basile AJ, Ao J, Horiuchi K, Semenova V, Steward-Clark E, Schiffer J Tags: J Virol Methods Source Type: research

Development and Evaluation of a One-Step Quantitative RT-PCR Assay for Detection of Lassa Virus.
Abstract Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. 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...
Source: Journal of Virological Methods - June 3, 2019 Category: Virology Authors: Dedkov VG, Magassouba N, Safonova MV, Naydenova EV, Ayginin AA, Soropogui B, Kourouma F, Camara AB, Camara J, Kritzkiy AA, Tuchkov IV, Shchelkanov MU, Maleev VV Tags: J Virol Methods Source Type: research

Ten-minute direct detection of Zika virus in serum samples by RT-LAMP.
Mendes Duarte GR Abstract Zika virus (ZIKV) is a current threat to global health. In most of cases, ZIKV infection has no symptoms; however in some cases, ZIKV can cause paralysis (Guillain-Barré syndrome), and in pregnant women, it can cause birth defects in infants. Rapid and accurate diagnosis can help improve disease control as well as being vital to prenatal care for women living in endemic areas. Molecular diagnostics based on isothermal amplification techniques are an excellent alternative to conventional methods of DNA amplification, such as PCR. Here, we develop and optimized a rapid and sensitive...
Source: Journal of Virological Methods - June 3, 2019 Category: Virology Authors: Neves Estrela PF, de Melo Mendes G, de Oliveira KG, Bailã AM, de Almeida Soares CM, Assunção NA, Mendes Duarte GR Tags: J Virol Methods Source Type: research

Detection and quantification of four viruses in Prunus pollen: implications for biosecurity.
Abstract Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detectio...
Source: Journal of Virological Methods - June 3, 2019 Category: Virology Authors: Beaver-Kanuya E, Harper SJ Tags: J Virol Methods Source Type: research

A Flow-Through Cell Counting Assay for Point-of-Care Enumeration of CD4 T-cells.
Abstract CD4 T-cell count is a priority for staging HIV disease and guiding clinical management as part of HIV care. Conventional CD4 T-cell enumeration methods based on flow cytometry are expensive, require well-trained personnel, and are challenging to use in rural, resource-scarce areas. A simple CD4 T-cell count test that can be used at point-of care, the Flow-Through cell Counting Assay (FTCA), is described in this article. The FTCA is based on the use of: 1) a special membrane that selectively retains white blood cells (WBCs); 2) a sample delivery system; and 3) optical signal detection. To show the feasibil...
Source: Journal of Virological Methods - May 27, 2019 Category: Virology Authors: Bystryak S, Bandwar RP, Santockyte R Tags: J Virol Methods Source Type: research

Rapid detection of Cucumber green mottle mosaic virus in watermelon through a recombinase polymerase amplification assay.
In this study, we developed a novel isothermal recombinase polymerase amplification (RPA) technique for detection of CGMMV in watermelon samples. A pair of CGMMV specific RPA primers was prepared based on the conserved CGMMV coat protein gene sequences. The result showed that this RPA detection method can be performed at 38 °C and completed in about 30 min, and there was no cross-reactivity with other common cucurbit viruses. Sensitivity assay showed that this RPA method was more sensitive compared with the regular RT-PCR. Using field-collected watermelon tissue samples, we have demonstrated that this new...
Source: Journal of Virological Methods - May 25, 2019 Category: Virology Authors: Jiao Y, Jiang J, Wu Y, Xia Z Tags: J Virol Methods Source Type: research

Development and evaluation of a one-step reverse transcription loop-mediated isothermal amplification for detection of Citrus leaf blotch virus.
In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed to detect CLBV; this technology has been widely used in the detection of various plant pathogenic microorganisms and exogenous genes. The sensitivity of the RT-LAMP method was increased 100-fold compared to that of the conventional RT-PCR. In addition, this method was fast, simple and specific; it could provide better technical support for field diagnosis, customs quarantine and the control measures of CLBV. To our knowledge, this is the first report detecting CLBV using RT-LAMP. PMID: 31132370 [PubMed - as sup...
Source: Journal of Virological Methods - May 24, 2019 Category: Virology Authors: Liu H, Wu W, Tan J, Li Y, Mi W, Jiang L, Wu Y Tags: J Virol Methods Source Type: research

Delivery of Maize mosaic rhabdovirus to planthopper vectors by microinjection increases infection efficiency and facilitates functional genomics experiments in the vector.
Abstract The corn planthopper, Peregrinus maidis, not only causes direct damage to plants by feeding, but also transmits maize mosaic virus (MMV) to the plant hosts. The virus is transmitted in a propagative manner but the acquisition of MMV by the vector feeding on infected plants can result in low acquisition and inoculation efficiency. Here, we increased the acquisition efficiency by delivering the virus directly into the hemocoel through microinjection, which resulted in efficient virus infection of the insect and transmission to maize. We found that delivery of virus by injection of 10 ng MMV (50&thins...
Source: Journal of Virological Methods - May 24, 2019 Category: Virology Authors: Yao J, Rotenberg D, Whitfield AE Tags: J Virol Methods Source Type: research

Corrigendum to "Genetic identification of two Acipenser iridovirus-European variants using high-resolution melting analysis" [J. Virol. Methods 265 (2018) 105-112].
Corrigendum to "Genetic identification of two Acipenser iridovirus-European variants using high-resolution melting analysis" [J. Virol. Methods 265 (2018) 105-112]. J Virol Methods. 2019 May 22;: Authors: Pallandre L, Lesne M, de Boisséson C, Charrier A, Daniel P, Tragnan A, Debeuf B, Chesneau V, Bigarré L PMID: 31128946 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - May 22, 2019 Category: Virology Authors: Pallandre L, Lesne M, de Boisséson C, Charrier A, Daniel P, Tragnan A, Debeuf B, Chesneau V, Bigarré L Tags: J Virol Methods Source Type: research

Evaluation of the Abbott RealTime quantitative CMV and EBV assays using the maxCycle protocol in a laboratory automation context.
In this study, we showed that both EBV assays exhibited a similar sensitivity but with a better precision for the EBV Abbott RealTime assay. For the CMV performances, the Abbott assay was more sensitive and more precise than our routine method. The use of WHO International Standards also indicated a slight underestimation of the viral loads (-0.25 log10 IU/mL and -0.21 log10 IU/mL for CMV and EBV assays respectively) while these were rather overestimated with the Starlet/Diagenode method (0.48 log10 IU/mL and 0.19 log10 IU/mL for CMV and EBV assays respectively). These trends were confirmed using relevant whole-blood clini...
Source: Journal of Virological Methods - May 20, 2019 Category: Virology Authors: Bontems S, Boreux R, Capraro V, Huynen P, Descy J, Melin P, Hayette MP, Meex C Tags: J Virol Methods Source Type: research

A reverse transcription-insulated isothermal PCR assay for the detection of duck hepatitis A virus type 3 based on the POCKIT ™ system.
In this study, a rapid reverse transcription insulated isothermal (RT-iiPCR) technique was developed for the on-site detection of DHAV-3 based on the POCKIT™ system in a convenient device. The concentration of primer pairs and probes were optimized for amplification of the DHAV-3 VP3 gene of DHAV-3, with no amplification of 12 other duck pathogens. The detection limit of viral RNA was 3.85 × 101 copies/µL, and the analytical sensitivity and specificity levels were both 100% in the detection of 40 liver samples. Furthermore, 97.5% of the RT-iiPCR results were in agreement with those of rRT-PC...
Source: Journal of Virological Methods - May 14, 2019 Category: Virology Authors: Ren Y, Yue H, Zhu L, Kan R, Tang C, Zhang B Tags: J Virol Methods Source Type: research

Development of a sandwich ELISA to detect virus-like-particles in enterovirus A71 vaccines.
Abstract The goal of this paper was to develop a sandwich ELISA that can detect intact human enterovirus A71 (EV-A71) virus-like particles (VLPs) in vaccines. This assay specifically detected EV-A71 viruses from different sub-genogroups as well as EV-A71 VLPs, and treatment of VLPs with high heat and low pH reduced or completely abolished detection of the VLPs suggesting that the ELISA detected assembled particles. Using a purified VLP as a reference standard, a quantitative sandwich ELISA (Q-ELISA) was established which was used to monitor the yield and purity of the VLPs during manufacturing. Coupled with immuno...
Source: Journal of Virological Methods - May 14, 2019 Category: Virology Authors: Lim PY, Cardosa MJ Tags: J Virol Methods Source Type: research

Production of monoclonal antibodies binding to the VP7 protein of African horse sickness virus.
Abstract Monoclonal antibodies (MAbs) against AHSV were produced immunising BALB/c mice with AHSV serotype 9 and six clones able to recognize specifically the VP7-AHSV with a strong reactivity were selected. The specificity of the MAbs was assessed in i-ELISA against a commercial VP7-AHSV and in immunoblot against a home-made VP7-AHSV was expressed by a Baculovirus expression system; potential cross-reactions with related orbiviruses (Bluetongue virus and Epizootic Haemorrhagic Disease virus) were investigated as well. One of the six MAbs selected, MAb 7F11E14, was tested in direct immunofluorescence and reacted w...
Source: Journal of Virological Methods - May 13, 2019 Category: Virology Authors: Luciani M, Armillotta G, Ciarelli A, Ulisse S, Teodori L, Bortone G, Giamboi A, Monaco F, Tittarelli M, Di Ventura M, Iannetti L, Vulpiani MP, Di Febo T Tags: J Virol Methods Source Type: research

PCR diagnostics: In silico validation by an automated tool using freely available software programs.
Abstract 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 diagn...
Source: Journal of Virological Methods - May 13, 2019 Category: Virology Authors: van Weezep E, Kooi EA, van Rijn PA Tags: J Virol Methods Source Type: research

Diagnostic comparison of serum and EDTA-stabilized blood samples for the detection of foot-and-mouth disease virus RNA by RT-qPCR.
GJ, Lohse L Abstract Foot-and-mouth disease (FMD) remains a globally important disease but there have only been occasional recent outbreaks in Europe, e.g. in the U.K. in 2001, U.K. 2007 and Bulgaria 2010/2011. However, this infection still poses a threat to Europe as the disease occurs close to its borders and incursions can occur through importation of contaminated animal products and through the air. To deal with a suspected outbreak, fast sampling, transportation and accurate laboratory diagnosis are critical; testing for FMDV is normally performed on epithelium samples or serum. Assessment of the use of stabi...
Source: Journal of Virological Methods - May 13, 2019 Category: Virology Authors: Fontél KS, Bøtner A, Belsham GJ, Lohse L Tags: J Virol Methods Source Type: research

A loop-mediated isothermal amplification coupling with a lateral flow dipstick for rapid and specific detection of fowl adenovirus serotype-4.
In this study, a loop-mediated isothermal amplification coupling with a lateral flow dipstick (LAMP-LFD) was developed for rapid and specific detection of fowl adenovirus serotype-4. The optimized LAMP-LFD can be completed in 60 min at 65 °C. The minimum detection limits of PCR, real-time PCR, nested PCR and LAMP-LFD are 1 × 104 copies/μl, 1 × 102 copies/μl, 10 copies/μl and 10 copies/μl respectively. Moreover, the specificity of the LAMP-LFD assay is satisfactory and does not produce cross reactions with other species. In field samples, 150 samples were...
Source: Journal of Virological Methods - May 1, 2019 Category: Virology Authors: Zhai X, Mei X, Wu X, Zuo L, Zhou L, Tian Y, Han X, Yang X, Wang H Tags: J Virol Methods Source Type: research

Analytical performances of the BD Veritor ™ System for the detection of respiratory syncytial virus and influenzaviruses A and B when used at bedside in the pediatric emergency department.
This study aims to evaluate the analytical performance of the BD Veritor™ rapid diagnostic assays (RDTs) for respiratory syncytial virus (RSV) and influenzaviruses when performed 24/7 at bedside by nurses in the pediatric emergency department (PED). The study was performed between 14/10/2015 and 19/03/2016 on nasopharyngeal aspirates (NPAs) collected from children consulting at the PED of the University Hospital of Saint-Etienne for bronchiolitis (RSV detection) or flu-like syndrome (influenzaviruses A/B detection). NPAs were tested 24/7 at the PED with the RDT and then sent to the Infectious Agents Department for ro...
Source: Journal of Virological Methods - April 29, 2019 Category: Virology Authors: Cantais A, Mory O, Plat A, Giraud A, Pozzetto B, Pillet S Tags: J Virol Methods Source Type: research

Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines.
In this study, we standardized a real-time quantitative RT-PCR duplex assay to be used during intermediate stages of the vaccine production. This assay was done for the quantification of vaccine strain rabies virus, targeting rabies nucleoprotein, and β-actin mRNA of BHK-21 cells as an internal endogenous control. The results showed specific amplification, with the analytical sensitivity ranged from 101 to 106 TCID50/mL with high repeatability rate for the quantification of rabies virus in inactivated vaccine samples. Global organizations are engaged to develop new approaches to determine viable residual virus, and th...
Source: Journal of Virological Methods - April 29, 2019 Category: Virology Authors: Correia Moreira BL, Pereira LA, Lappas Gimenez AP, Fernandes Inagaki JM, Raboni SM Tags: J Virol Methods Source Type: research

"Rapid diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay".
"Rapid diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay". J Virol Methods. 2019 Apr 29;: Authors: Srivastava N, Kapoor R, Kumar R, Kumar S, R K S, Kumar S, Baranwal VK Abstract Cucumber mosaic virus (CMV) is a widespread plant virus infecting important vegetables, plantation and flower crops. Currently, CMV is detected by enzyme-linked immunosorbent assays (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. ELISA requires polyclonal antibodies and is ti...
Source: Journal of Virological Methods - April 29, 2019 Category: Virology Authors: Srivastava N, Kapoor R, Kumar R, Kumar S, R K S, Kumar S, Baranwal VK Tags: J Virol Methods Source Type: research

WarmStart colorimetric LAMP for the specific and rapid detection of HPV16 and HPV18 DNA.
CONCLUSIONS: The newly established WarmStart colorimetric LAMP can be considered as a powerful molecular tool that it can be easily implemented in small clinical and research laboratories for a rapid and efficient identification of the most tumorigenic HPV genotypes. PMID: 31042552 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - April 28, 2019 Category: Virology Authors: Daskou M, Tsakogiannis D, Dimitriou TG, Amoutzias GD, Mossialos D, Kottaridi C, Gartzonika C, Markoulatos P Tags: J Virol Methods Source Type: research

Rapid detection of Tobacco streak virus (TSV) in cotton (Gossypium hirsutum) based on Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP).
Abstract Tobacco Streak Virus (TSV) belongs to the genus Ilarvirus of the family Bromoviridae an emerging pathogen posing threat to the crop species worldwide. Identification of symptoms due to TSV infection by visual observation of plants often results in misdiagnosis as symptoms produced by this virus can match with those reflecting physiological and nutritional disorders affecting cotton. Development of diagnostic tools with rapidity will have immense role to play in detection and management of the emerging virus. The protocol for rapid diagnosis of TSV infected samples by using Reverse Transcription-Loop Media...
Source: Journal of Virological Methods - April 23, 2019 Category: Virology Authors: Gawande SP, K P R, Monga D, Nagrale DT, Kranthi S Tags: J Virol Methods Source Type: research

Development of a droplet digital polymerase chain reaction for sensitive and simultaneous identification of porcine circovirus type 2 and 3.
In this study, a sensitive assay for simultaneous detection of porcine circovirus type 2 (PCV2) and type 3 (PCV3) was established using droplet digital PCR (ddPCR). Pairs of primers and probes were specifically designed to amplify PCV2 and PCV3. Specificity of the assay was confirmed by the failure of amplification of DNA of other relevant viruses. The detection limit for ddPCR was 2 copies/µL for PCV2 and 1 copy/µL for PCV3, a 3- and 10-fold greater sensitivity than TaqMan real-time PCR, respectively. Both methods showed a high degree of linearity, although TaqMan real-time PCR showed less sensitivity than ddP...
Source: Journal of Virological Methods - April 23, 2019 Category: Virology Authors: Liu Y, Meng H, Shi L, Li L Tags: J Virol Methods Source Type: research

Validation of quantitative real-time RT-PCR assays for the detection of six honeybee viruses.
te;ry R, Dubois E Abstract Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Varroa destructor virus-1 (VDV1) are the six main honeybee viruses reported in Europe. We assessed the accuracy (trueness and precision) of reverse transcriptase quantitative TaqMan® PCR methods (RT-qPCR) for quantifying ABPV, BQCV, DWV, VDV1 and SBV loads. Once the systematic bias in quantitative results had been corrected (overestimation in ABPV and BQCV quantification and underestimation in that of SBV and VDV1), measurements were...
Source: Journal of Virological Methods - April 23, 2019 Category: Virology Authors: Schurr F, Tison A, Militano L, Cheviron N, Sircoulomb F, Rivière MP, Ribière-Chabert M, Thiéry R, Dubois E Tags: J Virol Methods Source Type: research

Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus.
Abstract 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 NS5A genes were designed and used to detect PPgV. The optimal reaction parameter for RT-LAMP assay was 63℃ for 60 min. The detection limit of the RT-LAMP assay was 10 copies of PPgV genome, which was 100 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 wit...
Source: Journal of Virological Methods - April 22, 2019 Category: Virology Authors: Li H, Li K, Bi Z, Gu J, Song D, Lei D, Luo S, Huang D, Wu Q, Ding Z, Wang L, Ye Y, Tang Y Tags: J Virol Methods Source Type: research

Medium optimization and characterization of cell culture system from Penaeus vannamei for adaptation of white spot syndrome virus (WSSV).
Abstract The lack of shrimp cell lines and difficulty in establishing shrimp cell culture systems, with an appropriate medium is a major concern in the aquaculture sector. The present study attempts to address this issue by developing an in vitro cell culture system from various tissues (hemocytes, heart, lymphoid tissue, hepatopancreas, gill, eye stalk, and muscle) of Penaeus vannamei (P.vannamei) using commercially available L-15 medium. The cell culture medium was formulated using five different media such as HBSCM-1, HBSCM-2, HBSCM-3, HBSCM-4, and HBSCM-5 containing L-proline and glucose with fetal bovine seru...
Source: Journal of Virological Methods - April 19, 2019 Category: Virology Authors: Sivakumar S, Swaminathan TR, Anandan R, Kalaimani N Tags: J Virol Methods Source Type: research

Screening of novel drugs for inhibiting hepatitis E virus replication.
In conclusion, we constructed an HEV replicon system for anti-HEV drug screening and a novel cell-culture system to strictly evaluate the replication-inhibitory activities of the obtained anti-HEV candidates. Our findings suggested that interferon λ1-3 might be effective for treating hepatitis E. PMID: 31004661 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - April 17, 2019 Category: Virology Authors: Nishiyama T, Kobayashi T, Jirintai S, Kii I, Nagashima S, Prathiwi Primadharsini P, Nishizawa T, Okamoto H Tags: J Virol Methods Source Type: research

Comparison of nucleic acid extraction methods for next-generation sequencing of avian influenza A virus from ferret respiratory samples.
In this study, nasal wash samples from ferrets inoculated with different subtypes of AIV were collected on various days post-inoculation. Each nasal wash sample was aliquoted and extracted using five commercially available nucleic acid extraction methods. Extracted influenza virus RNA was amplified and NGS conducted using Illumina Mi-Seq. For each nasal wash sample, completeness of AIV genome segments and coverage depth were compared among five extraction methods. Nucleic acids extracted by MagNA pure compact RNA isolation consistently yielded codon complete sequences for all eight genome segments at the required coverage ...
Source: Journal of Virological Methods - April 17, 2019 Category: Virology Authors: Di H, Thor S, Trujillo AA, Stark T, Marinova-Petkova A, Jones J, Wentworth DE, Barnes J, Davis CT Tags: J Virol Methods Source Type: research

Comparison of multiplex-serology and ELISA based methods in detecting HPV16 L1 antibody responses in paired saliva and serum samples of healthy men.
In this study we show that anti-HPV16L1 antibodies, the IgA response being most abundant, can be measured in saliva of asymptomatic males. HPV16L1 specific multiplex serology and commercial ELISA methods were compared and also the total salivary IgA levels measured. The total salivary IgA concentrations varied from 36-163 ug/ml. All the assays could detect anti-HPV16 IgA from saliva, but the correlation between assays varied from non-significant 0.22 to highly significant 0.81, p
Source: Journal of Virological Methods - April 17, 2019 Category: Virology Authors: Loimaranta V, Sievi K, Werner J, Pawlita M, Waterboer T, Butt J, Syrjänen S Tags: J Virol Methods Source Type: research

Increased HPV detection by the use of a pre-heating step on vaginal self-samples analysed by Aptima HPV assay.
Abstract BACKGROUND: We recently reported a sensitivity of 85.5% to detect high-grade squamous intraepithelial lesions (HSIL)/adenocarcinoma in situ (AIS)/cancer by the use of self-collected vaginal samples analysed by the Aptima mRNA HPV assay (AHPV). OBJECTIVES: To increase detection of HPV among self-samples. STUDY DESIGN: We used a pre-heating step at 90 °C for 1 h on our previously AHPV-negative self-samples (N = 20) among women with AHPV-positive cervical samples. We also analysed AHPV results before and after the heating among a series of self-samples from women who...
Source: Journal of Virological Methods - April 16, 2019 Category: Virology Authors: Borgfeldt C, Forslund O Tags: J Virol Methods Source Type: research

Comparison of urine, self-collected vaginal swab, and cervical swab samples for detecting human papillomavirus (HPV) with Roche Cobas HPV, Anyplex II HPV, and RealTime HR-S HPV assay.
CONCLUSION: HPV tests using self-collected vaginal samples and urine showed substantial and moderate agreement compared with cervical samples, respectively, although HPV tests using these samples were still inferior to clinician-collected cervical samples. Further research is needed on the clinical performance of HPV testing using urine and self-collected vaginal samples as the screening method. PMID: 30998958 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - April 15, 2019 Category: Virology Authors: Cho HW, Ouh YT, Hong JH, Jin Min K, So KA, Kim TJ, Sun Paik E, Lee JW, Moon JH, Lee JK Tags: J Virol Methods Source Type: research

Development of a Bead-based Immunoassay Using Virus-like Particles for Detection of Alphaviral Humoral Response.
Abstract There is a pressing need for sustainable and sensitive immunodiagnostics for use in public health efforts to understand and combat the threat of endemic and emerging infectious diseases. In this proof-of-concept work, we describe an immunodiagnostic approach based on the utilization of virus-like particles (VLPs) in a magnetic bead-based platform for multiplexed detection of antiviral humoral response. A retroviral-based VLP, that presents Venezuelan equine encephalitis virus E1/E2 glycoprotein antigen on its surface, was synthesized and coupled to magnetic beads to create VLP-conjugated microspheres (VCM...
Source: Journal of Virological Methods - April 15, 2019 Category: Virology Authors: Ricks KM, Shoemaker CJ, Dupuy LC, Flusin O, Voorhees MA, Fulmer AN, Badger CV, Schmaljohn CS, Schoepp RJ Tags: J Virol Methods Source Type: research

A multiplex fluorescence microsphere immunoassay for increased understanding of Rift Valley fever immune responses in ruminants in Kenya.
Abstract Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen with devastating impacts on agriculture and public health. With outbreaks being reported beyond the continent of Africa to the Middle East, there is great concern that RVFV will continue to spread to non-endemic areas such as the Americas and Europe. There is a need for safe and high throughput serological assays for rapid detection of RVFV during outbreaks and for surveillance. We evaluated a multiplexing fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies in ruminant sera against the RVFV nucleocap...
Source: Journal of Virological Methods - April 8, 2019 Category: Virology Authors: Lindahl JF, Izabela Ragan I, Rowland RR, Wainaina M, Mbotha D, Wilson W Tags: J Virol Methods Source Type: research

Determination of the optimal method for the concentration and purification of 146S particles for foot-and-mouth disease vaccine production.
Abstract After the severe outbreak of foot-and-mouth disease (FMD) in South Korea in 2010, the Korean government implemented a vaccination policy and set out to develop an FMD vaccine using a local FMD virus (FMDV) strain. As a part of the basic research for domestic FMD vaccine development, three methods commonly used for the concentration and purification of FMDV to produce FMD vaccine antigens were compared. Among common concentration methods, including polyethylene glycol (PEG) precipitation, ammonium sulfate precipitation, and ultrafiltration, the most effective method both for concentrating 146S particles an...
Source: Journal of Virological Methods - April 8, 2019 Category: Virology Authors: Kim H, Kim AY, Kim JS, Lee JM, Kwon M, Bae S, Kim B, Park JW, Park CK, Ko YJ Tags: J Virol Methods Source Type: research

Development of an RT-LAMP assay for the detection of Lassa viruses in southeast and south-central Nigeria.
Abstract Lassa virus (LASV) causes Lassa fever (LF), a viral hemorrhagic fever endemic in West Africa. LASV strains are clustered into six lineages according to their geographic location. To confirm a diagnosis of LF, a laboratory test is required. Here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of LASV in southeast and south-central Nigeria using three primer sets specific for strains clustered in lineage II was developed. The assay detected in vitro transcribed LASV RNAs within 23 min and was further evaluated for detection in ...
Source: Journal of Virological Methods - April 8, 2019 Category: Virology Authors: Pemba CM, Kurosaki Y, Yoshikawa R, Oloniniyi OK, Urata S, Sueyoshi M, Zadeh VR, Nwafor I, Iroezindu MO, Ajayi NA, Chukwubike CM, Chika-Igwenyi NM, Ndu AC, Nwidi DU, Maehira Y, Unigwe US, Ojide CK, Onwe EO, Yasuda J Tags: J Virol Methods Source Type: research

One-step multiplex RT-qPCR for the detection and subtyping of influenza A virus in swine in Brazil.
This study describes the development and validation of a TaqMan based - one-step multiplex RT-qPCR to discriminate the hemagglutinin and neuraminidase genes of the three major IAV subtypes circulating in pigs in Brazil. The RT-qPCR assays presented 100% (95.7-100, CI 95%) of diagnostic sensitivity in the analysis of 85 IAVs, previously characterized by sequencing. The limits of detection ranged from 5.09 × 101 to 5.09 × 103 viral RNA copies/μL. For the analytical specificity, 73 pig samples collected during 2017 and 2018 were analyzed, resulting in the identification of the subtyp...
Source: Journal of Virological Methods - April 5, 2019 Category: Virology Authors: Haach V, Gava D, Egídio Cantão M, Franco AC, Schaefer R Tags: J Virol Methods Source Type: research

Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus.
Abstract 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...
Source: Journal of Virological Methods - April 5, 2019 Category: Virology Authors: Wang L, Eggett TE, Lanka S, Fredrickson RL, Li G, Zhang Y, Yoo D, Bowman AS Tags: J Virol Methods Source Type: research

Establishment of an immunofluorescence assay to detect IgM antibodies to Nipah virus using HeLa cells expressing recombinant nucleoprotein.
Abstract A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was r...
Source: Journal of Virological Methods - April 4, 2019 Category: Virology Authors: Kaku Y, Park ES, Noguchi A, Inoue S, Lunt R, Malbas FF, Demetria C, Neoh HM, Jamal R, Morikawa S Tags: J Virol Methods Source Type: research

Development of a cucumber green mottle mosaic virus-based expression vector for the production in cucumber of neutralizing epitopes against a devastating animal virus.
This study offers a promising solution to the production of a low cost, versatile and robust vaccine for oral administration against PRRSV through a chimeric virus particle display system. PMID: 30954462 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - April 4, 2019 Category: Virology Authors: Tran HH, Chen B, Chen H, Menassa R, Hao X, Bernards M, Hüner NPA, Wang A Tags: J Virol Methods Source Type: research

Extended direct lysis method for virus detection on berries including droplet digital RT-PCR or real time RT-PCR with reduced influence from inhibitors.
Abstract Detection of viruses on berries is a challenging task, often hampered by the presence of RT-qPCR inhibiting substances from berry juice. A direct extraction method for virus detection (murine norovirus and GA phage) on frozen raspberries was previously published. We expanded (different types of berries and viruses) and improved the method using MobiSpin S400 columns that filter nucleic acids based on size-exclusion chromatography. While no inhibition was detected in filtered RNA, unfiltered RNA needed from 1:2 to more than 1:8 dilution in order to remove inhibition. The modified method gave recoveries of ...
Source: Journal of Virological Methods - April 3, 2019 Category: Virology Authors: Sun B, Bosch A, Myrmel M Tags: J Virol Methods Source Type: research

A multiplex RT-PCR assay for rapid and simultaneous detection of four RNA viruses in swine.
Abstract A multiplex reverse transcription polymerase chain rection (mRT-PCR) was developed for simultaneous detection of four RNA viruses in swine. The conserved target sequences directed to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis coronavirus (TGEV) were selected based on alignments of genomic sequences and then specific primers were designed. The mRT-PCR assay was developed and evaluated for its specificity and sensitivity. The expected product from the single viral template was ampli...
Source: Journal of Virological Methods - April 2, 2019 Category: Virology Authors: Zhao Y, Liu F, Li Q, Wu M, Lei L, Pan Z Tags: J Virol Methods Source Type: research

Development and evaluation of real-time RT-PCR using ear hair for specific detection of sheep persistently infected with border disease virus (BDV).
Abstract The aim of this study was to develop an improved border disease virus (BDV) specific real time RT-PCR and to evaluate its performance on manually plucked hairs from sheep persistently infected with BDV that may act as a non-invasive alternate sample. The BDV real time RT-PCR assay reported here showed a high analytical sensitivity (100.6 TCID50/ml), specificity (no reactivity with BVDV-1, BVDV-2, HoBi-like pestivirus and CSFV) and reproducibility. When the assay was validated on 210 samples from BDV-infected and uninfected sheep, it showed a 100% diagnostic sensitivity and specificity with virus isolation...
Source: Journal of Virological Methods - April 2, 2019 Category: Virology Authors: Kalaiyarasu S, Mishra N, Rajukumar K, Behera SP, Jhade SK, Singh VP Tags: J Virol Methods Source Type: research

Molecular typing of Bluetongue virus using the nCounter ® Analysis System platform.
Molecular typing of Bluetongue virus using the nCounter® Analysis System platform. J Virol Methods. 2019 Apr 02;: Authors: Curini V, Marcacci M, Tonelli A, Di Teodoro G, Di Domenico M, D'Alterio N, Portanti O, Ancora M, Savini G, Panfili M, Camma' C, Lorusso A Abstract Bluetongue virus (BTV) is a segmented double-stranded RNA virus, existing in multiple serotypes, belonging to the genus Orbivirus of the family Reoviridae. BTV causes Bluetongue (BT), a major OIE-listed disease of ruminants. Identification of BTV serotype is accomplished using multiple typing assays and tends to be executed based on...
Source: Journal of Virological Methods - April 2, 2019 Category: Virology Authors: Curini V, Marcacci M, Tonelli A, Di Teodoro G, Di Domenico M, D'Alterio N, Portanti O, Ancora M, Savini G, Panfili M, Camma' C, Lorusso A Tags: J Virol Methods Source Type: research

Clinical evaluation of multiplex RT-PCR assays for the detection of influenza A/B and respiratory syncytial virus using a high throughput system.
In this study, we evaluated the analytical and clinical performance of a pre-designed primer/probe set for the simultaneous multiplex detection of both viruses on a high-throughput platform, the cobas 6800, using the "open channel" of the system for integration of lab-developed assays for the detection of influenza and RSV. RESULTS: Using the influenza/RSV qPCR Assay with swabs, LoD (95%) in TCID50/mL for influenza-A was 0.009, influenza-B 0.003, RSV-A 0.202, and RSV-B 0.009. Inter-run variability (3xLoD) was low (
Source: Journal of Virological Methods - April 1, 2019 Category: Virology Authors: Eigner U, Reucher S, Hefner N, Staffa-Peichl S, Kolb M, Betz U, Holfelder M, Spier G, Pfefferle S, Lütgehetmann M Tags: J Virol Methods Source Type: research

Simultaneous detection of Dengue virus, Chikungunya virus, Zika virus, Yellow fever virus and West Nile virus.
M, Melón S Abstract Although certain mosquito-borne virus, such as Dengue virus (DENV), Chikungunya virus (CHIKV), Zika virus (ZIKV), Yellow fever virus (YFV) and West Nile virus (WNV), are an important public health concern in those countries where transmitter mosquitoes are endemic, several cases of travelers from those endemic countries have been recently reported in Europe. Thus, early diagnosis of these viruses is essential for patient management and adoption of preventive measures. An assay for the simultaneous detection of DENV, CHIKV, ZIKV, YFV and WNV based on a multiplex real-time (RT)-PCR and its...
Source: Journal of Virological Methods - March 28, 2019 Category: Virology Authors: Boga JA, Alvarez-Arguelles ME, Rojo-Alba S, Rodríguez M, de Oña M, Melón S Tags: J Virol Methods Source Type: research