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Journal of Virological Methods

Nicole L Turnage, Kristen E Gibson
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...
June 17, 2017: Journal of Virological Methods
Susan Bennett, Rhona S Davidson, Rory N Gunson
Respiratory illness causes significant morbidity especially in children, the elderly and the immunocompromised. The sample type taken and the quality of that sample are of great significance in providing an accurate diagnosis. Gargle samples are easy to take and sample the same area as a throat swab (THS). In this study, we assessed the utility of gargle samples for the molecular detection of common respiratory infections. Paired gargle and THS samples collected on the same day from the same patient were compared...
June 17, 2017: Journal of Virological Methods
Sandi Radko, J Ian Stuart, George Zahariadis
BACKGROUND: Timely identification of respiratory virus infection is essential to mitigate inappropriate antibiotic use and to implement appropriate treatment and/or infection control procedures. As such, multiplexed PCR assays have become standard in many virology laboratories. OBJECTIVES: To compare the Seeplex RV15 (test of record) with two newer generation multiplex assays, the Anyplex II RV16 and the xTAG respiratory virus panels. STUDY DESIGN: Two hundred and three retrospective and 36 prospective respiratory samples were tested by all three assays...
June 17, 2017: Journal of Virological Methods
Mariana Leguia, Cristhopher D Cruz, Vidal Felices, Armando Torre, Gilda Troncos, Victoria Espejo, Carolina Guevara, Christopher Mores
We have developed methods for full-genome sequencing of Zika viruses (ZIKVs) based on a targeted amplification approach. We used alignments of publicly available complete genome data to design a primer set that selectively amplifies ZIKVs. The approach includes amplification strategies for templates present at both high- and low-copy number, and PCR cycling conditions that have been normalized across genome fragments in order to streamline laboratory handling. Abundant templates can be amplified using a strategy that uses 6 overlapping amplicons to cover the complete viral genome, whereas scarce templates can be amplified using a strategy that uses 11 overlapping amplicons of smaller size...
June 17, 2017: Journal of Virological Methods
Qing Xiong, Yuya Wang, Bingyu Xie, Xinyi Pei, Yihong Peng
A versatile single-step method is described for constructing a picornavirus replicon RNA with precise ends to facilitate improved understanding of viral genome function and mimic native virus replication in host cells as far as possible. The key innovation in this new approach is the use of a bridge primer to both introduce a ribozyme sequence for cis-cleavage of RNA to generate precise 5' ends of EV71 RNA and also mediate overlapping assembly of two fragments. Using an EV71 replicon as a test case, precise ends for the viral replicon were shown to be important for efficient virus replication...
June 16, 2017: Journal of Virological Methods
Brian J Morrison, Jessica A Roman, Thomas C Luke, Nishith Nagabhushana, Kanakatte Raviprakash, Maya Williams, Peifang Sun
This study describes an antibody-dependent NK cell degranulation assay, as a biomarker to assess antibody-dependent cellular cytotoxicity (ADCC) response in influenza plasma and for antibody therapies against influenza infection. The concentration of neutralizing antibodies (NAbs) against the hemagglutinin receptor of influenza viruses is a current determinant in protection against infection, particularly following receipt of the seasonal influenza vaccine. However, this is a limited assessment of protection, because: (i) NAb titers that incur full protection vary; and (ii) NAb titers do not account for the entire breadth of antibody responses against viral infection...
June 15, 2017: Journal of Virological Methods
Hana Langerová, Tomáš Ruml, Michaela Rumlová
To biochemically and structurally characterize viral intracytoplasmic particles (ICAPs), a sample of high purity and homogeneity is usually required. Production of ICAPs in the system closely related to their natural host cells is crucial for the analysis of host-cell binding proteins involved in ICAPs assembly, transport and budding. However, this approach is often hampered by problems with low yield of the ICAPs due to either low expression or fast release from the host cell. Another obstacle may be a low stability or fragility of the intracellular particles...
June 12, 2017: Journal of Virological Methods
Jaroslav Hollý, Margaréta Fogelová, Lucia Jakubcová, Karolína Tomčíková, Mária Vozárová, Eva Varečková, František Kostolanský
Infections caused by highly variable influenza A viruses (IAVs) pose perpetual threat to humans as well as to animals. Their surveillance requires reliable methods for their qualitative and quantitative analysis. The most frequently utilized quantification method is the titration by plaque assay or 50% tissue culture infectious dose estimation by TCID50. However, both methods are time-consuming. Moreover, some IAV strains form hardly visible plaques, and the evaluation of TCID50 is subjective. Employment of immuno-staining into the classic protocols for plaque assay or TCID50 assay enables to avoid these problems and moreover, shorten the time needed for reliable infectious virus quantification...
June 10, 2017: Journal of Virological Methods
Jennifer L DeCotiis, Noelle C Ortiz, Brian A Vega, David M Lukac
Reactivation of Kaposi's sarcoma-associated herpesvirus (KHSV; also known as Human herpesvirus (HHV)-8) from latency is associated with progression to disease. The primary experimental models for studying KSHV reactivation are B lymphocyte cell lines derived from patients with primary effusion lymphoma (PEL). PEL models have remained essential tools for understanding molecular details of latency and reactivation, yet they have shortcomings. In particular, PEL cells are difficult to transfect with plasmid DNA, which limits their routine use in studies that require introduction of exogenous DNA...
June 8, 2017: Journal of Virological Methods
Arun Parupudi, Flaviu Gruia, Samuel A Korman, Sonia Dragulin-Otto, Kuldip Sra, Richard L Remmele, Jared S Bee
Antigenic drift of the influenza A virus requires that vaccine production is targeted to the strains circulating each year. Live-attenuated influenza A vaccine manufacturing is used to produce intact virions with the surface antigens of the circulating strains. Influenza A typically contains a large percentage (>90%) of non-infective virions. The ribonucleoprotein (RNP) content, virion structure, and aggregation are factors that are thought to have an impact on infectivity. However, these factors are difficult to study because of the intrinsic variability in virion size, shape and overall structural integrity...
June 8, 2017: Journal of Virological Methods
Fan Yang, Guoping Wang, Wenxing Xu, Ni Hong
Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds...
June 3, 2017: Journal of Virological Methods
Paul J Wichgers Schreur, Janusz T Paweska, Jet Kant, Jeroen Kortekaas
Antibodies specific for Rift Valley fever virus (RVFV) can be detected by diverse methods, including ezyme-linked immunosortbent assay (ELISA) and virus neutralization test (VNT). The VNT is superior in sensitivity and specificity and is therefore considered the gold standard serological assay. Classical VNTs make use of virulent RVFV and therefore have to be performed in biosafety level-3 laboratories. Here, we report the development of a novel VNT that is based on an avirulent RVFV expressing the enhanced green fluorescent protein (eGFP), which can be performed safely outside level 3 biocontainment facilities...
June 2, 2017: Journal of Virological Methods
Binoy Babu, Brian K Washburn, Tülin Sarigül Ertek, Steven H Miller, Charles B Riddle, Gary W Knox, Francisco M Ochoa-Corona, Jennifer Olson, Yakup Zekai Katırcıoğlu, Mathews L Paret
Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories...
June 2, 2017: Journal of Virological Methods
Fengjiao Xu, Wen Yuan, Tongyuan Zhang, Yujun Zhu, Yuexiao Lian, Yu Zhang, Ren Huang, Pengju Guo
There are currently four rat parvoviruses including Kilham rat virus (KRV), Toolans H-1 parvovirus (H-1virus), rat parvovirus type 1a (RPV-1a) and rat minute virus (RMV). Virus detection methods are commonly based on conventional PCR - agarose gel electrophoresis or serological assay methods These methods are both time-consuming and lack specificity. In this study, we developed a bead array xTAG assay for the simultaneous detection and discrimination of four rat parvoviruses. The detection limits ranged from 100-1000 copies/μL of input purified plasmid DNA...
May 31, 2017: Journal of Virological Methods
Ed G Marins, Keerthi Bodinaidu, Matthew Lin, Alison Deforest
This study evaluated the use of dried blood spot (DBS) for HCV viral load quantification using the COBAS(®) AmpliPrep/COBAS(®) Taqman(®) HCV Quantitative Test v2.0 (CAP/CTM HCV v2), and compared two different procedures for preparation of DBS samples with a Specimen Pre-Extraction (SPEX) reagent (either heated [SPEX with SH] for 10min at 56°C on a thermomixer, or incubated for 1h at room temperature [SPEX at RT]) against the standard plasma input. Whole blood specimens from 48 patients with chronic HCV infection and Whatman(®) 903 Protein Saver Cards were used to prepare 35μL DBS...
May 30, 2017: Journal of Virological Methods
Marisa Kaspar, Kathrin Bohn-Wippert, Peter Bellstedt, Sabine Häfner, Matthias Görlach, Andreas Sauerbrei
Twenty amino acid substitutions in the thymidine kinase (TK) of clinical herpes simplex virus type 1 strains were assessed for conferring acyclovir (ACV) resistance. Site-directed mutagenesis, cell-free protein synthesis and protein expression in Escherichia coli were performed to obtain recombinant TK proteins, which were authenticated by Western blotting. A modified enzyme-linked immunosorbent assay (ELISA) was carried out to determine the phosphorylation activity of the mutants towards 5-bromo-2'-deoxyuridine (BrdU)...
May 30, 2017: Journal of Virological Methods
Muhammad Shafiq, Zafar Iqbal, Irfan Ali, Qamar Abbas, Shahid Mansoor, Rob W Briddon, Imran Amin
Cotton leaf curl disease (CLCuD) is the major biotic constraint to cotton production in Pakistan and northwestern India. The disease is caused by monopartite begomoviruses in association with a specific DNA satellite, Cotton leaf curl Multan betasatellite. The virus-betasatellite complex is also frequently associated with another DNA satellite-like molecule; an alphasatellite. A quantitative real-time PCR (qPCR) assay to detect all three components of the monopartite begomovirus/betasatellite/alphasatellite complex which causes CLCuD was established...
May 29, 2017: Journal of Virological Methods
Andrea Granados, Astrid Petrich, Allison McGeer, Jonathan B Gubbay
In this study, we aim to determine what effects prolonged storage and repeated freeze/thaw cycles have on the stability of influenza A(H1N1)pdm09 (influenza A/H1N1)RNA. Cloned influenza A/H1N1 RNA transcripts were serially diluted from 8.0-1.0 log10 copies/μl. RT-qPCR was used to measure RNA loss in transcripts stored at -80°C, -20°C, 4°C and 25°C for up to 84days or transcripts undergoing a total of 10 freeze/thaw cycles. Viral load was measured in clinical specimens stored at-80°C for three years (n=89 influenza A RNA extracts; n=35 primary specimens) and in 10 clinical specimens from the 2015/2016 influenza season that underwent 7 freeze/thaw cycles...
May 29, 2017: Journal of Virological Methods
Marc Hoferer, Anne Braun, Julia Skrypski, Sabine Bock, Sabine Thalheim, Reinhard Sting
Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively...
May 27, 2017: Journal of Virological Methods
Yueyang Yu, Xi Zhang, Baihui Zhao, Ying Sun, Xiaoguang Zhang, Tian Bai, Jian Lu, Zi Li, Liqi Liu, Dayan Wang, Yuelong Shu, Jianfang Zhou, Kun Qin
Although exhibiting no or low virulence in poultry, avian influenza virus H7N9 has caused around 1400 confirmed human infections in China with a case-fatality rate of 30% since 2013. A highly pathogenic H7N9 virus (HP-H7), with the HA antigenicity distinct from the previous, were recently detected in patients and poultry. Therefore, convenient rapid diagnosis with reliability will allow early antiviral use and management for H7N9 infection. Here, a sandwich ELISA targeting the conserved viral antigen, neuraminidase (NA) was developed...
May 25, 2017: Journal of Virological Methods
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