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Journal of Virological Methods

Tinka Jelsma, Fimme J van der Wal, Helmi Fijten, Nicolas Dailly, Evert van Dijk, Willie L Loeffen
Most serological assays detect antibody responses in biological samples through affinity of serum antibodies for antigens provided in the assay. Certain antigens, however, may be difficult to produce and/or may contain unwanted epitopes. In these cases, a practical alternative may be the use of peptides as representatives for specific epitopes. Peptides can be obtained after purification in large quantities for a modest price, but screening of a large set of peptides during development may be relatively expensive...
May 22, 2017: Journal of Virological Methods
Louis A Altamura, Lisa H Cazares, Susan R Coyne, James G Jaissle, Alyssa M Jespersen, Sundus Ahmed, Leonard P Wasieloski, Jeff Garrison, David A Kulesh, Ernst E Brueggemann, Tara Kenny, Michael D Ward, David E Harbourt, Timothy D Minogue
Research involving biosafety level 3 pathogens such as West Nile virus (WNV) is often limited by the limited space and technical constraints of these environments. To conduct complex analytical studies outside of high containment, robust and reliable inactivation methods are needed that maintain compatibility with downstream assays. Here we report the inactivation of WNV in spiked serum samples using a commercially available SDS-PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification proteins differentially expressed in the serum of mice experimentally infected with WNV...
May 19, 2017: Journal of Virological Methods
Fuquan Zhang, Eva Perez-Martin, Nick Juleff, Bryan Charleston, Julian Seago
Bioluminescence is a powerful tool in the study of viral infection both in vivo and in vitro. Foot-and-mouth disease virus (FMDV) has a small RNA genome with a limited tolerance to foreign RNA entities. There has been no success in making a reporter FMDV expressing a luciferase in infected cell culture supernatants. We report here for the first time a replication-competent FMDV encoding Nanoluciferase, named as Nano-FMDV. Nano-FMDV is genetically stable during serial passages in cells and exhibits growth kinetics and plaque morphology similar to its parental virus...
May 19, 2017: Journal of Virological Methods
Verna Federica, Giorda Federica, Miceli Ilaria, Rizzo Giovanna, Pautasso Alessandra, Romano Angelo, Iulini Barbara, Pintore Maria Domenica, Mignone Walter, Grattarola Carla, Bozzetta Elena, Varello Katia, Dondo Alessandro, Casalone Cristina, Goria Maria
Morbillivirus genus comprises several members related to specific hosts, such as canine distemper virus (CDV) and cetacean morbillivirus (CeMV) in which the dolphin morbillivirus (DMV) is included. Both CDV and DMV are able to cause serious outbreak associated with high morbidity and mortality representing an important conservation threat for terrestrial and aquatic mammalian species. This paper describes a new RT-PCR RFLP technique based on a RT-PCR with degenerate primers targeting a 287 bp fragment located on the conserved N terminus of the morbillivirus NP gene, followed by MseI RFLP, in order both to confirm the detection of the virus and to distinguish DMV from CDV...
May 17, 2017: Journal of Virological Methods
Geane Lopes Flores, Helena Medina Cruz, Denise Vigo Potsch, Silvia Beatriz May, Carlos Eduardo Brandão-Mello, Marcia Maria Amendola Pires, Jose Henrique Pilotto, Lia Laura Lewis-Ximenez, Elisabeth Lampe, Livia Melo Villar
Influence of HIV status in HBV markers detection in saliva and dried blood spots (DBS) was not well established. This study aims to evaluate the performance of optimized commercial immunoassay for identifying HBsAg and anti-HBc in saliva and DBS according HIV status. A sum of 535 individuals grouped as HIV(+), HBV(+), HIV/HBV(+) and HIV/HBV- were recruited where 347 and 188 were included for HBsAg and anti-HBc evaluation, respectively. Serum, DBS collected in Whatman 903 paper and saliva obtained using salivette device were analyzed using EIA...
May 12, 2017: Journal of Virological Methods
Craig Pouliott, Michelle Dupuis, Kim Appler, Scott Brunt, Robert Rudd, April Davis
Maintaining a healthy, continuous immortalized cell line is essential for rabies laboratories that perform virus isolation assays and test for the presence of viral neutralizing antibodies. Individuals who routinely work with rabies virus, such as rabies laboratory employees, or those who may have a high potential for exposure to rabies virus, including veterinarians, should be tested for the presence of anti-rabies viral neutralizing antibodies (VNA) every 6 to 24 months, depending on potential exposure level...
May 12, 2017: Journal of Virological Methods
Jayaseelan Boobalan, Andrea Torti, Thongadi Ramesh Dinesha, Sunil Suhas Solomon, Pachamuthu Balakrishnan, Shanmugam Saravanan
Virological monitoring through plasma viral load (PVL) quantification is essential for clinical management of HIV patients undergoing antiretroviral treatment (ART), and for detecting treatment failure. Quantitative PCR (qPCR)-based tests are the gold standard for measuring PVL. Largely because of their high cost, however, implementation of these tests in low- and middle-income countries fails to cover the testing demand. In this study, we aimed at reducing the running cost of the commercially available Abbott RealTime™ HIV-1 assay by minimizing the reagent consumption...
May 12, 2017: Journal of Virological Methods
Ratthaphol Charlermroj, Manlika Makornwattana, Orawan Himananto, Channarong Seepiban, Sudtida Phuengwas, Nuchnard Warin, Oraprapai Gajanandana, Nitsara Karoonuthaisiri
To employ a microsphere immunoassay (MIA) to simultaneously detect multiple plant pathogens (potyviruses, Watermelon silver mottle virus, Melon yellow spot virus, and Acidovorax avenae subsp. citrulli) in actual plant samples, several factors need to be optimized and rigorously validated. Here, a simple extraction method using a single extraction buffer was successfully selected to detect the four pathogens in various cucurbit samples (cucumber, cantaloupe, melon, and watermelon). The extraction method and assay performance were validated with inoculated and field cucurbit samples...
May 11, 2017: Journal of Virological Methods
Cui Yang, Yu Xu, Renyong Jia, Pengfei Li, Lei Zhang, Mingshu Wang, Dekang Zhu, Shun Chen, Mafeng Liu, Zhongqiong Yin, Anchun Cheng
Duck circovirus (DuCV), as a causative agent of long-term immunosuppressive disease, has caused heavy damage to waterfowl breeding worldwide. In this study, the full-length Cap (capsid) gene of DuCV was expressed in Escherichia coli (E. coli) for the first time by optimizing the codons in its nuclear localization signal (NLS) regions. The recombinant protein was expressed as inclusion bodies, and the quantification of purified Cap protein could reach 0.29mgmL(-1). Moreover, an indirect ELISA method (Cap-ELISA) was established based on the recombinant Cap protein...
May 10, 2017: Journal of Virological Methods
Matthew B Frankel, Kinnari Pandya, Jeffrey Gersch, Sarah Siddiqui, George J Schneider
Zika virus is an arthropod-borne flavivirus that has rapidly developed into a world-wide concern. Discovered in 1947, the virus was relatively obscure until an outbreak occurred in 2007 in the Yap islands and spread eventually to the Americas in 2015. Only 20% of patients infected with Zika virus develop symptoms. However, there can be serious consequences of infection including birth defects in developing fetuses and links to Guillain-Barré syndrome. The swift rise in infections has necessitated the development of diagnostic tests for both the detection of viral RNA and the presence of virus-specific antibodies...
May 4, 2017: Journal of Virological Methods
Cyril C Y Yip, Siddharth Sridhar, Andrew K W Cheng, Ami M Y Fung, Vincent C C Cheng, Kwok-Hung Chan, Kwok-Yung Yuen
BACKGROUND: HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar(®) HHV-6 PCR Kit. METHOD: The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated...
May 2, 2017: Journal of Virological Methods
Susanna K Tan, Malaya K Sahoo, Stephen Milligan, Nathaniel Taylor, Benjamin A Pinsky
BACKGROUND: Detection of Zika virus (ZIKV) RNA in urine is of increasing interest for the diagnosis of ZIKV infection. Pre-analytical variables can significantly impact the stability of RNA in urine. METHODS: To determine optimal specimen processing protocols that would maximize detection of ZIKV RNA in urine by real-time, reverse transcriptase PCR, we investigated the effect of temperature, initial ZIKV concentration, use of nucleic acid stabilizers, and time on ZIKV RNA levels...
May 1, 2017: Journal of Virological Methods
Lyudmyla G Glushakova, Barry W Alto, Myong Sang Kim, Andrea Bradley, Ozlem Yaren, Steven A Benner
Chikungunya virus (CHIKV) represents a growing and global concern for public health that needs inexpensive and convenient methods to collect mosquitoes as potential carriers so that they can be preserved, stored and transported for later and/or remote analysis. Reported here is a cellulose-based paper, derivatized with quaternary ammonium groups ("Q-paper") that meets these needs. In a series of tests, infected mosquito bodies were squashed directly on Q-paper. Aqueous ammonia was then added on the mosquito bodies to release viral RNA that adsorbed on the cationic surface via electrostatic interactions...
April 27, 2017: Journal of Virological Methods
Frank Vandenbussche, David J Lefebvre, Ilse De Leeuw, Steven Van Borm, Kris De Clercq
The 3D and 5UTR real-time RT-PCR assays (RT-qPCR) from Callahan et al. (2002) and Reid et al. (2002) are commonly used reference methods for the detection of foot-and-mouth disease virus (FMDV). For an optimal detection of FMDV in clinical samples, it is advised to use both assays simultaneously (King et al., 2006). Recently, Vandenbussche et al. (2016) showed that the addition of 5'-tails to the FMDV-specific primers enhances the detection of FMDV in both the 3D and the 5UTR RT-qPCR assay. To validate the 3D and 5UTR RT-qPCR assays with 5'-tailed primers for diagnostic purposes, both assays were run in parallel in a triplex one-step RT-qPCR protocol with beta-actin as an internal control and synthetic RNA as an external control...
April 27, 2017: Journal of Virological Methods
Rui Lin, Darren Heeke, Hui Liu, Eileen Rao, Jason D Marshall, Vera Chio, Floro Cataniag, Li Yu, Fengrong Zuo, Michael P McCarthy
The goal of most prophylactic vaccines is to elicit robust and effective neutralizing antibodies against the human pathogen target. The titer of neutralizing antibodies to Epstein-Barr Virus (EBV) is a useful biomarker for evaluating EBV vaccines. Here, the development and optimization of a 96-well micro-neutralization fluorescent imaging assay (FIA) using an EBV virus-encoding green fluorescent protein (GFP) to infect adherent EBV recipient cells is reported. The conditions were optimized for generating reproducible EBV-GFP virus, for maintaining viral infectivity for months, and for efficient viral infection of recipient cell culture...
April 27, 2017: Journal of Virological Methods
Wasun Chantratita, Keum-Soo Song, Satish Balasaheb Nimse, Viroj Pongthanapisith, Nipa Thongbaiphet, Garanyuta Wongtabtim, Ekawat Pasomsub, Kanokwan Angkanavin, Mukesh Digambar Sonawane, Shrikant Dasharath Warkad, Taisun Kim
According to EASL guidelines and WHO recommendations, the accurate detection of HCV genotypes such as HCV 1a, HCV1b, HCV 2, HCV 3, HCV 4, and HCV 6 (6a, 6f, 6i, 6n) is crucial for the efficient treatment of hepatitis C. HCV Genotyping 9G test allows simultaneous genotyping of HCV 1a, 1b, 2, 3, 4, and 6 (6a, 6f, 6i, and 6n) in clinical samples in 30min. The performance of the test was evaluated by comparison with sequence analysis. Serum samples (n=152) from HCV-infected patients (n=110) and healthy individuals (n=42) were processed under blinded codes...
April 27, 2017: Journal of Virological Methods
Eric Sarcey, Aurélie Serres, Fabrice Tindy, Audrey Chareyre, Siemon Ng, Marine Nicolas, Emmanuelle Vetter, Thierry Bonnevay, Eric Abachin, Laurent Mallet
Spontaneous reversion to neurovirulence of live attenuated oral poliovirus vaccine (OPV) serotype 3 (chiefly involving the n.472U>C mutation), must be monitored during production to ensure vaccine safety and consistency. Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) has long been endorsed by the World Health Organization as the preferred in vitro test for this purpose; however, it requires radiolabeling, which is no longer supported by many laboratories. We evaluated the performance and suitability of next generation sequencing (NGS) as an alternative to MAPREC...
April 26, 2017: Journal of Virological Methods
Lila M Zarski, Emily A High, Rahul K Nelli, Steven R Bolin, Kurt J Williams, Gisela Soboll Hussey
Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract...
April 25, 2017: Journal of Virological Methods
J C Chandler, J W Schaeffer, M Davidson, S L Magzamen, A Pérez-Méndez, S J Reynolds, L D Goodridge, J Volckens, A B Franklin, S A Shriner, B Bisha
The detection of aerosolized viruses can serve as an important surveillance and control tool in agriculture, human health, and environmental settings. Here, we adapted an anion exchange resin-based method, initially developed to concentrate negatively charged viruses from water, to liquid impingement-based bioaerosol sampling. In this method, aerosolized viruses are collected in a 20ml liquid sample contained within widely used impingers, BioSamplers (SKC Inc., Eighty Four, PA), and further concentrated via adsorption to an anion exchange resin that is suspended within this liquid...
April 24, 2017: Journal of Virological Methods
Sharton Vinicius Antunes Coelho, Rômulo Leão Silva Neris, Michelle Premazzi Papa, Laila Castro Schnellrath, Lana Monteiro Meuren, Diogo A Tschoeke, Luciana Leomil, Brunno Renato Farias Verçoza, Milene Miranda, Fabiano L Thompson, Andrea Thompson Da Poian, Thiago Moreno L Souza, Fabiana Avila Carneiro, Clarissa R Damaso, Iranaia Assunção-Miranda, Luciana Barros de Arruda
The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols...
April 23, 2017: Journal of Virological Methods
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