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Journal of Virological Methods

Elisa Gibert, Gerard Martín-Valls, Enric Mateu
The detection of porcine reproductive and respiratory syndrome virus (PRRSV) in oral fluids (OF) by quantitative real-time polymerase chain reaction (qRT-PCR) is gaining increasing popularity. However, the different steps leading to a result have not been extensively evaluated. The aim of the present study was to examine the effect on the performance of qRT-PCR with different sampling materials, conditions of storage of the OF, the need for centrifuging OF, as well as to compare RNA extraction methods and PCR mixes...
February 14, 2017: Journal of Virological Methods
Hayley Cassidy, Alasdair MacLean, Rory Gunson
BACKGROUND: Non-specific amplification can arise in real-time PCR when temperatures are above 4°C during PCR set up. Pressure of high throughput tests, particularly in a clinical setting, can lead to short cuts being taken during PCR set up. OBJECTIVES: This study set out to evaluate the outcome of exposing a real-time PCR assay to increasing durations of room temperature prior to PCR amplification. STUDY DESIGN: A real-time PCR assay was exposed to increasing durations of room temperature prior to PCR amplification...
February 10, 2017: Journal of Virological Methods
Andre Ten Haaf, Johannes Kohl, Sibylle Pscherer, Hans-Peter Hamann, Hans Ulrich Eskens, Max Bastian, Stefan Gattenlöhner, Mehmet Kemal Tur
Bluetongue is an infectious viral disease which can cause mortality in affected ruminants, and tremendous economic damage via impacts upon fertility, milk production and the quality of wool. The disease is caused by bluetongue virus (BTV) which is transmitted by species of Culicoides biting midge. Rapid detection of BTV is required to contain disease outbreaks and reduce economic losses. The purpose of this study was to develop a monoclonal sandwich ELISA for direct detection of BTV in infected animals. Phage display technology was used to isolate BTV specific antibody fragments by applying the human scFv Tomlinson antibody libraries directly on purified BTV-8 particles...
February 10, 2017: Journal of Virological Methods
Lih-Chiann Wang, Ya-Ting Kuo, Ling-Ling Chueh, Dean Huang, Jiunn-Horng Lin
Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip...
February 9, 2017: Journal of Virological Methods
Hui-Qiong Yin, Chang-Fu Ji, Xi-Qin Yang, Rui Wang, Shu Yang, He-Qiu Zhang, Jin-Gang Zhang
A gold nanoparticle probe-based assay (GNPA) was developed for ultrasensitive detection of Hepatitis C virus (HCV) core antigen. In the GNPA, after anti-HCV core antigen polyclonal antibodies and single-stranded barcode signal DNA were labeled on gold nanoparticle probe (NP), DNA enzyme was used to degrade the unbound barcode DNAs. The anti-HCV core antigen monoclonal antibodies were coated on magnetic microparticles probe (MMP). Then the NP-HCV core antigen-MMP sandwich immuno-complex was formed when the target antigen protein was added and captured...
February 8, 2017: Journal of Virological Methods
Wen-Lu Fan, Zi-Wei Wang, Yue Qin, Chao Sun, Zhong-Mei Liu, Yan-Ping Jiang, Xin-Yuan Qiao, Li-Jie Tang, Yi-Jing Li, Yi-Gang Xu
In this study, a specific and sensitive method for simultaneous detection of human astrovirus, human rotavirus, norovirus, sapovirus and enteric adenovirus associated with acute enteritis was developed, based on the specific dual priming oligonucleotide (DPO) system and the sensitive high-performance liquid chromatography (HPLC) analysis. The DPO system-based multiplex reverse transcription-polymerase chain reaction (RT-PCR) combined with HPLC assay was more sensitive than agarose gel electrophoresis analysis and real-time SYBR Green PCR assay, and showed a specificity of 100% and sensitivity of 96%-100%...
February 6, 2017: Journal of Virological Methods
Guanghui Wu, David Selden, Anthony R Fooks, Ashley Banyard
Rabies virus is a notifiable pathogen that must be handled in high containment facilities where national and international guidelines apply. For the effective inactivation of rabies virus, a number of reagents were tested. Virkon S (1%) solution caused more than 4log reduction of rabies virus in culture medium supplemented with 10% foetal calf serum within 1min. Isopropyl alcohol (70%) treatment resulted in >3log reduction of rabies virus within 20s when applied at a ratio of 19:1, making it a suitable agent for surface decontamination whereas 70% ethanol was ineffective...
February 5, 2017: Journal of Virological Methods
Weiwei Zeng, Wei Yao, Yingying Wang, Yingying Li, Sven M Bermann, Yan Ren, Cunbin Shi, Xinjian Song, Qiwen Huang, Shuchen Zheng, Qing Wang
Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons...
February 4, 2017: Journal of Virological Methods
Martin Faye, Laurent Dacheux, Manfred Weidmann, Sylvie Audrey Diop, Cheikh Loucoubar, Hervé Bourhy, Amadou Alpha Sall, Ousmane Faye
Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates...
February 4, 2017: Journal of Virological Methods
Myong Cheol Lim, Do-Hoon Lee, Sang-Hyun Hwang, Na Rae Hwang, Bomyee Lee, Hye Young Shin, Jae Kwan Jun, Chong Woo Yoo, Dong Ock Lee, Sang-Soo Seo, Sang-Yoon Park, Jungnam Joo
BACKGROUND: Human papillomavirus (HPV) testing based on cervical samples is important for use in cervical cancer screening. However, cervical sampling is invasive. Therefore, non-invasive methods for detecting HPV, such as urine samples, are needed. OBJECTIVES: For HPV detection in urine samples, two real-time PCR (RQ-PCR) tests, Roche cobas 4800 test (Roche_HPV; Roche Molecular Diagnostics) and Abbott RealTime High Risk HPV test (Abbott_HPV; Abbott Laboratories) were compared to standard cervical samples...
February 1, 2017: Journal of Virological Methods
S De Grazia, F Bonura, A Pepe, S Li Muli, V Cappa, A Collura, D M Terranova, N Urone, F Di Bernardo, D Matranga, G M Giammanco
Group A rotaviruses (RVAs) are the primary cause of acute gastroenteritis (AGE) in young children worldwide. Several commercial tests including latex agglutination, enzyme-linked assays (ELISA) and immunochromatographic tests (ICT) have been developed for the diagnosis of RVA infection. In the present study, the performance of two commercially available one-step chromatographic immunoassays, CerTest Rotavirus+Adenovirus (Biotec S.L, Zaragoza, Spain) and Vikia Rota-Adeno (bioMerieux SA, Lyon, France) were retrospectively evaluated using Real-time PCR as reference test...
January 31, 2017: Journal of Virological Methods
Issam Hmila, Manoosak Wongphatcharachai, Nacira Laamiri, Rim Aouini, Boutheina Marnissi, Marwa Arbi, Srinand Sreevatsan, Abdeljelil Ghram
H9N2 Influenza subtype has emerged in Tunisia causing epidemics in poultry and resulting in major economic losses. New mutations in their hemagglutinin and neuraminidase proteins were acquired, suggesting their potential to directly infect humans. Effective surveillance tools should be implemented to help prevent potential spillover of the virus across species. We have developed a highly sensitive real time immuno-polymerase chain reaction (RT-I-PCR) method for detecting H9N2 virus. The assay applies aptamers as ligands to capture and detect the virus...
January 31, 2017: Journal of Virological Methods
Po-An Tu, Jia-Shian Shiu, Shu-Hwae Lee, Victor Fei Pang, De-Chi Wang, Pei-Hwa Wang
Caprine arthritis-encephalitis (CAE) in goats is a complex disease syndrome caused by a lentivirus. This persistent viral infection results in arthritis in adult goats and encephalitis in lambs. The prognosis for the encephalitic form is normally poor, and this form of the disease has caused substantial economic losses for goat farmers. Hence, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed in the present study for detecting the proviral DNA of caprine arthritis-encephalitis virus (CAEV)...
January 31, 2017: Journal of Virological Methods
Silvia Faccini, Aurora De Mattia, Chiara Chiapponi, Ilaria Barbieri, Maria Beatrice Boniotti, Carlo Rosignoli, Giuliana Franzini, Ana Moreno, Emanuela Foni, Arrigo Daniele Nigrelli
The occurrence of virus belonging to the putative genus Influenzavirus D, has been demonstrated all-around the world arousing interest within the scientific community. Most of the published virological surveys are based on the first described Real-Time PCR method, designed on the PB1 gene of the first isolate. The necessity of extending investigation to different animal species and geographic areas, requires a continuous update of molecular tests, considering newly sequenced strains. Moreover, the availability of an alternative assay, is essential either to confirm data, or for ensuring the detection of the widest number of strains...
January 30, 2017: Journal of Virological Methods
Massimiliano Bergallo, Paola Montanari, Katia Mareschi, Marco Rassu, Ilaria Galliano, Paolo Ravanini
TaqMAMA is an allele-specific PCR-based (ASPCR) method that may be suitable for broad and cost-effective genotyping applications in all types of laboratories. There is evidence that interactions between some toll like receptors (TLRs) with viruses influence both the immune response and outcome of HCMV infection. We developed a TaqMAMA genotyping assay for the detection of rs352140 TLR9 polymorphism in transplant recipients with and without HCMV infections. Performance parameters to ensure a solid pre-validation protocol have been here argued...
January 28, 2017: Journal of Virological Methods
Laura Hughes, Kimberly Wilkins, Cynthia S Goldsmith, Scott Smith, Paul Hudson, Nishi Patel, Kevin Karem, Inger Damon, Yu Li, Victoria A Olson, P S Satheshkumar
Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols...
January 26, 2017: Journal of Virological Methods
Naidong Wang, Yan Zhang, Xinnuo Lei, Wanting Yu, Yang Zhan, Dongliang Wang, Jiaxin Zhang, Aibing Wang, Lehui Xiao, Ping Jiang, Yi Yang
Although porcine circovirus type 2 (PCV2) virus-like particles (VLPs) have been successfully harvested from various protein expression systems, conditions to promote their stability and integrity during long-term storage have not been well defined since only the intact VLPs, instead of the monomeric capsid protein (Cap), can induce neutralizing antibodies in pigs in previous studies. In this study, freshly prepared PCV2 VLPs were stored in several media (various concentrations of NaCl, sorbitol, sucrose and trehalose) at three temperatures (4°C, -20°C and -80°C) and their stability and integration were evaluated after 7 month...
January 25, 2017: Journal of Virological Methods
Jian-Chang Wang, Wan-Zhe Yuan, Qing-An Han, Jin-Feng Wang, Li-Bing Liu
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases...
January 22, 2017: Journal of Virological Methods
Xin Ji, Mohammad Zafrullah, Nicholas Wiese, Tonya Hayden-Mixon, Joseph C Forbi, Chong-Gee Teo, Michael A Purdy
A cloned stable cell line, HepG2-HBVE6, was established following transfection of HepG2 cells with a retroviral plasmid into which a 1.1-fold genomic construct of hepatitis B virus (HBV) belonging to genotype E (HBV/E) was inserted. The cell line retains the entire HBV/E insert, and produces episomal HBV DNA. It expresses HBV pregenomic, preS1 and preS2/S transcripts, and sheds hepatitis B surface and e antigens as well as structures resembling HBV-subviral and Dane particles. The HepG2-HBVE6 cell line, in permitting recapitulation of the HBV life cycle, may be used for studying viral characteristics, therapeutic and preventative outcomes and for preparing reagents specific to HBV genotype E...
January 22, 2017: Journal of Virological Methods
B Mubemba, P N Thompson, L Odendaal, P Coetzee, E H Venter
Rift Valley fever (RVF), caused by an arthropod borne Phlebovirus in the family Bunyaviridae, is a haemorrhagic disease that affects ruminants and humans. Due to the zoonotic nature of the virus, a biosafety level 3 laboratory is required for isolation of the virus. Fresh and frozen samples are the preferred sample type for isolation and acquisition of sequence data. However, these samples are scarce in addition to posing a health risk to laboratory personnel. Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are safe and readily available, however FFPE derived RNA is in most cases degraded and cross-linked in peptide bonds and it is unknown whether the sample type would be suitable as reference material for retrospective phylogenetic studies...
January 22, 2017: Journal of Virological Methods
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