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Journal of Virological Methods

Andrey N Anisenko, Ekaterina S Knyazhanskaya, Maria G Isaguliants, Marina B Gottikh
The post-integrational gap repair is a critical and poorly studied stage of the lentiviral life cycle. It might be performed by various cellular DNA repair pathways but the exact mechanism of the repair process has not yet been described. One of the reasons for that is the lack of a functional quantitative assay that could precisely measure the amount of integrated viral DNA that has completed the post-integrational gap repair stage. Here, we present an approach that is based on a widely used Alu-specific PCR for the estimation of integrated viral DNA but includes several steps that allow discrimination between integrated-repaired and integrated-unrepaired viral DNA forms...
September 13, 2018: Journal of Virological Methods
Pankaj Singh, Gagandeep Singh, Jenna Karsky, Eric Nelson, Sheela Ramamoorthy
Neonatal enteritis caused by the porcine epidemic diarrhea virus (PEDV) is an important cause of high mortality and economic losses to the swine industry. Virus neutralization (V/N) assays are commonly requested in diagnostic laboratories for the assessment of protective antibodies. However, the visual assessment of viral cytopathic effects by operators to determine antibody titers or for viral quantification is a tedious, subjective and time-consuming process, especially when high volume testing is involved...
September 12, 2018: Journal of Virological Methods
Sanjay Chhibber, Prabhjot Kaur, Vijay Singh Gondil
Phage enumeration is a basic prerequisite for application of phages in industrial, medical and other processes. Double layer agar (DLA) plaque assay is the classical method employed for isolation, detection as well as enumeration of phage particles in a solution. However, DLA method is considered cumbersome due to its specific temperature requirements and need for one petriplate with two agar layers for each phage sample. We are proposing a drop cast method for enumeration of phages which is comparatively easier and cost effective than classical DLA method as single layer of agar without any specific temperature condition is required...
September 10, 2018: Journal of Virological Methods
Deepa Upreti, Natalia Cernicchiaro, Juergen A Richt, William C Wilson, Alfonso Clavijo, A Sally Davis
Rift Valley fever virus (RVFV), a mosquito-borne, zoonotic pathogen, is endemic to sub-Saharan Africa and has spread beyond the continent to the Arabian Peninsula. The high likelihood of RVFV's spread to other non-endemic countries spurs the need for development and implementation of rapid diagnostic tests and surveillance programs. In this preliminary evaluation, we assessed the diagnostic accuracy and precision of a recombinant RVFV nucleoprotein based competitive ELISA (cELISA) assay to detect RVFV antibodies compared to a plaque reduction neutralization test (PRNT80 ), using sera of cattle and sheep that were experimentally infected with either the MP-12 RVFV vaccine or a wild-type RVFV strain, as well as using known RVFV negative sera...
September 8, 2018: Journal of Virological Methods
Lila Poiteau, Alexandre Soulier, Maud Lemoine, Zameer Mohammed, Mélanie Wlassow, John Rwegasha, Jean-Michel Pawlotsky, Stéphane Chevaliez
Rapid diagnostic tests (RDTs) represent an attractive alternative to conventional diagnostic methods for hepatitis C virus (HCV) infection. The aim of the present study was to assess the clinical performance of the new CE-marked Advanced Quality™ Rapid Anti-HCV Test for the detection of HCV antibodies in various patient populations. A total of 396 individuals, including 178 patients with chronic HCV infection, 26 patients with resolved HCV infection, and 192 subjects not infected with HCV, were studied. The clinical sensitivity and specificity in serum samples of the Advanced Quality™ Rapid Anti-HCV Test were both 99%...
August 31, 2018: Journal of Virological Methods
Jingjing Ren, Lu Zhang, Peng Cheng, Fan Zhang, Zehui Liu, Suresh K Tikoo, Rui Chen, Enqi Du
BACKGROUND AND OBJECTIVE: Bovine adenovirus type 3 (BAdV3) has been widely used as a vector for vaccine research and development, whereas BAdV1 biology and BAdV1-based vectored vaccine have been less frequently reported. We aimed to construct an infectious BAdV1 clone and explore the functions of BAdV1 genes. METHODS: First, the infectious clone of pUCBAdV1 containing the full-length BAdV1 DNA and the recombinant plasmid pUCBAV1-EYFP expressing the marker gene EYFP were constructed...
August 31, 2018: Journal of Virological Methods
Jun Zhang, Xiaowei Liu, Wei Li, Jing Zhang, Zhixin Xiao, Zhicheng Zhou, Tianbo Liu, Ying Li, Fenglong Wang, Songbai Zhang, Jinguang Yang
Milk vetch dwarf virus (MDV) is a member of the genus Nanovirus, and its genome is composed of multiple circular 1-kb ssDNA components. In this study, we first determined that the diseased tobacco samples obtained in Zhucheng, Shandong Province were naturally infected with MDV using a polymerase chain reaction (PCR) assay. Subsequently, loop-mediated isothermal amplification (LAMP) was developed for the detection of MDV for the first time. The Mg2+ and dNTP concentrations and the reaction temperature and time of the LAMP were optimized to 8 mM, 1...
August 31, 2018: Journal of Virological Methods
Emad Beshir Ata, Ahmed Zaghawa, Alaa A Ghazy, Ahmed Elsify, Khaled Abdelrahman, Samy Kasem, Mohamed Nayel
Equine herpesvirus-1 (EHV-1) is an important pathogen, which infects horses worldwide with high morbidity but low mortality rates. The respiratory disorders and abortions are the most common indicators. Ab4p (an abortigenic and paralytic virus) is one of the most important and virulent strains. The development and functional characterization of the open reading frame-68 (ORF68) negative EHV-1 Ab4p mutants and an assessment of their roles in the infection at the cellular level were the main targets of the current study...
August 27, 2018: Journal of Virological Methods
Revanaiah Yogisharadhya, Amit Kumar, Veerakyathappa Bhanuprakash, Sathish Bhadravati Shivachandra
Orf or contagious ecthyma, is a highly contagious transboundary disease of sheep and goats. For sero-diagnosis of orf, recombinant antigen based assays are considered as alternatives to conventional approaches such as serum neutralization test (SNT) and counter-immuno-electrophoresis (CIE). A major envelope protein of orf virus (ORFV), F1L, is highly immunogenic and is a candidate for use in these assays. In this study, the F1L gene of the ORFV-59/05 strain encoding a recombinant mature F1L protein (1 M-D302 aa) with a C- terminal truncation, was produced as a fusion protein (∼50 kDa) in Escherichia coli...
August 24, 2018: Journal of Virological Methods
Lu Xiao, Yin Wang, Runmin Kang, Xulong Wu, Hua Lin, Yonggang Ye, Jifeng Yu, Jianqiang Ye, Jing Xie, Ye Cao, Yong Wei, Dangjin Liao, Meng Pan, Yi Lin, Zhuojian Dai, Xingyu Li
The aim of this study was to develop a multiple PCR assay based on the suspension array system for the simultaneous detection of respiratory and reproductive pathogens in swine. Pseudorabies virus (PRV), Japanese encephalitis virus (JEV), classic swine fever virus (CFSV), African swine fever virus (ASFV), porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) are the major respiratory and reproductive viral pathogens in pig farms. Seven pairs of specific primers and probes were designed, and the multiple PCR was performed, with the PCR products hybridized to beads coupled to probes, which were then detected by Bio-Plex suspension array system...
August 24, 2018: Journal of Virological Methods
Eun-Mee Park, Sun-Whan Park, Ye-Ji Lee, Won-Ja Lee, Wooyoung Choi
In this study, we generated recombinant virus-like particles (VLPs) against family Filoviridae, genus Ebolavirus, species Zaire ebolavirus, strain Makona (EBOV) in Drosophila melanogaster Schneider 2 (S2) cells using the EBOV Makona. S2 cells were cotransfected with four viral plasmids encoding EBOV Makona proteins and protein expression was analyzed by immunoblotting. We confirmed that EBOV Makona proteins were successfully expressed in S2 cells. Additionally, we further examined the formation of intracellular and extracellular VLPs by electron microscopy...
August 23, 2018: Journal of Virological Methods
Felix Fingas, Antje Rückner, Kristin Heenemann, Daniela Volke, Michael Sieg, Petra Bielefeldt, Thomas Grunwald, Thomas W Vahlenkamp, Rayk Hassert, Ralf Hoffmann
Precise health monitoring of laboratory animals is a critical factor for surveillance and accuracy of animal experiments. Rotavirus epizootic diarrhea of infant mice (EDIM) leads to infections in mice that can influence animal studies, e.g., by altering the intestinal physiology. Thus, the aim of this study was establishing a highly sensitive and specific ELISA for the serological detection of EDIM infections in rodents. First, virus proteins were separated by SDS-PAGE and immunogenic proteins were visualized by immunoblotting and identified after in-gel digestion by tandem mass spectrometry...
August 22, 2018: Journal of Virological Methods
Somayeh Shokri, Mohammad Kazem Shahkarami, Abbas Shafyi, Ashraf Mohammadi, Fatemeh Esna-Ashari, Amir Hamta
Live attenuated viral vaccines are difficult to handle and often sensitive to temperature. The viral titer may drop during the processing and storing stage, especially at high temperatures. Using live attenuated viral vaccines successfully depends on keeping the sufficient potency required for an immune response. Although freeze-drying makes the vaccine more stable, in the absence of appropriate stabilizer the process may affect the structure and viability of the viruses. Therefore, the formulation of vaccine by means of an appropriate stabilizer plays a crucial role in the stability of viral structure and potency of the vaccine...
August 22, 2018: Journal of Virological Methods
Qing Fan, Zhixun Xie, Zhiqin Xie, Liji Xie, Jiaoling Huang, Yanfan Zhang, Tingting Zeng, Minxiu Zhang, Sheng Wan, Sisi Luo, Jiabo Liu, Xianwen Deng
Mycoplasma bovis (MB) and bovine herpes virus 1 (BHV-1) are two important pathogens that cause bovine respiratory disease in the beef feedlot and dairy industries. The aim of this study was to develop and validate a duplex fluorescence-based loop-mediated isothermal amplification (DLAMP) assay for simultaneous detection of MB and BHV-1. Two sets of specific primers for each pathogen were designed to target the unique sequences of the MB uvrC gene and the BHV-1 gB gene. The inner primer for BHV-1 was synthesized with the fluorophore FAM at the 5' end to detect the BHV-1 gB gene, and the inner primer for MB was synthesized with the fluorophore CY5 at the 5' end to detect the MB uvrC gene...
August 22, 2018: Journal of Virological Methods
Cheryl Jennings, Brian Harty, Salvatore R Scianna, Suzanne Granger, Amy Couzens, Daniel Zaccaro, James W Bremer
BACKGROUND: Commercially-available kits for HIV-1 detection include instructions for detecting HIV-1 in plasma and DBS, but don't support other specimen types. OBJECTIVES: Show quantitative stability of HIV-1 total nucleic acid (TNA) in blood and improved HIV-1 detection in alternative specimen types. STUDY DESIGN: Whole blood and DBS specimens, tested as part of an external quality assurance program for qualitative HIV-1 detection, were used to evaluated error rates (false negative [FN], false positive [FP] and indeterminant [IND] results) across assays (internally developed [IH], Roche Amplicor [RA], and Roche TaqMan Qual [TQ]) and specimen types (frozen whole blood [BLD], DBS and cell pellets [PEL])...
August 17, 2018: Journal of Virological Methods
Chunhe Wan, Cuiteng Chen, Longfei Cheng, Guanghua Fu, Shaohua Shi, Rongchang Liu, Hongmei Chen, Qiuling Fu, Yu Huang
Recently, a novel duck adenovirus (designated as duck adenovirus 3, DAdV-3) was discovered in Muscovy ducks, China. Here, we developed a TaqMan-based real-time PCR assay (qPCR) for the detection of DAdV-3 infection. After the optimization of the qPCR conditions, the lower limit of detection for DAdV-3 infection was 40.9 copies/μl. No cross-reactivity was observed with other duck-derived pathogens. Intra- and inter-assay variability was less than 2.30%. DAdV-3 was detected in embryos and newly hatched ducklings by qPCR assay, the findings provided evidence of possible vertical transmission of DAdV-3...
August 13, 2018: Journal of Virological Methods
Niroshan Thanthrige-Don, Oliver Lung, Tara Furukawa-Stoffer, Cody Buchanan, Tomy Joseph, Dale L Godson, John Gilleard, Trevor Alexander, Aruna Ambagala
Respiratory and enteric diseases continue to be two major causes of economic losses to the cattle industry worldwide. Despite their multifactorial etiology, the currently available diagnostic tests for bovine respiratory disease complex (BRDC) and bovine enteric disease (BED) are single-pathogen-tests. DNA microarray when combined with multiplex polymerase chain reaction (PCR) is a powerful tool in detection and differentiation of multiple pathogens in a single sample. This study reports development and initial validation of two independent highly sensitive and specific multiplex PCR-electronic microarray assays, one for the detection and differentiation of pathogens of the BRDC and the other for detection and differentiation of pathogens of the BED...
August 10, 2018: Journal of Virological Methods
Lyudmyla G Glushakova, Barry W Alto, Myong-Sang Kim, Keenan Wiggins, Bradley Eastmond, Patricia Moussatche, Nathan D Burkett-Cadena, Steven A Benner
Previously (Glushakova et al. 2017), a cellulose-based cationic (Q) paper derivatized with quaternary ammonium groups was shown to be a convenient platform to collect, preserve, and store nucleic acids (NAs) derived from mosquito vectors infected with pathogens for surveillance. NAs bind electrostatically to Q-paper, but the quantity of NA bound depends on the paper's binding capacity. To optimize the original technology for mosquito surveillance, factors that affected NA absorbance on Q-paper were evaluated...
August 9, 2018: Journal of Virological Methods
N Golender, V Y Bumbarov, O Erster, M Beer, Y Khinich, K Wernike
Simbu serogroup viruses induce acute clinical diseases and abnormal courses of pregnancies in livestock. In Israel, two members of this serogroup, namely Akabane virus (AKAV) and Shuni virus (SHUV), were recently detected and, in Europe, Schmallenberg virus (SBV) poses a threat to ruminants. To address this emerging problem, a universal S-segment-based real-time RT-PCR assay (Uni-S) for the detection of Simbu serogroup viruses was established, which, additionally, enabled species identification of the detected viruses by subsequent sequencing...
August 8, 2018: Journal of Virological Methods
Brian R McMinn, Eric R Rhodes, Emma M Huff, Pauline Wanjugi, Michael M Ware, Sharon P Nappier, Mike Cyterski, Orin C Shanks, Kevin Oshima, Asja Korajkic
Coliphages are alternative fecal indicators that may be suitable surrogates for viral pathogens, but majority of standard detection methods utilize insufficient volumes for routine detection in environmental waters. We compared three somatic and F+ coliphage methods based on a paired measurement from 1 L samples collected from the Great Lakes (n = 74). Methods include: 1) dead-end hollow fiber ultrafilter with single agar layer (D-HFUF-SAL); 2) modified SAL (M-SAL); and 3) direct membrane filtration (DMF) technique...
August 7, 2018: Journal of Virological Methods
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