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Journal of Virological Methods

Kazuya Shirato, Shohei Semba, Sherif A El-Kafrawy, Ahmed M Hassan, Ahmed M Tolah, Ikuyo Takayama, Tsutomu Kageyama, Tsugunori Notomi, Wataru Kamitani, Shutoku Matsuyama, Esam Ibraheem Azhar
Clinical detection of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) in patients is achieved using genetic diagnostic methods, such as real-time RT-PCR assay. Previously, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of MERS-CoV [Virol J. 2014. 11:139]. Generally, amplification of RT-LAMP is monitored by the turbidity induced by precipitation of magnesium pyrophosphate with newly synthesized DNA. However, this mechanism cannot completely exclude the possibility of unexpected reactions...
May 12, 2018: Journal of Virological Methods
Min Wang, Qian Ren, Zhenjie Zhang, Lehai Zhang, Michael J Carr, Juan Li, Hong Zhou, Weifeng Shi
Hand, foot and mouth disease (HFMD) is a pediatric disease associated with infection by enterovirus (EV) genotypes. The major HFMD EV pathogens are enterovirus A71 (EVA71) and coxsackievirus A16 (CVA16); however, recently, coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) have also emerged. EV genotypes cannot be distinguished on clinical grounds and a new methodology for the rapid detection of the four major HFMD EV genotypes is urgently required. In the present study, a multiplex real-time PCR assay was established for the simultaneous detection of CVA6, CVA10, CVA16 and EVA71...
May 11, 2018: Journal of Virological Methods
B D Haokip, D Alice, R Selvarajan, K Nagendran, L Rajendran, S K Manoranjitham, G Karthikeyan
Bud necrosis and chlorotic spots causing virus affecting chilli crop in Tamil Nadu (India) was identified as Capsicum chlorosis virus (CaCV). Specific primers were used for amplification and sequencing of the nucleocapsid protein (NP) gene. Polyclonal antibody against the bacterially expressed NP from the CaCV-TN-CBE isolate was produced using recombinant DNA technology. NP gene was subcloned into the pET-28a (+) vector and expressed by transformation in BL21 (DE3) pLysS. The expressed protein was about ~34 kDa and was confirmed through western blot analysis using Groundnut bud necrosis virus (GBNV) polyclonal antiserum from ICRISAT, India...
May 10, 2018: Journal of Virological Methods
S Arous, C L Harmon, H M Capobianco, J E Polston
The Potyvirus genus is one of the largest genera of plant viruses and encompasses many economically important pathogens. While a number of degenerate primers for use in broad spectrum RT-PCR assays have been published, it is not clear which of these primers would be the most useful for use by plant diagnostic laboratories. Twelve sets of primers were tested for their ability to detect nine potyviruses in a two-step RT-PCR. Viruses were extracted from different host backgrounds and were selected to represent eight clades plus one species between clades (sensuGibbs and Ohshima, 2010)...
May 10, 2018: Journal of Virological Methods
Eliana De Luca, Paolo Emidio Crisi, Marco Di Domenico, Daniela Malatesta, Giacomo Vincifori, Morena Di Tommaso, Giovanni Di Guardo, Gabriella Di Francesco, Antonio Petrini, Giovanni Savini, Andrea Boari, Alessio Lorusso
The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed...
May 3, 2018: Journal of Virological Methods
Rusheng Zhang, Dong Yao, Jingfang Chen, Wen Ye, Xinhua Ou, Tianmu Chen, Biancheng Sun
As of Aug 25, 2017, 17 incidences of human infection and 6 deaths due to the novel H5N6 virus have been reported in China. Genetic analysis of the viral genome revealed that this reassortant virus is highly pathogenic to poultry, and that the virus has a risk of transmission to humans. Accordingly, the development of a rapid, sensitive, and specific molecular diagnostic assay is critical for public health. In this study, a real-time reverse-transcription PCR (RT-PCR) assay was developed to specifically detect the novel H5N6 virus, with primer pairs targeting the hemagglutinin and neuraminidase gene sequences of this virus...
May 2, 2018: Journal of Virological Methods
Yongjuan Wang, Pingfu Cui, Shanyuan Zhu, Ting Meng, Fuxing Hao, Guoqiang Zhu, Weiyong Zuo
To construct phage antibody library for VP3 protein of duck hepatitis virus type 1(DHAV-1) and pan the specific single-chain variable fragment antibody (scFv), total RNA was extracted from the protein VP3- immunized mice spleen., vp3 gene encoding VP3 protein was amplified from the genome of DHAV-1 by RT-PCR method for the following recombinant pET-VP3 construction, immunogenic VP3 expression and purification, and combined with SOE-PCR method to complete the assembly of scFv. The scFv gene was cloned into pCANTAB5E vector for phage antibody library construction...
May 1, 2018: Journal of Virological Methods
Xiao-Hui Zou, Zhi-Xiang Bi, Xiao-Juan Guo, Zun Zhang, Yang Zhao, Min Wang, Ya-Lu Zhu, Hong-Ying Jie, Yang Yu, Tao Hung, Zhuo-Zhuang Lu
Plasmid bearing adenovirus genome is generally constructed with the method of homologous recombination in E. coli BJ5183 strain. Here, we utilized Gibson gene assembly technique to generate infectious clone of fowl adenovirus 4 (FAdV-4). Primers flanked with partial inverted terminal repeat (ITR) sequence of FAdV-4 were synthesized to amplify a plasmid backbone containing kanamycin-resistant gene and pBR322 origin (KAN-ORI). DNA assembly was carried out by combining the KAN-ORI fragment, virus genomic DNA and DNA assembly master mix...
April 24, 2018: Journal of Virological Methods
Eduardo J M Nascimento, James W Huleatt, Marli T Cordeiro, Priscila M S Castanha, James K George, Eduard Grebe, Alex Welte, Monique Brown, Donald S Burke, Ernesto T A Marques
Dengue virus (DENV) infections elicit antibody responses to the non-structural protein 1 (NS1) that are associated with protection against disease. However, the antibody isotypes and subclasses involved, and their kinetics have not been extensively studied. We characterized the antibody responses to DENV NS1 by enzyme-linked immunosorbent assay (ELISA) in a longitudinal cohort of 266 confirmed dengue cases in Recife, Northeast Brazil. Samples were collected during the febrile phase and up to over 3 years after onset of symptoms...
April 20, 2018: Journal of Virological Methods
Saurabh Majumdar, Tapan K S Chauhan, K Dhama, Purushottam P Goswami, A K Tiwari, B P Mishra, Deepak Kumar
No abstract text is available yet for this article.
April 13, 2018: Journal of Virological Methods
M Aloisio, M Morelli, V Elicio, P Saldarelli, B Ura, B Bortot, G M Severini, A Minafra
The detection of the four grapevine viruses (GLRaV-1, GLRaV-3, GFLV and ArMV) regulated in European Union plant material certification, requires sensitive and specific diagnostic tools. A strategy of simultaneous detection in a real-time single tube amplification was developed, based on the EvaGreen binding dye. The melting curve analysis (MCA) of the amplicons allows a qualitative detection of the four different virus targets in multiplex analysis. A plasmid dilution assay calculated an analytical sensitivity with an amplification threshold up to 100 copies of the target sequences...
April 11, 2018: Journal of Virological Methods
Jeffrey J DeStefano, Irani Alves Ferreira-Bravo
Although many new assays for HIV have been developed, several labs still use simple and reliable radioactivity-based reverse transcriptase (RT) nucleotide incorporation assays for detection and quantification. We describe here a new assay for detection and quantitation of HIV RT activity that is based on a high affinity DNA aptamer to RT. The aptamer is sequestered on 96-well plates where it can bind to RT and other constituents can be removed by extensive washing. Since the aptamer mimics a primer-template, upon radiolabeled nucleotide addition, bound RT molecules can extend the aptamer and the radioactive signal can be detected by standard methods...
April 6, 2018: Journal of Virological Methods
Narges Mashkour, Alicia Maclaine, Graham W Burgess, Ellen Ariel
The number of reptilian viruses detected are continuously increasing due to improvements and developments of new diagnostic techniques. In this case we used primary cell culture and qPCR to describe the first Australian Chelonia mydas papillomavirus. Commercial chelonian cell lines are limited to one cell line from a terrestrial turtle (Terrapene Carolina). To establish primary cultures from green turtles (Chelonia mydas), turtle eggs were collected form Heron Island, Queensland, Australia. From day 35 of incubation at 29°, the embryos were harvested to establish primary cultures...
April 6, 2018: Journal of Virological Methods
Larissa J Osterbaan, Corinne Schmitt-Keichinger, Emmanuelle Vigne, Marc Fuchs
One of the greatest hindrances to the study of grapevine fanleaf virus (GFLV) is the dearth of robust protocols for reliable, scalable, and cost-effective inoculation of host plants, especially methods which allow for rapid and targeted manipulation of the virus genome. Agroinoculation fulfills these requirements: it is a relatively rapid, inexpensive, and reliable method for establishing infections, and enables genetic manipulation of viral sequences by modifying plasmids. We designed a system of binary plasmids based on the two genomic RNAs [RNA1 (1) and RNA2 (2)] of GFLV strains F13 (F) and GHu (G) and optimized parameters to maximize systemic infection frequency in Nicotiana benthamiana via agroinoculation...
April 6, 2018: Journal of Virological Methods
Galina Burmakina, Kirill Bliznetsov, Alexander Malogolovkin
Conventional methods, which quantitatively assess virus replication, are based on direct examination of viral cytopathic effect (CPE), which is time consuming, tedious and based on endpoint reading. The Real-Time Cell Analysis (RTCA) xCELLigence® system offers an alternative approach to evaluate virus-induced CPE, and here was evaluated as a means to dynamically assess CPE caused by African swine fever virus (ASFV). RTCA was used to identify optimum time for ASFV infection based on cell index (CI) and to evaluate ASFV CPE kinetics in COS-1 cells...
April 5, 2018: Journal of Virological Methods
M A Vissani, M S Tordoya, Y-L Tsai, P-Y A Lee, Y-H Shen, F-C Lee, H-T T Wang, V Parreño, M Barrandeguy
Equine coital exanthema (ECE) is an infectious, venereally transmitted muco-cutaneous disease affecting mares and stallions, caused by equid alphaherpesvirus 3 (EHV3). Diagnostic tools for rapid identification of EHV3 are of primary importance to diminish the risk of EHV3 dissemination at the time of breeding. In the last years, it has been shown that the performance of the insulated-isothermal polymerase chain reaction (iiPCR) is comparable to virus isolation, nested PCR and real-time PCR (qPCR) in detecting pathogens of various animal species...
April 5, 2018: Journal of Virological Methods
Le Wang, Yu-Chang Hu, Chang-Yi Xiao, Fei Wang, Yu-Fei Liu, Li-Hua Tang, Rong-Shuang Xiao
AIMS: To determine the value of a monoclonal antibody panel against a C-terminal conserved sequence polypeptide of human papillomavirus (HPV) L1 (a major capsid protein) for the detection of HPV in cervical exfoliated cells, as well as the potential of this antibody panel to be developed into an assay kit for the clinical screening of cervical cancer. METHODS: Cervical exfoliated cells were collected at a gynecology clinic. One part of each sample was sent to the Department of Pathology for HPV genotyping, and the other part was sent to the Department of Pathology for cytologic testing and then to the laboratory for immunological histological chemistry (IHC) assay in which an HPV L1 C-terminal conserved sequence polypeptide-induced mouse monoclonal antibody panel was used to detect HPV L1...
March 31, 2018: Journal of Virological Methods
Anapolino Macedo de Oliveira, Antônio Augusto Fonseca, Marcelo Fernandes Camargos, Lívia Maria Orzil, Mateus Laguardia-Nascimento, Anna Gabriella Guimarães Oliveira, Jacqueline Gomes Rodrigues, Mariana Lázaro Sales, Tatiana Flávia Pinheiro de Oliveira, Cristiano Barros de Melo
Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity and analytical specificity, analytical performance (repeatability and reproducibility criteria) and the uncertainty of the measurement...
March 27, 2018: Journal of Virological Methods
Quentin Le Hingrat, Marine Perrier, Gilles Collin, Suzon Drumard, Alexandre Storto, Mélanie Bertine, Lucile Larrouy, Sophie Matheron, Florence Damond, Charlotte Charpentier, Diane Descamps, Benoit Visseaux
In-depth study of HIV often requires large stock of patients-derived viruses obtained through viral cultures. HIV cultures are currently limited by low recovery rates, especially when viral load is below 100,000 copies per mL. This is problematic for HIV-2 as most patients have spontaneously low to undetectable viremia. New approaches have been developed to enhance viral recovery rates but they are complex or costly to implement. We tested the impact of µMACSTM VitalVirus Isolation Kit (Miltenyi), a HIV virions capture method using paramagnetic microbeads directed against CD44, a human glycoprotein present in HIV envelope...
March 24, 2018: Journal of Virological Methods
Laxmi Narayan Sarangi, Naveena Thodangala, Samir Kumar Rana, Kota Sri Naga Leela Surendra, Rachamreddy Venkata Chandrasekhar Reddy, Bajibabu Putla, Ponnanna Nadikerianda Muthappa, Girish Kumar Sharma, Villuppanoor Srinivasan
The extended frozen semen (EFS) batches produced from infectious bovine rhinotracheitis (IBR) sero-positive cattle and buffalo bulls housed in various semen stations in India are transported to the testing laboratory in liquid nitrogen (LN2) for screening bovine herpesvirus-1 (BoHV-1). This procedure is laborious and poses LN2 related hazards. An alternative logistics for transportation of samples was investigated. Use of Flinders Technology Associates (FTA® ) elute card was evaluated for transportation of extended bovine semen to screen BoHV-1 DNA by real-time PCR targeting gB gene and the method was compared with the OIE approved Chelex resin based method...
March 24, 2018: Journal of Virological Methods
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