Detection of African swine fever virus in cell culture and wild boar tissues using a commercially available monoclonal antibody.
In this study two sensitive tests were developed for the detection of the p72 major capsid protein of ASFV both in cell culture with an immunocytochemical (IC) and in tissue samples with an immunohistochemical (IHC) method using a commercially available mouse monoclonal antibody (clone 1BC11). The IC test was able to detect the virus at high virus dilutions in cell culture and the IHC test indicated the presence of ASFV in all formalin-fixed and paraffin-embedded tissue samples collected from two wild boars. The reported IC and IHC methods were found to be useful ancillary laboratory tests for research purposes and for the...
Source: Journal of Virological Methods - May 22, 2020 Category: Virology Authors: Szeredi L, Bakcsa E, Zádori Z, Mészáros I, Olasz F, Bálint Á, Locsmándi G, Erdélyi K Tags: J Virol Methods Source Type: research

Effective production of recombinant Δ60VP1 Chicken anemia virus protein in Escherichia coli and its application to a serodiagnostic indirect ELISA.
In this study, a 60 amino acid N-terminally truncated VP1 (Δ60VP1) which removes the toxic region was expressed in Escherichia coli and the resultant insoluble recombinant protein was purified by Ni-NTA affinity chromatography with anionic denaturing detergents. The high amounts of purified Δ60VP1 produced (150 mg/L) retained appropriate antigenicity and the antigen was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of CAV. One hundred fifty-two chicken serum samples (n = 152) were evaluated using the newly developed Δ60VP1 indirect ELI...
Source: Journal of Virological Methods - May 20, 2020 Category: Virology Authors: Wanganurakkul S, Smith DR, Chintapitaksakul L, Assavalapsakul W Tags: J Virol Methods Source Type: research

Ion torrent-based nasopharyngeal swab metatranscriptomics in COVID-19.
Abstract Herein, we describe the detection of a SARS-CoV-2 genome through metatranscriptome next-generation sequencing directly from the nasopharyngeal swab of a suspected case of local transmission of Covid-19, in Brazil. Depletion of human ribosomal RNA and use of an optimized in-house developed bioinformatics strategy contributed to successful detection of the virus. PMID: 32445875 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - May 20, 2020 Category: Virology Authors: Campos GS, Sardi SI, Falcao MB, Belitardo EMMA, Rocha DJPG, Rolo CA, Menezes AD, Pinheiro CS, Carvalho RH, Almeida JPP, Aguiar ERGR, Pacheco LGC Tags: J Virol Methods Source Type: research

Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) assays for the detection of two novel viruses infecting ginger.
Abstract Our recent studies have shown the association of two novel viruses namely, ginger chlorotic fleck-associated virus 1 (GCFaV-1) and ginger chlorotic fleck-associated virus 2 (GCFaV-2) with chlorotic fleck disease of ginger. As ginger is propagated through vegetative means, the development of diagnostics would aid in the identification of virus-free plants. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) assays were developed and validated for the quick detection of GCFaV-1 and GCFaV-2. The d...
Source: Journal of Virological Methods - May 19, 2020 Category: Virology Authors: Naveen KP, Bhat AI Tags: J Virol Methods Source Type: research

Development of RT-PCR degenerate primers to overcome the high genetic diversity of grapevine virus T.
Abstract Grapevine virus T (GVT) is a new member of the genus Foveavirus and has been reported to infect grapevines in several European countries. In 2018, GVT was detected for the first time in California in a domestic selection of wine grape, cv. Lambrusca di Alessandria, via high-throughput sequencing (HTS). To further investigate the presence of GVT in other grapevine plants, a two-step reverse transcription (RT)-PCR assay involving degenerate primers was developed. In order to cover the high genetic diversity of GVT, the sequences of available isolates were aligned to identify a conserved region in the coat p...
Source: Journal of Virological Methods - May 15, 2020 Category: Virology Authors: Diaz-Lara A, Golino D, Preece JE, Al Rwahnih M Tags: J Virol Methods Source Type: research

Improved detection of dengue and Zika viruses using multiplex RT-qPCR assays.
This study aimed to develop: (i) an one-step duplex real-time reverse transcription polymerase chain reaction (RT-qPCR) assay to efficiently and simultaneously detect and quantify DENV and ZIKV; (ii) a fourplex RT-qPCR to differentiate and quantify the four DENV serotypes. The detection limit of the duplex assay was 0.028 and 0.065 FFU (focus forming unit)/ml for DENV and ZIKV respectively. The lower limit of analytical sensitivity of fourplex assay was 0.01 FFU/ml for DENV-1 and 0.1 FFU/ml for DENV-2,-3 and -4. The assessment of specificity indicated both assays were highly specific to targeted viruses with negative resul...
Source: Journal of Virological Methods - May 14, 2020 Category: Virology Authors: Ou TP, Yun C, Auerswald H, In S, Leang R, Huy R, Chhoeung R, Dussart P, Duong V Tags: J Virol Methods Source Type: research

Corrigendum to "Development and evaluation of silver amplification immunochromatography kit for foot-and-mouth disease virus antigen detection" [J. Virol. Methods 275C (2019) 113736].
Corrigendum to "Development and evaluation of silver amplification immunochromatography kit for foot-and-mouth disease virus antigen detection" [J. Virol. Methods 275C (2019) 113736]. J Virol Methods. 2020 May 14;:113860 Authors: Morioka K, Urayama K, Wada A, Yoshida K, Kato T, Kitano R, Nishi T, Kanno T, Yamada M, Yamakawa M, Ulziibat G, Makino Y, Nakamura K, Hojo E, Matsumoto T, Fukai K PMID: 32418753 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - May 14, 2020 Category: Virology Authors: Morioka K, Urayama K, Wada A, Yoshida K, Kato T, Kitano R, Nishi T, Kanno T, Yamada M, Yamakawa M, Ulziibat G, Makino Y, Nakamura K, Hojo E, Matsumoto T, Fukai K Tags: J Virol Methods Source Type: research

Design, Validation and Evaluation of a SYBR Green-based Quantitative PCR Array for Comprehensive Analysis of Adenovirus Type 5 Transcriptional Patterns.
Abstract The adenoviral genome encodes coordinately expressed early and late gene transcriptional units that specify a complex collection of extensively spliced overlapping mRNAs. These complexities confound the generation of compatible, validated and optimized qPCR assays that permit comprehensive evaluation of adenoviral transcription. We have developed and evaluated a compilation of qPCR assays that represents the majority of the human adenovirus 5 (hAdV5) genome and allows for absolute and relative quantification of transcriptional activity. A panel of specific adenovirus gene primer pairs was designed through...
Source: Journal of Virological Methods - May 12, 2020 Category: Virology Authors: Nebeluk N, Foster TP Tags: J Virol Methods Source Type: research

BlueTYPE - A low density TaqMan-RT-qPCR array for the identification of all 24 classical Bluetongue virus serotypes.
Abstract Bluetongue virus is a double-stranded RNA virus with 10 genome segments. VP2 is the primary target for neutralising antibodies and defines the serotype. Today, more than 27 serotypes are known, 24 are defined as "classical", and new serotypes are under investigation. Beside group-specific BTV-genome detection, additional serotype characterisation is important for disease control and epidemiological investigations. Therefore, a low-density RT-qPCR array representing a panel of group- and serotype-specific assays, was combined with an internal control system. For BTV serotype detection, both publi...
Source: Journal of Virological Methods - May 12, 2020 Category: Virology Authors: Ries C, Beer M, Hoffmann B Tags: J Virol Methods Source Type: research

Establishment of a longitudinal pre-clinical model of lyssavirus infection.
In this study, we established a novel non-invasive mouse model of lyssavirus pathogenesis, which consists of longitudinal imaging of a luciferase-expressing Australian bat lyssavirus (ABLV) reporter virus. In vivo bioluminescence imaging (BLI) in mice revealed viral spread from a peripheral site of inoculation into the central nervous system (CNS), with kinetically and spatially distinct foci of replication in the footpad, spinal cord, and hindbrain. Detection of virus within the CNS was associated with onset of clinical disease. Quantification of virus-derived luminescent signal in the brain was found to be a reliable mea...
Source: Journal of Virological Methods - May 11, 2020 Category: Virology Authors: Mastraccio KE, Huaman C, Warrilow D, Smith GA, Craig SB, Weir DL, Laing ED, Smith IL, Broder CC, Schaefer BC Tags: J Virol Methods Source Type: research

Digital PCR Method for Detection and Quantification of Specific Antimicrobial Drug-Resistance Mutations in Human Cytomegalovirus.
vec M Abstract Antimicrobial drug resistance is one of the biggest threats to human health worldwide. Timely detection and quantification of infectious agents and their susceptibility to antimicrobial drugs are crucial for efficient management of resistance to antiviral drugs. In clinical settings, viral drug resistance is most often associated with prolonged treatment of chronic infections, and assessed by genotyping methods; e.g., sequencing and PCR. These approaches have limitations: sequencing can be expensive and does not provide quantification; and qPCR quantification is hampered by a lack of reference mater...
Source: Journal of Virological Methods - May 4, 2020 Category: Virology Authors: Košir AB, Cvelbar T, Kammel M, Grunert HP, Zeichhardt H, Milavec M Tags: J Virol Methods Source Type: research

A Simple, Two-Step, Small-Scale Purification of Recombinant Adeno-Associated Viruses.
Abstract Recombinant adeno-associated viruses (rAAVs) are robust and versatile tools for in vivo gene delivery. Natural and designer capsid variations in rAAVs allow for targeted gene delivery to specific cell types. Low immunogenicity and lack of pathogenesis also add to the popularity of this virus as an innocuous gene delivery vector for gene therapy. rAAVs are routinely used to express recombinases, sensors, detectors, CRISPR-Cas9 components, or to simply overexpress a gene of interest for functional studies. High production demand has given rise to multiple platforms for the production and purification of rAA...
Source: Journal of Virological Methods - May 1, 2020 Category: Virology Authors: Chen SH, Papaneri A, Walker M, Scappini E, Keys RD, Martin NP Tags: J Virol Methods Source Type: research

Performance evaluation of the polyclonal anti-rabies virus ribonucleoprotein IgG antibodies produced in-house for use in direct fluorescent antibody test.
In conclusion, the in-house produced polyclonal anti-RNP IgG-FITC proved suitable for rabies virus antigen detection by dFAT. PMID: 32360663 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - April 30, 2020 Category: Virology Authors: da Silva GH, Santos da Silva JH, Iamamoto K, de Arruda TS, Katz ISS, Fernandes ER, Guedes F, Rodrigues da Silva AC, Silva SR Tags: J Virol Methods Source Type: research

Evaluation of seven commercial African swine fever virus detection kits and three Taq polymerases on 300 well-characterized field samples.
This study confirms that all commercial kits and two Taq polymerases are suitable for ASFV detection in diagnostic laboratories. Furthermore, the use of endogenous controls is emphasized when testing field samples harvested on carcasses in an advanced stage of decomposition, in order to avoid false negative results. PMID: 32360149 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - April 28, 2020 Category: Virology Authors: Schoder ME, Tignon M, Linden A, Vervaeke M, Cay AB Tags: J Virol Methods Source Type: research

A method for the unbiased quantification of reassortment in segmented viruses.
Abstract Reassortment of segmented viruses can be an important source of genetic diversity underlying viral evolution and emergence. Methods for the quantification of reassortment have been described but are often cumbersome and best suited for the analysis of reassortment between highly divergent parental strains. While it is useful to understand the potential of divergent parents to reassort, outcomes of such heterologous reassortment are driven by differential selection acting on the progeny and are typically strain specific. To quantify reassortment robustly, a system free of differential selection is needed. ...
Source: Journal of Virological Methods - April 27, 2020 Category: Virology Authors: Hockman MR, Phipps KL, Holmes KE, Lowen AC Tags: J Virol Methods Source Type: research

Development of dipstick enzyme linked immunosorbent assay for on-site sero-diagnosis of Japanese encephalitis in swine.
Abstract Japanese encephalitis (JE) is an important viral zoonotic disease in Asia, especially in rural and suburban areas where rice cultivation and pig farming coexist. Pigs serve as a suitable sentinel model, the surveillance of which could predict a potential JE outbreak in human population in the immediate vicinity. However, existing diagnostics like ELISA and VNT require sophisticated laboratory facilities which are more often not available in field conditions. In the present study, we aimed at developing recombinant non-structural (NS1) protein-based dipstick IgG ELISA as an on-site assay for sero-diagnosis...
Source: Journal of Virological Methods - April 27, 2020 Category: Virology Authors: Chauhan J, Dhanze H, Kumar C, Suman Kumar HBM, Bhilegaonkar KN Tags: J Virol Methods Source Type: research

Development of a recombinase-aided amplification assay for detection of orf virus.
Abstract Orf, caused by orf virus (ORFV), is an important zoonotic disease that infects goat and sheep, leading to huge economic losses. ORFV can also cause cutaneous lesions in people who come in close contact with the diseased animals. Although accurate diagnostic methods for ORFV infection exist, there is a need for a rapid, specific, and sensitive method for easy clinical application. Here, we successfully established a recombinase-aided amplification (RAA) assay for rapid detection of ORFV. The analytical sensitivity of the assay for ORFV detection is 1 × 101 copies per reaction. Moreover,...
Source: Journal of Virological Methods - April 25, 2020 Category: Virology Authors: Wang Y, Cui Y, Yu Z, Li Y, Bai C, Sun P, Zhu W, Li Y Tags: J Virol Methods Source Type: research

Development of a triplex real-time PCR assay for detection and differentiation of gene-deleted and wild-type African swine fever virus.
In this study, a triplex real-time PCR assay was developed to detect and differentiate the gene-deleted and wild-type ASFV strains. Three pairs of primers and probes were designed to target the conserved region of B646 L gene (p72), MGF_360-14 L gene (located in the middle of MGF360-505R gene) and CD2v gene, respectively. Gene-deleted (with MGF360-505R and / or CD2v genes deletion) and wild-type ASFV strains were detected specifically and simultaneously by the assay developed without cross-reactions with other nucleic acids of PCV-2, CSFV, PRRSV, FMDV or SVA. The detection limits of the triplex rPCR were 7.9 ...
Source: Journal of Virological Methods - April 22, 2020 Category: Virology Authors: Lin Y, Cao C, Shi W, Huang C, Zeng S, Sun J, Wu J, Hua Q Tags: J Virol Methods Source Type: research

Screening for protective antigens of Cyprinid herpesvirus 2 and construction of DNA vaccines.
CONCLUSION: The results showed that the DNA vaccine constructed based on the ORF25 gene had a greater immune protective effect and can be used as a candidate vaccine for immunoprotection against CyHV-2. PMID: 32333944 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - April 22, 2020 Category: Virology Authors: Yuan X, Shen J, Pan X, Yao J, Lyu S, Liu L Tags: J Virol Methods Source Type: research

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses.
n Oberste M Abstract Next-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. The Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms were evaluated using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of ass...
Source: Journal of Virological Methods - April 14, 2020 Category: Virology Authors: Marine RL, Magaña LC, Castro CJ, Zhao K, Montmayeur AM, Schmidt A, Diez-Valcarce M, Fan Ng TF, Vinjé J, Burns CC, Allan Nix W, Rota PA, Steven Oberste M Tags: J Virol Methods Source Type: research

Rapid protocol of dot-immunnoassay for orthopoxviruses detection.
Abstract The aim of the work was to create a sensitive and fast immunochemical test for the detection of orthopoxviruses (OPXV) in the "point of care" format. This work presents the results of the comparative evaluation of a single-stage (rapid version) and two-stage protocol of dot-immunoassay based on plane protein array for detection of vaccinia virus (VACV), cowpoxvirus (CPXV) and ectromelia virus (ECTV) in viral culture materials with different degrees of purification. It has been established that rabbit polyclonal VACV-antibodies can be used in a one-stage dot-analysis, both as a capture agent immo...
Source: Journal of Virological Methods - March 21, 2020 Category: Virology Authors: Poltavchenko AG, Ersh AV, Filatov PV, Yakubitskiy SN Tags: J Virol Methods Source Type: research

Virus isolation and full-length genome sequencing of a representative Canine Distemper Virus wild type strain of the South America 2 clade.
lo CM Abstract Canine Distemper Virus (CDV) is a highly contagious pathogen of dogs that causes severe respiratory, gastrointestinal and nervous signs. Although vaccines have been used to prevent infections, CDV has been reported worldwide, even in vaccinated animals. In the present study, a representative wild type CDV strain (Arg24) was isolated from a sick vaccinated dog and its genome was completely sequenced using Illumina technology. This strain produced a strong cytopathic effect in Vero SLAM (Signaling Lymphocyte Activation Molecule) cells with a higher titer of 1.1 × 105 Median Tissue ...
Source: Journal of Virological Methods - March 20, 2020 Category: Virology Authors: Romanutti C, Keller L, La Torre J, Panzera Y, Fuques E, Pérez R, Gallo CM Tags: J Virol Methods Source Type: research

Development of a Multiplex RT-PCR Assay for the Routine Detection of Seven RNA Viruses in Apis Mellifera.
Abstract Colony losses in apiaries are frequently one of the most important problems in beekeeping. Colony loss is multifactorial with many reported disorders, Colony Collapse Disorder (CCD), is an increasingly recognised phenomenon which is thought to be caused by many pathogens, including viruses. The aim of this study was to develop a multiplex RT-PCR (mRT-PCR) test to obtain faster results in routine diagnostic laboratories for seven crucial bee viruses. Specific primers for seven RNA viruses, including Israeli acute bee paralysis virus (IAPV), deformed wing virus (DWV), sacbrood virus (SBV), acute bee paralys...
Source: Journal of Virological Methods - March 20, 2020 Category: Virology Authors: Cagirgan AA, Yazici Z Tags: J Virol Methods Source Type: research

Enhancement of transduction efficiency using Adeno-associated viral vectors by chemical pretreatment to mice bladder urothelium.
Abstract Adeno-associated virus (AAV) vectors have been recognized as promising tools for gene delivery. The bladder is a seemingly ideal organ for virus transfer, with easy access through the urethra enabling organ-specific delivery. However, achieving adequate transduction efficiency in the urothelium has been a major challenge because of the barrier function of the glycosaminoglycan (GAG) layer. We investigated optimal pretreatments of the bladder urothelium to maximize transduction efficiency by AAV vectors in vivo. Murine bladders were pretreated with five different chemical agents followed by transurethral i...
Source: Journal of Virological Methods - March 17, 2020 Category: Virology Authors: Hamada A, Kita Y, Murakami K, Matsumoto K, Sakatani T, Sano T, Ogawa O, Kobayashi T Tags: J Virol Methods Source Type: research

Bacteriophage amplification - a comparison of selected methods.
The objective of this study was to compare five commonly applied methods of phage amplification: (i) from a single plaque, (ii) the plate wash method, (iii) the agar culture method, (iv) the two-stage culture method, and (v) in liquid culture. All methods were tested for fifteen different phages. The results described herein indicate that there is no optimal, universal method for phage amplification, and the most effective method has to be established individually for each phage. These observations confirm the enormous diversity of bacterial viruses. PMID: 32198027 [PubMed - as supplied by publisher] (Source: Journal ...
Source: Journal of Virological Methods - March 17, 2020 Category: Virology Authors: Skaradzińska A, Ochocka M, Śliwka P, Kuźmińska-Bajor M, Skaradziński G, Friese A, Roschanski N, Murugaiyan J, Roesler U Tags: J Virol Methods Source Type: research

Development of a rapid immunochromatographic strip test for the detection of porcine epidemic diarrhea virus specific SIgA in colostrum.
Abstract Porcine epidemic diarrhea virus (PEDV) causes very high mortality in newborn piglets. The mucosal immune system in the gut must eliminate potential pathogens while maintaining a mutually beneficial relationship with the commensal microbiota. Antibodies derived from the secretory immunoglobulin A (SIgA) class, act as the first line of antigen-specific immunity in the gut by recognizing both pathogens and commensals. Therefore, the measurement of SIgA levels is an important index in evaluating PEDV infections and immune status. A simple and rapid method for the detection of PEDV-specific SIgA using an immun...
Source: Journal of Virological Methods - March 12, 2020 Category: Virology Authors: Liu J, Gao R, Shi H, Cong G, Chen J, Zhang X, Shi D, Cao L, Wang X, Zhang J, Ji Z, Jing Z, Feng L Tags: J Virol Methods Source Type: research

Attenuated porcine-derived type 2 bovine viral diarrhea virus as vector stably expressing viral gene.
Abstract Infectious bovine viral diarrhea virus (BVDV) cDNA clones have been used for the expression of classical swine fever virus (CSFV) genes for immune prevention and control. However, can it be used for the expression of an allogenetic fragment? To answer this question, a BVDV chimeric virus expressing the spike (S) antigen fragment of porcine epidemic diarrhea virus (PEDV) was constructed. Antigen S499-602 was inserted into pig-derived BVDV-2 infectious cDNA clone pASH28 in tandem by overlapping PCR, located between the seventh and eighth amino acids at the N-terminus of the capsid (C) protein of BVDV. Indir...
Source: Journal of Virological Methods - March 2, 2020 Category: Virology Authors: Tao J, Li B, YingShi, Chen J, Zhu G, Shen X, Liu H Tags: J Virol Methods Source Type: research

Performance of AmpFire HPV assay on Neck Cervical Lymph Node Aspirate and Oropharyngeal Samples.
The objectives were to evaluate the performance of a new rapid isothermal nucleic acid amplification point of care HPV test (AmpFire HPV) on fine needle neck aspirates (FNA) of cervical lymph nodes and oropharyngeal swabs and saliva (OPS) which had been previously tested by the cobas HPV assay. The comparison was performed on 56 FNA and 81 OPS. The two assays showed strong agreement (94.6%, K = 0.88) on FNA and fair agreement (65.4%, K = 0.34) on OPS. AmpFire HPV performed on FNA demonstrated a sensitivity of 76.7% and specificity of 81.8% for the prediction of p16 antigens in OPSCC with results...
Source: Journal of Virological Methods - February 24, 2020 Category: Virology Authors: Jang D, Shah A, Arias M, Ratnam S, Smieja M, Chen X, Wang Y, Speicher DJ, Chernesky M Tags: J Virol Methods Source Type: research

Rapid differential detection of Genotype I and III Japanese Encephalitis Virus from clinical samples by a novel duplex TaqMan probe-based RT-qPCR assay.
This study aimed to establish a rapid JEV genotyping method using novel duplex TaqMan RT-qPCR assay.specific primer and probes located in the PrM/M gene that were able to specifically differentiate GI and GIII JEV, was selected as the duplex TaqMan RT-qPCR target.The specificity, sensitivity and reproducibility test of this assay were validated. The sensitivity of the assay was 10 genomic RNA copies for both GI and GIII JEV in field mosquito and pig samples,and more sensitive than the current methods. In addition, the novel assay can be completed in less than 1 h. Therefore, This duplex TaqMan RT-qPCR assay is a pro...
Source: Journal of Virological Methods - February 24, 2020 Category: Virology Authors: Wang X, Guo S, Hameed M, Zhang J, Pang L, Li B, Qiu Y, Liu K, Shao D, Ma Z, Zhong D, Wei J, Li P Tags: J Virol Methods Source Type: research

Comparison of two Immunoassays for Concurrent Detection of HCV Antigen and Antibodies among HIV/HCV Co-infected Patients in Dried Serum/Plasma Spots.
In this study the performance of the Murex Ag/Ab (Murex, Abbott) and Monolisa Ag/Ab Ultra (Monolisa, Bio-Rad) ELISAs was compared for the use of filter dried serum/plasma spots (DS/PS) with a focus on the sensitivity and the percentage of correct positive test results. Correct positive ELISA results were assumed for samples that subsequently tested positive for HCV RNA by RT-qPCR, or RNA negative samples that tested positive in a Western blot (confirmed ELISA results). Sensitivity was evaluated from DS/PS eluates using HCV seroconversion panels [plasma samples of subtypes-(St) 1a, 2b)] and longitudinal HCV antibody-positiv...
Source: Journal of Virological Methods - February 19, 2020 Category: Virology Authors: Eshetu A, Hauser A, Schmidt D, Bartmeyer B, Bremer V, Obermeier M, Ehret R, Volkwein A, Bock CT, Bannert N Tags: J Virol Methods Source Type: research

Development of RT-qPCR assays for the detection of three latent viruses of pome.
In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), and Apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays demonstrated specificity by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 101 copies per reaction. The assays also demonstrated that both the choice of extraction method and the reage...
Source: Journal of Virological Methods - February 19, 2020 Category: Virology Authors: Beaver-Kanuya E, Harper SJ Tags: J Virol Methods Source Type: research

A multiplex RT-PCR assay for the simultaneous detection of prevalent viruses infecting pepper (Capsicum annuum L.).
nki K Abstract The aim of this work was to create an easy, fast and sensitive method for the simultaneous detection of the most frequent viruses known to infect pepper (Capsicum annuum L.) crops. A multiplex RT-PCR assay was developed that successfully achieved this aim. Using specifically designed primer pairs, the assay could simultaneously amplify the genomes of members of the two subgroups (I and II) of cucumber mosaic virus (CMV), two tobamoviruses, tobacco mosaic virus (TMV) and pepper mild mottle virus (PMMoV), potato virus Y (PVY), and tomato spotted wilt virus (TSWV) in a single assay. The multiplex RT-PC...
Source: Journal of Virological Methods - February 13, 2020 Category: Virology Authors: Nemes K, Salánki K Tags: J Virol Methods Source Type: research

Development of monoclonal antibodies against melon necrotic spot virus and their use for virus detection.
In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10-3 - 10-4 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as ...
Source: Journal of Virological Methods - February 12, 2020 Category: Virology Authors: Miras M, Torre C, Gómez-Aix C, Hernando Y, Aranda MA Tags: J Virol Methods Source Type: research

Inactivation of foot-and-mouth disease virus A/IRN/8/2015 with commercially available lysis buffers.
In this study, three commercial lysis buffers (AL, AVL, and MagMAX CORE) were tested in two laboratories for their ability to inactivate FMDV A/IRN/8/2015 in different sample matrices (cell culture supernatant, epithelial tissue suspension and milk). Residual infectivity after the addition of lysis buffer was evaluated by inoculating susceptible cell cultures. No cytopathic effect was observed for all three lysis buffers, indicating that the buffers are capable of reducing viral infectivity (estimated range 3.1 to>5.1 Log10). These results highlight the capacity of lysis buffers to decrease FMDV infectivity; however, ad...
Source: Journal of Virological Methods - February 5, 2020 Category: Virology Authors: Wood BA, Mioulet V, Henry E, Gray A, Azhar M, Thapa B, Diederich S, Hoffmann B, Beer M, King DP, Eschbaumer M Tags: J Virol Methods Source Type: research

Combining multiplex pcr and high-resolution melting for the detection and discrimination of arthropod transmitted viruses of cereals.
COMBINING MULTIPLEX PCR AND HIGH-RESOLUTION MELTING FOR THE DETECTION AND DISCRIMINATION OF ARTHROPOD TRANSMITTED VIRUSES OF CEREALS. J Virol Methods. 2020 Jan 22;:113823 Authors: Rydzak P Abstract The Great Plains of the United States is a region comprised of approximately 45 million hectares of grasslands where several economically important cereal crops are grown. Arthropod-transmitted, cereal-infecting viruses vary in incidence from year-to-year and are often difficult to detect in large acreages. To facilitate the detection of economically important viruses of cereals that often exist in co-infec...
Source: Journal of Virological Methods - January 22, 2020 Category: Virology Authors: Rydzak P Tags: J Virol Methods Source Type: research

Development of TaqMan real-time RT-PCR for sensitive detection of diverse Raspberry ringspot virus isolates.
In this study, a TaqMan real-time RT-PCR assay was developed for the rapid and sensitive detection of RpRSV. Primers and probes targeting the most conserved region of the movement protein gene were designed to amplify a 229 bp fragment of RpRSV RNA-2. The assay was able to amplify all RpRSV isolates tested. The detection limit of the RpRSV target region was estimated to be 61-98 copies, depending on the RpRSV strain. The sensitivity was about 100 times greater than the conventional RT-PCR assay using the same primers as the real-time RT-PCR assay. A comparison with published conventional RT-PCR assays indicated that both p...
Source: Journal of Virological Methods - January 17, 2020 Category: Virology Authors: Tang J, Ng F, Kanchiraopally D, Ward L Tags: J Virol Methods Source Type: research

A simple and rapid approach to prepare Sindbis and West Nile viral RNA controls for differentiation between positive samples and laboratory contamination.
This study demonstrates how simple recombinant technology can be used to produce a positive control that has application in the laboratory for surveillance studies or as a diagnostic tool using synthetic genes to abrogate the requirement for handling infectious virus. PMID: 31954734 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - January 16, 2020 Category: Virology Authors: Dimaculangan M, Wiid SC, Bester PA, Sekee TR, Burt FJ Tags: J Virol Methods Source Type: research

Evaluation of the cobas ® HCV Test for quantifying HCV RNA in dried plasma spots collected using the cobas® Plasma Separation Card.
CONCLUSIONS: PSC samples correlate well with plasma viral load and demonstrate a LoD below 1000 IU/mL and good stability at ambient temperature for 28 days. A correction factor would allow quantification of HCV viral RNA load from samples collected using a PSC. PMID: 31945390 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - January 13, 2020 Category: Virology Authors: Marins E, Krey N, Becker A, Melzer S, Hoppler M Tags: J Virol Methods Source Type: research

Field-based method for assessing duration of infectivity for influenza A viruses in the environment.
Abstract Understanding influenza A virus (IAV) persistence in wetlands is limited by a paucity of field studies relating to the maintenance of infectivity over time. The duration of IAV infectivity in water has been assessed under variable laboratory conditions, but results are difficult to translate to more complex field conditions. We tested a field-based method to assess the viability of IAVs in an Alaska wetland during fall and winter which incorporated physical and chemical properties of the waterbody in which samples were held. Filtered pond water was inoculated with avian fecal samples collected from the en...
Source: Journal of Virological Methods - January 7, 2020 Category: Virology Authors: Reeves AB, Ramey AM, Koch JC, Poulson RL, Stallknecht DE Tags: J Virol Methods Source Type: research

An advanced loop-mediated isothermal amplification assay for the rapid detection of beak and feather disease virus in psittacine birds.
Abstract Swarm primer-applied loop-mediated isothermal amplification (sLAMP) assay was developed for the rapid and specific detection of the ORF V1 gene of beak and feather disease virus (BFDV). The amplification can be completed in 40 min at 62 °C, and the results can be visually detected by the naked eye. The assay specifically amplified BFDV DNA and not amplified other viral nucleic acids. The limit of detection of the assay was 5 × 102 DNA copies/reaction, which was lower than that of the previously described LAMP (preLAMP) assay and comparable to that of a previously repo...
Source: Journal of Virological Methods - January 7, 2020 Category: Virology Authors: Chae HG, Lim DR, Kim HR, Park MJ, Park CK Tags: J Virol Methods Source Type: research

An efficient, reproducible and accurate RT-qPCR based method to determine mumps specific neutralizing antibody.
Abstract INTRODUCTION: A resurgence of mumps among fully vaccinated adolescents and young adults globally has led to questions about the longevity of vaccine derived specific immunity. Unfortunately, the ideal serological correlate of immunity to mumps has yet to be identified. However, neutralising antibody titres in serum are used extensively as a surrogate marker of immunity to mumps. Conventional neutralisation tests are technically challenging, thus we developed and validated a high throughput, RT-qPCR microneutralisation (RT-qPCR-MN) method to determine serum neutralising antibody levels to mumps virus strai...
Source: Journal of Virological Methods - January 3, 2020 Category: Virology Authors: Sikazwe CT, Levy A, Speers D, Smith DW Tags: J Virol Methods Source Type: research

Analysis of preferred codon usage in the coronavirus N genes and their implications for genome evolution and vaccine design.
In this study, the variations in the N proteins among 13 different coronaviruses (CoVs) were analysed at the nucleotide and amino acid levels in an attempt to reveal how these viruses adapt to their hosts relative to their preferred codon usage in the N genes. The results revealed that, overall, eighteen amino acids had different preferred codons and eight of these were over-biased. The N genes had a higher AT% over GC% and the values of their effective number of codons ranged from 40.43 to 53.85, indicating a slight codon bias. Neutrality plots and correlation analyses showed a very high level of GC3s/GC correlation in po...
Source: Journal of Virological Methods - January 3, 2020 Category: Virology Authors: Sheikh A, Al-Taher A, Al-Nazawi M, Al-Mubarak AI, Kandeel M Tags: J Virol Methods Source Type: research

Evaluation of a portable nanopore-based sequencer for detection of viruses in water.
Abstract The newly emerged nanopore sequencing technology such as MinIONTM allows for real-time detection of long DNA/ RNA fragments on a portable device, yet few have examined its performance for environmental viromes. Here we seeded one RNA virus (bacteriophage MS2) and one DNA virus (bacteriophage PhiX174) into 10 L well water at three levels ranging from 1 to 21100 plaque-forming units (PFU)/mL. Two pipelines were established to maximize the number of sequencing reads of DNA and RNA viruses using MinIONTM. With dead-end ultrafiltration, PEG precipitation, and random amplification, MinIONTM was capable o...
Source: Journal of Virological Methods - December 28, 2019 Category: Virology Authors: Ji P, Aw TG, Van Bonn W, Rose JB Tags: J Virol Methods Source Type: research

Development of a simple and rapid immunochromatographic strip test for detecting duck plague virus antibodies based on gI protein.
Abstract A colloidal gold strip (CGS) for detecting antibodies to duck plague virus (DPV) was developed. Colloidal gold-labeled DPV gI protein and goat anti-rabbit IgG were dispensed on a conjugate pad as tracers. The recombinant DPV gI protein and rabbit IgG were used as capture reagents at the test line and control line, respectively. The detection limit of this assay was 1:256. Additionally, the CGS did not react with antisera from other common duck diseases, only reacting with anti-DPV serum and yielding a specific and strong red signal. 123 serum samples were tested by CGS and enzyme-linked immunosorbent assa...
Source: Journal of Virological Methods - December 18, 2019 Category: Virology Authors: Zhou X, Wang M, Cheng A, Yang Q, Ying W, Jia R, Liu M, Zhu D, Chen S, Zhang S, Zhao XX, Huang J, Mao S, Xumin O, Gao Q, Liu Y, Yanling Y, Zhang L, Tian B, Pan L, Rehman MU, Chen X Tags: J Virol Methods Source Type: research

Development and validation of a multiplex-PCR based assay for the detection of 18 pathogens in the cerebrospinal fluid of hospitalized children with viral encephalitis.
CONCLUSIONS: Our findings suggest that the ABI3500-based multiplex-PCR detection kit could be a valuable diagnostic tool for pathogen detection in CSF of children with suspected viral encephalitis. PMID: 31863863 [PubMed - as supplied by publisher] (Source: Journal of Virological Methods)
Source: Journal of Virological Methods - December 18, 2019 Category: Virology Authors: Wang L, Chen F, You D, Ma G, Guo Y, Wu Y, Zeng X, Sun S, Li G Tags: J Virol Methods Source Type: research

A real-time reverse-transcription isothermal recombinase polymerase amplification assay for the rapid detection of genotype III grass carp (Ctenopharyngodon idella) reovirus.
In this study, a real-time reverse-transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed to detect the genotype III grass carp reovirus GCRV-104. The assay was based on the detection of the vp55 gene which encodes the outer fiber protein of the virus. A portable ESE-Quant Tube scanner, with a dimension of 17.4 x 18.8 cm, weighing about 1 kg, and equipped with temperature settings to amplify the DNA isothermally and spectral devices to detect the amplified products using fluorescence, was used to complete the assay. Under the optimal conditions, the assay took ...
Source: Journal of Virological Methods - December 13, 2019 Category: Virology Authors: Wang H, Zhou S, Wen J, Sun M, Jiang Y, Lu L, Xie J Tags: J Virol Methods Source Type: research

A Clinic-Based Direct Real-time Fluorescent Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Influenza Virus.
Abstract Seasonal influenza virus causes acute respiratory tract infections, which can be severe in children and the elderly. At present, rapid influenza diagnostic tests (RIDTs) are popular at clinical sites because they enable early diagnosis and avoid unnecessary use of antibiotics; in addition, high risk patients with underlying disease can be given antiviral drugs. However, the sensitivity and specificity of some of those tests are relatively poor. To overcome these problems, nucleic acid-based molecular point-of-care tests have been developed; however, they are significantly more expensive than RIDTs. Previo...
Source: Journal of Virological Methods - December 12, 2019 Category: Virology Authors: Ngan LT, Takayama I, Binh NG, Phuong TT, Tuong Van VT, Vuong BM, Co DX, Dung LT, Phuong PT, Cuong DD, Thach PT, Van Thanh D, Phuong Thuy PT, Chau NQ, Tuan DQ, Takasaki J, Semba S, Odagiri T, Nakajima N, Kageyama T Tags: J Virol Methods Source Type: research

An Immunoperoxidase Monolayer Assay (IPMA) for the detection of lumpy skin disease antibodies.
Abstract During this study a new Immunoperoxidase Monolayer Assay (IPMA) was developed for the detection of antibodies against lumpy skin disease virus (LSDV) in an easy and low tech setting. Using two dilutions (1:50 and 1:300) in a duplicate format, the test was shown to be highly sensitive, specific and repeatable. In comparison to the VNT and a commercial ELISA, the LSDV-IPMA was able to detect the LSDV antibodies earlier in infected, vaccinated and vaccinated/infected animals. The assay is very flexible as it can be easily adapted for the detection of sheeppox or goatpox antibodies and it can be scaled-up to ...
Source: Journal of Virological Methods - December 11, 2019 Category: Virology Authors: Haegeman A, De Leeuw I, Mostin L, Van Campe W, Aerts L, Vastag M, De Clercq K Tags: J Virol Methods Source Type: research

A versatile dual-use RT-PCR control for use in assays for the detection of peste des petits ruminants virus.
Abstract Peste des petits ruminants (PPR) is an acute and highly contagious disease with high mortality in small ruminants and significant socioeconomic impact in developing countries. The causative agent is peste des petits ruminants virus (PPRV). The Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (OIE) have set up a goal for the global eradication of PPR by 2030. To assist in this effort, an easily produced, specific, non-pathogenic Bacteriophage Qβ based real-time RT-PCR (qRT-PCR) PPRV positive control was developed. This control is compatible for...
Source: Journal of Virological Methods - December 11, 2019 Category: Virology Authors: Lucas J, Holder D, Dodd K, Wei J Tags: J Virol Methods Source Type: research

Evaluation of accuracy of hepatitis B virus antigen and antibody detection and relationship between epidemiological factors using dried blood Spot.
This study aims to evaluate the diagnostic accuracy of HBsAg, anti-HBc and anti-HBs detection in DBS in clinical settings and field studies and to evaluate demographic and risk behaviour according the presence of HBsAg and anti-HBc. Paired sera and DBS samples were obtained from 2,309 individuals from 3 groups, defined as follows: G1: clinical setting (n = 509), G2: general population (n = 1,305) and G3: vulnerable individuals that could be more exposed to blood contact (n = 485). Sera and DBS were tested using commercial enzyme immunoassay (EIA), with some modifications added. Usi...
Source: Journal of Virological Methods - December 11, 2019 Category: Virology Authors: Cruz HM, de Paula VS, Cruz JCM, do Ó KMR, Milagres FAP, Bastos FI, da Mota JC, Cruz MS, de Andrade TM, Flores PP, Leal E, Motta-Castro ARC, Ivantes CAP, Bezerra CS, Barbosa JR, da Cruz JNM, Ximenez LLL, Villar LM Tags: J Virol Methods Source Type: research