Dataset Information

A SYBR green I-based quantitative RT-PCR assay for bovine ephemeral fever virus and its utility for evaluating viral kinetics in cattle.

ABSTRACT: We developed a SYBR green I-based reverse-transcription quantitative PCR (RT-qPCR) assay for bovine ephemeral fever virus (BEFV). Analytical sensitivity of the assay was ~ 100 times higher than conventional RT-PCR. The precision of the RT-qPCR established for RNA standards was high, with intra-assay and inter-assay coefficients of variation of 0.23-0.89% and 0.23-1.02%, respectively. The test was highly specific for BEFV strains, with no cross-reactivity with other viruses of veterinary significance. The assay detected BEFV RNA as early as 1 d post-infection (dpi) and up to 7-8 dpi in the blood samples of experimentally infected cattle. The most stable reference gene, peptidylprolyl isomerase A (PPIA), was selected for the quantification of BEFV. Viral RNA loads reached peak level at 3-5 dpi and then decreased rapidly through 7-8 dpi. Our assay provides a reliable approach for the detection of BEFV in the early infection stage and for use in the profiling of BEFV kinetics in vivo.

SUBMITTER: Gao S

PROVIDER: S-EPMC7003230 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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A SYBR green I-based quantitative RT-PCR assay for bovine ephemeral fever virus and its utility for evaluating viral kinetics in cattle.

Gao Shandian S Du Junzheng J Tian Zhancheng Z Niu Qingli Q Huang Dexuan D Wang Jidong J Luo Jianxun J Liu Guangyuan G Yin Hong H

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 20191217 1

We developed a SYBR green I-based reverse-transcription quantitative PCR (RT-qPCR) assay for bovine ephemeral fever virus (BEFV). Analytical sensitivity of the assay was ~ 100 times higher than conventional RT-PCR. The precision of the RT-qPCR established for RNA standards was high, with intra-assay and inter-assay coefficients of variation of 0.23-0.89% and 0.23-1.02%, respectively. The test was highly specific for BEFV strains, with no cross-reactivity with other viruses of veterinary signific ...[more]

PMID: 31845623

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Project description:Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that causes a debilitating disease of cattle in Africa, Asia, and Australia; however, its global geodynamics are poorly understood. An evolutionary analysis of G gene (envelope glycoprotein) ectodomain sequences of 97 BEFV isolates collected from Australia during 1956 to 2012 revealed that all have a single common ancestor and are phylogenetically distinct from BEFV sampled in other geographical regions. The age of the Australian clade is estimated to be between 56 and 65 years, suggesting that BEFV has entered the continent on few occasions since it was first reported in 1936 and that the 1955-1956 epizootic was the source of all currently circulating viruses. Notably, the Australian clade has evolved as a single genetic lineage across the continent and at a high evolutionary rate of ∼10(-3) nucleotide substitutions/site/year. Screening of 66 isolates using monoclonal antibodies indicated that neutralizing antigenic sites G1, G2, and G4 have been relatively stable, although variations in site G3a/b defined four antigenic subtypes. A shift in an epitope at site G3a, which occurred in the mid-1970s, was strongly associated with a K218R substitution. Similarly, a shift at site G3b was associated primarily with substitutions at residues 215, 220, and 223, which map to the tip of the spike on the prefusion form of the G protein. Finally, we propose that positive selection on residue 215 was due to cross-reacting neutralizing antibody to Kimberley virus (KIMV). This is the first study of the evolution of BEFV in Australia, showing that the virus has entered the continent only once during the past 50 to 60 years, it is evolving at a relatively constant rate as a single genetic lineage, and although the virus is relatively stable antigenically, mutations have resulted in four antigenic subtypes. Furthermore, the study shows that the evolution of BEFV in Australia appears to be driven, at least in part, by cross-reactive antibodies to KIMV which has a similar distribution and ecology but has not been associated with disease. As BEFV and KIMV are each known to be present in Africa and Asia, this interaction may occur on a broader geographic scale.

Dataset Information

A SYBR green I-based quantitative RT-PCR assay for bovine ephemeral fever virus and its utility for evaluating viral kinetics in cattle.

Publications

A SYBR green I-based quantitative RT-PCR assay for bovine ephemeral fever virus and its utility for evaluating viral kinetics in cattle.

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