Abstract
Bovine rhinitis B virus (BRBV) has been frequently identified in cattle diagnosed with bovine respiratory disease complex (BRDC) in recent years, suggesting its potential contribution to BRDC. The goal of this study was to develop a TaqMan-based real-time quantitative RT-PCR assay for efficient BRBV detection. A pair of primers and a probe were designed based on the 3D gene of the BRBV genome. The assay was specific for BRBV and able to exclude bovine rhinitis A virus, foot-and-mouth disease virus and Senecavirus A. The limit of detection of the assay was 4.46 copies per reaction. A standard curve was plotted, with a coefficient of determination of 0.999 in the concentration range of 100-108 copies/μl. The reproducibility of the assay was acceptable, with the standard deviations of cycle threshold values lower than 1.00 in both intra- and inter-assay. Of 200 samples collected from 150 head of cattle in recent years in China, 11% (22/200) of the samples tested positive in the assay, i.e., 4.6% (7/150) of the cattle were BRBV positive. This study provides an efficient diagnostic tool for the epidemiological investigations of BRBV.
Highlights
Bovine rhinitis B virus (BRBV), previously designated as bovine rhinovirus 2, is a non-enveloped single-stranded positive-sense RNA virus
The nine complete coding sequences (CDSs) were aligned, and it was found that the alignment of the 3D gene showed better consistency with fewer mutations, so it was chosen for the initial design templates
This means that the positive cattle were likely infected by an isolate that differed from BRBV-CHN1 and BRBV-SWE1, and the isolate was identified with the assay developed in this study
Summary
Bovine rhinitis B virus (BRBV), previously designated as bovine rhinovirus 2, is a non-enveloped single-stranded positive-sense RNA virus. BRBV was first isolated in 1971 in England and inferred to be a kind of BRAV (previously classified as bovine rhinovirus). It was differentiated from BRAV because of the absence of crossreactions in the neutralization test against BRAV (2). It was found that BRBV infection could lead to RT-qPCR for BRBV Detection slight pneumonia in specific pathogen-free calves (3). No detection assay for BRBV has been reported so far. To better understand the epidemiology of BRBV, and to provide a useful detection tool for further pathogenesis research, this study aimed to establish a rapid and reliable TaqMan probe-based real-time RT-qPCR assay. Evaluation tests were conducted, including sensitivity test, specificity test, reproducibility tests, plotting of the standard curve, and tests on clinical samples
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