青海海北地区牦牛病毒性腹泻病毒流行病学调查及5'-UTR遗传进化分析

doi:10.16431/j.cnki.1671-7236.2025.03.026

摘要/Abstract

摘要： 【目的】了解青海省海北地区牦牛群中牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)的流行情况及遗传学分子特征。【方法】对采集自青海海北地区的148份腹泻牦牛粪便样品提取病毒RNA并反转录合成cDNA，以其为模板扩增BVDV 5'-UTR保守区域，建立特异性检测牦牛BVDV的实时荧光定量PCR方法，并以该方法对青海海北地区牦牛养殖场BVDV感染情况进行检测。选取4份BVDV阳性样品，对BVDV 5'-UTR区域序列进行PCR扩增和测序，与NCBI中不同亚型BVDV以及同属病毒毒株进行相似性比对，并基于BVDV 5'-UTR区域进行遗传进化分析。【结果】本研究基于BVDV 5'-UTR成功建立实时荧光定量PCR检测方法，标准曲线相关系数(R²)为0.997，标准品浓度为2.69×10²～2.69×10⁹拷贝/μL时具有良好的线性关系，最小检出限为2.69×10²拷贝/μL。重复性试验结果显示，变异系数均＜1.7％，具有良好的重复性和稳定性。采用建立的实时荧光定量PCR方法对青海省海北地区门源县、祁连县、海晏县和湟源县的腹泻牦牛粪便样品进行检测，结果显示，BVDV阳性率分别为45.00%、36.00%、30.77%和29.00%。BVDV 5'-UTR序列比对结果显示，分离获得的4株牦牛源BVDV与伊朗、美国的BVDV-1a亚型毒株聚集在同一个独立分支，核苷酸序列相似性为76.6%～99.7%。【结论】本研究明确了BVDV-1a型是导致青海省海北地区牦牛腹泻的重要病原之一，为该地区牦牛养殖场及时、有效地采取防控措施提供了科学依据，同时丰富了当地BVDV的分子流行病学调查数据，为该地区牛病毒性腹泻病的防控及疫苗研发提供了理论基础。

关键词: 牦牛; 牛病毒性腹泻病毒(BVDV); 流行病学; 实时荧光定量PCR; 遗传进化分析

Abstract: 【Objective】 The aim of this study was to understand the prevalence and molecular characteristics of Bovine viral diarrhea virus (BVDV) in yaks in Haibei,Qinghai province.【Method】 Viral RNA was extracted from 148 diarrhea yak feces samples collected from Haibei,Qinghai province,and cDNA was synthesized by reverse transcription.BVDV 5'-UTR conserved region was amplified with the template,and a Real-time quantitative PCR method was established to detect BVDV infection in yak farms in Haibei,Qinghai province.4 BVDV positive samples were selected for PCR amplification and sequencing of the BVDV 5'-UTR region sequence,similarity comparison with different subtypes of BVDV and strains of the same genus in NCBI,and genetic evolution analysis was conducted based on the BVDV 5'-UTR region.【Result】 In this study,Real-time quantitative PCR detection method was successfully established based on BVDV 5'-UTR.The correlation coefficient (R²) of standard curve was 0.997,and the linear relationship was good when the standard concentration was 2.69×10²-2.69×10⁹ copies/μL,and the minimum detection limit was 2.69×10² copies/μL.The results of repeatability test showed that the coefficient of variation was less than 1.7%,which had good repeatability and stability.Yak diarrhea feces samples from Menyuan,Qilian,Haiyan and Huangyuan counties in Haibei area of Qinghai province were detected by Real-time quantitative PCR method，the positive rates of BVDV were 45.00%,36.00%,30.77% and 29.00%,respectively.The results of BVDV 5'-UTR sequence comparison showed that the 4 yak BVDV isolates were clustered in the same independent branch with the BVDV-1a subtype strains from Iran and United States,and the nucleotide sequence similarity was 76.6%-99.7%.【Conclusion】 This study identified BVDV-1a as one of the important pathogens causing yak diarrhea in Haibei area of Qinghai province,and provided scientific basis for yak farms in this area to take timely and effective prevention and control measures.At the same time,the molecular epidemiological data of local BVDV were enriched,which provided a theoretical basis for the prevention and control of bovine viral diarrhea disease and vaccine research and development in this area.

Key words: yak; Bovine viral diarrhea virus (BVDV); epidemiology; Real-time quantitative PCR; genetic evolution analysis

中图分类号:

S852.65⁺3

林伟山, 马豆豆, 雷萌桐, 魏斌, 李国才, 王光华, 王戈平, 简莹娜, 李秀萍. 青海海北地区牦牛病毒性腹泻病毒流行病学调查及5'-UTR遗传进化分析[J]. 中国畜牧兽医, 2025, 52(3): 1241-1249.

LIN Weishan, MA Doudou, LEI Mengtong, WEI Bin, LI Guocai, WANG Guanghua, WANG Geping, JIAN Yingna, LI Xiuping. Epidemiological Investigation and Genetic Evolution Analysis of 5'-UTR of Bovine Viral Diarrhea Virus in Yaks in Haibei，Qinghai Province[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(3): 1241-1249.

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