摘要
流行性出血病(EHD)是由流行性出血病病毒(EHDV)感染反刍动物引起的一种虫媒病毒病,为了解中国云南EHDV的感染情况和病毒遗传特征,笔者课题组在云南省师宗县以EHDV血清学和核酸阴性的牛、羊为哨兵动物,拟对流行于云南省的EHDV进行分离与鉴定。将采集于哨兵动物的EHDV核酸阳性血液接种BHK-21细胞进行病毒分离;通过血清中和试验与病毒Seg-2、Seg-3 ORF区的序列分析,确定病毒的血清型与遗传特征;采用C-ELISA和qRT-PCR方法对EHDV感染动物血液中的抗体水平与病毒核酸进行监测。结果如下:从2013年8月采集自云南师宗县哨兵牛的血样中分离出一株EHDV(毒株号YNSZ/V269/2013),血清中和试验结果表明分离的病毒为血清型7型(EHDV-7);Seg-2、Seg-3序列分析表明分离的病毒属EHDV-7 Eastern型,与日本毒株和澳大利亚EHDV-7型毒株具有最近的亲缘关系。哨兵动物病毒核酸转阳后,血清抗体迅速上升,3周后达最高点并能在该水平持续较长时间,而病毒核酸含量却迅速下降,7周后已经检测不到。本研究首次报道了EHDV-7型毒株在中国的分离、毒株序列特征以及在动物上的感染特性。研究结果可为进一步开展中国EHDV-7型病毒的全基因组测序、流行病学调查、诊断方法的建立和致病性等相关研究提供基础。
Epizootic haemorrhagic disease (EHD) is an infectious, non-contagious insect transmitted disease of ruminants. To investigate the infection of epizootic hemorrhagic disease virus (EHDV) among ruminants in Yunnan Province of China and study the virus genetic characteristics, monitoring points were set up in Shizong. Virus isolation was carried out through the inoculating BHK-21 cells with EHDV nucleic acid positive blood samples collected from the sentinel herds. Serotype of the isolated virus was identified by serum neutralization test;ORF of Seg -2 and Seg -3 of the virus were amplified with specific primers, then cloned and sequenced;antibody levels and viral nucleic acid in the EHDV infected animals throughout the year were tested by C-ELISA and qRT-PCR. Results were as follows: In August 2013, an EHDV strain (YNSZ/V269/2013) was isolated from Sentinel Cattle in Shizong, Yunnan. The serum neutralization test showed that the YNSZ/V269/2013 was an EHDV-7 virus. Sequence analysis of Seg -2 and Seg -3 showed that the isolated virus belonged to EHDV-7 Eastern type and had the closest genetic relationship with the Serotype 7 strain in Japan or Australia. The animal serum antibody increased rapidly after being infected by EHDV-7 and the antibody reached the highest point after 3 weeks and lasted for a longer time;meanwhile the nucleic acid content of the virus declined rapidly, 7 weeks later could not be detected in blood samples. This study reported the first isolation of EHDV serotype 7 in China and the virus sequence characterization of Seg -2 and Seg -3. The research results provide the basis for further development of the whole genome sequencing of Chinese EHDV-7 virus, epidemiological investigation, establishment of diagnostic methods and pathogenicity research.
作者
杨振兴
孟锦昕
肖雷
朱建波
廖德芳
高林
李占鸿
杨恒
李华春
YANG Zhenxing;MENG Jinxin;XIAO Lei;ZHU Jianbo;LIAO Defang;GAO Lin;LI Zhanhong;YANG Heng;LI Huachun(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Yunnan Veterinary and Animal Science Institute,Kunming 650224,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2019年第3期602-610,共9页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家重点研发计划(2016YFD0500908)
公益性行业(农业)科研专项(201303035)
云南省中青年学术和技术带头人后备人才培养项目(2017HB055)
关键词
流行性出血病病毒
病毒分离鉴定
血清型
序列分析
epizootic haemorrhagic disease virus (EHDV)
virus isolation and identification
serotype
sequence analysis
