3株牛病毒性腹泻病毒的分离鉴定及其基因分型
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摘要
为确定牛呼吸道传染病的病原以及牛病毒性腹泻病毒(BVDV)在临床健康牛群中是否存在持续性感染,本研究采集了内蒙古自治区和黑龙江省两个牛场牛鼻拭子46份及肺脏样品8份,采用MDBK细胞进行病毒分离培养,经BVDV特异性引物RT-PCR检测有3份样品为阳性。利用间接免疫荧光检测3株阳性样品,可以观察到特异性荧光,表明分离得到3株BVDV,分别命名为480、A0583和Lung-6。进一步研究显示480、A0583和Lung-6均为非致细胞病变型。对3株分离病毒的5'端非编码区和N~(pro)基因进化树分析显示3株病毒均属于BVDV1型,其中480株属于BVDV1c亚型,A0583株和Lung-6株同属于BVDV1m亚型。本研究首次在国内同一个牛场分离到BVDV1m(A0583)和BVDV1c(480)两个基因亚型。本研究为我国BVDV-1型不同基因亚型的抗原关系分析及BVDV疫苗的研制奠定了基础。
To investigate the pathogens related with bovine respiratory infectious diseases and to study whether there is persistent infection of bovine viral diarrhea virus(BVDV) in clinical healthy cattle, forty-six nasal swabs and eight lung samples were collected from two cattle farms from Inner Mongolia Autonomous region and Heilongjiang province, respectively, and 3 virus isolates were isolated by MDBK cell cultures which were identified as BVDV by PCR detection with BVDV-specific primers and indirect immunofluorescences assay, named 480, A0583 and Lung-6. Further studies showed that these isolates were noncytopathic. The 5' non-coding region of the 3 isolates and the Nprogene phylogenetic tree analysis showed that all three viruses belonged to BVDV1 type. Further analysis showed that 480 strains belonged to BVDV1c subtype; While, the isolates of A0583 and Lung-6 belonged to BVDV1m subtype. For the first time, this study isolated two subtypes of the BVDV1m(A0583) and BVDV1c(480) simultaneously existed in the same cattle farm. In addition, the successful isolation of two different genotypes of BVDVs provides a material basis for studying the antigenic relationship of different genetic subtypes of BVDV1 and the development of BVDV vaccine.
To investigate the pathogens related with bovine respiratory infectious diseases and to study whether there is persistent infection of bovine viral diarrhea virus(BVDV) in clinical healthy cattle, forty-six nasal swabs and eight lung samples were collected from two cattle farms from Inner Mongolia Autonomous region and Heilongjiang province, respectively, and 3 virus isolates were isolated by MDBK cell cultures which were identified as BVDV by PCR detection with BVDV-specific primers and indirect immunofluorescences assay, named 480, A0583 and Lung-6. Further studies showed that these isolates were noncytopathic. The 5' non-coding region of the 3 isolates and the Nprogene phylogenetic tree analysis showed that all three viruses belonged to BVDV1 type. Further analysis showed that 480 strains belonged to BVDV1c subtype; While, the isolates of A0583 and Lung-6 belonged to BVDV1m subtype. For the first time, this study isolated two subtypes of the BVDV1m(A0583) and BVDV1c(480) simultaneously existed in the same cattle farm. In addition, the successful isolation of two different genotypes of BVDVs provides a material basis for studying the antigenic relationship of different genetic subtypes of BVDV1 and the development of BVDV vaccine.
引文
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[3]Xue Fei,Zhu Yuan-mao,Li Jiao,et al.Genotyping of bovine viral diarrhea viruses from cattle in China between 2005 and2008[J].Vet Microbiol,2010,143(2-4):379-383.
[4]Gao Shan-dian,Luo Ji-hua,Du Jun-zheng,et al.Serological and molecular evidence for natural infection of Bactrian camels with multiple subgenotypes of bovine viral diarrhea virus in Western China[J].Vet Microbiol,2013,163(1-2):172-176.
[5]Nelson D D,Duprau J L,Wolff P L,et al.Persistent bovine viral diarrhea virus infection in domestic and wild small ruminants and camelids including the mountain goat(Oreamnos americanus)[J].Front Microbiol,2016,6:1415.
[6]Giammarioli M,Ceglie L,Rossi E,et al.Increased genetic diversity of BVDV-1:recent findings and implications thereof[J].Virus Genes,2015,50(1):147-151.
[7]Mahony T J,McC arthy F M,Gravel J L,et al.Genetic analysis of bovine viral diarrhoea viruses from Australia[J].Vet Microbiol,2005,106:1-6.