一株牦牛源牛病毒性腹泻病毒毒株的分离鉴定及其遗传特性分析

Isolation,identification and analyses for genetic features of one bovine viral diarrhea virus strain from yak

通过病毒分离培养技术,从牦牛血清中分离获得一株致细胞病变的牛病毒性腹泻病毒(BVDV),命名为GSTZ毒株。经电镜观察,GSTZ毒株的直径在50~60 nm。按Karber法测算,该病毒滴度为5.0×10^(6.5) mL^(-1)[以组织半数感染量(TCID_(50))计]。利用反转录PCR(RT-PCR)测定GSTZ毒株的完整开放阅读框(ORF),全长11691 nt,将其与参考毒株ORF在核酸序列和同义密码子使用模式等方面进行分析,确定GSTZ毒株在遗传学特性上属于基因1型家族成员。在GSTZ毒株的完整ORF中,A+U的出现频率要高于G+C,而且,病毒同义密码子的使用模式体现出对U/A结尾同义密码子使用偏嗜性高的遗传学特征。GSTZ毒株明显降低了使用含有CpG二联核苷酸的同义密码子的频率。这有助于降低病毒对宿主细胞免疫系统的刺激强度,从而促进病毒的复制增殖。研究成果可为进一步研究BVDV的相关分子机制提供试验材料,并为BVDV不同基因型的抗原关系分析与BVDV疫苗的研制奠定基础。

In this study,one bovine viral diarrhea virus(BVDV)strain was successfully isolated and termed as GSTZ.By electron microscopy,several 50-60 nm viral particles were observed.GSTZ strain displayed the TCID_(50)(50%tissue culture infection dose)value of 5.0×10^(6.5) mL^(-1) as revealed by Karber method.Via reverse transcription-PCR(RT-PCR),the whole open reading frame(ORF)of GSTZ strain was sequenced(whole length of 11691 nt)and classified into the genotype 1 group at either nucleotide level or synonymous codon usage level.The appearance frequency of A+U was higher than that of G+C in GSTZ ORF,and the synonymous codon usage pattern of GSTZ strain displayed the obvious usage bias in synonymous codons with U/A.It was worth noting that,this strain had a tendency of avoiding the selection of the synonymous codons containing CpG dinucleotides,which strongly suggested that the GSTZ strain might weaken the immune responses from host in order to successfully generate its viral particles.The isolated BVDV strain and the above findings could provide biological materials for the related mechanism study of BVDV,and lay foundations for the analysis of antigen relationship of different genotypes of BVDV and vaccine development.