牛流行热病毒分离株HN1/2012的全基因组序列测定及演化分析

The Complete Genome Sequence Determination and Evolutionary Analysis of Bovine Ephemeral Fever Virus Strain HN1/2012

旨在解析我国牛流行热病毒(BEFV)分离株HN1/2012的基因组特征,为阐明我国BEFV毒株的演化规律提供数据。根据GenBank中收录的BEFV毒株的全基因组信息,设计引物,以RT-PCR扩增11段相互部分重叠的DNA片段,克隆至pGEM T-easy载体,分别进行测序,利用DNAStar软件进行序列拼接获得HN1/2012株的全基因组序列。根据BEFV编码基因的转录起始(TI)和转录终止/多聚腺苷酸化(TTP)序列分析获得各基因序列及其开放阅读框(ORF),与参考毒株比对序列相似性,并以G基因序列构建系统发生树,分析其遗传演化关系。结果显示:HN1/2012株基因组全长为14 899nt,包含前导序列(50nt)、N基因(1 328nt)、P基因(858nt)、M基因(691nt)、G基因(1 896nt)、GNS基因(1 785nt)、α1α2基因(638nt)、β基因(459nt)、γ基因(400nt)、L基因(6 470nt)和尾随序列(70nt)。病毒9个基因分别由26、43、47、53、37、39、30、-21nt的基因间隔区(IGR)连接。在全基因组水平,HN1/2012株与我国2002年浙江分离株JT02L的相似性最高,但G基因与同期分离株LYC11、LS11相似性最高。重组分析表明HN1/2012株未发生基因重组。HN1/2012株的演化可能与免疫选择压力导致的基因突变有关。本研究测定了HN1/2012株的基因组序列,为我国BEFV分离株的全基因组分子演化研究奠定了基础。

This study focused on determination of the genomic character of bovine ephemeral fever virus(BEFV)strain HN1/2012,which provided data for clarifying the genetic evolution of domestic strains of BEFV in China.Based on the complete genome of BEFV strains deposited in GenBank,eleven sets of primers were designed and used for RT-PCR for eleven overlapped DNA fragments.The obtained fragments were cloned to pGEM T-easy for sequencing and then assembled to obtain the complete genome sequence of the strain HN1/2012,using the DNAstar software.Individual genes and the corresponding open reading frames(ORF)were determined based on the transcription initiation(TI)and transcription termination/polyadenylation(TTP)sequences.The evolutionary relationships between the strain HN1/2012 and the referenced BEFV strains were analyzed based on the sequence similarities and phylogenetic tree constructed based on the G gene sequences.The results showed that the genome of HN1/2012 strain is 14 899 nt in length,comprising a leader sequence(50 nt),N gene(1 328 nt),P gene(858 nt),M gene(691 nt),G gene(1 896 nt),G NS gene(1 785 nt),α1α2 gene(638 nt),βgene(459 nt),γgene(400 nt),L gene(6 470 nt),and a trailer sequence(70 nt).The nine genes were separated by intergenic regions(IGRs)of 26,43,47,53,37,39,30 and-21 nt.At genomic level,HN1/2012 demonstrated the highest similarity with JT02L that was isolated form Zhejiang in 2002,but its G gene had highest similarity with epidemic strains LYC11 and LS11 that circulated over the same period.Gene recombination was not found in HN1/2012 by recombination analysis,indicating that gene mutation promoted by immune selection pressure may account for the evolution of HN1/2012.The genomic sequence of bovine ephemeral fever virus strain HN1/2012 was determined in the present study,which may lay a foundation for evolution analysis of BEFV based on complete genome in China.