狐源犬瘟热病毒HBF-1株全基因组序列测定及其分析

Full-length genome sequencing and analysis of Vero-dSlam cell-adapted canine distemper virus HBF-1 strain

通过比对GenBank中已发表的21株犬瘟热病毒全基因组序列,利用Primer Premier5.0设计了首尾重叠的11对特异性引物,对1株适应Vero-dSlam细胞的犬瘟热病毒强毒HBF-1的全基因组进行RT-PCR扩增。将扩增获得的11个特异性目的片段分别连接到pEASY-Blunt载体,筛选出阳性克隆子。经过反复测序后,对11个片段的序列进行拼接最终获得了HBF-1株全基因组序列。利用DNAstar和MEGA6.0软件,分析HBF-1株与其他已公布CDV序列遗传进化关系。结果显示,适应Vero-dSlam细胞的犬瘟热病毒强毒HBF-1基因组全长为15690bp,与最初分离株Hebei株的同源性高达为99.8%,与国内黑龙江分离株HLJ-06的亲缘关系较近,同源性为98.5%,同经典疫苗株的亲缘关系相对较远,同源率为92.7%。HBF-1是分离株Hebei的Vero-dSlam细胞适应株,对二者基因编码区的序列比对,共出现13处氨基酸突变。其中,P蛋白突变率为0.39%,H蛋白突变率为0.33%,是2个变异率最高的结构蛋白。这些变异很有可能是病毒在适应Vero-dSlam细胞过程中形成的,但它们对HBF-1的致病性和毒力强弱是否存在影响,后续我们将深入研究。因此,开展细胞适应株HBF-1的全基因组序列测定及序列分析,对未来研究强毒株与Slam受体互作关系,及强毒株的致弱机制提供基础。

Based on the comparisons of the full-length genome of published 21pieces of canine distemper viruses in GenBank,11pairs of specific primers with overlapping were designed with sofeware Primer Premier5.0in this study.Eleven specific PCR fragments were obtained by amplification RT-PCR amplification for a full-length genome of a Vero-dSlam cell-adapted canine distemper virus HBF-1.11purified PCR fragments were cloned to pEASY-Blunt vector,and the right recombinant plasmids were sequencing repeatedly to ensure the mostly possible sequences.These quenced 11fragments were spliced to obtain the full-length genome sequences of HBF-1strainabout 15690bp.Alignment analysis of the full-length genome sequences of HBF-1strain and other CDV sequences published in GenBank with DNAstar and MEGA6.0software,the results showed that Vero-dSlam cell-adapted HBF-1,had the highest homology of 99.8%with the original isolate variant of Hebei strain,and was close to the domestic HLJ-06isolated from Heilongjiang Province with homology of 98.5%.However,the relationships with the classical vaccine strains were far with the homology of 92.7%.HBF-1was a Vero-dSlam cell-adapted CDV isolated Hebei Province.The sequence alignment of the coding regions between the two strains revealed that 13amino acid mutations occurred with the max variation of 2mutation(0.39%)in P protein and 2mutations(0.33%)in H protein.These mutations were mostly happened during the adaptation to VerodSlam of the HBF-1strain.However,whether these mutations have an effect on the pathogenicity and virulence of HBF-1strain in vivo,would be investigated in further study.Thus,the full-length genome sequencing and sequence analysis of the cell-adapted HBF-1strainprovided a basis for the future study of the interaction between virus and Slam receptors,and the weakening mechanism of wild type CDV strains.