大熊猫源犬瘟热病毒基因组遗传特征分析

【目的】对首次暴发的大熊猫源性犬瘟热病毒(panda derived-canine distemper virus,P-CDV)全基因组进行克隆测序,以了解大熊猫源CDV的全基因组遗传变异情况,进一步追踪感染源,为大熊猫犬瘟热防控提供理论依据。【方法】根据Gen Bank公布的CDV全基因组序列设计17对特异性引物,利用RT-PCR技术,从感染犬瘟热病毒的大熊猫肺渗出液中分片段扩增CDV全基因序列,并克隆到p MD19-T载体中;经测序、拼接,获得第一个P-CDV全长c DNA序列;利用DNAman生物学分析软件分别对全基因组序列、H蛋白基因序列、F蛋白基因序列、P蛋白基因序列、M蛋白基因序列等进行遗传变异分析,构建系统进化树。【结果】经序列测序和拼接,大熊猫源犬瘟热病毒全基因组长15 690 nt,Gen Bank登录号为KP677502,主要编码6种蛋白,分别是N蛋白、P蛋白、M蛋白、F蛋白、H蛋白和L蛋白,在各个基因及间隔区中未发现碱基插入和缺失。遗传进化分析显示,P-CDV全基因组与20株代表性CDV全基因组序列同源性为91.5%—98.7%,P-CDV与强毒株MKY-KM08(HM852904)、PS、HLJ1-06(JX681125)、Hebei(KC427278)以及AC96I-H358(AB753776)在一个大的分支上,与PS株(JN896331)亲缘关系最近,同源性为98.7%,与标准野毒株Strain A75-17(AF164967)同源性为95.7%,与疫苗株CDV3(EU726268)亲缘关系较远,同源性为91.5%。H蛋白氨基酸序列分析和系统进化树表明,大熊猫源犬瘟热病毒属于强毒株,基因型为Asia-I型。P-CDV各蛋白基因与20株代表性的毒株相比有9处氨基酸发生了变异,与Gen Bank上现有的CDV序列相比,其中3处是独有的,分别是:F蛋白基因的208位由N(Asn,天冬酰胺,强毒株多为N)或K(Lys,赖氨酸,弱毒株多为K)变成了S(Ser,丝氨酸),第215位由S变成了A(Ala,丙氨酸),P蛋白基因的58位由Q(Gln,谷氨酰胺)变成K(Lys,赖氨酸)。【结论】成功克隆了大熊猫源犬瘟热病毒的全基因,并完成了序列分析,发现了在F、P基因上碱基的重要变异。这些数据将为研究大熊猫犬瘟热的遗传变异和流行特征提供分子生物学依据。

Genomic Characterization of the Newly Emerged Canine Distemper Virus in Giant Panda

【Objective】 The objective of this study is to analyze the genetic variation of the newly emerged giant panda canine distemper virus by sequencing its full-length genome, thereby to track the source of infection and provide a theoretical basis for the prevention and control of giant panda CDV infection. 【Method】 According to the CDV genomic sequences published in GenBank, 17 pairs of oligonucleotide primers were designed, the overlapping fragments spanning whole genome of CDV were amplified by RT-PCR from lung exudate of the infected giant panda and then cloned into vector pMD19-T and sequenced, respectively, the determined sequences were spliced and assembled by using biological software DNAman. Based on above efforts, the first complete genome sequence of P-CDV isolated from giant panda was accomplished, and was further characterized by alignment with 20 published CDV whole-genome sequences from GenBank. Moreover, to track the source of infection, different phylogenetic trees were constructed according to complete genome as well as genes H, F, P and M, respectively. 【Result】 The genome of the giant panda CDV is 15 690 nucleotides in length (GenBank accession No. KP677502), and mainly encodes six genes in the order of 3′-N-P-M-F-H-L-5′. No nucleotides insert and deletion were observed in encoding and intergenic regions compared to other CDV complete genome sequences. Phylogenetic analysis of the giant panda CDV genome showed 91.5%-98.7% nucleotide identity with 20 representative CDV isolates. Along with virulent strains MKY-KM08 (accession no. HM852904), PS, HLJ1-06 (accession no. JX681125), Hebei (accession no. KC427278) and AC96I-H358 (accession no. AB753776), P-CDV belongs to one large homogeneous clade, and is phylogenetically most closely related to the isolate PS with 98.7% identity at nucleotide. It shares 95.7% nucleotide identity with wild strain A75-17, and exhibits the lowest nucleotide similarity with standard vaccine strain CDV3 (91.5%). Sequence analysis based on H gene indicates that the giant panda CDV is a virulent strain, it belongs to genotype Asia-I. There are a total of nine amino acid (aa) substitutions, among them three amino acid substitutions were firstly found and are unique to the giant panda CDV. Of these 3 aa substitutions, 2 substitutions (N/K208S, S215A) occur in F open reading frame (ORF), one substitution (Q58K) occurs in P ORF.【Conclusion】 The complete genomic sequence of this newly emerged giant panda CDV was successfully characterized, the key site mutations in F and P genes were recognized. The data will provide valuable theoretical reference for the prevention and control of giant panda CDV infection.