浙江省野生动物鼬獾狂犬病毒全基因组序列测定分析

目的 测定浙江省分离的2株野生动物鼬獾狂犬病毒株全基因组序列,从分子水平进行遗传变异特征分析,了解狂犬病毒在浙江省的流行和变异情况.方法 RT-PCR测定鼬獾狂犬病毒株全基因组核苷酸序列,并进行基因序列和编码蛋白相似性比较及种系发生分析.结果 测序获得2株鼬獾狂犬病毒全基因组核苷酸序列信息:基因组全长11 923 nts,leader长58 nts,由5个编码区组成:NP(1353 nts)、PP(894 nts)、MP(609 nts)、GP(1575 nts)、LP(6386 nts),N-P-M-G间隔序列长2、5、5 nts;G-L基因间伪基因ψ长423 nts;trailer长70 nts.核酸BLAST及多序列比对显示,浙江省鼬獾狂犬病毒株全基因组序列的组成和结构符合弹状病毒科狂犬病毒属特征;鼬獾病毒株负链RNA基因组5个基因编码氨基酸的长度没有变异,编码区基因没有发生重组,编码蛋白仅表现较少的序列变化,多数只发生碱基的替代;中国病毒株之间特别是同种动物狂犬病毒之间各个基因区域核苷酸与氨基酸序列相似性最高,鼬獾狂犬病毒基因组序列相似性在氨基酸水平明显高于核苷酸水平,蛋白质编码基因的核苷酸变异大多属于同义突变.结论 鼬獾狂犬病毒与研究中选择的代表性疫苗株或者街毒株的变异位点和变异类型相似,多序列相似性比较和N基因种系发生分析显示,鼬獾狂犬病毒均属于基因1型,具有中国地域性特点,2株野生动物鼬獾狂犬病毒极有可能是存在于自然界中固有的街毒株.

Complete genome sequencing and analyses of rabies viruses isolated from wild animals (Chinese Ferret-Badger) in Zhejiang province

Objective Based on sequencing the full-length genomes of two Chinese Ferret-Badger, we analyzed the properties of rabies viruses genetic variation in molecular level to get information on prevalence and variation of rabies viruses in Zhejiang,and to enrich the genome database of rabies viruses street swains isolated from Chinese wildlife.Methods Overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses of the N genes from Chinese Ferret-Badger,sika deer,vole,dog.Vaccine strains were then determined.Results The two full-length genomes were completely sequenced to find out that they had the same genetic structure with 11 923 nts including 58 nts-Leader, 1353 nts-NP,894 nts-PP,609 nts-MP,1575 nts-GP,6386 nts-LP, and 2,5,5 nts-intergenic regions (IORs),423 nts-Pseudogene-like sequence(ψ),70 nts-Trailer.Conclusion The two full-length genomes were in accordance with the propertms of Rhabdoviridae Lyssa virus by blast and multi-sequence alignment.Th nucleotide and amino acid sequences among Chinese strains had the highest similarity,especially among animals of the same species.Of the two full-length genomes,the similarity in amino acid level was dramatically higher than that in nucleotide level, so that the nucleotide mutations happened in these two genomes were most probably as synonymous mutations.Compared to the referenced rabies viruses,the lengths of the five protein coding regions did not show any changes or recombination,but only with a few-point mutations.It was evident that the five proteins appeared to be stable.The variation sites and types of the two ferret badgers genomes were similar to the referenced vaccine or street strains.The two strains were genotype 1 according to the multi-sequence and phyiogenetic analyses,which possessing the distinct geographyphic characteristics of China.All the evidence suggested a cue that these two ferret badgers rabies viruses were likely to be street virus that already circulating in wildlife.