4株狂犬病街毒的分离及其核蛋白和糖蛋白序列分析

目的以4株狂犬病街毒分离株及我国南北方不同地区狂犬病病毒街毒为研究对象,通过其核蛋白及糖蛋白基因的同源性及进化分析,比较我国狂犬病流行毒株与人用及兽用狂犬病疫苗株间的基因及抗原性差异,为相关疫苗的研究奠定理论基础。方法以直接免疫荧光法(DFA)检测疑似狂犬病的犬脑标本,以乳鼠颅内接种法(MIT)和细胞培养分离技术(CIT)分离狂犬病病毒,并进行TCID50和LD50测定;以RT-PCR法扩增其核蛋白(N)和糖蛋白(G)基因,克隆入T载体并测序后,下载GenBank内已有的狂犬病病毒数据资源,以(Clustal和MEGA3软件)进行基因同源性比对及系统发生分析。结果从广东、河北两省分离到狂犬病病毒街毒4株,分别命名为GN07、WJ07-1、WJ07-2、GC07;序列分析表明4株狂犬病病毒均为基因1型,其N基因与G基因核苷酸的同源性分别为89.5%~99.9%和87.9%~99.9%,其中GN07与WJ07-1、WJ07-2、GC07的同源性较低。与己知的基因1型狂犬病毒相比,WJ07-1、WJ07-2、GC07与湖南、湖北、云南、浙江等地分离株及印度尼西亚分离株核苷酸同源性最高,分别为97.8%~99.4%和92.2%;GN07与CTN疫苗株核苷酸同源性最高,为94.5%。系统发育分析表明,4株病毒与CTN疫苗株同源性较高,与人用狂犬病疫苗3aG、PV株及兽用狂犬病疫苗ERA株和Flury疫苗株的同源性相对较远。适应细胞培养后,四株街毒的LD50和TCID50最高者为GN07,而WJ07-1较低,差别较大。结论4株狂犬病分离株与我国南北方不同地区狂犬病流行毒株的基因和氨基酸序列存在一定差异,在进化关系上分属于不同的分支。

Isolation and sequence analysis of four street strains of rabies virus isolated from China

The genetic homology of the genes encoding nucleoproteins and glycoproteins in 4 street strains of rabies virus isolated from Guangdong and Hebei province of China and their evolutionary relationship were investigated in order to compare the genetic and antigenic differences of the epidemic rabies strains in China and the rabies vaccine strains used in humans and animals to lay the foundation for the related studies for vaccine production. In the present study, the rabies viral antigens in canine brain were detected by direct immunofluorescence assay and the rabies viruses were isolated through mouse inoculation technique(MIT) and cell-culture isolation technique(CIT) respectively. Accordingly, their TCID50 and LD50 were determined. The nucleic acid gene(N) and glycoprotein gene(G) were amplified by RT-PCR, cloned into T vector and then sequenced. Meanwhile, the genetic homology analysis was done with Clustal and MEGA3 softwares and the phylogenetic tree was constructed for analysis of genetic properties of rabies virus. In this way, 4 street strains of rabies virus were isolated from Guangdong and Hebei provinces, designated as GN07, WJ07-1, WJ07-2 and GC07 respectively. Data on the sequence analysis indicated that these 4 street virus strains all belonged to the genotype 1 and the nucleotide and amino acid identities between N and G genes of these strains of virus were 89.5%, 99.9%, 87.9% and 99.9% respectively. In comparison with other known rabies viruses isolated in China, WJ07-1, WJ07-2 and GC07 strains shared closer identity with strains isolated from Hunan, Hubei, Yunnan, Zhejiang and Indonesia, and the rate of nucleotide homology of N and G genes were 97.8%-99.4% and 99.2% respectively. The nucleotide homology between GN07 and the CTN vaccine strain was the highest, accounting up to 94.5%. Furthermore, the phylogenetic analysis revealed that these 4 strains of isolates were also closely related to the CTN vaccine strain and the Flury strain than other known street viruses and vaccine strains. Among these 4 strains of rabies viruses, GN07 had highest values on LD50 and TCID50 and those in WJ07-1 were the lowest. It is concluded that certain nucleotide and amino acid differences exist in these 4 street strains of rabies virus isolated in different parts of China and they belong to the different branches in the evolutionary relationship.