河南省8株狂犬病毒磷蛋白基质蛋白基因分析

目的分析河南省8株狂犬病毒磷蛋白P及基质蛋白M的基因序列,探讨狂犬病毒流行株毒力与致病性变异的可能原因。方法以免疫荧光法检测2006年采自河南省的121份犬脑,取阳性犬脑组织悬液接种鼠脑分离病毒,以RT-nest-edPCR法扩增病毒P与M基因,克隆测序后进行生物信息学分析。结果分离到8株狂犬病毒,序列分析表明8株病毒均为基因1型狂犬病毒,8株病毒彼此之间P基因与M基因核苷酸序列同源性均为98.9%～99.8%,推导的氨基酸序列同源性分别为98.0%～99.3%和97.0%～99.5%。8株病毒与我国宁夏分离株CNX8511、CNX8601的同源性最高,P基因与M基因核苷酸同源性分别为89.1%～89.7%和91.1%～92.0%,氨基酸同源性分别为93.3%～94.3%和96.6%～98.0%。在核苷酸及氨基酸水平上,8株病毒与CTN疫苗株的P与M同源性均明显高于与其它疫苗株的相应同源性。系统发育分析表明,8株病毒与我国宁夏街毒株、CTN疫苗株、泰国及菲律宾等东南亚株进化关系最近,而与CTN以外的其它疫苗株、标准攻击毒CVS株,以及我国80年代初分离的DRV、MRV株进化关系较远。氨基酸对位分析表明,较之参比的其它基因1型毒株,河南省8株狂犬病毒的P与M出现多处变异。结论8株狂犬病毒仍属基因1型狂犬病毒,但其P基因及M基因在核苷酸及推导的氨基酸水平上均出现了明显变异。

Analysis on the P and M gene sequence of eight rabies virus isolates in Henan province

To analyze the genetic characteristics of 8 rabies virus isolates from Henan province,in order to investigate their relatedness with known rabies virus isolates and RV vaccine strain aG,CTN,PV,ERA,etc.the immunofluorescence assay was used to detect the presence of rabies virus in 121 samples of canine brains collected from Henan province.Rabies virus was isolated by inoculating the suspensions of brain tissues into mouse brain.Phosphoprotein P gene and matrix protein M gene were amplified by RT-nested PCR,and then sequenced.The phylogenetic trees were constructed for the analysis of genetic characteristics of rabies virus.In this way,8 rabies virus were obtained.Sequence comparison revealed their identity of P and M were both 98.9%-99.8% on nucleotide level,98.0%-99.3% and 97.0%-99.5% respectively on amino acid level.The isolates were more homogeneous with street strains CNX8511,CNX8601 found in Ningxia,China.The identity of P gene and M gene was 89.1%-89.7% and 91.1%-92.0% on nucleotide level,93.3%-94.3% and 96.6%-98.0% on amino acid level respectively.Eight isolates shared much higher identity with CTN than with other vaccine strains on both nucleotide level and amino acid level.Phylogenetic analysis showed that these 8 isolates were more closely related to vaccine strain CTN and strains from Thailand,Philippines and Ningxia province in China than to any other known street viruses and vaccine strains.It was surprised that the 8 isolates shared the lowest identity in amino acid with two street isolates DRV,MRV established in 1980s',China.Compared deduced amino acid sequences of the 8 isolates with other genotype 1 rabies virus,multiple amino acid residues were substituted in P and M.It is concluded that these 8 strains of rabies virus isolated from Henan province belong to genotype 1,but,both P and M gene of them diverged from known street viruses and vaccine strains on either nucleotide level or on amino acid level.