盐城市狂犬病毒核蛋白及糖蛋白基因序列分析

目的对江苏省盐城市狂犬病毒核蛋白及糖蛋白的基因进行遗传学分析,揭示流行于该地区的狂犬病毒与人用及兽用狂犬病疫苗株间的差异。方法以直接免疫荧光法检测犬脑标本,用阳性犬脑组织悬液接种鼠脑分离病毒。以RT-PCR法扩增病毒核蛋白及糖蛋白全基因片段,克隆测序后进行遗传学分析。结果从58份犬脑中发现两份样品狂犬病毒抗原阳性,分别命名为Jiangsu Yc87与Jiangsu Yc88。从两份阳性样品均扩增到全N基因与G基因序列。阳性犬脑组织接种乳鼠后,从Jiangsu Yc88样品分离到狂犬病毒。分析发现两株病毒均为基因1型狂犬病毒,两株病毒间N基因与G基因的核苷酸及氨基酸的同源性分别为99.8%和99.7%,氨基酸的同源性分别为99.3%和98.8%。与已知的狂犬病毒相比,两株病毒与我国宁夏、河南、湖南、印度尼西亚病毒株的同源性较高,N基因的核苷酸及氨基酸同源性分别为86.3%～98.7%和95.4%～99.8%,G基因核苷酸及氨基酸同源性分别为82.1%～92.1%和94.1%～95.7%;Jiangsu Yc87与Jiangsu Yc88N基因的氨基酸序列分别发生了4和2处氨基酸的替换,G基因的氨基酸序列发生了29和26处氨基酸的替换。与当前使用的疫苗株相比,两株病毒与CTN疫苗株同源性较高,N基因与G基因核苷酸同源性分别为90.2%和87.1%～87.3%,氨基酸同源性分别为98.4%和91.7%～92.3%,但G基因膜外区与所有疫苗株无显著差异;N基因的氨基酸序列分别发生了5和6处氨基酸的替换,G基因的氨基酸序列发生了31和28处氨基酸的替换。结论两株狂犬病毒为基因1型狂犬病毒,其N基因和G基因的核苷酸序列以及推导出来的氨基酸序列与已知的1型狂犬病毒株及疫苗株均有一定的差异。

Genetic analysis of N and G gene of rabies viruses carried by dogs in Yancheng City

To characterize rabies viruses, dogs were captured and brain tissues were collected in Yancheng city of Jiangsu province. Rabies virus antigens were detected in two out of 58 dogs by direct immunofluorescence assay with anti-nucleoprotein monoclonal antibodies, designed Jiangsu Yc87 and Jiangsu Yc88. One isolate from sample Jiangsu Yc88 was obtained by inoculating the suspension of positive brains into suckling mice. The N and G genes of rbies virus were amplified by RT-PCR, and then sequenced. Genetic analysis indicated that these two strains belonged to genotype 1, and the homogeny of N and G genes were 99.8% and 99.7% at nucleotide level, and 99.3% and 98.8% at amino acid level between the two strains,respectively. Comparison of N and G genes with previously published sequences of other known rabies viruses revealed the higher homology with the isolates from Ningxia, Henan, Hunan in China and the isolates form Indonesia than other strains, with 92.8%-98.4% of nucleotide identity and 98.4%-99.3% of amino acid identity for N gene, and 87.4%-91.9% of nucleotide identity and 94.1%-95.7% of amino acid for G gene. Further analysis of the deduced amino acid sequences indicated 4 and 2 of amino acid residues of N gene, 16 and 12 of amino acid residues of Gene, were substituted for Jiangsu Yc87 and Jiangsu Yc88, respectively. When compared the two strains with vaccine strains used in China, Jiangsu Yc87 and Jiangsu Yc88 had a relatively high level of identity with Chinese CTN vaccine strain (90.2% and 87.1%-87.3% of nucleotide identity for N gene, and 98.4% and 91.7%-92.3% of amino acid identity for G gene, respectively), but there were no significantly differences at amino acid level of ectodomain of G gene between the two strains and vaccine strains. Furthermore, 5 and 6 of amino acid residues substitution occurred in N gene, at least 32 and 28 of amino acid residues substitution in G qenes for Jiangsu Yc87 and Jiangsu Yc88 respectively. These results suggested that the two rabies viruses belong to genotype 1, and both N and G gene diverge from known street viruses and vaccine strains at either nucleotide level or amino acid level.