2005--2010年贵州省狂犬病病毒与疫苗株核蛋白遗传差异分析

Analysis of the genetic differences in the nucleoprotein between rabies virus and its vaccine strains in Guizhou province from year 2005 to 2010

目的分析2005--2010年贵州省狂犬病病毒（RABV）与疫苗株核蛋白基因的差异。方法对贵州省不同地区2005--2010年收集的4份狂犬病患者标本和28份狂犬病病犬脑组织标本进行分析。采用直接免疫荧光法（DFA）和巢式RT—PCR检测，测定病毒N基因序列全长，同时，根据同源性及系统进化树比较，分析贵州省近年流行的RABV与GenBank数据库收录的RABV疫苗株N基因序列之间的差异。结果经检测，21份病犬脑组织和4份患者标本RABV抗原和核酸阳性。经N基因序列测定拼接均得到长度为1353bp的核苷酸序列。同源性分析显示，25株RABV毒株基因序列与GenBank数据库收录的RABV基因1型毒株的N基因核苷酸和氨基酸序列同源性较高，分别为89％～100％和98％-100％；与疫苗株核苷酸和氨基酸序列同源性分别为86％～95％和96％-100％，在兽用疫苗株中，与HEP．Flury株N基因核苷酸和氨基酸的同源性最高（分别为88％-89％和98％-99％），而在人用疫苗株中，与CTN株的核苷酸和氨基酸同源性最高（分别为86％～100％和96％-100％）。N基因系统进化树分析显示，25株毒株与RABV基因1型毒株和疫苗株CTN株单独构成一群，与人用疫苗株CTN的差异最小。结论贵州省2005--2010年流行的RABV与兽用疫苗株HEP—Flury和人用疫苗株CTN在N基因水平的差异最小。

Objective This study was to explore the differences in the nucleoprotein gene between rabies virus （RABV） and its vaccine strains in Guizhou province from year 2005 to 2010. Methods Samples from 4 rabies patients and cerebral tissue samples of 28 rabies infected dogs were collected from different districts in Guizhou province between year 2005 and 2010. Direct Immunofluorescence Assay （DFA） and RT-nested PCR assay were applied to detect the overall length of N gene sequence. Meanwhile, based on the comparison between the homology and phylogenetic tree, the differences in N gene sequence between the prevalent RABV and the RABV vaccine strains collected from NCBI database in these years. Results According to DFA and RT-nested PCR assay, the antigen and nucleic acid of the 21 dogs and 4 human samples were both confirmed positive; whose full length of N gene sequences were both 1353 bp. The homological analysis showed that the 25 strains of RABV virus and the RABV type I virus stored by GenBank database shared a high homology in N gene nucleotide and amino acid sequences, which were 89% - 100% and 98% - 100% , respectively. Besides, the homology between the 25 strains of RABV virus and its vaccines in nucleotide and amino acid sequences were separately 860/o - 95% and 96% - 100% . The N gene of vaccines for livestock shared the highest homology with HEP-Flury strain in the nueleotide and amino acid, which were 88% - 89% and 98% - 99%,respectively. The vaccines for human use showed its greatest homology with the CTN strain in nucleotide （86% -100% ） and amino acid （96% -100% ）. The phylogenetic tree analysis indicated that the 25 strains of RABV virus, RABV type I virus and the CTN vaccine strains constituted one individual cluster, which was least different from the CTN vaccine for human use. Conclusion The prevalent RABV virus, the vaccine HEP-Flury for livestock and the vaccine CTN for human use were found to be highly similar in N gene expression in Guizhou province from 2005 to 2010.