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目的对2013年武汉市新发生的一例疑似狂犬病死亡病例的脑组织进行病毒分离及鉴定。方法采用直接荧光抗体法(DFA)和ELISA法检测狂犬病病毒(rabies virus,RABV)抗原;提取病毒RNA,设计12对引物,分段扩增全基因组,克隆后测序,应用DNAStar软件包中的MegAlign模块对基因所编码的氨基酸进行对位分析;Clustal X 1.83软件和GENEDOC软件用于序列比对;MEGA 4.1软件以Kimura two-parameter模型邻位相连法(Neighbor-Joining,NJ)构建系统进化树(Bootstrap-l000),并与国内外疫苗株及近期湖北省及周边地区分离的代表性街毒株进行同源性分析。结果分离的街毒株13WH10的RABV抗原呈阳性;13WH10街毒株基因组全长11 924 bp,属基因Ⅰ型狂犬病病毒;13WH10与湖北省及周边街毒株处于同一亚群,与CTN-1疫苗株及东南亚地区的街毒株同源性较高,高于欧美国家疫苗株。结论成功对武汉市2013年新发生的一例疑似狂犬病死亡病例的脑组织进行了病毒分离及鉴定;CTN-1与RABV国内分离株同源性较高,系统进化关系较近,提示采用CTN-1疫苗株生产的狂犬病疫苗可有效预防中国狂犬病的流行。
Objective To isolate and identify a new case of suspected rabies death from brain in Wuhan in 2013. Methods Rabies virus (RABV) antigen was detected by direct fluorescent antibody method (DFA) and ELISA. The viral RNA was extracted and 12 pairs of primers were designed. The whole genome was amplified by segmented amplification and sequenced. The MegAlign The alignment of the amino acids encoded by the gene was carried out by using the Clustal X 1.83 software and GENEDOC software. The MEGA 4.1 software was used to construct the phylogenetic tree based on the Kimura two-parameter model (Neighbor-Joining, NJ) Bootstrap-l000), and homologous analysis was carried out with domestic and foreign vaccine strains and representative street strains isolated recently in Hubei Province and surrounding areas. Results The 13WH10 isolates showed positive RABV antigen. The 13WH10 strain was 11 924 bp in length and belonged to genotype I rabies virus. 13WH10 was in the same subpopulation with that in Hubei and surrounding areas, Strain strains in Southeast Asia and higher homology, higher than Europe and the United States vaccine strains. Conclusions A new case of suspected rabies death in Wuhan was successfully isolated from the brain in 2013. CTN-1 was highly homologous to RABV domestic isolates and had a close phylogenetic relationship, suggesting that CTN-1 Vaccine strains of rabies vaccine can effectively prevent the epidemic of rabies in China.