禽痘病毒活疫苗中网状内皮组织增生病病毒的分离鉴定及其序列比较  

ISOLATION OF RETICULOENDOTHELIOSIS VIRUS FROM A FOWLPOX LIVE VACCINE AND ENV GENE SEQUENCE ANALYSIS

作　　者：王景艳[1] 李中明[1] 赵鹏[1] 陈浩[1] 崔治中[1] 

机构地区：[1]山东农业大学动物科技学院,泰安271018

出　　处：《中国动物传染病学报》2010年第1期35-40,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家科技支撑计划(2006BAD06A08)

摘　　要：为了从怀疑污染禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)的禽痘病毒(Fowlpoxvirus,FPV)活疫苗中分离病毒,将市场上购买的某些厂家的禽痘弱毒疫苗接种7日龄SPF雏鸡。在5d后采其抗凝血浆接种鸡胚成纤维细胞(chicken embryo fibroblast,CEF),在连续传2代后,用REV特异性单抗做间接免疫荧光试验,确认REV感染,命名为JS0809。提取感染细胞的DNA,采用PCR扩增env基因。测序分析表明,该毒株的env基因开放阅读框同大部分REV毒株一致,为1 761 bp。JS0809的囊膜蛋白基因(envelope protein gene,env)与国内已发表株的同源性高达96.0%～99.%。相对而言,它与早年分离到的毒株的同源性只有96%～97.3%,但与近几年的分离珠的同源性则均高达99.5%～99.8%。它与整合进FPV野毒株中的全基因组REV的env基因同源性很高为99.8%,而与整合进FPV疫苗株中全基因组REV的env基因同源性较低仅为97.3%。本研究证实了在我国生产的某些FPV疫苗中存在REV污染,所污染的REV的env基因更接近最近的流行野毒,这是国内第一次报道从禽痘弱毒疫苗中分离到REV活毒。Seven days old SPF chickens were inoculated with fowl pox live vaccines from different manufactures to isolate reticuloendotheliosis virus （REV）. Five days later, plasma samples were collected and inoculated into chicken embryo fibroblast （CEF） monolayer. The CEF cultures were trypsinized a week later and cell suspensions were replated into fresh dishes and incubated for another week. After two passages, CEF monolayers were stained in indirect fluorescence antibody test with REV-specific monoclonal antibody and REV infection was demonstrated in one CEF culture. The REV isolate was designated as JS0809. Genomic DNA was extracted from the infected CEF and used as template for amplification of the env gene. Sequence analysis indicated that the env ORF contained 1761 bp identical to most published strains and had 96%-99.8% sequence identity with all compared strains. It had 96%-97.3 identity with earlier isolated reference strains but had 99.5%-99.8% identity with the recent isolates. Interestingly, it had 97.% identity with REV integrated into a fowl pox vaccine virus reported in Australia and 99.8% identity with REV integrated into a fowl pox virus （FPV） field strain reported in US. The study demonstrated the REV contamination in fowl pox live vaccine made in China and env sequence of the contaminated REV was very close to FPV field strains isolated recently. This is the first report to isolate REV from fowl pox live vaccine in China

关 键 词：禽痘病毒活疫苗 网状内皮组织增片病病毒 鉴定 序列分析 

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