貉源犬瘟热病毒CDV-ZC株分离鉴定、全基因测序与分析  

Isolation,identification and genomic sequencing analysis of CDV-ZC strain of canine distemper virus from raccoon dog

作　　者：戴秀美[1] 常维山[1] 张永兵[1] 王敏[1] 王蕾[1] 郭树源[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2015年第5期711-717,共7页Chinese Journal of Veterinary Science

基　　金：山东省教育厅基金资助项目(J08LF60)

摘　　要：应用非洲绿猴肾细胞(Vero)从山东省诸城市疑似犬瘟热(canine distemper,CD)感染貉的肝脏、脾脏、肺脏等组织病料的研磨上清液中分离出1株病毒。分离株经Vero细胞传至第4代出现典型的细胞病变效应(CPE),经毒力测定、血清学、RT-PCR检测及测序、回归动物试验证明为犬瘟热病毒(Canine distemper virus,CDV),命名为CDV诸城分离株(CDV-ZC)。采用RT-PCR方法分段克隆分离株的全基因序列并测序,测序成功的各序列依次拼接得到全基因序列并进行序列比对。结果表明,CDV-ZC株全基因(KJ994343)与标准美洲型犬瘟热强毒株A75/17核苷酸同源性高达96.5%,而与疫苗株Onderstepoort、CDV3等亲缘关系较远,同源性为91.6%~92.0%。血凝素(H)基因序列分析表明,CDV-ZC株与国内野毒株LN(13)2、GP株等以及日本野毒株UENO、HAMA等共同归属于Asia-1型,在H蛋白信号淋巴细胞激活因子(SLAM)受体结合区即542~544位增加了1个潜在N-连接糖基化位点(N-X-S/T)。致病性强毒株CDV-ZC的成功分离及全基因序列测定加深了我们对当前中国CDV流行株遗传变异情况的了解,为CD的有效预防、诊断及控制提供理论依据。A virus strain was isolated by Vero cells from supernatant of cell extracts of liver,spleen,lung that were collected from a raccoon dog with suspected canine distemper virus infection in Zhucheng city,Shandong province.Typical cytopathic effect was observed in Vero cells after four passages.It was identified as CDV by virulence investigation,serology neutralization test,RT-PCR and animal regression experiment and named CDV Zhucheng strain.Full-length genome of strain CDV-ZC was amplified,sequenced and aligned with other CDV strains by RT-PCR.The complete genome nucleotide sequence of CDV-ZC(KJ994343)exhibited highly homology to the standard virulent strain A75/17(96.5%at nt level),while it was identity with the vaccine strains Onderstepoort,CDV3 and so on ranged from 91.6%-92.0%.Phylogenetic analysis based on the hemagglutinin gene demonstrated that CDV-ZC strain belonged to Asia-1genotype,along with Chinese wild-type strain LN(13)2,GP and Japanese strain UENO,HAMA and so on.In inspecting the H protein alignment,a novel potential N-linked glycosylation sites was observed at position 542-544,which located at H protein receptor(SLAM)binding region.The successful isolation and complete sequences determination of highly pathogenic and virulent strain CDV-ZC provided a better understanding of the genetic variations of CDVs in China and is likely to be helpful to guide efficient preventive,diagnostic,and control strategies for CD.

关 键 词：犬瘟热病毒 CDV-ZC株 分离与鉴定 全基因测序 血凝素基因 

分 类 号：S852.65[农业科学—基础兽医学]