大熊猫犬瘟热病毒LG株的分离鉴定

Identification of Canine Distemper Virus LG Strain Isolated from Giant Panda

为获得大熊猫犬瘟热病毒株,采集死亡大熊猫的心脏、肺、肝病料,研磨并反复冻融后收集上清液接种Vero细胞,待出现细胞病变(CPE)后,收集病毒液。用逆转录-聚合酶链反应(RT-PCR)鉴定病毒分离株,测定病毒TCID_(50),动物回归试验测定其毒力。结果显示,盲传到第5代时,Vero细胞出现圆缩、聚集、脱落等病变;PCR扩增出287bp片段,与预期相符;测序结果显示,该毒株与已发表的CDV SD(14)11毒株(亚洲-Ⅰ型)的同源性为98%;毒株的TCID_(50)为10^(-5.2)/mL;幼犬感染分离毒株后出现体温升高、鼻头发干、拉稀、眼鼻分泌出水样分泌物等症状。本研究成功分离出大熊猫源犬瘟热病毒株。

To isolate a wild strain of canine distemper virus （CDV）, after 3 times repeated grinding and freezing, the supernatants of tissue samples of giant panda including heart,lung and liver were inoculated in monolayer Vero cells, and harvested when viruses cause apperently cytopathic effect （CPE）. The isolate was identified by reverse transcription polymerase chain reaction （RT-PCR） TCIDs0 of CDV was calculated by the Reed-Muench algorithm. The virulence of this isolate was assessed by animal regression assay. The result showed that the CPE such as shrink and pyknosis were observed in Vero cells after blindly passaging 5 times. The length of PCR amplification fragment was 287 bp, which coincided with the expected result. The nucleotide homology between the isolate and CDV SD （14） 11 strain （Asia- I ） was 98%. The result of TCID50 was 10^-5.2 TCID50/mL. The clinical symptoms of CD like body temperature increasing,nose appearing dry, diarrhea, watery discharge secreted in eyes and nose and so on were observed after inoculation with this isolate. A CDV,named LG strain,was successfully obtained in this study from giant panda.