兔出血症病毒2型的分离鉴定与序列分析  

Isolation,identification and sequence analysis of rabbit hemorrhagic disease virus type 2

作　　者：魏后军[1] 胡波[1] 范志宇[1] 宋艳华[1] 仇汝龙 陈萌萌[1] 薛家宾[1] 王芳[1] WEI Hou-jun;HU Bo;FAN Zhi-yu;SONG Yan-hua;QIU Ru-long;CHEN Meng-meng;XUE Jia-bin;WANG Fang(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory for Veterinary Bio-Product Engineering,Ministry of Agriculture,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院兽医研究所/农业部兽用生物制品工程重点实验室,江苏南京210014

出　　处：《江苏农业学报》2020年第2期404-409,共6页Jiangsu Journal of Agricultural Sciences

基　　金：现代农业产业技术体系建设专项资金资助项目(CARS-43-C-1);国家重点研发计划项目(2018YFD0502203)。

摘　　要：兔出血症(Rabbit hemorrhagic disease,RHD)是由兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)引起的一种具有高度传染性、急性致死性兔病。兔出血症病毒2型(RHDV2)引起的RHD主要在欧洲流行,病兔的死亡率高达90%,而用经典RHDV毒株制备的疫苗几乎不能预防家兔感染RHDV2,从而给世界养兔业造成了巨大的经济损失。2020年4月,中国四川省某兔场首次发生由疑似RHDV2引起的RHD,笔者所在实验室通过红细胞凝集试验(Hemagglutination,HA)、逆转录-聚合酶链式反应(Reverse transcription-polymerase chain reaction,RT-PCR)扩增、vp60基因序列分析和病毒复制试验,对采集的病死家兔肝脏样本进行病原鉴定。HA结果显示,部分病死家兔的肝脏样本不能引起血凝现象;RT-PCR结果显示,样品中出现了RHDV2的特异性条带。对新发现毒株cDNA的RT-PCR产物进一步分析发现,所扩增vp60基因核苷酸序列与经典RHDV vp60基因核苷酸序列的一致性为77.5%~80.1%,与RHDV2 vp60基因核苷酸序列的一致性为93.6%~96.1%,并且与RHDV2处于同一个进化分支。病毒复制试验结果显示,用病死家兔肝脏组织与磷酸缓冲盐溶液(Phosphate buffer saline,PBS)按1 g∶10 ml混合后制成的悬液分别人工感染5只非免疫25日龄未断奶仔兔和5只2月龄兔,在24~48 h全部死亡。基于以上试验结果,确定本研究新发现的RHDV毒株为RHDV2,并将其命名为SC2020,这是在中国首次发现RHDV2毒株,应引起高度重视。Rabbit hemorrhagic disease(RHD)is a highly contagious and acute fatal disease caused by rabbit hemorrhagic disease virus(RHDV).RHL caused by rabbit hemorrhagic disease virus type 2(RHDV2)is mainly prevalent in Europe,and the mortality rate is as high as 90%.Rabbits can hardly be protected from infecting RHDV2 by classical RHDV vaccine,resulting in huge economic loss to world rabbit-raising industry.In April 2020,suspected RHDV2-induced RHD cases were found in a rabbitry of Sichuan province,China.Hemagglutination(HA)test,reverse transcription-polymerase chain reaction(RT-PCR),vp60 gene sequence analysis and virus replication experiment were conducted to identify the pathogen in the collected livers of dead rabbits in our lab.The results of HA showed that the liver samples of some dead rabbits could not induce hemagglutination.In addition,specific bands of RHDV2 were detected by RT-PCR in the samples.Further analysis on RT-PCR product of newly discovered strain indicated that the nucleotide sequence of amplified vp60 gene shared 77.5%-80.1%and 93.6%-96.1%nucleotide sequence consistency with classic RHDV vp60 gene and RHDV2 vp60 gene,respectively.The amplified segment and RHDV2 were on the same branch of the evolutionary tree.The results of virus replication showed that five unweaned rabbits(25-day-old)and five rabbits(two-month-old)died in 24 h to 48 h after being infected with suspension of 10 ml phosphate buffer saline(PBS)versus 1 g liver tissues from dead rabbits.These results indicated that the newly discovered RHDV strain was RHDV2,and it was named as SC2020.This is the first RHDV2 strain detected in China,and it should be carefully monitored.

关 键 词：兔出血症 兔出血症病毒2型(RHDV2) VP60基因 鉴定 序列分析 

分 类 号：S855.3[农业科学—临床兽医学]