1株新型鸭呼肠孤病毒(SL株)的全基因组测定分析及其致病性  

Whole genome analysis and pathogenicity of a novel duck reovirus(SL strain)

作　　者：杨晓伟 陆婧 王凯民 庄鸿琨 田宇森 李俊锋 朱盈名 刘霞[3] 张立武 赵光伟 YANG Xiaowei;LU Jing;WANG Kaimin;ZHUANG Hongkun;TIAN Yusen;LI Junfeng;ZHU Yingming;LIU Xia;ZHANG Liwu;ZHAO Guangwei(College of Veterinary Medicine,Southwest University,Rongchang,Chongqing 402460,China;Animal,Plantand Food Test Center of Nanjing Customs,Nanjing 210095,China;Guizhou Provincial Center for Animal Disease Control and Prevention,Guiyang 550008,China;Chongqing Sanjiezhongxin Bioen-gineering Co.Ltd.,Rongchang,Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆荣昌402460 [2]南京海关动植物与食品检测中心,江苏南京210095 [3]贵州省动物疫病预防控制中心,贵州贵阳550008 [4]重庆三杰众鑫生物工程有限公司,重庆荣昌402460

出　　处：《中国兽医学报》2022年第11期2195-2201,共7页Chinese Journal of Veterinary Science

基　　金：贵州省科技支撑资助项目(2020-1Y032)。

摘　　要：为明确我国川渝地区鸭疑似呼肠孤病毒病的致病原,本试验对患病鸭进行病毒的分离、鉴定、全基因组测序分析及致病性试验。首先针对临床肝脾肿大、出血坏死为主要特征的病例,利用鸭胚传代法进行病毒的分离,结合RT-PCR方法鉴定分离毒株;其次利用二代测序技术对分离毒株进行全基因组测序,利用MEGA X软件对序列进行比对和分析;最后将分离毒接种7日龄健康雏鸭,观察其病死率及肝脏、脾脏组织的病理变化。结果发现F6代病毒能够稳定适应鸭胚,RT-PCR鉴定为呼肠孤病毒,对鸭胚的半数致死量(ELD50)为10-5.32/0.2 mL,分离毒命名为SL株。全基因测序显示该毒株全长23580 bp,GC含量49.62%,共分10个节段,分别为L1~L3、M1~M3和S1~S4(GenBank登录号:MW196679~MW196688)。序列分析显示SL株与新型鸭呼肠孤病毒(novel duck reovirus,NDRV)具有较高的相似性,为87.02%~99.48%;与番鸭呼肠孤病毒(MDRV)的相似性次之,为80.09%~98.77%;而与鸡源呼肠孤病毒(ARV)相似度最低,为70.04%~91.15%。系统进化树显示SL株10个基因片段均与NDRV位于同一分支,与GX-Y7和XT182个NDRV毒株具有较近的亲缘关系,说明SL株为一新型呼肠孤病毒。同时发现SL毒株中M1片段和L1片段与MDRV也具有较高的进化地位,推测SL株或许存在MDRV和NDRV基因重组现象。致病性试验显示SL株能导致雏鸭死亡,肝脏、脾脏出血肿胀,与临床表现一致。病理组织学观察发现肝细胞以出血、肿胀和空泡变性为主而脾脏以白髓萎缩、淋巴细胞坏死和出现血栓等为特征病变。本试验结果为鸭呼肠孤病毒病的防控及其特异性生物制品的研发提供技术参考。In order to identify the causative agent of suspected duck reovirus disease in Sichuan Province,the virus isolation,identification,whole genome sequencing(WGS)and pathogenicity test were carried out with the sick ducks.Firstly,duck embryo passage method was used to isolate the virus,and RT-PCR was utilized to identify the isolated virus strain;Secondly,the whole genome of the isolate was sequenced by next-generation sequencing technology,and the sequences were compared and analyzed by MEGA X software;Finally,the isolated virus was inoculated into 7-day-old healthy ducklings,and the mortality and pathological changes of liver and spleen were observed.The results showed that the F6 virus could stably adapt to duck embryos.It was identified as reovirus by RT-PCR.The median lethal dose(ELD50)to duck embryos was 10-5.32/0.2 mL.The isolated virus was named SL strain.WGS results showed that SL strain had a total length of23580bp and a GC content of 49.62%.It was divided into 10segments,L1-L3,M1-M3 and S1-S4,respectively(GenBank accession number:MW196679-MW196688).Sequence analysis showed that SL strain had high similarity with novel duck reovirus(NDRV),which was 87.02%-99.48%.The similarity with muscovy duck reovirus(MDRV)was 80.09%-98.77%while 70.04%-91.15%with chicken reovirus(ARV).Phylogenetic tree showed that the 10 gene fragments of SL strain were located in the same branch as NDRV,and had close genetic relationship with GX-Y7 and XT18 NDRV strains,indicating that SL strain was a novel reovirus.Meanwhile,it was found that M1 fragment and L1 fragment of SL strain also had a high evolutionary status with MDRV,suggesting that SL strain might have genomic recombination of MDRV and NDRV.The pathogenicity test showed that SL strain could cause the death of ducklings,bleeding and swelling of liver and spleen,which was consistent with the clinical manifestations.Histopathological observation showed that bleeding,swelling and vacuolar degeneration was mainly found in the liver,while pulp atrophy,lymphocyte necrosis and thro

关 键 词：新型鸭呼肠孤病毒 分离鉴定 全基因组测序 系统进化树 

分 类 号：S852.65[农业科学—基础兽医学]