安徽地区新型鸭呼肠孤病毒的分离鉴定及其σC基因序列分析  

Isolation,identification andσC gene sequence analysis of a novel duck reovirus from Anhui Province

作　　者：胡晓苗[1] 李薇 李婷 许夏澎 薄宗义 郭梦娇 张小荣[3] 吴艳涛[3] 戴银[1] 张成成 HU Xiao-miao;LI Wei;LI Ting;XU Xia-peng;BO Zong-yi;GUO Meng-jiao;ZHANG Xiao-rong;WU Yan-tao;DAI Yin;ZHANG Cheng-cheng(Institute of Animal Husbandry and Veterinary Medicine,Anhui Academy ofAgricultural Sciences/Livestock and Poultry Epidemic Diseases Research Center of Anbui Province/Anhui province Key laboratory ofLivestock and Poultry Product Safety Engineering,Hefei 230031,China;Yangzhou Vocational University,Yangzhou 225009,China;College of Veterinary Medicine,Yangzhou University,Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses,Yangzhou 225009,China;Agriculture and Rural Bureau of Dingzhou,Dingzhou 073000,China)

机构地区：[1]安徽省农业科学院畜牧兽医研究所/安徽省畜禽疫病研究中心/畜禽产品安全工程安徽省重点实验室,安徽合肥230031 [2]扬州市职业大学,江苏扬州225009 [3]扬州大学兽医学院/江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [4]河北省定州市农业农村局,河北定州073000

出　　处：《中国预防兽医学报》2024年第8期805-811,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：财政部和农业农村部:国家现代农业产业技术体系(CARS-40)资助;江苏高校优势学科建设工程资助项目;安徽省科技重大专项(202003a06020012);安徽省家禽产业技术体系(AHCYJSTX-06);安徽省农业科学院平台项目(2023YL013)。

摘　　要：为掌握新型鸭呼肠孤病毒(NDRV)的最新流行特征,本研究收集安徽某地区鸭场出现肝组织肿胀、出血等临床症状的25份雏鸭肝脏及脾脏样品,利用RT-PCR方法检测NDRV。将RT-PCR检测为阳性的病料样品接种SPF鸡胚分离病毒,收获第3代尿囊液进行PCR检测及病毒纯净性鉴定;同时对NDRV分离株的全长σC基因进行克隆并测序,将测序所得的分离株σC基因序列与GenBank中登录的21株禽源呼肠孤病毒参考株的相应基因序列及推导氨基酸序列进行相似性比对分析,利用Mega 7.0软件构建分离株基于σC基因的系统进化树,并进行σC基因编码氨基酸序列的突变分析。将分离株接种Vero细胞观察其体外培养特性,感染雏鸭进行致病性试验。结果显示,25份病料样品中检出3份NDRV阳性样品,将阳性病料样品接种鸡胚3 d内,鸡胚全部死亡,并伴有水肿、严重充血等现象;病毒纯净性试验结果显示,病料样品仅NDRV呈阳性,其他病原如鸭甲型肝炎病毒(DHV)、鸭坦布苏病毒(DTMUV)、禽呼肠孤病毒(ARV)、番鸭呼肠孤病毒(MDRV)、H9亚型禽流感病毒(H9N2 AIV)及禽腺病毒(FAdV)均为阴性。将3株分离株经测序分析,结果显示,其目的基因序列完全一致,为同一株病毒,并将其命名为NDRV AH株。对NDRV AH株σC基因进行序列相似性分析发现,NDRV AH株与其他NDRV参考株σC核苷酸序列的相似性为89.5%~99.0%,氨基酸序列的相似性为85.3%~98.9%。σC基因的遗传演化结果显示,NDRV AH株属于基因2型。σC氨基酸比对分析结果显示,与灭活疫苗TH11株(KC493571.1)相比,NDRV AH株aa67、aa93、aa100、aa132、aa151、aa158、aa212、aa253和aa298均发生了突变。将该分离株接种Vero细胞,可观察到细胞出现形态圆缩、细胞核聚集等细胞病变。致病性试验结果显示,感染雏鸭死亡率为40%,剖检发现雏鸭肾脏、肝脏出血,脾脏肿大,有轻微坏死灶或大面积坏死灶,有些脾脏质地较硬。可见大�In order to understand and grasp the latest epidemic characteristics of a new type of duck reovirus(NDRV),liver samples of ducks with clinical symptoms such as liver swelling and bleeding were collected from a duck farm in Anhui Province,and NDRV was detected by RT-PCR.Positive samples were inoculated into SPF chicken embryo to isolate the virus,and the third generation allantoic fluid was harvested for PCR detection and virus purity identification.Meanwhile,the full-lengthσC gene of NDRV isolates was cloned and sequenced.TheσC gene sequence of NDRV AH strain was compared with the gene sequence and derived amino acid sequence of 21 avian reovirus reference strains downloaded from GenBank.Meanwhile,the phylogenetic tree of isolates was constructed using theσC gene sequence as the object by using Mega 7.0 software,referencing sequences from GenBank.Finally,the isolates were inoculated with Vero cells to observe their culture characteristics in vitro,and the chicks were infected for pathogenicity test.The result showed,inoculated of chicken embryoswith positive material resulted in embryos death within 3 days,accompanied by edema and severe congestion.Virus purity test results confirmed that the samples were positive for NDRV,but negative for other pathogens such as duck hepatitis A virus(DHV),duck Tambusu virus(DTMUV),avian reovirus(ARV),Muscovy duck reovirus(MDRV),H9 subtype avian influenza virus(H9N2 AIV)and avian adenovirus(FAdV).Sequencing and analysis showed that the three isolates had identical target genes and belonged to one strain,named NDRV AH strain.When Vero cells was inoculated with the strain,the pathological changes such as morphological rounding and nucleus aggregation were observed.Genetic evolution analysis ofσC gene of NDRV AH strain showed a similarity of 89.5%-99.0%σC in gene sequence and 85.3%-98.9%in amino acid sequence compared to other NDRV reference strains.The evolutionary tree of theσC gene showed that the NDRV AH strain isolated in this study belonged to genotype 2.Compared with t

关 键 词：新型鸭呼肠孤病毒 分离鉴定 σC基因 进化分析 致病性试验 

分 类 号：S852.65[农业科学—基础兽医学]