新型鸭呼肠孤病毒分离鉴定及其σC基因序列分析  

Isolation and Identification of Novel Duck Reovirus and Its σC Gene Sequence Analysis

作　　者：张薇[1] 武华 阴雅洁 李松励[1] 侯绍华[1] ZHANG Wei;WU Hua;YIN Yajie;LI Songli;HOU Shaohua(Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China;College of Veterinary Medicine,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]河北农业大学动物医学院,保定071000

出　　处：《中国畜牧兽医》2022年第9期3520-3529,共10页China Animal Husbandry & Veterinary Medicine

基　　金：水禽重要疫病免疫防控新技术研究项目(2017YFD0500800)。

摘　　要：【目的】了解并掌握新型鸭呼肠孤病毒(Novel duck reovirus, NDRV)流行特点及生物学特性,为NDRV的防治提供理论基础和技术支持。【方法】采集河北某鸭场疑似发生鸭呼肠孤病毒感染的组织,无菌处理后接种SPF鸡胚分离病毒,收获第3代尿囊液进行血凝试验,通过PCR、透射电镜观察、间接免疫荧光法(IFA)鉴定病毒,对分离得到的病毒进行体外细胞培养和动物回归试验,并采用Mega 7.0对其σC基因进行遗传进化分析。【结果】分离得到的病毒不能凝集鸡红细胞,可致死鸡胚,死亡鸡胚出血、充血严重;经PCR鉴定,病毒呈NDRV阳性,其他病原(禽呼肠孤病毒、鸭病毒性肝炎、鸭坦布苏病毒、鸭瘟病毒、禽腺病毒血清4型)均呈阴性,将分离得到的病毒命名为BD/CHN/2020株;病毒纯化后,经电镜观察可见直径为60~80 nm、无囊膜的球形病毒粒子,该毒株可在BHK和LMH细胞上稳定增殖并产生细胞融合的细胞病变效应(CPE);IFA结果显示,BD/CHN/2020株接种BHK细胞后在激光共聚焦显微镜下观察可见特异性绿色荧光;BD/CHN/2020株经皮下接种后,发病鸭出现精神沉郁、排白色稀粪等临床症状,剖检可见脾脏出血、肿大、有白色坏死灶等病理变化;序列比对发现,BD/CHN/2020株与NDRV毒株(TH11、091等毒株)在同一分支,与NDRV SY株核苷酸和氨基酸序列相似性最高,均为99.7%,属于NDRV。与灭活疫苗TH11株(KC493571.1)和弱毒疫苗JS01-105P株(V202168)相比,BD/CHN/2020株第93、120、132、158、253、298位氨基酸处发生了位点突变。【结论】成功分离得到1株NDRV BD/CHN/2020株,分离毒株对北京鸭有较强的致病性,与国内疫苗株相比,BD/CHN/2020株的σC蛋白已经发生了氨基酸位点的突变,结果可为新型鸭呼肠孤病毒病的流行病学及疫苗研发奠定基础。【Objective】 The aim of this study was to understand and master the epidemiological characteristics and biological characteristics of Novel duck reovirus(NDRV),and to provide theoretical basis and technical support for the prevention and treatment of NDRV.【Method】 The tissues suspected of Duck reovirus infection was collected from a duck farm in Hebei, and after aseptic treatment, it was inoculated with SPF chicken embryos to isolate the virus, and the third-generation allantoic fluid was harvested for hemagglutination test and in vitro cell culture.The virus was identified by observation and indirect immunofluorescence assay(IFA).The isolated virus was subjected to animal regression test, and its σC gene was analyzed by Mega 7.0.【Result】 The isolated virus couldn’t agglutinate chicken erythrocytes, and could kill chicken embryos.The dead chicken embryos were hemorrhagic and congested.The virus was NDRV positive by PCR identification, and other pathogens(Avian reovirus, Duck hepatitis virus, Duck Tembusu virus, Duck plague virus and Fowl adenovirus 4) were all negative, and the isolated virus was named BD/CHN/2020 strain.Spherical virus particles with a diameter of 60-80 nm were observed by electron microscopy and no envelope could be seen after viruses were purificated.The strain could be proliferated stably on BHK cells and LMH cells and produced a cytopathic effect(CPE) of cell fusion.IFA results showed that specific green fluorescence could be observed under a laser confocal microscope after BD/CHN/2020 strain was inoculated in BHK cells.After the BD/CHN/2020 strain was subcutaneously inoculated, the diseased ducks showed clinical symptoms such as depression and white loose stools.The autopsy showed pathological changes such as spleen hemorrhage, enlargement, and white necrotic foci.Sequence alignment showed that the BD/CHN/2020 strain and NDRV(TH11,091 and other strains) were in the same branch, and had the highest similarity of nucleotide and amino acid sequences to the NDRV SY strain, which w

关 键 词：新型鸭呼肠孤病毒(NDRV) 分离鉴定 σC基因 

分 类 号：S858.32[农业科学—临床兽医学]