河南地区青年鹅源细小病毒分离鉴定和全基因组序列分析

Isolation,identification and genomic characterization of goose parvovirus from young goose flock in Henan Province

小鹅瘟(goose plague,GP)是由鹅细小病毒(goose parvovirus,GPV)感染引起的一种急性、致死性、高度接触性水禽传染病,主要感染3周龄以内的雏鹅和雏番鸭,发病率和死亡率可达100%,给多个国家的水禽养殖业带来了巨大的经济损失。青年鹅对GPV具有一定抵抗力,多表现为隐性感染,临床上常常忽视该群体GP的防控。2020年6月,河南开封某地养鹅场雏鹅群和青年鹅群相继发病。为了确定其病因和致病原分子流行学情况,本研究无菌采集濒死青年鹅肝脏、脾脏和小肠等组织样品,通过PCR/RT-PCR检测样品中鹅细小病毒(GPV)、鹅星状病毒(GAstV)、禽流感病毒(AIV)、禽副黏病毒1型(APMV-1)和禽呼肠孤病毒(GRV)等病原,并针对致病原进行分离鉴定、全基因组测序和遗传演化分析。结果表明,通过PCR/RT-PCR方法对其常见鹅致病病原核酸进行检验,发现病死鹅组织检测到GPV(3/3)和GAstV(1/3),AIV、APMV-1和GRV等病原的核酸检测阴性。挑选单独感染的病鹅病料组织,研磨处理后接种健康鹅胚,成功分离到1株青年鹅源GPV命名为GPV/HNKF-2020株。随后通过重叠PCR方法对GPV/HNKF-2020株进行全基因组测序,序列分析发现,与经典GPV毒株Virulent B株和疫苗株SYG61v株相比,GPV/HNKF-2020株ITRs区域70~83bp和280~293bp间存在两段14bp基因缺失。GPV/HNKF-2020株与2016年以来国内GPV分离株序列相似性高,尤其是与DY16株、RC16株和鸵鸟源HB-2019株相似性高达99.5%。基于全基因组序列和VP3结构蛋白序列的遗传演化分析结果均显示GPV/HNKF-2020株与2016年以来国内GPV分离株亲缘关系最近,属于DY-16like分支的毒株。与经典GPV毒株、疫苗株和NGPV毒株属于同一个大的分支,遗传关系较近。此外,GPV/HNKF-2020株含有7个特有的碱基突变位点,其中G942A突变导致NS1发生R145K氨基酸突变。综上所述,本研究确定了该鹅场青年鹅发病致死系感染GPV所致,并进一步分离鉴定了流行毒株和分析了国内GPV的流行情况,为该病的生物学特性研究和科学防控奠定了一定的理论基础。

Goose plague is an acute,fatal and highly contagious waterfowl infectious disease caused by goose parvovirus(GPV).It mainly infected goslings and Muscovy ducklings within three weeks,with up to 100%morbidity and mortality,which has caused great economic losses in waterfowl industry worldwide.The prevention and control of GP in young goose flocks was often neglected,since most young gooses infected with GPV performed silent infection in clinical,and did not become ill.In June 2020,a continuous emergence of fatal disease occurred in goslings and young geese from a commercial goose farm in Kaifeng City of Henan province.To identify the causative agent of this outbreak,we examined the anatomical changes of moribund geese,and collected the samples of lungs,tracheas and kidneys.The potential causative viral pathogens,including GPV,goose astrovirus(GAstV),avian influenza virus(AIV),avian paramyxovirus-1(APMV-1)and Tembusu virus(TMUV),were detected by PCR and RT-PCR assays.The results showed that the samples were positive for GPV(3/3)and GAstV(1/3),and no corresponding nucleotide fragments were observed for AIV,APMV-1and GRV.Single GPV positive samples were chosen for homogenization and then inoculated into healthy goose embryos for viral isolation,and a young goose derived virulent GPV strain,designated as GPV/HNKF-2020,was isolated.The whole genome of the newly isolated GPV was further determined by overlapping PCR.Sequencing analysis of the genome of GPV/HNKF-2020revealed that the newly identified GPV contained two 14bp deletion in the 70-83bp and 280-293bp of ITRs region compared with that of classical GPV B and vaccine SYG61vstrains.Multiple sequence alignment results shown that the GPV/HNKF-2020 strain shared the high homology with the Chinese GPV isolates since 2016,and in particular shared up to 99.5%nt identity with DY16,RC16and Struthio camelus GPV HB-2019strain.Phylogenetic analysis based on complete genome and VP3protein were all revealed that the GPV/HNKF-2020 strain was clustered together with Chinese GPV isolates since 2016and grouped into the DY-16 subclade.These subclade strains were relatively close to classical GPV,vaccine strains and NGPV,and classified into a branch.Moreover,seven nucleotide mutations were only found in GPV/HNKF-2020genome and the G942A mutation induced the special amino acid mutation R145Kin NS1protein.In conclusion,this study identified that the moribund geese were infected with GPV,and further investigated the molecular epidemiology of the newly GPV isolate and other Chinese GPV strains,which provided a theoretical basis for the study on biological characteristics of GPV and the development of scientific control strategies for this disease.