一株鸭细小病毒的分离鉴定与ns基因序列分析

采用余姚某鸭场濒死鸭肝组织悬液，接种鸭胚尿囊腔增殖病毒，蔗糖梯度离心法纯化病毒，电镜观察见直径约20nm的球形病毒粒子，免疫琼脂扩散实验结果显示与鸭细小病毒（duck parvovirus，DPV）抗血清有明显沉淀线；SDS-PAGE电泳可见3条结构蛋白带，与DPV毒株一敛；参照GenBank DPVns基因序列979～1566bp设计引物，PCR扩增反应获得其目的片段．克隆到载体pMD18-T后测序，该序列与GenBank中的番鸭细小病毒（Muscovy duck parvovirus，MDPV）以及巴比里鸭细小病毒（Barbarie duck parvovirus，BDPV）的ns基因序列同源性为98％．病鸭肝组织匀浆液雏鸭感染试验显示雏鸭发病症状及剖解病理变化明显，死亡率为75％．利用血清学实验与鸭肝炎Ⅰ型、禽流感和新城疫病毒感染举别．由此确定引起本次鸭场疫病的病原为DPV，命名为04NY株．

Isolation and Identification of a Duck Parvovirus and Sequence Analysis of ns Gene

An acute and contagious disease broke out in several duck farms in the region of Ningbo of Zhejiang Province in 2004.In the research,the suspension of the dead ducks' liver tissues was inoculated into the allantoic cavity of 11-day-old duck embryos.After the purification of virus by the sucrose density gradient centrifugation,a typical Duck Parvovirus(DPV) particle was observed as the size of 20 nm by(electron) microscopy.In the test of double agar gel immunodiffusion,a line of precipitation was visible.Through SDS-PAGE,three main segments of structural proteins were observed which were consistent with the segments of the standard Duck Parvovirus.With a pair of primers designed according to the sequence from 979 bp to 1 566 bp of the genes of ns of DPV in GenBank,a fragment was amplified by PCR as expectation.The amplified DNA was cloned into pMD18-t vector and sequenced.Sequence indicated that both DNAs of the amplified and the DPV sequence in genbank displayed 98% identity.The result of the animal test indicated that the mortal rate is 75%.The syndrome of the sick young duck and the symptom of anatomical pathology were obvious.The infection of duck hepatitis virus type I,avian influenza virus or newcastle disease virus was excluded by the antiserum reaction.From the above results,the pathologic agent was diagnosed as Duck parvovirus.