鹅细小病毒中国分离株HG5/82的回顾性研究
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摘要
小鹅瘟（Gosling Plague，GPa）是由细小病毒科、细小病毒属中的鹅细小病毒（Goose Parvovirus，GPV）引起的一种烈性传染病。主要发生于1月龄内雏鹅和雏番鸭，以急性肠炎及肝、肾、心等实质脏器炎症为特征。该病病程短，传染性强，死亡率高。在欧洲、亚洲等许多国家相继都有发生，给水禽养殖造成了巨大的经济损失。
本实验旨在研究1982年一株鹅细小病毒流行株基因的分子生物学特征，并与国内外分离株相应基因进行分析比较。为进一步研究1982年流行株HG5/82和国内外流行株间的基因遗传变化规律、GPV的基因功能、及病毒分子致病机理提供了一定的分子基础。
1．用鹅胚分离到鹅细小病毒，测定了第五代病毒尿囊液HG5/82对12日龄鹅胚的ELD50为10-5.92/0.1ml。雏鹅接种试验表明该病毒对12日龄雏鹅的致病性较弱。用套式PCR检测雏鹅肛拭子病毒的体外排放情况，发现三个试验组在接种病毒后1—20天均可检测到病毒。PCR产物序列与GPV参考毒株B的相应序列同源性达93.6%。
2．提取病毒DNA，根据国外已发表的GPV 基因序列，设计并合成引物，用聚合酶链式反应技术扩增得到GPV中国分离株HG5/82的非结构基因与结构基因，片段大小分别约为1.9kb、2.2kb的片段。将该片段分别进行克隆及序列测定，并与GPV国内外部分已发表的毒株及番鸭细小病毒的对应序列比较。结果表明，我国地方分离株与国内外鹅细小病毒相比，NS基因和VP基因均表现出较高的同源性，并且具有共同的分子特征。结构基因VP3间变异较小，这是目前GPV只有一个血清型的分子基础，为基因工程苗的研制提供了可行性。HG5/82与番鸭细小病毒FM株相应序列比较发现，与细小病毒其它成员相比两者具有较近的亲源关系，但这种同源性明显低于鹅细小病毒之间的同源性。
Goose parvovirus (GPV) infection is a highly fatal disease of goslings and Muscovy ducklings under 1 month old .The causative agent of the disease has been classified as a member of the Parvovirus genus of the Parvoviridae, goose parvovirus. The disease is characterized by acute enteritis and high mortality. The outbreak of GPV has been reported in many countries including Europe and China, which result heavy economic losses.
This study is to research the molecular characteristics of the virus that popular in 1982.Mean- while,the NS and VP gene were compared with other strains. This study offers a good base for further investigations that including genome function, mutation regulation of genome and pathogeny of the virus et al.
1. A field strain of GPV was isolated successfully from goose embryos in this study. We detected the ELD50 of the fifth passage virus(HG5/82) to 12-day-old goose embryos was 10-5.92/0.1ml. No typical clinical signs were observed after inoculation of GPV-HG5/82 strain. We detected the GPV in the anal swab of the inoculated gosling with nested-PCR and sequencing the product of PCR. The nucleotide identity of the PCR product between HG5/82 and GPV strain B was 93.6%.
2. Non-structure gene and structure gene of goose parvovirus (GPV) HG5/82 strain were amplified using polymerase chain reaction (PCR) method and cloned into pMD18-T vector, respectively. The two fragments were sequenced and analyzed with reference GPV strains and Muscovy duck parvovirus(MDPV) strains. The results showed that the
identity of NS and VP gene between HG5/82 and GPV-YG strain isolated in China is higher than HG5/82 and B that isolated in Hungary. The identity of partial VP3 genes between HG5/82 and MDPV is higher than the identity between GPV and other members of parvovirinae. Furthermore, these results were also proved by the analysis of VP3 gene. This high identity between GPV HG5/82 and MDPV indicated that they would be came from the same ancestor.
本实验旨在研究1982年一株鹅细小病毒流行株基因的分子生物学特征，并与国内外分离株相应基因进行分析比较。为进一步研究1982年流行株HG5/82和国内外流行株间的基因遗传变化规律、GPV的基因功能、及病毒分子致病机理提供了一定的分子基础。
1．用鹅胚分离到鹅细小病毒，测定了第五代病毒尿囊液HG5/82对12日龄鹅胚的ELD50为10-5.92/0.1ml。雏鹅接种试验表明该病毒对12日龄雏鹅的致病性较弱。用套式PCR检测雏鹅肛拭子病毒的体外排放情况，发现三个试验组在接种病毒后1—20天均可检测到病毒。PCR产物序列与GPV参考毒株B的相应序列同源性达93.6%。
2．提取病毒DNA，根据国外已发表的GPV 基因序列，设计并合成引物，用聚合酶链式反应技术扩增得到GPV中国分离株HG5/82的非结构基因与结构基因，片段大小分别约为1.9kb、2.2kb的片段。将该片段分别进行克隆及序列测定，并与GPV国内外部分已发表的毒株及番鸭细小病毒的对应序列比较。结果表明，我国地方分离株与国内外鹅细小病毒相比，NS基因和VP基因均表现出较高的同源性，并且具有共同的分子特征。结构基因VP3间变异较小，这是目前GPV只有一个血清型的分子基础，为基因工程苗的研制提供了可行性。HG5/82与番鸭细小病毒FM株相应序列比较发现，与细小病毒其它成员相比两者具有较近的亲源关系，但这种同源性明显低于鹅细小病毒之间的同源性。
Goose parvovirus (GPV) infection is a highly fatal disease of goslings and Muscovy ducklings under 1 month old .The causative agent of the disease has been classified as a member of the Parvovirus genus of the Parvoviridae, goose parvovirus. The disease is characterized by acute enteritis and high mortality. The outbreak of GPV has been reported in many countries including Europe and China, which result heavy economic losses.
This study is to research the molecular characteristics of the virus that popular in 1982.Mean- while,the NS and VP gene were compared with other strains. This study offers a good base for further investigations that including genome function, mutation regulation of genome and pathogeny of the virus et al.
1. A field strain of GPV was isolated successfully from goose embryos in this study. We detected the ELD50 of the fifth passage virus(HG5/82) to 12-day-old goose embryos was 10-5.92/0.1ml. No typical clinical signs were observed after inoculation of GPV-HG5/82 strain. We detected the GPV in the anal swab of the inoculated gosling with nested-PCR and sequencing the product of PCR. The nucleotide identity of the PCR product between HG5/82 and GPV strain B was 93.6%.
2. Non-structure gene and structure gene of goose parvovirus (GPV) HG5/82 strain were amplified using polymerase chain reaction (PCR) method and cloned into pMD18-T vector, respectively. The two fragments were sequenced and analyzed with reference GPV strains and Muscovy duck parvovirus(MDPV) strains. The results showed that the
identity of NS and VP gene between HG5/82 and GPV-YG strain isolated in China is higher than HG5/82 and B that isolated in Hungary. The identity of partial VP3 genes between HG5/82 and MDPV is higher than the identity between GPV and other members of parvovirinae. Furthermore, these results were also proved by the analysis of VP3 gene. This high identity between GPV HG5/82 and MDPV indicated that they would be came from the same ancestor.
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