鸭肝炎病毒抗原性鉴定及相关基因分析
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摘要
鸭病毒性肝炎(DVH)是雏鸭的一种急性致死性传染病,其病原是鸭肝炎病毒(DHV)。在2000年前,国际病毒分类委员会(ICTV)将DHV分为3个独立血清型,其中1型DHV(DHV-1)的流行范围最为广泛,致病性最强。现已证明2型DHV(DHV-2)及3型DHV(DHV-3)均为禽星状病毒。近年来,在中国、韩国、台湾地区等均有新型鸭肝炎病毒(N-DHV)发病流行的报道。为更好地防制DVH的广泛流行,对现有DHV的抗原性进行鉴定具有重要意义。
本研究使用易感鸭胚和鸭胚原代肝细胞(DELC)对在我国不同年代不同地区分离的9株DHV进行传代培养。传代适应后的病毒具有较稳定的滴度,能够致死鸭胚并产生特异性病变和产生明显的细胞病变。利用血清交叉中和试验和8日龄雏鸭被动保护试验,对病毒的血清型进行鉴定参照同属微RNA病毒的口蹄疫病毒的血清型及亚型划分标准,计算不同毒株之间的亲缘值(R值),对其抗原相关性进行判定。结果表明,GD株与DHV-1标准株的R值<0.47%,YN29株与DHV-1标准株的R值为100%;GD株为N-DHV,YN29为DHV-1。用标准DHV-1抗血清和N-DHV抗血清对国内分离的7株DHV进行交叉中和试验,结果表明,PLK株和YB3株为DHV-1,YB1、YB2、YB5、HY2和HY3株为N-DHV。同时对病毒的抗原相关VP1基因进行扩增和测序。对VP1基因的核苷酸序列与推导的氨基酸序列分析表明,PLK株等3个DHV-1毒株的氨基酸序列同源性为95%左右,GD株等6个N-DHV毒株的氨基酸同源性为98%左右；DHV-1毒株与N-DHV毒株间氨基酸同源性为72-77%。参照同属微RNA病毒的人肠病毒的血清型分型标准,通过对DHV VP1基因序列同源性比较,判定9株病毒的血清型。依据VP1基因序列同源性进行的血清型分型结果与根据抗原性鉴定进行的血清型分型结果一致。同血清型病毒株的VP1基因变异很小,说明DHV的抗原性稳定。同型不同毒株VP1基因的差异,可以导致病毒在致病力、细胞感染能力等方面的差异。目前我国有两种独立的血清型DHV流行,尚未见血清型亚型的流行。
本试验对在我国分离的3株N-DHV进行了全基因组序列测定和分析,参考GenBank公布的DHV-1及N-DHV各毒株的基因组全序列,对DHV遗传进化关系进行分析。我国的N-DHV与韩国N-DHV的VP1基因核苷酸序列同源性在94-95%,属同一基因型；而与DHV-1及台湾N-DHV毒株的同源性分别为72-77%和80%,亲缘关系较远,属不同的基因型。对病毒3D(RNA依赖的RNA聚合酶)保守序列绘制的遗传进化树得到同样的结论,我国N-DHV毒株与韩国N-DHV处于同一进化群。对DHV基因组结构进行分析表明,3株N-DHV基因组结构具有典型的微RNA病毒科病毒的结构特征,同时还具有许多独特之处。
Duck hepatitis virus (DHV) is the causative agent of duck viral hepatitis, which is an acute and fatal infectious disease of ducklings. Three distinct serotypes of DHV (DHV-1, DHV-2 and DHV-3) had been described by ICTV before 2000. Compared with the other two serotypes, DHV-1 is more wildly distributed and higher pathogenic. Today, it is proved that both DHV-2 and DHV-3 belong to avian astrovirus. Recently, outbreaks of new serotypes of DHV were reported in China, Korea, and elsewhere. In order to prevent the distribution of DHV, it is significant to identify the antigenicity of DHV strains isolated in China.
Nine DHV strains isolated at different time and in different areas of China were cultured and passaged on duck embryos and duck embryo liver cells. During the passage, the adapted viruses had the relatively stable titers and caused the death of the embryos with the specific pathological changes and obvious CPE on the cells. Serum cross neutralization test and passive protection test of 8-day-old ducklings were employed to make identification on antigenicity of viruses. Referring on the classification standard on serotype and sero-subtype of Foot and mouth disease virus belonging to Picornaviridae, the judgment on the correlation of antigenicity was carried out by calculating the related value (R) between the different strains. The results showed that, compared to the type strain of DHV-1, the R value of strain GD was less than 0.47%, however the value of strain YN29 was 100%. It is suggested that the strain GD was N-DHV and strain YN29 was DHV-1. According to the cross-neutralization test among the seven strains isolated in China with the antisera of standard DHV-1 and N-DHV, Strains PLK and YB3 were all DHV-1, while Strains YB1, YB2, YB5, HY2 and HY3 were N-DHV. Meanwhile, the VP1 genes of about strains were amplified and sequenced. By analyzing the nucleic acid sequence and induced amino acid sequence, the homology of amino acid sequence among the DVH-1 strains was about 95%, while the homology of amino acid sequence among the N-DVH strains was about 98%. The homology of amino acid sequence between the DVH-1 and N-DVH strains was 72-77%. Referring on the serotyping criteria of human enterovirus belonging to Picornaviridae, the serotypes of the strains were classified by the homology of the VP1 genes. The results of serotyping by the homology of the VP1 genes were correspondent with that of serotyping by the identification of antigenicity. VP1 gene mutations among the strains belonging to the same serotypes were limited and it was proved that the antigenicity of the same serotype strains was stable. The small mutations in VP1 gene of the different strains with the same serotypes could lead to the different pathogenicity and infection. The results indicated that two serotypes of DHV were prevalent in China, but no mutations of sero-subtype were found.
The complete genomes of three strains of N-DHV isolated in China were sequenced and analyzed. The analysis on the genetic evolution of DHV was carried out with the reference on complete genome of DHV-1 and other strains of N-DHV from Genbank. The homogeneity of nucleic acid sequence of the VP1 gene between the N-DHV strains isolated in China and Korea was 94-95% and these strains belonged to the same genotype. The identities of the N-DHV strains isolated in China and Korea aligned with DHV-1 and the N-DHV strains in Taiwan were 72-77% and 80%, respectively, and they belonged to the different genotypes. Drawing a phylogenetic tree based on 3D (RNA-dependent RNA polymerase) conserved nucleic acid sequence, identical conclusion could be drawn that N-DHV in China and Korea belonging to same genetic group. Analyzing on the genome structures of three strains of N-DHV, typical features of genome structures of Picornaviruses and other special features were shown.
本研究使用易感鸭胚和鸭胚原代肝细胞(DELC)对在我国不同年代不同地区分离的9株DHV进行传代培养。传代适应后的病毒具有较稳定的滴度,能够致死鸭胚并产生特异性病变和产生明显的细胞病变。利用血清交叉中和试验和8日龄雏鸭被动保护试验,对病毒的血清型进行鉴定参照同属微RNA病毒的口蹄疫病毒的血清型及亚型划分标准,计算不同毒株之间的亲缘值(R值),对其抗原相关性进行判定。结果表明,GD株与DHV-1标准株的R值<0.47%,YN29株与DHV-1标准株的R值为100%;GD株为N-DHV,YN29为DHV-1。用标准DHV-1抗血清和N-DHV抗血清对国内分离的7株DHV进行交叉中和试验,结果表明,PLK株和YB3株为DHV-1,YB1、YB2、YB5、HY2和HY3株为N-DHV。同时对病毒的抗原相关VP1基因进行扩增和测序。对VP1基因的核苷酸序列与推导的氨基酸序列分析表明,PLK株等3个DHV-1毒株的氨基酸序列同源性为95%左右,GD株等6个N-DHV毒株的氨基酸同源性为98%左右；DHV-1毒株与N-DHV毒株间氨基酸同源性为72-77%。参照同属微RNA病毒的人肠病毒的血清型分型标准,通过对DHV VP1基因序列同源性比较,判定9株病毒的血清型。依据VP1基因序列同源性进行的血清型分型结果与根据抗原性鉴定进行的血清型分型结果一致。同血清型病毒株的VP1基因变异很小,说明DHV的抗原性稳定。同型不同毒株VP1基因的差异,可以导致病毒在致病力、细胞感染能力等方面的差异。目前我国有两种独立的血清型DHV流行,尚未见血清型亚型的流行。
本试验对在我国分离的3株N-DHV进行了全基因组序列测定和分析,参考GenBank公布的DHV-1及N-DHV各毒株的基因组全序列,对DHV遗传进化关系进行分析。我国的N-DHV与韩国N-DHV的VP1基因核苷酸序列同源性在94-95%,属同一基因型；而与DHV-1及台湾N-DHV毒株的同源性分别为72-77%和80%,亲缘关系较远,属不同的基因型。对病毒3D(RNA依赖的RNA聚合酶)保守序列绘制的遗传进化树得到同样的结论,我国N-DHV毒株与韩国N-DHV处于同一进化群。对DHV基因组结构进行分析表明,3株N-DHV基因组结构具有典型的微RNA病毒科病毒的结构特征,同时还具有许多独特之处。
Duck hepatitis virus (DHV) is the causative agent of duck viral hepatitis, which is an acute and fatal infectious disease of ducklings. Three distinct serotypes of DHV (DHV-1, DHV-2 and DHV-3) had been described by ICTV before 2000. Compared with the other two serotypes, DHV-1 is more wildly distributed and higher pathogenic. Today, it is proved that both DHV-2 and DHV-3 belong to avian astrovirus. Recently, outbreaks of new serotypes of DHV were reported in China, Korea, and elsewhere. In order to prevent the distribution of DHV, it is significant to identify the antigenicity of DHV strains isolated in China.
Nine DHV strains isolated at different time and in different areas of China were cultured and passaged on duck embryos and duck embryo liver cells. During the passage, the adapted viruses had the relatively stable titers and caused the death of the embryos with the specific pathological changes and obvious CPE on the cells. Serum cross neutralization test and passive protection test of 8-day-old ducklings were employed to make identification on antigenicity of viruses. Referring on the classification standard on serotype and sero-subtype of Foot and mouth disease virus belonging to Picornaviridae, the judgment on the correlation of antigenicity was carried out by calculating the related value (R) between the different strains. The results showed that, compared to the type strain of DHV-1, the R value of strain GD was less than 0.47%, however the value of strain YN29 was 100%. It is suggested that the strain GD was N-DHV and strain YN29 was DHV-1. According to the cross-neutralization test among the seven strains isolated in China with the antisera of standard DHV-1 and N-DHV, Strains PLK and YB3 were all DHV-1, while Strains YB1, YB2, YB5, HY2 and HY3 were N-DHV. Meanwhile, the VP1 genes of about strains were amplified and sequenced. By analyzing the nucleic acid sequence and induced amino acid sequence, the homology of amino acid sequence among the DVH-1 strains was about 95%, while the homology of amino acid sequence among the N-DVH strains was about 98%. The homology of amino acid sequence between the DVH-1 and N-DVH strains was 72-77%. Referring on the serotyping criteria of human enterovirus belonging to Picornaviridae, the serotypes of the strains were classified by the homology of the VP1 genes. The results of serotyping by the homology of the VP1 genes were correspondent with that of serotyping by the identification of antigenicity. VP1 gene mutations among the strains belonging to the same serotypes were limited and it was proved that the antigenicity of the same serotype strains was stable. The small mutations in VP1 gene of the different strains with the same serotypes could lead to the different pathogenicity and infection. The results indicated that two serotypes of DHV were prevalent in China, but no mutations of sero-subtype were found.
The complete genomes of three strains of N-DHV isolated in China were sequenced and analyzed. The analysis on the genetic evolution of DHV was carried out with the reference on complete genome of DHV-1 and other strains of N-DHV from Genbank. The homogeneity of nucleic acid sequence of the VP1 gene between the N-DHV strains isolated in China and Korea was 94-95% and these strains belonged to the same genotype. The identities of the N-DHV strains isolated in China and Korea aligned with DHV-1 and the N-DHV strains in Taiwan were 72-77% and 80%, respectively, and they belonged to the different genotypes. Drawing a phylogenetic tree based on 3D (RNA-dependent RNA polymerase) conserved nucleic acid sequence, identical conclusion could be drawn that N-DHV in China and Korea belonging to same genetic group. Analyzing on the genome structures of three strains of N-DHV, typical features of genome structures of Picornaviruses and other special features were shown.
引文
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