不同禽类A型流感病毒H5N1亚型全基因克隆及序列分析
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摘要
本文对来自河南省的鸡源(A／chick／Henan／88／02以下简称CCK)、鸭源(A／duck／Henan／89／02以下简称CDK)及鸵鸟源(A／Ostrich／Henan／90／02以下简称COR)禽流感病毒(AIV)分离株进行了深入研究，包括致病性、感染谱、免疫学特性、生物学和分子生物学特性、遗传变异及进化情况等，从而为我省乃至我国AIV流行病学监控体系研究、快速诊断、基因工程疫苗的研制及综合防治等提供了重要参考依据。血凝试验、血凝抑制试验、ELD_(50)测定、动物致病性试验、攻毒保护试验等结果显示，所分离的三株AIV均为高致病力H5N1亚型毒株，其中鸵鸟流感病毒株对鸡的致病性明显低于其余两分离株，说明AIV存在明显宿主特异性。
用无特定病原体(SPF)鸡胚增殖上述3株禽流感病毒，提取病毒基因组总RNA，参考Genebank中对应基因片段的核苷酸序列设计合成8对特异性引物，用反转录-聚合酶链反应(RT-PCR)技术分别扩增各分离株的8个基因片段的全序列cDNA。并将其分别克隆到PMD18-T载体上，转化JM-109感受态菌，获得阳性质粒，并进行了鉴定和序列测定。将测定的序列与参照序列对比，视其同源性高低来判定该序列是否为所测定的基因片段的核苷酸序列。结果表明从每个毒株扩增到的8个基因片段均包含病毒相应的完整开放阅读框架、起始和终止密码子。
运用序列分析软件对各毒株的相关序列进行了同源性和遗传进化分析。结果表明：①三个河南地方株(CK、DK、OR)的HA基因与欧亚种系代表株Ire／83的同源性分别为86.9％、86.0％、86.1％，而与北美种系代表株PA／83同源性为77.5％、76.9％、76.7％，表明三毒株均起源于欧亚种系；三个毒株HA裂解位点的氨基酸序列均为PQRERR-RKKR，且均多于5个碱性氨基酸，说明三个毒株均为高致病力禽流感病毒。②三者NA之间
的同源性在95.6%一98%之间，其中以DK和OR的同源性最高，为98%，
而CK和两者的同源性仅分别为95.670和96%。同时DK和OR在165一224
处均缺少了59个核昔酸，二者在结构上相似，由此可以看出，DK和0R亲
缘关系极为密切，再结合HA基因和生物学特性，提示0R是由DK变异而
来;各毒株之间的NP基因核昔酸同源性差异较大(91.170一99.5%)，而氨
基酸的差异较小(94%一99.4%)，提示AIV具有型特异性;三株分离株M
基因的核昔酸的同源性高达99%以上，尤其是DK和0R的同源性为99.4
%，而且各分离株与同种动物流感毒株的同源性也均在98%以上:推导的
氨基酸序列分析结果也进一步证实了M基因高度保守。三株分离株之间以
及与其他毒株NS基因核昔酸的同源性及推导的氨基酸序列同源性也都在
98%以上，说明NS基因高度保守;聚合酶基因(PA、PBI、PBZ遗传进化树
分析发现遗传进化路线非常清晰，PA基因、PBI基因、PBZ基因的遗传进
化树中DK和0R分离株均同属一个相对独立的分支，两个分离株聚合酶的
同源性高达98%一99.4%，与香港禽流感病毒分离株形成的分支以及北美
群系代表毒株形成的两个分支并行。
In the present study, three avain influenza viruse (AIV) isolates (A/chick/Henan/88/02 (CK), A/duck/Henan/89/02(DK)and A/Ostrich/Henan/90/02(OR)were studied by testing their pathogenicity , infection spectrum, immunology specificity, biologic and molecular biologic specificity, genetic variation and evolution etc. The results of hemagglutination(HA) test, hemagglutination inhibition(HI) test,ELD50 assay,animal pathogenicity and experimental challenge indicated that three isolates are all high pathogenic avain influenza(HPAI) virus belonging to H5N1 subtype, and the strain OR shown lower pathogenicity to chicken than the other two strains , which reveals that AIV have great host specificity
The isolates were replicated in SPF chicken embryo.Full-length cDNAs of eight genes from each isolate were amplified by RT-PCR and then successfully cloned into vector pMDT-18. All cDNA segments of eight genes from each isolate were sequenced and compared with each other and also with some published sequences of AIVs for their homology .The sequencing result shown that all eight cDNA fragments of each strain included the completed open reading frame, initiator and terminator codon corresponding to AIV genome,respectively.
Analysis of homology and genetic evolution shown that l)The sequence homologies for HA gene between Eurasia lineage strain Ire83 and isolates Ck,Dk and OR were 6.9%,86.0%
and 86.1 % respectively,but only 77.5%, 76.9% and 76.7% between North American lineage strain PA83 and three isolates.All three isolates have a same deduced amino acid(AA) sequence PQRERRRKKR in HA lysing site, with more than 5 Basic amino acids, suggested that the strains are all HPAI viruses.2)The homology of NA gene between DK and OR was 98%, and 95.6% and 96% between Ck and the other two strains, respectively.A deletion of 59 nucleic acid was found at the position 165-224 both in DK and OR. implied that they have very intimate relationship.The homologic analysis of NP gene between three isolates shown great difference in nucleic acid sequenceC 91.1 %-99.5%), but less difference in amino acids homology (94%-99.4%) , suggested that AIV is type-specific.The nucleic acid sequences of M genes from all isolates shown a high homology up to 99%, especially a 99.4% homology between DK and OR.The nucleic acid sequence homology of NA genes between the isolated strains and the published strains from isotype
animals was more than 98% Analysis of deduced AA sequence further confirmed that M gene is highly conservative The homologies between NS gene of the three strains and some published strains were more than 98%, suggested that NS gene is highly conservative.Cladogram analysis of PA PB1 PB2 genes suggested that the genetic evolution route of three isolates is very clear: strains DK and OR belong to the same branch different from which formed by Hongkong and North America strains.Homologies of polymerases between the strains DK and OR were 98%-99.4%.
用无特定病原体(SPF)鸡胚增殖上述3株禽流感病毒，提取病毒基因组总RNA，参考Genebank中对应基因片段的核苷酸序列设计合成8对特异性引物，用反转录-聚合酶链反应(RT-PCR)技术分别扩增各分离株的8个基因片段的全序列cDNA。并将其分别克隆到PMD18-T载体上，转化JM-109感受态菌，获得阳性质粒，并进行了鉴定和序列测定。将测定的序列与参照序列对比，视其同源性高低来判定该序列是否为所测定的基因片段的核苷酸序列。结果表明从每个毒株扩增到的8个基因片段均包含病毒相应的完整开放阅读框架、起始和终止密码子。
运用序列分析软件对各毒株的相关序列进行了同源性和遗传进化分析。结果表明：①三个河南地方株(CK、DK、OR)的HA基因与欧亚种系代表株Ire／83的同源性分别为86.9％、86.0％、86.1％，而与北美种系代表株PA／83同源性为77.5％、76.9％、76.7％，表明三毒株均起源于欧亚种系；三个毒株HA裂解位点的氨基酸序列均为PQRERR-RKKR，且均多于5个碱性氨基酸，说明三个毒株均为高致病力禽流感病毒。②三者NA之间
的同源性在95.6%一98%之间，其中以DK和OR的同源性最高，为98%，
而CK和两者的同源性仅分别为95.670和96%。同时DK和OR在165一224
处均缺少了59个核昔酸，二者在结构上相似，由此可以看出，DK和0R亲
缘关系极为密切，再结合HA基因和生物学特性，提示0R是由DK变异而
来;各毒株之间的NP基因核昔酸同源性差异较大(91.170一99.5%)，而氨
基酸的差异较小(94%一99.4%)，提示AIV具有型特异性;三株分离株M
基因的核昔酸的同源性高达99%以上，尤其是DK和0R的同源性为99.4
%，而且各分离株与同种动物流感毒株的同源性也均在98%以上:推导的
氨基酸序列分析结果也进一步证实了M基因高度保守。三株分离株之间以
及与其他毒株NS基因核昔酸的同源性及推导的氨基酸序列同源性也都在
98%以上，说明NS基因高度保守;聚合酶基因(PA、PBI、PBZ遗传进化树
分析发现遗传进化路线非常清晰，PA基因、PBI基因、PBZ基因的遗传进
化树中DK和0R分离株均同属一个相对独立的分支，两个分离株聚合酶的
同源性高达98%一99.4%，与香港禽流感病毒分离株形成的分支以及北美
群系代表毒株形成的两个分支并行。
In the present study, three avain influenza viruse (AIV) isolates (A/chick/Henan/88/02 (CK), A/duck/Henan/89/02(DK)and A/Ostrich/Henan/90/02(OR)were studied by testing their pathogenicity , infection spectrum, immunology specificity, biologic and molecular biologic specificity, genetic variation and evolution etc. The results of hemagglutination(HA) test, hemagglutination inhibition(HI) test,ELD50 assay,animal pathogenicity and experimental challenge indicated that three isolates are all high pathogenic avain influenza(HPAI) virus belonging to H5N1 subtype, and the strain OR shown lower pathogenicity to chicken than the other two strains , which reveals that AIV have great host specificity
The isolates were replicated in SPF chicken embryo.Full-length cDNAs of eight genes from each isolate were amplified by RT-PCR and then successfully cloned into vector pMDT-18. All cDNA segments of eight genes from each isolate were sequenced and compared with each other and also with some published sequences of AIVs for their homology .The sequencing result shown that all eight cDNA fragments of each strain included the completed open reading frame, initiator and terminator codon corresponding to AIV genome,respectively.
Analysis of homology and genetic evolution shown that l)The sequence homologies for HA gene between Eurasia lineage strain Ire83 and isolates Ck,Dk and OR were 6.9%,86.0%
and 86.1 % respectively,but only 77.5%, 76.9% and 76.7% between North American lineage strain PA83 and three isolates.All three isolates have a same deduced amino acid(AA) sequence PQRERRRKKR in HA lysing site, with more than 5 Basic amino acids, suggested that the strains are all HPAI viruses.2)The homology of NA gene between DK and OR was 98%, and 95.6% and 96% between Ck and the other two strains, respectively.A deletion of 59 nucleic acid was found at the position 165-224 both in DK and OR. implied that they have very intimate relationship.The homologic analysis of NP gene between three isolates shown great difference in nucleic acid sequenceC 91.1 %-99.5%), but less difference in amino acids homology (94%-99.4%) , suggested that AIV is type-specific.The nucleic acid sequences of M genes from all isolates shown a high homology up to 99%, especially a 99.4% homology between DK and OR.The nucleic acid sequence homology of NA genes between the isolated strains and the published strains from isotype
animals was more than 98% Analysis of deduced AA sequence further confirmed that M gene is highly conservative The homologies between NS gene of the three strains and some published strains were more than 98%, suggested that NS gene is highly conservative.Cladogram analysis of PA PB1 PB2 genes suggested that the genetic evolution route of three isolates is very clear: strains DK and OR belong to the same branch different from which formed by Hongkong and North America strains.Homologies of polymerases between the strains DK and OR were 98%-99.4%.
引文
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