【作者】 郭亮；

【导师】 华育平；

【作者基本信息】 东北林业大学 ， 生理学， 2009， 硕士

【摘要】 多年来本实验室开展了野鸟流行病学监测工作。2006年秋季,从黑龙江省三江国家级自然保护区采集了93个样品,其中分别由两只健康绿头鸭体内分离到2株具有血凝性的病毒,经本实验室确认并经国家禽流感参考实验室鉴定,确定其均为禽流感病毒H3N8亚型,并命名为A/Mallard/SanJiang/98/2006（H3N8）和A/Mallard/SanJiang/167/2006（H3N8）。本论文对这两株分离株进行了全基因组序列测定,并研究其基因特征及遗传演化关系。本研究采用无特定病原体（SPF）鸡胚增殖禽流感病毒野鸭源分离株MSJ98/06和MSJ167/06,从鸡胚尿囊液中提取病毒基因组总RNA,参考GeneBank已发表的H3N8亚型AIV各段基因的核苷酸序列,利用分子生物学软件设计并合成了14对特异性引物,用RT-PCR技术分别扩增MSJ98/06和MSJ167/06分离株的8个基因节段的cDNA（其中HA、PA、PB1、PB2基因均分为两段扩增）。PCR产物经回收纯化后连接到PMD18-T载体上,将连接产物转入DH5-a感受态细胞中,重组质粒经PCR鉴定为阳性后,进行序列测定。序列测定正确后,与国内外H3N8亚型的部分分离株的相应基因及其推导的氨基酸序列进行同源性比较和遗传进化分析。序列分析结果表明,所得到的两株AIV的8个节段均含有完整的开放阅读框架以及5′端和3′端的非编码区序列。根据测得的基因序列绘制遗传进化树发现两个分离株的大部分基因与韩国地区分离株、日本北海道分离株以及中国大陆南昌分离株的亲缘关系密切,有着相似的遗传演化过程,可能具有共同祖先。与北美和澳大利亚的禽源分离株亲缘关系较远,在地理分布上表现出明显的差异。且与所有马源分离株的核苷酸同源率仅有70%左右,说明H3N8亚型流感病毒具有明显的种属差异性。而两株H3N8亚型禽流感分离株的NA基因核苷酸同源率仅有77.3%,差异较大。NS基因分析表明,本研究的两个分离株应属于欧亚禽一马群。HA基因裂解位点处的氨基酸序列分别为³⁴⁰PEKQTR↓SLF³⁴⁸和³⁴⁰PEKQTR↓GLF³⁴⁸,与低致病性禽流感的HA的裂解位点相一致,且已有的研究不支持H3N8亚型禽流感病毒对禽类具有高度致病力,可以推断分离株MSJ98/06和MSJ167/06为非高致病力毒株。由于从野生中分离出H3N8亚型禽流感病毒在我国尚无报道,所以本研究的两株H3N8亚型禽流感病毒全基因的克隆及序列分析结果,对于禽流感的深入研究,特别是对野生水禽禽流感病毒的携带状况和分子流行病学的研究具有重要的参考价值。更多还原

【Abstract】 We Surveyed the epidemiology of wild birds for years.93 samples were captured in Sanjiang National Natural Reserve of Hei Longjiang province in October,2006.Two mallardorigin influenza viruses were sequenced and molecular evolutionarily analyzed in this study. The identification confirmed them as H3N8 subtype by National Influenza Reference Laboratory and our laboratory,and were named as A/Mallard/SanJiang/98/2006（H3N8） and A/Mallard/SanJiang/167/2006（H3N8） respectively.In this research,the isolated strains MSJ98/06&MSJ167/06 were propagated in specific pathogen free（SPF） chicken embryos.14 pairs of primers were designed and synthesized according to the relevant nucleotide sequences in GeneBank,which were used for amplifying the eight full-length genes of isolated strains MSJ98/06 and MSJ167/06.Thereinto,HA,PA, PBI and PB2 genes were divided into two fragments to amplified.The viral RNAs and mRNAs were extracted and used Reverse Transcription-Polymerase Chain Reaction（RT-PCR） to amplify the eight full-length cDNAs of the virus genes.The purified cDNAs were inserted into PMD 18-T vector,and the recombinant plasmid was transformed into competent DH5-a E.Coli, then sequenced when the PCR result was positive for amplifying the inserted gene in recombinant plasmid.The nucleotide sequences and their deduced amino acid sequences were compared and analyzed for the homology and phylogenetic relationships between the isolated strains MSJ98/06&MSJ167/06 and some other isolated strains of H3N8 subtype by the Lasergene Sequence Analysis Package.Sequence analysis-results showed that all of the eight segments of AIV contained completed open-reading-frames and 5’-3’-untranslated regions.Analysis of the results of homology comparison and phylogenetic relationships indicated that the strains were homology with Korea strains,Hokkaido strains and Nan Chang strains from mainland of China. Conspicuous species differences existed among different species and different areas where the virus isolated from.But the homological rates of amino acid was 77.3%for NA gene between MSJ98/06 and MSJ167/06.Analysis of NS gene indicated that they belonged to Eurasian avian-equine Group.Nucleotide sequence of HA genes pointed that there were similar cleavage sites with low pathogenic AIV HA gene which amino acid were ³⁴⁰PEKQTR↓SLF³⁴⁸ and ³⁴⁰PEKQTR↓GLF³⁴⁸ respectively and considered that none of the H3N8 subtype isolates have been found which were highly pathogenic strains to poultry,it should be concluded that isolated strains MSJ98/06&MSJ167/06 were not highly pathogenic avian influenza strains.Because it was the first time of H3N8 subtype AIV to be isolated from wild waterfowls to report in China,the results of molecular clone and analysis of the entire genes of the isolated strains MSJ98/06&MSJ167/06 will have great value for the further researches of avian influenza,especially for the researches of perpetuation status of AIV among wild waterfowls and the researches of molecular epidemiology of avian influenza.更多还原