【作者】 杨彩然；

【导师】 马学恩； 陈化兰； 赵振华；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2005， 博士

【摘要】 本论文对来自中国安徽、福建、浙江、江苏省的 14 株鸭源和来自中国四川的 2株鸡源的禽流感病毒(AIV)分离株进行了亚型鉴定、全基因序列测定和分析,从中挑选出 8 个毒株用于鸡的致病性试验研究。 16 株禽流感病毒的序列分析:经亚型鉴定,确认 16 株流感病毒中,12 株为 H3亚型流感病毒,其中 1 株 H3N2 亚型, 1 株 H3N6 亚型,10 株 H3N1 亚型;4 株为 H4N6亚型。通过提取病毒的核酸、设计特异性引物,用 RT-PCR 方法扩增所有毒株的各个基因片段、克隆到 pMD18-T 载体、测序、拼接序列等一系列步骤,最后绘制进化树。 H3 亚型 HA1 基因进化分析结果:本研究 10 个 H3N1 亚型分离株和 1 个 H3N2 亚型分离株聚成一支,它们与 1963 年乌克兰病毒分离株 A/Duck/Ukraine/1/63(H3N8)同源率最高,达 87.7%～88.7%;分离株 DK/SZH/1/02(H3N6)另成一支,其与 A/Aquartic Bird/Hong Kong/399/99 (H3N8) 同源率最高,达 95.4%。两分支间同源率为 86.6%～87.5%。 10 个 H3N1 亚型流感病毒分离株的 N1 基因形成一个独立的小分支,它们之间同源率达 98.7%～100%,与基因库中的 Env/HK/437-6/99(H5N1) 同源率最高,在93.3%～93.8%之间。DK/AH/271/02(H3N2)毒株的 N2 基因与基因库中的A/Ostrich/South Africa/9508103/95 (H9N2)的同源率最高,达 94.3%。 12 个 H3 亚型毒株的 NP 基因中,DK/AH/271/02(H3N2)、 DK/SZH/1/02(H3N6)与 AQUB/HK/399/99(H3N8)、DK/GD/40/00(H5N1)聚在一起,两个分离株 DK/AH/271/02(H3N2)与 DK/SZH/1/02(H3N6)的 NP 基因同源率为 96.1%,其余 10 个 H3N1 亚型的NP 基因与 SW/Fujian/1/03(H5N1)的形成一小分支,此小分支的 H3N1 亚型分离株之间 NP 基因同源率为 96.6%～100%。 H3 亚型分离株 M 基因分为两个分支,其 中 DK/YZH/314/03(H3N1)、DK/YZH/341/03(H3N1) 、 DK/SZH/1/02(H3N6) 聚 成 一 支 , 它 们 与WDk/ST/1737/00(H6N8)的 M1 基因核苷酸的同源率最高;其余分离株另成一支,它们与 DK/Primorie/2633/01(H5N3)的 M1 基因核苷酸的同源率最高。两支分离株之间,M1 基因的同源率为 93.3%～94.9% H3 分离株的 NS 基因分为 A 和 B 两组,本研究的 DK/YZH/314/03(H3N1)、DK/YZH/341/03(H3N1)、和 DK/SZH/1/02(H3N6) 分离株属于 B 组,而其它分离株归 A组。 H3 亚型分离株的 PB2 基因分为 4 个小支。DK/SZH/1/02 为一支,DK/AH/271/02为另一支,DK/YZH/314/03 和 DK/YZH/341/03 在一起,其余 H3N1 分离株为一支。后两支间的同源率仅为 87.9%～88.3%。更多还原

【Abstract】 14 duck-origin and 2 chicken-origin influenza viruses isolated from the China continent were subtyped,sequenced and molecular evolutionarily analyzed in this study. These viruses were isolated from different regions,including Anhui province, Fujian province, Zhejiang province, Sichuan province and Jiangsu province.8 strains were chosen out and evaluated biological characteristics. Sequence analysis:The 16 isolates were identified as 12 H3(including 1 H3N2,1 H3N6,10 H3N1) and 4 H4N6 viruses. The viral RNA of the all influenza viruses conducted in the research were extracted from the viruses propagated in specific-pathogen-free chicken embryos. Viral RNA was reverse transcribed using an oligodeoxynucleotide primer(5’AGCAAAAGCAGG).PCR was performed with gene-specific primers.Amplified products of the expected sizes were purified and cloned into pMD-T plasmid.The insert in the recombinant plasmid was sequenced with specific primers.Finally, the Lasergene Sequence Analysis Package were used for data analysis, drawing phylogenetic tree and molecular evolution analysis. Phylogenetical analysis of HA1 region of H3 HA found that it involved two subgroups.DK/SZH/1/02(H3N6) isolate together with A/Aquartic Bird/Hong Kong/399/99 (H3N8) belonged to one subgroup.The rest of H3 viruses belonged to another subgroup which had the highest homology with A/Duck/Ukraine/1/63(H3N8), with 87.7%～88.7%. The phylogenetic tree of 10 N1 gene of H3N1 subtype influenza viruses involved one subgroup with homology between 98.7% and 100%.They had the highest homology with Env/HK/437-6/99(H5N1),with 93.3%～93.8%. DK/AH/271/02(H3N2)strain had the most relation to A/Ostrich/South Africa/9508103/95 (H9N2),with 94.3% identity. NP gene sequence data showed DK/AH/271/02(H3N2)and DK/SZH/1/02(H3N6) isolates clustered with AQUB/HK/399/99(H3N8) and DK/GD/40/00(H5N1). DK/AH/271 /02(H3N2)and DK/SZH/1/02(H3N6) are relevant each other, with 96.1% similarity.NP genes 0f 10 H3N1 were put together with that of SW/Fujian/1/03(H5N1),with 96.6%～100%. The M genes of 12 H3 subtype viruses divided into 2 Supgroups: DK/YZH/314/03(H3N1), DK/YZH/341/03(H3N1) and DK/SZH/1/02(H3N6) isolates together with WDk/ST/1737/00(H6N8) belonged to one subgroup. The rest of 12 M genes belonged to another subgroup which had the highest homology with DK/Primorie/2633/01(H5N3).M1 gene homology was 93.3%～94.9% between two subgroups. The NS gene had two separate groups. DK/YZH/314/03(H3N1), DK/YZH/341/03 (H3N1),DK/SZH/1/02(H3N6) isolates belonged to NS group A and the rest of isolates belonged to group B. The PB2 genes Of H3 subtype were divieded into 4 sublineages. DK/SZH/1/02 isolate belonged to one sublineage. DK/AH/271/02 strain was in the second sublinage. DK/YZH/314/03 and DK/YZH/341/03 viruses formed the third group.The rest of H3N1 subtype PB2 genes formed the last group. PB2 genes homology was 87.9%～88.3% between the third and the fourth subgroup. All of the PB1 genes Of the subtype H3 were most homologous to that of DK/Guangxi/53/02(H5N1) virus with identities 94.5%-95.1%. The PA genes Of the subtype H3 had three subgroups. DK/SZH/1/02(H3N6) had the highest homology with DK/GX/35/01(H5N1),with 96.3%. DK/YZH/314/03(H3N1) and DK/YZH/341/03(H3N1) formed another group.The rest of H3 was one subgroup.The homology of PA genes was 91.4%～91.8% between the last two groups. Phylogenetic analysis of H3 revealed that HA,NA,PB1 and NP genes of all 10 subtype H3N1 viruses were similar. Difference were seen ,however,in PB2,PA,M and NS. H3N1 viruses in this study were divided into two genetypes according to 95% similarity. Phylogenetic analysis of H4 showed that 2 duck-origin isolates clustered each other,and 2 chicken-origin isolates were together. Pathogenicity for SPF chickens of H3, H4 viruses.8(1 H3N2 virus,1 H3N6 virus,2 of H3N1 viruses and 4 H4N6 viruses) of 16 isolates were used. Sixty-four 4-week-old SPF chickens were randomly divided into eight groups with 8 chickens in each group.Birds were challenged by intravenous(wing vein)inoculation of 0.1ml of virus. The addi更多还原