| 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...
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