| The H9N2 subtype avian influenza virus was first isolated from chickens in southern China in 1994, since then it had circulated in Southeast Asia including China and many other countries for several years. However, the pandemic of H9N2 subtype AI, which started in summer of 1998, spread very rapidly to more than 20 provinces within several months, Which raised great concerns for its economic importance to poultry industry and potential threat to the public health. In order to investigate the molecular mechanism affecting route of transmisson for H9N2 subtype AIV. So in this study, the H9N2 subtype avian influenza virus strain A/Chicken/Guangdong/SS/94(C/GD/SS/94), isolated from mainland China was chosen for sequence analysis of its inner genes, And also, its pathogenicity test along with another H9N2 subtype Avian Influenza Virus strain, A/Chicken/Shanghai/7/01, were performed here to investigate the property of transmisson and replication for H9N2 subtype avian influenza viruses.1 Sequence analysis of the six full-length inner genes of an H9N2 subtype AIVA previously characterized AIV, C/GD/SS/94(H9N2), was propagated in embryonated 9~10 days old SPF chicken eggs and virus in the allantoic fluid was examined by HI and HA tests. Viral RNAs were extracted and RT-PCR was performed to amplify the six inner genes segments (PB1, PB2, PA, NP, M and NS) with several sets of specific primers designed according to published sequences and the reference of Hoffmann.Diffierent length of inner genes sequences were downloaded from the genebank. Subsequently, homology comparison and phylogenetic analysis were performed with the six inner genes sequences from C/GD/SS/94. Phylogenetically, significant difference was observed between C/GD/SS/94 and Q/HK/G1/97, a typical H5N1 subtype virus responsible for 1997 Hong Kong Bird flu incident and associated with human infection. The protein sequences of C/GD/SS/94 were highly homology with C/GD/5/97 strain isolated 3 years before C/GD/SS/94, which suggested that the inner genes of C/GD/5/97 probably come from the strain C/GD/SS/94. By the homology comparison and phylogenetic analysis, we found that the six inner genes of C/GD/SS/94 shared 95.7%~99.4% homology with C/BJ/1/94, indicated that these two viruses may derive from the same ancestor virus.2 Pathogenicity in 4 week SPF Chickens with H9N2 subtype AIVsIn order to investigate the property of transmisson and replication for H9N2 subtype avian influenza viruses, strains of A/Chicken/Guangdong/SS/94(H9N2)(SS) and A/Chicken/Shanghai/7/01(H9N2)(62) were compared in their pathogenicity. Based on the fact that SS strain could transmitted in chickens only by direct contact, the pathogentic difference in 4 week SPF chickens between these two strains were examined.Two H9N2 subtype AIVs of SS and 62 mentioned above were propagated in embryonated 9~10 days old SPF chicken eggs and virus in the allantoic fluid was examined by HI and HA tests. The HA-and HI-positive allantoic fluids were diluted and inoculated 4 weeks SPF chickens throug tranasal, trachea and eyes with the dose of 200μl per chicken. Two chickens were killed respectively for every group at day 3, 5, 7, 9 and 11 post-inoculation, and then the organs such as trachea, small intestine, lung, liver, pancreas, spleen, bursa, kidney were taken and fixed in the 10 percent formalin. Subsequently, paraffin sections were made for pathological examination by the optical microscope and for immunohistochemical detection. The results showed that viral antigens could only be detected in kidney of the chickens inoculated with SS, Whereas viral antigen only presented on the lungs in 62 group, which indicated that the transmission style might have altered between these two isolates.
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