Phylogenetic analysis of eight genes of H9N2 subtype influenza virus: a mainland China strain possessing early isolates' genes that have been circulating.

H9N2 subtype influenza virus has become worldwide and prevalent in China. Previous studies illustrated that at least three sublineages had been established in terrestrial poultry of Eurasian avian. In this presentation, eight full-length genes of an H9N2 strain, A/Chicken/Shanghai/F/98 (Ck/SH/F/98) were obtained. Sequence analysis and phylogenetic studies were conducted by comparing eight genes with those of all the available H9N2 strains from the GenBank. The results showed that four genes (HA, NA, M and NS genes) of Ck/SH/F/98 were incorporated into the sublineage represented by the early mainland China strain, Ck/BJ/1/94. However, the other four of RNP genes of Ck/SH/F/98 did not show close relationship with those of the three known sublineages' viruses. Therefore, Ck/SH/F/98 was a natural reassortant between different sublineages. In addition, comparison showed that Ck/SH/F/98 could be a putative precursor of a later isolate from southern China, Dk/ST/1605/01, with at least six genes of both closely related, indicating genes of Ck/SH/F/98 and early isolates had ever been circulating. Further comparison in terms of molecular markers of species specificity of HA1 revealed that DK/ST/1605/01 also resembled Ck/SH/F/98 more than a common earlier duck strain. The results supported the idea of two-way transmission between terrestrial and aquatic birds that emphasized the importance to raise concerns on the natural evolution of all the eight genes of H9N2 avian influenza viruses.

[Generation of attenuated H5N1 and H5N2 subtypes of influenza virus recombinants by reverse genetics system].

The HA connecting peptide at cleavage site, PQRERRKKR / GL, of an H5N1 subtype avian influenza virus (AIV) was replaced with PQRESR / GL, and then the modified HA gene was cloned into the transcription/expression vector, pHW2000, constructing a plasmid named pHW524-HA. The NA (N1) gene from the H5N1 virus and the NA (N2) gene from an H9N2 AIV were also cloned into pHW2000 separately, resulting in plasmids pHW506-NA and pHW206-NA. With the organization of pHW524-HA, pHW506-NA or pHW206-NA, and six plasmids containing internal genes from A/WSN/33 backbone virus, two transfectants, H5N1/WSN and H5N2/WSN, were subsequently generated by eight-plasmid system. After 15 consecutive passages in embryonated eggs, the two recombinants grew up to high titers of 1:2(9) in hemagglutination test with no changes in nucleotide sequences of the surface genes detected. Both the recombinant viruses belonged to mildly pathogen when evaluated by the pathogenicity test in six-week-old SPF chickens. H5N2/WSN recombinant virus was obviously less pathogenic than H5N1/WSN virus for embryonated chicken eggs. This presentation showed that the reverse genetics system is a very useful tool for studying the construction and function of individual genes and for the generation of virus as vaccine candidate.

[Construction of recombinant fowlpox virus expressing chicken IL-2 and assay of biologic activity of the product in vitro].

In order to determine the adjuvant effects of the chicken IL-2 (ChIL-2) on new generation vaccines, ChIL-2 gene was amplified from ConA-stimulated chicken spleen cells by RT-PCR and was directionally inserted into fowlpox virus (FPV) transferring vector p1175 under the control of FPV early/late promoter (PE/L), resulting in recombinant transferring vector p1175IL2. Then the p1175IL2 plasmid was transfected into chicken embryo fibroblasts (CEF) pre-infected with wild type FPV to generate recombinant fowlpox virus expressing ChIL-2 (rFPV-IL2). By selection of blue plaques on the CEF, overlaid with agar containing X-gal, rFPV-IL2 was obtained and purified. The supernatant from CEF monolayer infected with rFPV-IL2 (M.O.I2.0) after 72 hours was detected for the production of ChIL-2 by XTT/PMS colorimetric assay. About 3.6 x 10(5) u/mL of specific ChIL-2 activity was determined. The results show that rFPV-IL2 can express ChIL-2 effectively. rFPV-IL2 provides us with an effective tool for studying avian immunology as well as a potential vaccine-enhancing agent.