Baculovirus Expression The HA And NA Genes Of Swine Influenza Viruses And The Establishment Of Indirect ELISA For H1 And H3 Subtype

Swine influenza is an acute respiratory disease caused by influenza A virus within the orthomyxoviridae family. The primary clinical manifestations of viral infection are fever and acute respiratory disease. Swine influenza was first observed in 1918 at the time of the human pandemic and the virus was isolated and identified in 1930 by Shope. Pigs may act as an intermediate host of interspecies transmission or as mixing vessels for the emergence of new isolate with human pandemic potential. Human influenza viruses that infect pigs may become a new swine influenza virus. Currently, three main subtypes of influenza viruses are circulating in the swine population throughout the world: H1N1, H3N2, and H1N2.The hemagglutinin (HA) and neuraminidase (NA) gene fragment of swine influenza virus A/Swine/Guangdong/LM/05(H1N1) and A/Swine/Henan/S4/04(H3N2) was amplified with HA and NA gene's specific primers and cloned into baculovirus transfer plasmid pFASTBacGP67. The recombinant plasmid pFastBacGP67-HA and pFastBacGP67-NA was identified by restriction enzyme digestion and gene sequencing. Following the transformation of DH10Bac Escherichia coli component cells by pFastBacGP67- HA and pFastBacGP67-NA, recombinant bacmids rBacmid- HA and rBacmid- NA were identified by blue/white selection and PCR analysis. Then recombinant baculovirus r-Bv was rescued by lipofectant reagent Cellfectin induced rBacmid- HA and rBacmid- NA DNA transfection of the long-phage sf9 insect cells. The recombinant HA and NA protein was characterized by hemagglutination test (just for the target HA protein), SDS-PAGE, western-blot and immunohistochemistry (IHC).An indirect enzyme-linked immunosorbent assay (ELISA) was assessed to detect pigs IgG against H1 and H3 subtype SIV present in Inner Mongolia, Liaoning and Heilongjiang provinces. 31.15 per cent (29 of 93) serum samples tested from swine reared in commercial herds were positive for H1 SIV subtype; 46.24 per cent (43 of 93) serum samples tested from swine reared in commercial herds were positive for H3 SIV subtype; also, 18.28% per cent (17 of 93) serum samples tested from swine reared in commercial herds were positive for H1 and H3 SIV subtype. However, all irrelevant control sera tested were negative. We conclude, therefore, that ELISA performed with recombinant HA as coating antigen is a better tool for swine influenza surveillance in China.