Evolution Of H1N2 Swine Influenza Viruses And Preparation Of Monoclonal Antibodies Against HA Protein Of H1 Influenza

Swine influenza virus (SIV) is one of important pathogens that cause respiratory disease in pigs. At present, the subtypes of SIV includes H1N1, H1N2, H1N7, H2N3, H3N1, H3N2, H3N6, H4N6, H5N1 and H9N2. Several reassortant H1N2 SIV strains were isolated from some provinces in recent years, since the first H1N2 SIV strain was isolated in China in 2004. Whether or not an epidemic swine influenza disease induced by these viruses would break out in pigs should be further studied.In the present study, the specific primers for RT-PCR and sequencing were designed based on the preference sequences of H1N2 viruses. All the eight gene fragments of SW/HeB/10/06 and SW/TJ/1/07 are sequenced and genetically analysised with bioinformatic softwares. Results of genetic analysis showed that SW/HeB/10/06 and SW/TJ/1/07 are far in relatives. Homology of eight gene fragments of the two strains is: PB2 81.4%,PB1 83.2%,PA 83.0%,HA 90.1%,NP 81.6%,NA 91.7%,M 84.1%,NS 80.4%. HA and NA gene fragments of SW/TJ/1/07 shows highest homology with corresponding genes of SW/ZJ/1/04 (H1N2), and other internal gene fragments with two H1N1 SIV strains in europe. HA, NA and NS gene fragments of SW/HeB/10/06 shows highest homology with A/Turkey/MO/24093/99,and other internal gene fragments with H1N1 human influenza virus, H3N2 and H1N2 swine influenza strains. SW/HeB/10/06 is proved to be a new reassorted virus different from other H1N2 SIV, which is the first H1N2 SIV including human,avian and swine influenza genes in mainland.We proceed HI test among 4 H1 influenza viruses and serum. According to the results, SW/HeN/11/05 was selected to be propagated in embryonated eggs. Allantoic fluids were harvested and puried. Mixed with Freunds adjuvant, the viruses were injected into BALB/c female mice. Two monoclonal antibody(McAb)hybridoma cell strains were developed by fusing SP2/0 cells with spleen cells of mouse that had been immunized with SIV SW/HeN/11/05 (H1N1).Cloned several times, positive hybridoma cells strains 4E6B2,3E11C4 were determined by HI and ELISA . In HI test, The two antibodies show specific activity in HI, no activity with H3, H5 and H9 swine influenza virus and other pathogens of swine.In summary, genetic relationship of these two SIV stains were analysed in the study. The results showed that the two new reassorted SIV strains were far in relatives. The strains SW/TJ/1/07 and SW/HeB/10/06 provides evidence for producing new reassorted influenza viruses with pig as a vessel. Meanwhile, two monoclonal antibody(McAb)hybridoma cell strains against HA protein were developed, providing materials and conditions for detecting H1 SIV.