The Research Of Biological Characteristics And Genome Evolution Of Swine Influenza Virus Isolated In Henan Province

This paper studied the research of biological characteristics andgenome evolution of swine influenza virus isolated in Henan province.Swineinfluenza (SI) is caused by swine influenza virus (SIV)that can have significanteconomic impact on the swine industry worldwide.The primary clinicalmanifestations of viral infection are fever and acute respiratory distress. Swineinfluenza virus (SIV), the causative agent is known as influenza A virus within theOrthomyxoviridae family with a segmented RNA genome.The virus contains eightsegments of single-stranded RNA (ssRNA) which encode11proteins, PB2, PB1,PB1-F2, PA,HA, NP,NA,M1,M2,NS1and NS2. Currently, three main sub-types ofinfluenza viruses are circulating in the swine population throughout theworld:subtypes H1N1, H3N2and H1N2.These include classical swineH1N1,avian-like H1N1,human-like or avian-like H3N2, reassortant H3N2andvarious genotype H1N2viruses.To further understand the prevalence of swine influenza in Henan province,360porcine nasal swabs and35swine lung and spleen samples of pigs that showed clinicsigns of respiratory symptoms were collected from some cities in Henan province.From2010to2012, we carried out swine influenza virus surveillance in Henanprovince. In this study, we reported classical H1N1and novel ressortant H1N2influenza viruses from pigs in Henan province.Homology and phylogenetic analysesshowed that the H1N1viruses, A/Swine/Henan/01/2012(H1N1) andA/Swine/Henan/02/2012(H1N1) was closely to classical H1N1viruses and seemedto be derivedfrom the American swine H1N1viruses.and the three H1N2viruses(A/Swine/HeNan/01/2010(H1N2),A/Swine/HeNan/02/2010(H1N2),A/Swine/Henan/01/2012(H1N2)were novel ressortant H1N2influenza viruses containinggenes from the classical swine, human and avian lineages, which indicted that thereassortment among human, avian, and swine influenza viruses had taken place inpigs in Henan and resulted in the generation of new viruses.And the biological characterizations of swine influenza virus were tested andanalysed. At last, three strains of swine influenza virus were isolated by the chicken embryo inoculation method. Results showed that two swine influenza virusesisolated were H1N1subtype and one swine influenza virus isolated was H1N2subtype, namedas:A/Swine/Henan/01/2012(H1N2),A/Swine/Henan/01/2012(H1N1)andA/Swine/Henan/02/2012(H1N1) respectively. On this basis, these isolation strainswere purified by limited dilution cloning. Biological characteristics research showed:the isolates had hemagglutinin to1%red cells of chicken, rabbit, mouse and bovinerespectively.Hemagglutinin thermostability test results displayedA/Swine/Henan/01/2012(H1N1) EID50=10-4.6,A/Swine/Henan/02/2012(H1N1)EID50=10-8.22,A/Swine/Henan/01/2012(H1N2)EID50=10-5.64,A/Swine/Henan/01/2010(H1N2)EID50=10-5.86,,A/Swine/Henan/02/2010(H1N2)EID50=10-3.83,A/Swine/Henan/01/2012(H1N1)ELD50=10-4.46,A/Swine/Henan/02/2012(H1N1)ELD50=10-6.16,A/Swine/Henan/01/2012(H1N2)ELD50=10-5.54,A/Swine/Henan/01/2010(H1N2)ELD50=10-6.54,A/Swine/Henan/02/2010(H1N2)ELD50=10-2.43.A/Swine/HeNan/01/2010(H1N2)showed higher pathogenicity to chicken embryo, of which EID50and ELD50were10-7.16and10-6.54.In addition, the isolates were sensitive to the chemical reagent ofEthyl ether, chloroform and acids of pH3.0. And the different strains of EID50andELD50were different b iological characteristics with each other, infection of micewere ill after the onset of4days.To analyze the genetic variation and recombination of the virues issolated inHenan, Full-length sequence of typical H1N1and H1N2influenza virus wasdownloaded from NCBI database. MEGA5.0software was used to connect andalign the eight fragments of the virus. Then the fragments of different subtypes suchas H1N1and H1N2of the historical strains from different hosts, including human,poultry and pigs, were connected and aligned in the same way. The phylogenetictrees were constructed by NJ method The trees showed that Full length sequence ofthe H1N2swine influenza viruses had a highest similarity with swine H1N2virusisolated from North American ressortant swine influenza viruses.While the H1N1swine influenza viruses belonged to the Classical swine influenza viruses. In thispaper, we describe, for the first time, the isolation and genetic analysis of threequatriple-reassortment viruses from pigs in a slaughterhouse of Henan province. Inaddition,we also depict the isolation and genetic analysis of two swine H1N1influenza viruses. It provides further evidence that pigs serve as intermediate hosts or “mixing vessels”, and swine influenza virus surveillance in Henan should be given ahigh priority.