Isolation And Identification Of Swine Influenza Virus In Henan And Study On The Biological Charactistics

Swine influenza viruses, members of the family Orthomyxoviridae,arenegative-stranded, segmented RNA viruses. A、B and C all three types of influenza can infect pigsand cause the pandemic of Swine influenza(SI). Currently, three main subtypes of influenzaviruses are circulating in the swine population worldwide:H1N1,H3N2and H1N2.Pigs aresusceptible to both human and avian influenza virus and have been proposed to be intermediatehosts or mixing vessels because earlier studies have shown that they have receptors for both avian(SA-alpha-2,3) and swine/human (SA-alpha-2,6) influenza virus in the upper respiratory tractwhich low the species barrier of pigs. The pig has become more and more different subtypes ofinfluenza virus’ intermediate host. Many different subtypes of influenza virus long-termcoexistence in herds, which create conditions to produce rearrangement viruses, will be a potentialthreat to the pig industry and human public health. this gives the SI a pivotal position in theetiology, ecology and epidemiology of human and animal influenza. The international academiccommunity has generally believe that pigs can produce the virus strains with pandemic potentialin the crowd. Therefore, the impact of swine flu, not only in its obvious significance of theVeterinary Epidemiology but also in its far-reaching public health implications.In this study,Nasal swabs samples of pigs with influenza-like clinical symptoms were collectedfrom Henan province in2010.with specific-pathogen-free,embryonated chicken eggs, twoinfluenza viruses were isolated from nasal swabs samples of pigs with influenza-like clinicalsymptoms from Henan province and identified by HA and HI test, meanwhile, All eight genesegments of isolated strains were amplified by RT-PCR and sequenced. The isolation resultshows two stbtypes influenza viruses were isolated and designated A/swine/Henan/1/2010（H3N2）（SW/HN/1/10）、A/swine/Henan/405/2010（H1N1）（SW/HN/405/10）respectively.in order to understand the molecular epidemiology of the two isolated virus strains, homology ofnucleotide and deduced amino acid sequence based on the nucleotide sequence were compared tothe relatived strains which avaliable in GeneBank. The cleavage site,receptor binding sites,potential glycosylation sites and HB sites of HA and the enzymatically active site, glycosylationsites and disulfed bonds and the antigen sites of NA were analyzed to learn the variation and thephylogenetic.SW/HN/1/10virus strain including eight genes, HA consists1728nucleotides, encoding567amino acids. deduced amino acid sequence based on the nucleotide sequence of the HA gene, andfound that its cleavage site sequence is PEKQTR↓G. Do not meet the molecular characteristicshighly of pathogenic strain, suggesting that the isolates is non-highly pathogenic strains.There are 11potential glycosylation sites, which are the same with classic human origin strains such asA/Moscow/10/99, A/Sydney5/97, A/NewYork/333/99and so on. NA consists1477nucleotides,encoding470amino acids. deduced amino acid sequence based on the nucleotide sequence of theNA gene found there was no amino acid insertion or deletion, the enzymatically active site,glycosylation sites and disulfed bonds were strictly conserved, but the antigen sites varied. Alleight gene segments of SW/HN/1/10were sequenced and compaired with H3N2human influenzavirus, classic swine influenza virus and avain influenza virus, which are available in GeneBank.The SW/HN/1/10showed above98.5%sequence homology with human influenza Virus;Phylogenetic analysis showed that SW/HN/1/10was clustered in the same groups as the humaninfluenza viruses. These results indicated that SW/HN/1/10probably was originated from humaninfluenza virusesSW/HN/405/10virus strain including eight genes, HA consists1701nucleotides, encoding566amino acids. deduced amino acid sequence based on the nucleotide sequence of the HA gene,andfound that its cleavage site sequence is IPSIQSR↓G, consistent with the sequence of allreference strains and no basic amino acids consist. There are7potential glycosylation sites, whichare the same with European strains (Sw/cd/1999), compaired to the Hong Kong strain(Sw/hh/2001) and the Inter-American strains (Swlwi/1998), and domestic Guangdong strains(Sw/gd/2001),there are three points difference. NA consists1477nucleotides, encoding470amino acids. deduced amino acid sequence based on the nucleotide sequence of the NA genefound there was no amino acid insertion or deletion, the enzymatically active site, glycosylationsites and disulfed bonds were strictly conserved, but the antigen sites varied. There are6potentialglycosylation sites, which are the same with Avian-like H1N1SIV. All eight gene segments ofSW/HN/405/10were sequenced and compaired with H1N1human influenza virus, classic H1N1swine influenza virus and avain-like influenza virus, which are available in GeneBank. TheSW/HN/405/10showed a high sequence homology(92.1％-99.4％) with avian-like H1N1influenza Virus; Phylogenetic analysis showed that SW/HN/405/10was clustered in the samegroups as the avian-like H1N1virus. These results indicated that SW/HN/405/10probably wasoriginated from avian-like H1N1SIV.In this study, two different subtypes of swine influenza virus were Isolated from Henan and thewhole comLete genes were sequenced,the genetic evolutionary relationships were analysised andexplored the source of the virus’s genes from the molecular level. Theose results are very usefullto the further research of antigen changes of SIV in Henan region even in the whole country andprovide a scientific basis for influenza prevention and control work in China.