The Research Of The Biological Characteristics Of Monoclonal Virus H5N1,H5N6,H6N6 Derived From A Mixed Infection Virus And The Recombination Mechanism Study Of H5N6 Avian Influenza Virus

For years,the avian influenza virus has been a kind of very harmful virus,especially the highly pathogenic avian influenza virus(HPAIV)which can cause a pandemic,not only brings a serious negative impact on the development of animal husbandry,but also cause a great threat to human health.The pandemic of influenza virus has a great relationship with the new emergence of a recombinant virus,influenza virus can acquire the advantages in terms of replication efficiency,virulence and host adaptation by recombinant with another different subtype influenza virus.Therefore,the investigation of genome characterization and recombination mechanism of influenza virus will provide important theoretical basis for people to understand and prevent influenza virus.The world's first human infected with the H5N6 avian influenza virus and death cases was reported in Sichuan province in May,2014.The virus was formed by the recombinant between H5N1 and H6N6 virus.H5N1 and H6N6 avian influenza viruses have long existed in the poultry markets in our country,which provides the condition for virus recombinant.We isolated a mixed subtype virus of H5N1 and H6N6 from Yueyang,Hunan province in December 2011,in which the H5 copy number is correspond with H6.This indicates that the virus is in the middle of the process of restructuring.In order to understand the presence situation,the composition of the genome segments and biological characteristics of the different subtypes viruses,we selected the H5N1/H6N6 mixed-subtype virus named(A/duck/Hunan/747/2011(mixed))for our study.The virus is isolated and purified by the methods of plaque-purification assay and specific serum neutralization test.We finally acquired 95 monoclonal viruses,including 11 H5N1 influenza viruses,6 H6N6 viruses,and it is noteworthy that we get 78 H5N6 influenza viruses.The data reveals that H5N6 virus has an advantage in the replication process.Then the whole genome of 18 H5N6 viruses,9 H5N1 viruses and 6 H6N6 viruses were sequenced and the phylogenetic trees was dralfted.The genome and phylogenetic trees analysis indicated that the homology of 27 H5HA is 94.3%-100%,all the H5HA belongs to calade 2.3.2.1 and the homology of 6 H6HA is 99.8%-100%,H6 belongs to the group ? lineage.NA genes are similar with HA genes.While the internal genes present a diversity of restructuring,for example,8 PB1 segments of H5N6 viruses are highly homologous with H5N1 PB1 segment,and the other 10 are highly homologous with H6N6 PB1 segment.We compared the amino acid sequence and found that individual strains appeared important amino acid residues substitution or deletion.For further investigation,we selected three H5N1 viruses,two H6N6 viruses,four H5N6 viruses from the monoclonal strains,plus two mixed-subtype virus and two H5N6 viruses isolated from Yueyang,Hunan in 2013 and 2014 respectively.To evaluate the replication capacity and virulence of these viruses,in vivo experiments were performed.The growth curve reflected that H5N6 virus are showing growth advantage;the mice that infected with H5 virus showed significant clinical symptoms,a larger proportion of weight loss and varying degrees of lethality.While the mice infected with H6N6 virus showed no apparent clinical symptoms,weight loss is not obvious and no lethality.In summary,H5N6 virus acquires better replication capacity and higher pathogenicity by gene restructuring and mutation,which brings challenge for the prevention and control of this virus.Our study will provide important theoretical basis for the prevention and control of H5N6 avian influenza virus.