Objective To understand the possible origins,genetic re-assortment and molecular characterization of 4 highly pathogenic avian influenza A(H5N1)viruses isolated from humans in Hunan province,between 2006 and 2009,

Methods H5N1 PCR test-positive specimens were inoculated in embryonated eggs while H5N1 virus was isolated and genomes sequenced.Genome homology and genetic molecular characterization were analyzed by BLAST and MEGA 4.0.

Results All gene segments of the 4 viruses were avian in origin.No re-assortment was found between avian influenza A(H5N1)viruses and human seasonal influenza viruses.Virnses that isolated from domestic poultry shared high similarity with the 4 human viruses in gene homology.Data from the whole genome phylogenetic analysis showed that the 4 viruses were in clade 2.3.4,while 2 viruses belonged to genotype V,and another 2 were new genotypes.Results from molecular characterization showed that amino acid sequences of HA cleavage site of the 4 viruses were PLRERRKR/G.All 4 viruses had A160T mutation in HA,a 20 amino acid deletion in the neuraminidase(NA)stalk at position 49-68,and a 5 amino acid deletion in the non-structural protein 1(NS1).Most sites in the HA molecules showed that the viruses preferentially bound to avian influenza virus receptor.However,T192I mutation that might enhance the α2,6-linked sialic acid human influenza receptor binding had emerged in HN/1/09 and HN/2/09.D701N mutation of PB2 that increased the virulence in mice was found in HN/1/08.Analysis on drug resistance gene amino acid showed that all 4 viruses were sensitive to amantadine and oseltamivir.

Conclusion Highly pathogenic avian influenza A(H5N1)viruses isolated from humans in Hunan province from 2006 to 2009 were avian in origin,and the 4 viruses belonged to different genotypes.Some mutations that related to virulence and receptor binding positions had emerged in some of the strains.