ABSTRACT
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.
ABSTRACT
Objective To study the genetic characteristics of avian influenza virus strain A/Zhejiang/16/2006 which was isolated from the case first reported in Zhejiang province.Methods Complete genome of A/Zhejiang/16/2006 including eight segments were sequenced and compared on the genetic homogeneity with sequences of the similar strains provided through domestic and overseas sources.Results There were 11 amino acids showing differences on HA between A/Zhejiang/16/2006 and the H5N1 isolates of neighboring countries,but these differences had not affected the stability of glycosylation sites.In the NA region,20 amino acids located in the 49th to 68th position were found absent in the isolates obtained after 1997.Eight segments of H5N1 isolates,circulating in the mainland of China in the recent years,formed a separate branch compared to the strains in neighboring countries and there was also obviously different from the strains isolated in Hong Kong and Guangzhou in 1996 and 1997.However,several Chinese strains were close to the Hong Kong strains isolated in 1997 but diffferent from the current strains in the phylogenetic tree.Conclusion The influenza virus strain A/Zhejiang/16/2006 formed a separate branch with the strains isolated in the mainland of China in the past years but it presented an obvious difference with the isolates from the neighboring countries.
ABSTRACT
The 1.7 kb full-length hemagglutinin (HA) gene fragment of H5N1 subtype avian influenza virus was amplified by RT-PCR and then cloned into the pFastBacHT donor plasmids. The recombinant plasmid pFastBac-H5 was identified by restriction enzyme digestion and sequencing. Following the transposition pFastBac-H5 into the bacmid in DH10Bac E.coli competent cells, the colonies were identified by blue and white selection. The recombinant bacmid (rBacmid-H5) was verified by PCR analysis. Transfection of rBacmid-H5 DNA into sf9 cells using Cellfectin reagent results in the production of recombinant viral stock. Cells were harvested 72h post infection and analyzed by SDS-PAGE, Western-blot, hemagglutination and hemagglutination inhibition test;the expressed HA protein (rH5)shows hemaggluting activity and can be inhibited by H5N1 virus immunized chicken sera. On the other hand, immunization of chickens with rH5 protein results in high titers of H5N1 virus specific hemagglutation inhibition antibodies, which proved its biological activity.