Molecular characterization of highly pathogenic avian influenza A (H5N1) viruses isolated from humans in Hunan province, 2006 - 2009

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. Viruses 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 α 2, 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.

Conclusions: 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.