Objective: To elucidate the genetic diversifications of avian influenza subtype H5N1 viruses in the boundary regions of Yunnan province during 2009 to July, 2011.
Methods: Swab samples were collected from foreign poultry and wild birds in boundary regions of Yunnan province during 2009 to July, 2011 and tested by H5/N1 subtype-specific multiplex RT-PCR. The HA genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. Both alignment and phylogenetic analysis were performed with sequences of the known reference strains.
Results: Fifteen different HA sequences were obtained from 36 representative positive samples and could be divided into 2 distinct Clades (2.3.2 and 2.3.4). Through phylogenetic analysis, Clade 2.3.2 and 2.3.4 could then be further divided into 3 (II-1 to II-3) and 2 smaller clades (I-1 and I-2), respectively. The viruses of Clade 2.3.2 II-1 and II-2 were new variant strains of H5N1 virus. The cleavage sites of HA from positive samples all possessed molecular characterization of highly pathogenic avian influenza virus. Mutation of key amino acids had been found among receptor binding sites, potential glycosylation sites, neutralizing epitopes and others.
Conclusion: It seemed evident that the H5N1 subtype viruses showed genetic diversifications and had undergone the evolution progress of multi-clade (2.3.2, 2.3.4) to single calde (2.3.2) in the boundary regions of Yunnan province, during 2009 to July, 2011.