Molecular Epidemiology Investigation And Variation Analysis Of H5Subtype Avian Influenza Of Somewhere In China

Over the past two decades, the poultry sector in China went through a phase of tremendous growth as well as rapid intensification and concentration. Highly pathogenic avian influenza virus (HPAIV) subtype H5N1was first detected in Guangdong province in1996. It had seriously affected the development of Asian poultry industry since their recurrence in2003. Since then, control of the disease in China has relied heavily on wide-scale preventive vaccination combined with quarantine and stamping out. This strategy has been successful in drastically reducing the number of outbreaks in recent years. However, H5N1AIV is still circulating and is regularly isolated in poultry in China, which represent a public health hazard for human health. Therefore, we must further investigate the genetic variation and epidemiology of H5N1avian influenza viruses strains in poultry.In this study, cloacal and tissue samples were collected from live poultry marketes and farms in7regions in China during2011-2012. H5N1AIV was detected from2110samples by reverse transcription-PCR (RT-PCR). We found that the total positive rate was7.25%. There was a very high infection rate in duck (approximately14.83%), followed by chicken (3.32%) and dove (2.50%). The samples of quail and goose were negative. Furthermore, we analyzed the relation between the AIV-infection rate and different seasons. The positive rate of samples which were collected during the spring and autumn was7.50%and7.96%, which was higher than other seasons. In addition, the infection of different geographical regions was also further analyzed. There was higher infection rate in regions (1and3), when compared with other regions. Meanwhile, the positive rate of different live poultry marketes and farms was also analyzed. We found that the positive rate of live poultry marketes was more higher.To further understand the genetic variation of H5N1avian influenza viruses strains in this region,26positive samples were subjected to the RT-PCR amplification of the whole-genome sequences and the complete HA gene sequences using the universal primers, and then sequenced. Twenty-four reference sequences were downloaded from GenBank database. Sequence data was analyzed using both Lasergene DNAstar (version7.1) and DNAMAN (version6.0). The phylogenetic tree was constructed by using the neighbor-joining method with MEGA5.1. The results showed that all the HA gene sequences from26H5N1virus strains sharing91.2-99.9%nucleotide sequence identity. Simultaneously, the NA, NS, M, NP, PA, PB1and PB2of5strains gene sequences were highly homologous to each other, sharing96.9%-99.8%,96.3%-100.0%, 98.6%-99.8%,96.5%-99.9%,93.1%-99.8%,97.8%-99.8%and98.7%-99.9%nucleotide sequence identity, respectively. The amino acid sequence of HA genes from26H5N1strains had the cleavage site R-R-R-K/-/R-K-R which represents the molecular basis of high pathogenic AIV stain. Meanwhile, all the5virus strains had a20-amino-acid deletion in the NA stalk and3the potential glycosylation sites. In addition, they had a5-amino-acid deletion in the NS (residues81-85). The phylogenetic tree showed that all the5virus strains had a relatively close phylogenetic relation. The HA genes from5virus strains and A/Chicken/Jiangsu/K0402/2010(H5N1) were located in the same branch, sharing highly homologous to each other. The M, NA, NP, NS and PB1genes from5virus strains had a relatively close phylogenetic relation with A/Duck/Zhejiang/213/2011(H5N1) strain. The remaining PA gene and PB2gene had a relatively close phylogenetic relation with A/muscovy duck/Vietnam/LBM66/2011(H5N1) strain, A/muscovy duck/Vietnam/LBM66/2011(H5N1), respectively. Furthermore, the5virus strains were recombination strains. In addition, HA genes of26H5N1strains which were identified in this study had larger variation, when compared with early isolates. However, there was no variation in the host-binding sites of HA genes, which indicated that the infected host of AIV was unchanged. When compared with cleavage site of the strains collected from2008to2012, there was a mutation (K-R) in the strains collected from2001or2004. The dominant AIV strain in this region had experienced gradually variation (approximately1%rate per annum). Therefore, in order to prevention and control of AIV, the inactivated vaccine strains should be similar with the current epidemic strains.