Preliminary Study On Evolution And Drug Resistant Of Avian Influenza Viruses Isolated From Ducks In Poyang Lake

The Poyang Lake, located in Jiangxi Province, is the main habitat for migratory birds in the East Asia Australia migratory route. The area provides opportunities for interspecies transmission of avian influenza viruses between wild birds and domestic poultry. In this study, we performed surveys and environmental samples collections in farm households of seven counties around the area of Poyang Lake. Distribution, genetics and drug resistant of avian influenza viruses isolates from ducks were analyzed in this study.We performed sampling in farm households in seven counties around the area of Poyang Lake in 2014. Total of 3467 samples from feces or drinking water of ducks were collected. Samples were inoculated and grown in 9 to 10-day-old specific pathogen-free (SPF) embryonated chicken eggs. The full genomes of the virus isolates were amplified with a Qiagen OneStep RT-PCR Kit for sequencing (Qiagen, Germany). The PCR products were purified from an agarose gel with a QIAquick Gel Extraction Kit (Qiagen, Germany). Deep sequencing was performed using the Ion torrent PGM platform. The next-generation sequencing (NGS) reads were analyzed with a pipeline process using the CLC Genomics workbench 7.0.4. A maximum likelihood phylogenetic tree for the nucleotide sequences of each gene of the selected influenza viruses and molecular markers related with drug resistant were constructed with MEGA5.1.We isolated 109 avian influenza viruses (3.18%,109/3425) in this study.44.95%(49/109) of those viruses was positive for influenza A including 2 H10N3,2 H11N3,2 H12N5,5 H3N2,2 H3N3,8 H3N6, 5 H3N8,8 H4N6,3 H5N6,1 H6N1,4 H6N6.3 H7N7 and 4 mixed isolates. For the H5N6 virus which resulted in sporadic human infections in China, the HA,they contained hemagglutinin (HA) genes of H5 clade 2.3.4.4, neuraminidase (NA) genes of H6N6 viruses and internal genes from H5N6. All these three H5N6 isolates were susceptible with adamantanes and inhibitors of neuraminidase (NA).Diversified subtypes of avian influenza viruses including H10N3, H11N3, H12N5, H3N2, H3N3 , H3N6, H3N8, H4N6, H5N6, H6N1, H6N6, H7N7 and mixed viruses were detected in domestic ducks in Poyang Lake. Of those identified viruses, the H5N6 virus was evidenced with the threat to human infection.