Surveillance And Genetic Evolution Of Avian Influenza Virus In Wild Birds And Poultry In Dongying And Yancheng,2014-2016

Avian Influenza, the full name of the bird avian influenza. Avian influenza viruses(AIV) belongs to the influenza A virus genus in the family Orthomyxoviridae. The AIV genome consists of eight single-stranded, negative-sense RNA segments, comprising the PB2, PB1, PA, HA, NP, NA, M, and NS genes. Sixteen HA(H1-H16) and nine NA(N1-N9) subtypes have been identified among influenza A viruses which combined with each other to form a variety of AIV subtypes, such as H9N2 subtype, H5N1 subtype. Wild birds are regarded as the natural hosts for avian influenza viruses(AIV), however, AIV carried by wild birds have been seldom seen to infect humans directly. Recently, novel H7N9 and H10N8 AIV posed a serious threat to public health and caused a huge economic loss of poultry production in China.Previous studies have showed that both of them were low pathogenic AIV and the source of the surface proteins of these pathogens was associated with wild birds along the East Asia-Australia Flyway, whereas the internal genes were derived from poultry H9N2 influenza viruses circulating in China. Therefore,it is important to monitor the AIV of wild birds and poultry which in the wild birds habitats area. What's more,the research work of the genetic diversity of wild and domestic birds carring AIV and the spatial and temporal characteristics of AIV which spreading in different habitats is important.In the present study, Yellow River Delta and Yancheng Wetlands, indexed in The List of Wetlands of International Importance, were selected as the Northern and Southern research bases along the East Asia-Australia Flyway, respectively. In this study, there were 6 times samples collection from Dongying,Shandong Province and Yancheng, Jiangsu Province from December 2014 to January 2016. Wild bird samples were collected 3477 parts, parts of poultry samples were 3093, a total of 6570 parts. The presence of AIV RNA was evaluated by real-time PCR and the positivity rate of live bird markets were 8.8% to35.0%(22/250~210/600)while the positive rate of wild birds was 0.3% to 9.4%(2/688~78/826).H5 subtype of AIV was identified from Ct <30 samples by common PCR. If the samples were not H5 subtype,the supernatant was inoculated into the allantoic cavity of 9 or 10-day-old specific pathogen-free(SPF)chicken embryos for viral isolation. H5 subtype samples were sent to the CDC National Institute of Microbiology for virus isolation. We have isolated 29 H9N2 strains, 13 H5N1 strains, 2 H5N2 strains, 2H3N2 strains and 2 mixed infection of H5N1 + H3N2 strains. Three H9N2 strains were isolated from Yancheng, other strains were from Dongying. We obtained 50 complete gene sequence. 25 AIV strains obtained from the previous two samples in Dongying. H9N2 subtype which accounted for 88%(22/25),H5N1 subtype 4%(1/25). The 22 AIV strains obtained from Dongying third samples of H9N2 subtype accounted for 18.2%(4/22), H5N1 subtype accounted for 63.6%(14/22). This data shows that the predominant subtype Dongying area may change from H9N2 to H5N1 subtype.AIV whole genomes were generated and phylogenetically analysed. Our results demonstrate that all of the viruses belonged to the H9N2 subtype, and could be classified into nine novel genotypes based on the phylogenetic analysis of the eight gene segments of the AIV genomes. This revealed a high genetic diversity of H9N2 in this region and suggested that they might have undergone frequent genetic reassortment. In addition, the internal genes(PB2, etc.) of two viruses from wild birds and several viruses from poultry belonged to the same gene constellation, suggesting a potential inter-host transmission of AIV between wild birds and poultry in live markets along routes of migratory flyways.To study the continuing evolution and spread of H5N1 highly pathogenic avian influenza, 22 fecal samples were collected from a duck farm is Shandong Province. They were tested for type A influenza using real-time PCR, and the HA subtype of the positive samples were also determined. The RNA of the positive sample was extracted and was used for full-length genome sequencing. The complete viral genome was used for phylogenetic analyses and molecular characterizations were also studied. Our real-time PCR results showed that one sample was type A influenza positive and the HA subtype belonged to H5. This strain was named as A/Duck/Shandong/1-14/2014(H5N1). Phylogenetic analysis of the HA gene showed that it fell within the 2.3.2.1c lineage. Apart from the PB2 gene which was derived from the counterpart of avian H9N2 influenza virus, the remaining seven genes stemmed from H5N1. In particular, the Shandong strain, the strain causing the first human infection with H5N1 in North America, and those leading to the avian influenza virus outbreak in Sanmenxia of Henan Province in January, 2015, belonged to the same genotype. However, no substitutions have been found in the key amino acid sites of this genotype. The Shandong strain belonged to 2.3.2.1c and a novel genotype recently described. This genotype has been identified from different time points(2014, 2015), different geographic regions(Beijing, Shandong Province, and Henan Province), and different hosts(poultry and wild birds), suggesting that it might have gained certain fitness and continued spread and evolution. Therefore, continual surveillance is warranted to monitor its further spread in a larger geographic region.This study from November 2014 to February 2016 in the eastern part of migratory birds and local poultry AIV prevalence provide important monitoring data proved along the East Asian- Australian migratory routes AIV high genetic diversity. The prevention of human infection with low pathogenic avianinfluenza in advance to wild birds- poultry windows potential to infect humans AIV warning in advance.