Epidemiological Investigation Of Avian Influenza Viruses Isolated In Eastern China From2011to2013and Antigenic Analysis Of H9N2Subtype Avian Influenza Viruses

Avian Influenza A Viruses (AIVs) of H9N2subtype have been widely distributed in China and other countries of the world since90s of last century, causes not only a great loss to poultry industry, but also causes incalculable hazards to the public health. In China, the unique geographical environment and breeding patterns provide favorable condition for the occurrence and transmission of this disease. The immune pressure of vaccines and trade in live bird markets (LBMs) provide a necessary condition for reassortment and mutation of H9N2subtype avian influenza virus.Cloacal swabs collected from LBMs and specimens collected from diseased poultry in Eastern China during2011-2013were used to isolate AIVs. The whole genome of H9N2isolates were sequenced and phylogenetic analysis were performed. Two novel reassortant H10viruses H10N8and H10N9were isolated and their biological characteristics and genetic sources of each gene fragments were analyzed. In addition, we evaluated the immune efficacy of the vaccine candidates strains made from epidemic strains and the currently widely used F98vaccine. This study provides an epidemiological data and experimental basis for prevention and control of avian influenza and vaccine selection.1. Epidemiological investigation of avian influenza viruses in Eastern China from2011to2013.Three hundred and forty specimens of diseased poultry from Jiangsu and surrounding areas and3100cloacal swabs from LBMs in Eastern China were collected during2011-2013.31strains of H5AIVs,36strains of H9AIVs, and31strains of Newcastle disease viruses (NDVs) were isolated by inoculation of embryonated eggs and identified by HA and HI test combined with RT-PCR. The isolation rates of H5AIVs, H9AIVs, and NDVs from cloacal swabs were0.42%,0.52%, and0.45%, respectively, and that from specimens were5.29%,5.88%, and5.00%, respectively. The result of virus isolation from LBMs showed that eight subtypes of AIVs including H3, H4, H5, H6, H7, H9, H10, and H11were identified. There were six subtypes of AIVs isolated from duck-orgin and chicken-orgin samples, and three subtypes of AIVs isolated from goose-orgin samples, indicating that chickens also play an important role in viral mutant and reassortment.2. Phylogenetic analysis of36strains of H9N2subtypes AIVs.Phylogenetic analysis for nucleotide sequences of HA genes in the36H9N2AIVs indicated that34strains of Y280-Like AIVs (Clade h9.4.2.4) became the predominant strains in Eastern China, and there was only one strain of F98-Like AIV (Clade h9.4.2.1) and one strain of G1-Like AIV (Clade h9.4.1). It is noteworthy that there were mutations among different Clades of AIVs, even within same Clade of AIVs, especially for Y280-Like AIVs. Those Y280-like strains have evolved more small branches, in which the nucleotide homology of HA genes was about90.9%-94.7%. The residue226in HA protein of most H9N2AIVs were Leu, indicating a preference to bind to human cell receptor.NA genes of all H9N2AIVs belonged to F98-like branch of Eurasian-lineage, three amino acids in the position63-65of the NA stalk were deleted. The internal genes of H9N2AIVs were conserved. However, PA, NP, NS genes of AIV strain Qa/WX3/13were belonged to Y439-like, G1-like and G1-like branchs, respectively. Drug resistance analysis showed that there were no apparent antagonism mutations in AIVs for oseltamivir and zanamivir. All isolates were resistant to amantadine except for AIV strain Qa/WX3/13.3. Biological characteristics and derivation analysis of two reassortant H10subtype viruses.AIVs H10N9and H10N8were isolated from chickens and pheasants, respectively during the epidemiological surveillance during2011-2013, and their biological characteristics and genetic evolution were studied. The two H10viruses were both low pathogenic avian influenza virus to chickens according to the IVPI. Phylogenetic analysis of genome sequences showed that all the gene segments of the two viruses belonged to the Eurasian-lineage. The NA gene of the H10N9strain was closely related to that of H7N9AIV, suggesting that it is very likely that it could be the NA donor of the epidemic H7N9AIV. The H10N8strain is poorly related to H10N8strain Jiangxi JX346, which infected people in2013, especially for NA gene, indicating that the strains most likely to be a reassortment virus generated in the migratory birds.4. Antigenicity analysis and immune efficacy of H9N2AIV vaccine candidates.Based on phylogenetic analysis, two H9N2AIVs, Ck/yz4/12and Dk/Yc2/13, were further selected by antigenic analysis and biological characteristics analysis. Then, SPF chickens were immunized with inactiated Ck/yz4/12, Dk/Yc2/13and F98, and boosted once. On14days after last immunization, the chickens were inoculated intranasally with106EID5o doses of homologous and heterogonous avian influenza H9N2virus The results of viral shedding showed that the vaccine preparation of AIV strain Ck/yz4/12provided protection not only against the homologous H9N2AIV challenge, but also against heterologous H9N2AIV challenge, indicating that Ck/yz4/12may be the ideal vaccine candidate for inactivated vaccine against H9N2avian influenza.