The Phylogenetic And Antigenic Analysis Of Chicken H9N2 AIV In China & The Study Of Real-time RT-PCR Method For NA Subtyping

Avian Influenza (AI) is a disease caused by Avian Influenza Virus (AIV). Multiple species of animals are susceptible to AIV infection. AI is also a big problem for the global poultry industry. Furthermore, the end of last century saw the infection of AIVs of several subtypes (H5, H7 and H9) in humans, which demonstrate the capability of AIV to transmit directly from poultry to human being and arise a great public health concern. Therefore, it's crucial to take long-time surveillance of AIV to trace its evolution and prevalence in diiferent hosts. In this study, H9N2 subtype AIVs were collected from several intensive farming districts in China. Molecular epidemiology and HA antigenicity analysis were conducted to illuminate the genesis and evolution of these isolates. To meet the practical need for NA subtyping in AIV surveillance, one step real-time RT-PCR were developed for NA differentiation. The major work is as following:1. Phylogenetic and antigenic analysis of chicken H9N2 AIVs in ChinaH9N2 subtype is the most prevalent Avian Influenza virus in poultry in mainland China. Infection of chickens with H9N2 virus may cause respiratory syndrome, reduced egg production and variable rate of morbidity and mortality. The epidemiologic study of AIV in multiple poultry species was reinforced after human infection cases of H5N1 in Hongkong in 1997. However, systematical surveillance information for the evolution of H9N2 viruses in chicken flocks in northern and eastern China is limited, especially in the last few years, when the intensive farming accounts for more than 50 percent of the country's total chicken farming. In this study, seventeen H9N2 viruses isolated from northern and eastern China were analyzed phylogenetically and antigenically to reveal the genesis and evolution properties of H9N2 viruses in this region.The seventeen H9N2 viruses were collected in 1998-2008 during avian influenza (AI) outbreaks in Beijing, Tianjin, Hebei, Henan, Shandong, and Jiangsu provinces of northern and eastern China. To systematically track the genesis and evolution of H9N2 viruses in this region, whole genome sequences of these isolates were obtained and their phylogenetic properties were determined. Phylogenetic analysis revealed several newly emerged lineages of gene segments in addition to the A/chicken/Beijing/1/94 (BJ 94)-like and A/chicken/Shanghai/F/98(F 98)-like lineages, which are prevailing in northern and eastern China according to the previous reports. Reassortments among these gene segments generated five novel genotypes of H9N2 viruses (M, N,O, P and Q) that have not been reported before in China. The emerging genotypes of H9N2 viruses in this region indicate that H9N2 virus genes undergo active evolution, particularly their internal genes, which raises concern for their likely contribution to gene reassortment and production of AIVs with new properties. Our study provides valuable insight into the prevalence of H9N2 viruses in northern and eastern China and demonstrates the need of long-term monitoring of the evolution of H9N2 AIV.Hemagglutinin (HA) is the main antigen of AIV to induce neutralizing antibody in infected animals. HA is also the main determinants of virulence and host-specificity of AIV. In this section, Hyperimmune antisera against each of 12 H9N2 viruses were prepared. Antigenic analysis between these 12 H9N2 isolates was performed by cross HI test with the above panel of monofactorial sera. The test was repeated three times and the mean values were used for antigenic analysis. HA antigenic correlation between two certain viruses were calculated according to the formula R=√r1×r2. The cross HI test results of 12 selected viruses demonstrated that the rooster antisera panel successfully inhibitded each of the 12 isolates in HI test, although the titration varied when a certain serum was reacted with different isolates. Statistical analysis about the correlation of two certain HA antigens were performed and the results indicated that there is no significant antigenic difference between HA of the 12 viruses.The result also demonstrated that the point mutation of these HA gene havn't significantly influenced the antigenicity of HA proteins.Seventeen H9N2 isolates encode PB1-F2 protein of 5 different lengths. Molecular characterization of PB1-F2 gene of chicken H9N2 subtype avian influenza viruses (AIVs) in China was analyzed and the prevalence of the PB1-F2 genes of influenza A viruses (IAVs) prevailed in China was further investigated.337 PB1 genes of IAVs derived from avian (H5N1 and H9N2 subtypes), pig (H5N1, H9N2, H1N1 and H3N2 subtypes) and human beings (H5N1,H9N2, H1N1 and H3N2 subtypes) in China were downloaded from Genbank database and analyzed. The deduced sequences of PB1-F2 proteins have multifold lengths and the ratio of PB1-F2 genes encoding full-length PB1-F2 protein differed in the HA subtype and host origin of IAV isolates. Through this study, the molecular epidemiologic characterization of PB1-F2 gene of IAVs in China was illuminated, which can also works as the foundation of PB1-F2 gene function research in the infection process of IAVs.The HA proteins of fifteen H9N2 isolates contain 7 potential glycosylation sites, while the HA of CK SD B198 and CK BJ L105 have I at position 300 (C to T mutation at position 899 of their HA genes) and result in the loss of one potential glycosylation site. The four H9N2 isolates that belong to the G9-like lineage (CK ZB B1 08, CK HN L1 08, CK HN L208 and CK HN L308) possess full-length NA protein (469AA), while the NA of the Y280-like lineage contain deletion of three amino acids (62-64 AA) at the stalk region. An analysis of the viral amino acid sequence of M2 protein has revealed substitution of S3 IN in two isolates, which is the molecular characterization of amantadine resistance in AIVs.2. Development of Real Time RT-PCR Assays for Neuraminidase Subtyping of Avian Influenza VirusIn this study, nine pairs of neuramidinase (NA) subtype-specific primers were designed with Primer Hunter software and successfully used in real time RT-PCR with four primer-pool reactions to differentiate nine NA subtypes of AIV. The RRT-PCR assays are sensitive and can detect in vitro transcribed RNA of different NA subtypes ranging from 351 to 8000 copies per reaction, or 4-62fg of total RNA of AIV per reaction. The assays possess good specificity. There is no cross reaction between RNA of different NA subtypes in RRT-PCR with each subtype-specific primers. No ampliation were displayed when detect RNA of IBV, IBDV, NDV with the primer pools, either. This study validated the powerful function of Primer Hunter for the design of subtyping primers and also introduced a sensitive and specific method for NA subtyping in AIV epidemiological surveillance.