Studies On Ecological Evolution And Rapid Detection Of H3 Subtype Of Avian Influenza Virus In Guangxi Province

Avian Influenza is a world of infectious disease in avian. It has made not only huge economic losses for poultry in industries but damage for human being’s health. H3 subtype of Avian influenza virus (AIV) is a low pathogenic AIV, it can infect poultry, and can also be detected in the body of pigs, horses and dogs. In order to understand the ecological trend of H3 subtype of AIV in guangxi region, grasp the genetic evolution characteristics and analyze the relationship of H3 subtype AIV Transmission between different host like from swine, human, Canine and equine, and to get the aim of early diagnosis and prevention of H3 subtype AIV, two kinds of nucleic acids detection techniques was established.The study preliminary screening H3 subtype AIV by serological test in the samples collected from live poultry market from 2009 to 2014. The H3 subtype AIV was isolated and purified by dilution and Serum neutralization method, further confirmation the genotype to the HA and NA genes cloning, sequence determination, results show that the separated strains is H3N2, H3N6 and the H3N8 subtype. The results of EID50、ELD50、Receptor affinity test and pathogenic experiments on animals to 14 isolated stains were Low pathogenic and all preference poultry SAa-2,3-Gal receptor, not have potential ability to infect humans.Complete genomic sequences of eight gene segments(HA、NA、PB2、PB1、PA、NP、 NS and M) of H3 subtype of AIV were cloning, sequencing and analysis, The results show these 14 strains of H3 subtype of AIV only with one basic amino acid in The amino acid residues at the cleavage site of the HA molecule, which is characteristic of low pathogenic AIV; Compared with the H3 subtype influenza virus from different host and the highest nucleotide strains In the GenBank and build the molecular genetic evolutionary tree. It shows that all the 8 gene segments of these 14 strains of H3 subtype of AIV all belong to poultry Eurasian lineage, The gene segments of these 14 strains isolated nearly year have higher homology and the distribution location relatively concentrated in the evolutionary tree, and probably from the same ancestor. isolated not nearly year have lower homology and distant phylogenetic relationships, and is obvious at different time, the results show that the H3 subtype AIV may be constantly in evolution; Eight gene segments of the same strains complicated sources and probably by gene rearrangement from different subtypes of AIV; these 14 strains of H3 subtype of AIV have low homology with the H3 subtype influenza virus from different host, the relatives is far and there are clear differences between species.This study established two kinds of nucleic acid detection methods for the H3 subtype AIV:1) Multiple RT-PCR, including a duplex RT-PCR assay for detection of H3N2 AIV and multiplex RT-PCR assay for detection of H3N2 subtypes and M gene; 2) Real-time Fluorescent Quantitative RT-PCR, including a duplex real-time RT-PCR for detection of H3N2 AIV and One-step Real-time Fluorescent Quantitative RT-PCR for Detection of H3N8. These methods were implemented for more than a pipe inspectionnot, only improve the accuracy of the detection, but also save the time and the cost and provide a certain technical support to the test of clinical samples.