Research On Ecological Evolution Of H9 Subtype Avian Influenza Viruses Origin From Different Hosts In Guangxi And Rapid Detection Methods

Avian Influenza(AI)is a highly contagious disease of poultry and birds caused by influenza A viruses,a member of Influenza virus A genis.According to its surface glycoprotein haemagglutinin(HA)and neuraminidase(NA),the virus is divided into different HA and NA subtype combination.The H9 subtype of avian influenza is one of the most serious diseases to the poultry,it was scattered in various locations around the world,and H9N2 subtype AIV which is the most widely popular in chicken flocks in China.H9N2 subtype AIV also can cross the species barrier to infect humans directly,and that appeared H9N2 subtype AIV provide internal genes for H5N6,H10N8,H7N9 and H6N1 four different human infection with avian influenza in China since 2013,therefore H9N2 subtype AIV also has become a serious threat to human public health.In order to understand the prevalence of different host H9 subtype avian influenza viruses in Guangxi from 2014 to 2016 and genetic evolution of the internal characteristics of the isolates,we established rapid detection method for H9 subtype avian influenza virus,and some of the isolates were performed whole-genome sequencing and biological characterization.The results were as follows:(1)In recent years,different parts of the bird flu monitoring results show that low pathogenic AIV subtype H9 and H6 mixed infection situation in poultry production and sale link is relatively common.In this study H6 and H9 subtypes of AIV HA gene specific primers were designed to amplify 551 bp and 222 bp amplicon respectively.Amplification conditions were optimized including primer concentrations and annealing temperature for simultaneousdetection of H9 and H6 by double RT-PCR detection method.The detection limit of triplex PCR was 5×104 copies/?L.It suggests that this duplex RT-PCR assay is a specific,sensitive,stable and repeatable method for the detection of H9 and H6 subtype of AIV,can provide technical support for the monitoring of H9 and H6 subtype AIV.Using the newly developed method samples origonatong from nine different host 26 strains of H9 subtype AIV was detected and sequencing of their HA and NA gene showed that they all belong to H9N2 subtype AIV,cracking site also conforms to the low pathogenic AIV molecular features.Source separation of different host plants HA gene and reference strains respectively in the evolution of the different branches in the evolutionary tree.Genetic evolution analysis result of 26 strains representative isolates and reference strains of showed that most of the isolates eight gene segments with varying degrees of genetic recombination,a total of seven different restructuring mode.(2)In this study biological characterization of the isolates were also done along with their whole-genome sequencing.The results show that ELD5O range between 10-4.5 and 10-8,EID50 range between 10-6.2 and 10-8.5.Receptor affinity determination results showed that 17 strains of 26 strains have poultry at the same time SA? 2,3 and anthropogenic SA? 2,6 characteristics of two kinds of receptor affinity,and the other 9 cases with poultry SAa 2,3 receptor affinity characteristics.In order to evaluate different sources of host H9N2 subtype of avian influenza virus pathogenicity we used SPF and infected them with the isolateschicken,we according to different sources of host in recent years,Guangxi H9N2 subtype AIV HA selection genetic evolution analysis of the results in different branches of the evolution of the virus of SPF chicken pathogenic experiment was carried out.Test results show that isolates H9N2 subtype avian influenza viruses can infect SPF chicken,virus in chicken body there exist certain differences in tissue distribution in different organs.SPF chickens infected H9N2 virus showed certain clinical symptoms,can through the respiratory tract infection chicken and cloacal poison outward.Autopsy and histopathological observation results showed that the part of the airway infection chicken hyperemia lesions,lung tissue also showed certain degree of pathological damage,etc.Cohabitation infection test,moreover,that the two strains of the virus can be spread by infected group level of chicken to cohabitation group.Inoculation of H9N2 subtype avian influenza virus in SPF chicken demonstrated pathological lesions.And these viruses poses a big potential threat for chickens.We recommend to avoid contact of chickens with other species of birds.