Preliminary Study On Genetic Evolution And Inactivated Vaccine Of H9N2 Virus In Hunan Area In Recent 4 Years

The avian influenza virus belongs to the Orthomyxoviridae single-negative RNA virus,which can infect poultry and mammals.According to the virulence,it can be divided into three types:non-pathogenic avian influenza(NPAI),low pathogenic avian influenza(LPAI)and highly pathogenic avian influenza(HPAI).In China,low pathogenic avian influenza The H9N2 subtype is the main type.The widespread epidemic of avian flu has caused huge economic losses to the poultry breeding industry.The most common and effective prevention and control method adopted in China is vaccine immunization.However,recent studies have shown that the avian influenza virus is in the vaccine.Under the pressure of selection,recombination and mutation are constantly occurring to adapt to new hosts.In particular,mutations at some key sites of the low pathogenic H9N2 virus cause avian influenza to gradually break through the barrier of infecting mammals,making it a potential threat to humans and mammals.Therefore,it is especially important to monitor the epidemic situation of avian influenza in real time,and to understand its genetic evolution.This paper systematically analyzes the genetic evolution of 17 H9N2 representative strains isolated from Hunan in 2015-2017.The candidate vaccine strains were screened for evaluation of protection effects.Seventeen H9N2 representative strains isolated from 2015 to 2017 were selected for whole-genome sequencing,and HA and NA gene fragments were constructed to construct a phylogenetic tree for genetic analysis.The results showed that the H9N2prevalent in Hunan Province belonged to the HK/Y280/97/like sub-line during this period,but it had low homology with HK/Y280/97/like and the locally used vaccine strain YBF300.Many antigenic sites change,and the receptor binding site is 226.Based on the genetic evolution analysis of H9N2 virus,this study further tested the antigenicity and biological characteristics,and selected the H9N2 vaccine candidate XKY46.In January 2017,it was isolated from the farms and live poultry markets in Hunan Province.HA titer 10⁹,EID50 is 10^8.46,has good reproductive ability,no mixed infection,heat stable.The level of primary antibody can reach 9 or more,which is 2 titers higher than the commercial YBF300 vaccine.The overall protection rate is 93.3%,which is also higher than 86%of YBF300 vaccine.In summary,XKY46 is a good candidate vaccine strain.This study evaluated the immune effect of XKY46 inactivated in different ways and different white oils used to inactivate vaccine adjuvants.The virus was inactivated by using different concentrations of formaldehyde solution and?-propiolactone respectively.Three kinds of imported white oil,two kinds of domestic white oil and related physical and biochemical characteristics independently developed by the laboratory were tested to evaluate the performance of the white oil independently developed by the laboratory.The results showed that1/4000?-propiolactone,the antigen after inactivation at 4°C for 24 h,was inferior to other methods.The new adjuvant vaccine spreads easily in water,and the stratification is not serious after repeated freezing and thawing.The antibody production is fast,the antibody level is high,and the long duration of immunity can be used for the production of H9N2 subtype avian influenza vaccine adjuvant.