| H9N2 subtype of Avian Influenza(H9N2 AI)is a respiratory infectious disease caused by the H9N2 subtype of Avian Influenza Virus(H9N2 AIV),which mainly infects birds.H9N2 AIV has a strong transmissibility,a wide host range,and can infect humans across interspecific barriers,posing a serious threat to Chinese poultry industry and public health security.Previous research by the research team has proven that the H9N2 epidemic strain isolated in southwest China from 2013 to 2016 has undergone significant antigen drift,and can break through the immune protection of commercial vaccines.In order to monitor and prevent the prevalence of H9N2,this study analysed the epidemiological of H9N2 AIVs in China,including the evolution of antigenic site and pathogenicity characteristics of recent years’ isolates.The recombinant H9N2 virus r CQY-GHHA constructed in the early stage was used as the vaccine strain,and its immune protection efficacy was evaluated through animal immune protection tests.The research results have significant implications for preventing and controlling the spread of H9N2 AIV.The results are as follows:1.Epidemiological analysis of H9N2 AIVThis section analyzes the genetic evolution,antigenic site variation characteristics,and pathogenicity characteristics of H9N2 epidemic strains in Southwest China from 2017 to 2022.First,we constructed the Neighbor-Joining phylogenetic tree of the H9N2 HA and NA genes.The results showed that the HA and NA of the current H9N2 epidemic strains belonged to the h9.4.2.5 subline,and did not belong to the same subline with the commonly used commercial vaccine strains A/Chicken/Guangdong/SS/94,A/Chicken/Shandong/6/96,and the Genetic distance was increasing year by year.Secondly,the variation of 46 reported HA protein antigenrelated sites was analyzed.The results showed that compared with the 2014 strain,there were significant changes in amino acids at 35 loci(35/46,76%)in the 2019 to 2022 strain;Compared with the vaccine strain SS strain,there were 29 sites(29/46,63%)where the dominant amino acids of the epidemic strain were replaced from 2019 to 2022.The changes in antigen-related sites located at receptor binding sites may further lead to the occurrence of immune escape.Finally,the pathogenicity of the epidemic strain was studied.The results showed that the H9N2 epidemic strain still maintains its weak virulence characteristics,mainly affecting the trachea and lungs.A strain A/chicken/Chongqing/LXL/2019 with obvious symptoms was selected as the attacking strain for subsequent immune efficacy evaluation.2.Preparation and immune efficacy evaluation of H9N2 recombinant inactivated vaccineThis section uses the H9N2 recombinant strain r CQY-GHHA constructed in the laboratory as the vaccine strain.The recombinant strain was prepared as an oil emulsion inactivated vaccine according to industrial processes,and its quality was evaluated through dosage form,stability,and safety indicators.The results showed that the self-developed vaccine met the standards of veterinary biological products.In order to evaluate the specific antibody levels produced by the vaccine,r CQY-GHHA vaccine and SS commercial vaccine were administered to SPF chickens for first immunization at 7 days of age and secondary immunization at 28 days of age.Serum antibody monitoring showed that the r CQY-GHHA immunized group reached an effective protective level(HI ≥ 4 log2)on the 9th day after the first immunization,and reached a peak(HI ≥ 11 log2)on the 4th day after the second immunization.The antibody titer was still higher than 8 log2 on the 29 th day;The SS immune group reached an effective protective level on the 11 th day,with a peak of HI ≥ 10 log2.After secondary immunization,the antibody titer was only 5 log2 on the 29 th day.The results showed that the self-developed r CQY-GHHA vaccine achieved faster effective protection time,higher antibody peak,and longer duration than the SS commercial vaccine.To evaluate the immune protection efficacy of the vaccine,chickens were vaccinated 14 days after secondary immunization.The results showed that the r CQY-GHHA immune challenge group showed 12.5% of clinical symptoms on the 5th day after challenge,and disappeared after 1-2 days;No obvious pathological damage was observed through gross anatomy and pathological tissue observation;On the 5th day after the virus attack(47 days old),25% of the pharyngeal swabs turned negative,and 100% of the cloacal swabs were negative(both pharyngeal and cloacal swabs in the control group were 100% positive),and the virus load in the trachea and lungs was also much lower than that in the SS immune attack group.The incidence rate of SS immunized chickens was as high as 75%,and typical respiratory symptoms and pathological changes were observed;On the 5th day of the challenge,the pharyngeal swabs of the SS immune challenge group were still 100% positive,and the viral load of the trachea and lungs was similar to that of the challenge control group.The above data show that both r CQY-GHHA and SS vaccines can effectively inhibit cloacal excretion of infected chickens,but the performance of commercial vaccine strain SS in reducing incidence rate,pathological damage,throat swab excretion and load of sexual organs is not as good as the self-developed r CQY-GHHA vaccine strain.In summary,the current H9N2 epidemic strains in China belong to the h9.4.2.5 lineage,but there have been significant mutations in antigen-related sites of the epidemic strains in the 2019 to 2022 years;The strain is still weak and mainly damages the trachea and lungs of chickens;The developed recombinant oil emulsion inactivated vaccine r CQY-GHHA can provide ideal immune protection for recent H9N2 epidemic strains. |
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