Isolation Identification And Immunogenicity Analysis Of H9N2 Subtype Of Avian Influenza Virus

Avian influenza subtype H9N2 is an infectious disease caused by Avian influenza viru s（AIV）that infects avians and causes damage to the respiratory and digestive tracts,decre ased egg production and immunosuppression.In this study,one epidemic strain of H9N2 a vian influenza virus was isolated and identified,and the virulence and immunogenicity of t his strain were determined after being cultured in suspension MDCK cells.Swabs of throat and cloaca were collected from chickens suspected to be infected with avian influenza virus,and total RNA was extracted from specimens for RT-PCR identification.Supernatants of throat swab a nd cloaca swab of positive samples were inoculated into SPF chicken 10-day-old embryos.After the collected allantoic fluids was purified by limited dilution for three rounds,the allantoic fluid of chicken embryos with the highest agglutination titer was taken and inoculated into chicken embryos again to proliferate the isolated virus for further determinations,which included virus titration,RT-PCR identification,hemagglutination and serologic specificity by HI test.The virus titer was measured as 10^8.32 EID₅₀/0.1m L.When observed for morphology by transmission electron microscopy,the virus has capsule,spherical in shape and 80～120 nm in diameter.Serologic specific examination confirmed that the isolate was an H9 subtype avian influenza virus,and the virus was named A/Chicken/Hu Nan/20/2020（H9N2）,abbreviated as the Hu N20 strain.The purity test exhibited that Hu N20 strain was sterile and free from contaminations of mycoplasma and exogenous virus.Homology phylogenetic analysis of HA and NA genes of Hu N20 by using reference sequences of domestic isolates in recent years showed that both HA and NA genes of Hu N20 belonged to Y280-like subfamily.The pathogenicity of Hu N20 to SPF chickens was examined,including the minimum infection dose and viral shedding of infected chickens.Results showed that viral shedding from throat and cloaca was detected from 1 to 7 days after infection with Hu N20.The minimum infection dose to SPF chickens aged 42 days was 10^5.0 EID₅₀/0.1m L.The technology of virus culture and inactivation was optimized.The MDCK cell suspe nsion culture was adopted.When the cell density was 3×10⁶～4×10⁶ cells/m L,the virus was inoculated to the medium at 0.1%of the volume,and the concentration of trypsin was 60μg/m L.The hemagglutinin titer of the proliferated virus reached up to 11 log₂.The viruses in the harvested solution were completely inactivat ed when treated with formaldehyde at t he final concentrations of 0.1%,0.2%and 0.05%for 4 hours and 0.01%for 8 hours.The E xperiments for the immunogenicity of inactivated Hu N20 showed that the mean geometric titer of HI antibody of vaccinated chicken w as 2.9 log₂ on day 7 post immunization,the HI antibody titer was 10.5 log₂ on day 14 post immunization,and reached to 11.5 log₂ on day21 post immunization.The protection rate of immunized chickens against virus challenge21 days after the vaccination was 100%,which indicates that the inactivated vaccine prepa red by using Hu N20 cultivated in suspension MDCK cells has excellent immunogenicity.Vaccines prepared from inactivated virus solutions with different doses of antigen wer e used to immunize 21-day-old SPF chickens for 21 days to examine the serum HI antibod y titer and then to perform virus challenge at the same day.Results showed that when the antigen content was not less than 10^6.5 EID₅₀/0.1m L,the mean geometric titer of the HI ant ibody of the inactivated vaccine-immunized chicken was above 9.8 log₂,and the rates of pr otection against virus challenge were all 100%for chickens immunized with different dose s of vaccine.It was determined that the minimum antigen content for the preparation of the vaccine was 10^6.5 EID₅₀/0.1m L.The inactivated vaccines prepared from Hu N20 and a commercial vaccine strain HZ w ere used to immunize 21-day-old SPF chickens,and the cross HI antibody potency was det ermined at 7,14 and 21 d after immunization with these two antigens.Results showed that when Hu N20 was used as the antigen for HI test,the geometric mean titer of serum HI anti body was 11.5 log₂ in chickens 21 d after vaccination with Hu N20 and 7.8 log₂ in chickens21 d after vaccination with HZ.When the HZ strain was used as the HI test antigen,the ge ometric mean titer of serum HI antibody in chickens 21 d after vaccination with the Hu N20strain was 9.8 log₂,and the geometric mean value of serum HI antibody potency in chicke ns 21 d after vaccination with the HZ strain was 11.0 log₂.Furthermore,the Hu N20 strains were used for intravenous infection of chickens immunized with inactivated Hu N20 and H Z,respectively.The challenge protection rate of chickens immunized by the Hu N20 vaccin e was 100%,that of chickens immunized by HZ vaccine was 80%.All the unimmunized co ntrol chickens were infected and shedding the challenge virus.Therefore,chickens immuni zed by Hz vaccine did not completely resist the attack of epidemic strain Hu N20.In conclusion,in this study,a strain of H9N2 subtype avian influenza virus was identified after isolation,identification and purification of specimens from chickens suspected with infection of avian influenza virus.This strain had excellent immunogenicity when proliferated in MDCK suspension cells,which made immunized chickens resist to the challenge of Hu N20,and the immune protection rate was 100%.This study lays a foundation for the research and development of H9N2 subtype avian influenza vaccines and the manufacture of avian influenza virus using MDCK suspension cells.