Development On Inactivated Vaccine Against Avian Influenza (H9 Subtype)

H9 Subtype Avian Influenza is a type 3 disease that has occurred and been reported in many countries and regions throughout the world.Its prevention and control have become the focus of common concern in the world.In China,bird flu has also brought a lot of economic losses to China's poultry breeding industry.Bird flu viruses have been reported in many provinces and regions in China.H9 subtype avian influenza is a low-pathogenic bird flu,but it is also now endemic in China.Birds infected with H9 subtype avian influenza only cause respiratory symptoms,decreased feed intake,and slight clinical symptoms such as decreased egg production rate.The death rate is extremely low.However,infection with the H9 subtype of low pathogenic avian influenza,the body's immune system will be greatly reduced,susceptible to other diseases,if the secondary infection of other diseases,will greatly increase the mortality of diseased chickens.H9 subtype Avian Influenza has been mutated under new host environment and immune pressure due to its pathogenic characteristics,which makes it difficult to be effectively prevented and controlled.The gene of H9 subtype avian influenza virus can be used as gene carriers for a variety of highly pathogenic influenza Gene,through gene recombination,can produce new viruses that have pathogenic effects on humans,threatening public health security.The epidemiological investigation of H9 subtype AIV and its mastery of pathogenic evolution can help us to better prevent and control the H9subtype of low pathogenic avian influenza.In this study epidemiological survey of bird flu virus in Hebei Province was conducted,to understand the prevalence of H9 subtypes in Hebei Province.Meanwhile,the H9 subtype avian influenza virus were isolated to study its genetic evolution and pathogenicity to provide some theoretical basis and technical support for the.Prevention and control of this disease.From 2017 to 2018,a total of 139 H9 subtype avian influenza cases in Hebei Province were collected.Through the extraction of viral nucleic acid and PCR gel imaging,84 of the139 samples were bird flu,accounting for approximately 64.43%of the patients were classified by the hemagglutination inhibition test,65 H9 subtypes,accounting for 77.38%.The other nine are other subtypes.The onset season of H9 subtype AIV was mainly in 1,2,3,4 and 12 months.The onset age was widely distributed.Layers were concentrated in 1month of age and broilers were concentrated in 3 weeks of age.During the survey,it was found that some chicken farms lacked the immunity or immunity to H9,such as improper preservation of vaccines.65 H9 subtype avian influenza viruses were isolated and separated by SPF chicken embryos.The antigens were neutralized and mixed with other antigens for infection.The key dilution method was used for purification and propagation.Five generations were passed and two strains of the virus ZD5 were isolated.And SD,determination of blood coagulation value ZD5 29,SD 28,EID50,10-8.75 and 10-7.25,designed by HA gene primers,the target fragment is 550bp,sent for sequencing,compared with 27 Hebei reference strains The homology of the two isolates was 95.8%,the highest homology of ZD5 with A-chicken-hebei-0822-2007 and A-chicken-hebei-C6-2007 was 967%;SD and A-chicken-Thehighesthomologywas90.1%forhebei-C6-2007and A-chicken-hebei-0822-2007.Both isolates were closely related to the 2007 reference strain.Through an artificial infection test,ZD5 was intramuscularly injected into 21-day old SPF chickens to cause the disease.The clinical symptoms were runny nose,sneezing,mouth breathing,apathy,and decreased feed intake.The necropsy revealed that the trachea had mucus,lung lesions,glandular papillary swellings,and cecal tonsillar hemorrhage.Observed by histological observation of tracheal mucosal shedding,partial lumen filled with serous exudate and inflammatory cells;there are inflammatory exudates in the blood vessels of the lungs,alveoli filled with red blood cells,alveoli's outline is incomplete.The incubation period for isolate ZD5 is approximately 3 days,the cotton swab collected on the5th day after the challenge was separated by SPF chicken embryo and the separation rate reached 90%,allantoic fluid can agglutinate 1%chicken red blood cells,the HA reaches 2~6.ZD5 is used as a strain for the preparation of vaccines,inactivate it to prepare the aqueous phase,then mix and emulsify with the oil phase in a ratio of oil to water of 3:1 to prepare an oil emulsion inactivated vaccine.Vaccine formulation,stability,viscosity,sterility,safety,and efficacy tested in accordance with veterinary pharmacopoeia,The test results show that the prepared inactivated vaccine has a water-in-oil type,the stability was good,the viscosity was in line with the standard,and there was no adverse reaction in the injected animals.After immunization with SPF chickens,the level of HI antibody in chickens was significantly increased,the challenge was conducted 21 days after the immunization vaccine,and the protection rate was 100%.