Preparation method of multivalent inactivated vaccine for preventing H9 subtype avian influenza virus

Abstract

The invention relates to a preparation method of a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus. Comprising the following steps: S1, treating a large number of samples obtained in different regions in a long period by adopting isolation and identification, gene sequencing and genetic typing methods, and judging the variation direction of viruses; s2, carrying out a cross hemagglutination inhibition test on the whole sample, and screening a preferred strain with a wide protection spectrum from the whole sample; s3, using a gold standard immune challenge method for immunogenicity evaluation, and carrying out a challenge test on the whole sample size to verify the protection effectiveness of the screened strain on other strains of the same branch; s4, preparing an inactivated vaccine by adopting the optimized strains obtained by screening; and S5, carrying out clinical tests in poultry of different breeding varieties in different regions so as to evaluate the effectiveness of the product. The H9 subtype avian influenza virus is subjected to genetic typing, an H9 subtype avian influenza evolutionary tree is established, the genetic typing condition is analyzed, a cross hemagglutination inhibition test is carried out to screen out dominant strains in main epidemic branches, an antibody evaluation method and an immune challenge method are used for carrying out immunogenicity evaluation on the preferred strains, and the H9 subtype avian influenza virus is obtained. The H9 subtype avian influenza virus strain disclosed by the invention not only provides good protection for the attack of homologous strains, but also can effectively resist the attack of heterologous strains, thereby effectively coping with the infection of H9 subtype avian influenza and solving the core pain point problem of the current breeding industry.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary vaccines, and in particular relates to a method for preparing a polyvalent inactivated vaccine for preventing H9 subtype avian influenza virus disease, which is used for preparing a polyvalent inactivated vaccine for H9 subtype avian influenza virus disease. Background Art

[0002] Avian influenza virus belongs to influenza A virus. It is classified into three levels: high, medium, and low / non-pathogenic according to the different pathogenicity of avian influenza virus to chickens and turkeys. Due to the characteristics of the hemagglutinin structure of avian influenza virus, it generally infects poultry. When the virus undergoes gene reassortment during replication, the structure changes and acquires the ability to infect humans, it may cause human infection with avian influenza disease.

[0003] H9 subtype avian influenza virus is one of the important types. In the prior art, inactivated vaccines are usually used for the prevention and treatment of avian influenza. The existing inactivated vaccine preparation methods have the following problems and shortcomings: first, the sample size is too small, and the screening strains are not universal; second, except for the hemagglutination cross-inhibition test to screen the strains, no virus attack is performed to verify the immunogenicity of the screened strains; third, no clinical trials have been conducted in different regions and different breeds of poultry to evaluate the safety and effectiveness of the vaccine.

[0004] In summary, it is necessary to develop a method for preparing a polyvalent inactivated vaccine for preventing H9 subtype avian influenza virus disease, so as to form an inactivated vaccine of the dominant strain of H9 subtype avian influenza with better performance. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease, that is, with the support of a large amount of epidemiological survey data, H9 subtype avian influenza is subjected to genotyping, an H9 subtype avian influenza evolutionary tree is established, the genotyping situation is analyzed, a cross-hemagglutination inhibition test is carried out to screen out the dominant strains in the main epidemic branches, the immunogenicity of the preferred strains is evaluated by using an antibody evaluation method and an immune attack method, and finally an inactivated vaccine containing a bivalent or trivalent H9 subtype avian influenza dominant strain is developed, which not only provides good protection against the attack of homologous strains, but also can effectively resist the attack of heterologous strains, thereby effectively coping with the infection of H9 subtype avian influenza, solving the core pain points of the current breeding industry, ensuring animal health, and improving the economic benefits of breeding.

[0006] The technical solution adopted by the present invention is: a method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease comprises the following steps:

[0007] Step S1, using separation and identification, gene sequencing and genotyping methods to process a large number of samples obtained over a long period and from different regions to determine the mutation direction of the virus;

[0008] Step S2, performing a cross-hemagglutination inhibition test on all samples to screen out the preferred strains with a broad protection spectrum;

[0009] Step S3, using the gold standard immune challenge method for immunogenicity evaluation, a challenge test is conducted on the full sample size to verify the protective effectiveness of the screened strain against other strains in the same branch;

[0010] Step S4, using the selected strains obtained through screening to prepare an inactivated vaccine;

[0011] Step S5, conducting clinical trials on poultry of different breeds and in different regions to evaluate the effectiveness of the product.

[0012] Preferably, step S1 comprises the following steps:

[0013] Step S11, sample processing: collect tissues such as lung, larynx, trachea, liver, spleen, and oviduct of the diseased chicken, add them into an EP tube with an appropriate amount of physiological saline, fully grind the diseased tissue using a vibrating stainless steel strain method, centrifuge at 5000 rpm and 4°C for 10 minutes, and filter and sterilize the supernatant for later use;

[0014] Step S12, chicken embryo inoculation: the supernatant is inoculated into 5 10-day-old SPF chicken embryos through the allantoic cavity, 0.2 mL / embryo, and cultured in a 37°C incubator, and the embryos are inspected once a day, and the chicken embryos that die within 24 hours are discarded, and the chicken embryos that die after 24 hours are harvested at any time. After 96 hours, all chicken embryos are taken out, and the chicken embryo fluid is harvested respectively, and a sample is retained for a hemagglutination test;

[0015] Step S13, serological identification: taking a hemagglutination-positive sample to prepare 4HAU antigen, and conducting a hemagglutination inhibition test with chicken Newcastle disease HI positive serum, EDS76HI positive serum, avian influenza H9 subtype specific antiserum, H5, H7 subtype specific antiserum, and SPF chicken serum to carry out serological identification;

[0016] Step S14, RT-PCR test typing identification: take a hemagglutination-positive sample, extract RNA with EasyPure Viral DNA / RNA K, and reverse transcribe it into cDNA; use H9N2 subtype typing identification primers for PCR identification; after the PCR product is confirmed to be of correct size by electrophoresis, it is sent to a sequencing company for nucleotide sequence determination; use DNA STAR software to splice the sequencing results; obtain the representative HA gene sequence of the H9N2 subtype avian influenza virus from Gen Bank, and the HA gene sequence of the vaccine strain used in some domestic H9 subtype avian influenza inactivated vaccine products, and use software to perform genetic evolution analysis.

[0017] Preferably, step S2 comprises the following steps:

[0018] Step S21, preparing single factor serum: taking antigens without exogenous virus contamination and preparing antigens by conventional methods and emulsifying oil emulsion inactivated vaccine, immunizing 21-day-old SPF chickens with a dose of 0.3 mL per chicken, immunizing 5 chickens with the vaccine corresponding to each strain; setting up 5 SPF chickens that were not immunized as controls and raised under the same conditions; 21 days after immunization, blood was collected from 5 chickens in each immunization group and 5 chickens in the control group under the same conditions, and serum was separated;

[0019] Step S22, cross hemagglutination inhibition test: using the inactivated antigen of each evolutionary branch virus liquid as the hemagglutination inhibition test antigen, and conducting a cross hemagglutination inhibition test with the prepared single factor serum of each strain;

[0020] Step S23, screening of preferred strains: based on the results of the cross-hemagglutination inhibition test, the strain with the smallest dispersion between the detection antigen prepared from each strain and the single factor serum HI is selected as the preferred strain for each evolved molecule.

[0021] Preferably, step S3 comprises the following steps:

[0022] Step S31, preparation of inactivated vaccine of preferred strains and immunization: screening the preferred strains with broad serum protection spectrum of each evolutionary branch, and using the prepared inactivated vaccine to immunize 21-day-old SPF chickens, 0.3 mL per chicken;

[0023] Step S32, challenge test for each evolutionary branch: 21 days after vaccination, use other strains of each evolutionary branch for challenge, 10 chickens / group; the challenge method is: dilute the virus solution 10 times with PBS, and challenge by intravenous injection, 0.2 mL / chicken, and the challenge dose for each chicken is not less than 10 6.0 EID 50 ; On the 5th day after the virus attack, collect laryngeal and cloaca cotton swabs, mix the two cotton swabs collected from the same chicken as a sample, and inoculate into 10-day-old SPF chicken embryos for virus isolation; samples with negative virus isolation should be blindly propagated for one generation before judgment;

[0024] Step S33, determination of vaccine reserve strains: further verify whether the strains screened by the cross-hemagglutination inhibition test have a protection rate of more than 80% against other strains in the same evolutionary branch based on the challenge results, and finally determine whether the preferred strain can become a vaccine reserve strain.

[0025] Preferably, step S4 comprises the following steps:

[0026] Step S41, preparing an antigen solution of a preferred strain of H9 subtype avian influenza virus;

[0027] Step S42, oil phase preparation, 94 parts of white oil for injection and 2 parts of aluminum stearate are mixed evenly in an oil phase preparation tank, and then 6 parts of Siben-80 are added, and after mixing, they are sterilized by high pressure for standby use;

[0028] Step S43, aqueous phase preparation, mixing the antigen liquids of multiple H9 subtype avian influenza virus preferred strains that have passed the test in equal proportions, so that the total amount of each strain of avian influenza virus in the aqueous phase before inactivation is ≥10 7.5 EID 50 / 0.1mL; take 96 parts of the mixed antigen solution, add 4 parts of sterilized Tween-80, and stir thoroughly until the Tween-80 is completely dissolved;

[0029] Step S44, emulsification, take 2 parts of the oil phase and introduce them into an emulsification tank, slowly stir and slowly add 1 part of the water phase, mix at medium speed, and then emulsify at high speed; after emulsification, take 10mL of the vaccine and add it to a centrifuge tube, centrifuge at 3000r / min for 15min, and the water phase precipitated at the bottom of the tube should not exceed 0.5mL;

[0030] Step S45, quantitatively packing, sealing with a cover, and storing at 2-8°C.

[0031] Preferably, step S5 comprises the following steps:

[0032] Step S51, immunizing white-feathered broilers, laying hens, and yellow-feathered broilers in farms in three provinces and cities using the multivalent inactivated vaccines prepared from the reserve vaccine strains of each evolutionary branch and the control H9 subtype avian influenza vaccine, with each farm immunizing no less than 10,000 chickens;

[0033] Step S52, 7 days, 14 days, 21 days, 28 days, and 35 days after vaccination, randomly select 40 chickens from the multivalent vaccine group and the control vaccine group in each farm to collect blood and separate serum to measure HI antibodies. At the same time, pharyngeal swabs and cloacal swabs are collected from the 40 chickens, and the pharyngeal swabs and cloacal swabs of the same chicken are mixed into one tube to detect the positive rate of H9 influenza;

[0034] Step S53, analyzing the separation positive rates of HI antibodies and H9 influenza detected in the multivalent vaccine groups and the control vaccine groups of the three farms.

[0035] The advantages and positive effects of the present invention are:

[0036] The present invention provides a method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease. First, by adopting this method, the epidemiological survey is continuously carried out to control the mutation direction of H9 subtype avian influenza. The effectiveness of the current vaccine can be judged by genotyping and cross-hemagglutination inhibition test, providing a decision-making basis for developing new vaccines. Secondly, by screening a large sample, the broad spectrum of the product can be achieved as much as possible, and the product has universal applicability when promoted to different regions across the country. Furthermore, the hemagglutination inhibition test of the whole sample, the attack protection test, and the verification method of the clinical trial are used to ensure the effectiveness of the product. Furthermore, through clinical evaluation, both the antibody level and the protection effect are significantly better than the current vaccine, which can well solve the mutation problem of H9 subtype avian influenza virus.

[0037] The multivalent inactivated vaccine prepared by the method of the present invention not only provides good protection against attacks of homologous strains, but also can effectively resist attacks of heterologous strains, thereby effectively responding to infection of H9 subtype avian influenza virus, solving the core pain points of the current breeding industry, ensuring animal health, and improving the economic benefits of breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a simplified flow chart of the method of the present invention;

[0039] Figure 2 These are the results of PCR identification of 22 strains of H9 subtype avian influenza in 2020;

[0040] Figure 3 It is the genetic evolution tree of the nucleotide sequence of the HA gene of the H9 subtype avian influenza virus. DETAILED DESCRIPTION

[0041] In order to further understand the content, features and effects of the present invention, the following embodiments are given to explain in detail.

[0042] See also Figure 1 The present invention discloses a method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease, comprising the following steps:

[0043] Step S1, using separation and identification, gene sequencing and genotyping methods, a large number of samples obtained over a long period and from different regions are processed to determine the mutation direction of the virus.

[0044] This step specifically includes the following sub-steps:

[0045] Step S11, sample processing: collect tissues such as lung, larynx, trachea, liver, spleen, and oviduct of the diseased chicken, add them into an EP tube with an appropriate amount of physiological saline, fully grind the diseased tissue using a vibrating stainless steel strain method, centrifuge at 5000 rpm and 4°C for 10 minutes, and filter and sterilize the supernatant for later use;

[0046] Step S12, chicken embryo inoculation: the supernatant is inoculated into 5 10-day-old SPF chicken embryos through the allantoic cavity, 0.2 mL / embryo, and cultured in a 37°C incubator, and the embryos are inspected once a day, and the chicken embryos that die within 24 hours are discarded, and the chicken embryos that die after 24 hours are harvested at any time. After 96 hours, all chicken embryos are taken out, and the chicken embryo fluid is harvested respectively, and a sample is retained for a hemagglutination test;

[0047] Step S13, serological identification: taking a hemagglutination-positive sample to prepare 4HAU antigen, and conducting a hemagglutination inhibition test with chicken Newcastle disease HI positive serum, EDS76HI positive serum, avian influenza H9 subtype specific antiserum, H5, H7 subtype specific antiserum, and SPF chicken serum to carry out serological identification;

[0048] Step S14, RT-PCR test typing identification: take a hemagglutination-positive sample, extract RNA with EasyPure Viral DNA / RNA K, and reverse transcribe it into cDNA; use H9N2 subtype typing identification primers for PCR identification; after the PCR product is confirmed to be of correct size by electrophoresis, it is sent to a sequencing company for nucleotide sequence determination; use DNA STAR software to splice the sequencing results; obtain the representative HA gene sequence of the H9N2 subtype avian influenza virus from Gen Bank, and the HA gene sequence of the vaccine strain used in some domestic H9 subtype avian influenza inactivated vaccine products, and use software to perform genetic evolution analysis.

[0049] Step S2: Perform cross-hemagglutination inhibition test on all samples to select the preferred strains with broad protection spectrum.

[0050] This step specifically includes the following sub-steps:

[0051] Step S21, preparing single factor serum: taking antigens without exogenous virus contamination and preparing antigens by conventional methods and emulsifying oil emulsion inactivated vaccine, immunizing 21-day-old SPF chickens with a dose of 0.3 mL per chicken, immunizing 5 chickens with the vaccine corresponding to each strain; setting up 5 SPF chickens that were not immunized as controls and raised under the same conditions; 21 days after immunization, blood was collected from 5 chickens in each immunization group and 5 chickens in the control group under the same conditions, and serum was separated;

[0052] Step S22, cross hemagglutination inhibition test: using the inactivated antigen of each evolutionary branch virus liquid as the hemagglutination inhibition test antigen, and conducting a cross hemagglutination inhibition test with the prepared single factor serum of each strain;

[0053] Step S23, screening of preferred strains: based on the results of the cross-hemagglutination inhibition test, the strain with the smallest dispersion between the detection antigen prepared from each strain and the single factor serum HI is selected as the preferred strain for each evolved molecule.

[0054] Step S3, using the gold standard immune challenge method for immunogenicity evaluation, conduct a challenge test with the full sample size to verify the effectiveness of the screened strain in protecting against other strains in the same branch.

[0055] This step specifically includes the following sub-steps:

[0056] Step S31, preparation of inactivated vaccine of preferred strains and immunization: screening the preferred strains with broad serum protection spectrum of each evolutionary branch, and using the prepared inactivated vaccine to immunize 21-day-old SPF chickens, 0.3 mL per chicken;

[0057] Step S32, challenge test for each evolutionary branch: 21 days after vaccination, use other strains of each evolutionary branch for challenge, 10 chickens / group; the challenge method is: dilute the virus solution 10 times with PBS, and challenge by intravenous injection, 0.2 mL / chicken, and the challenge dose for each chicken is not less than 10 6.0 EID 50 ; On the 5th day after the virus attack, collect laryngeal and cloaca cotton swabs, mix the two cotton swabs collected from the same chicken as a sample, and inoculate into 10-day-old SPF chicken embryos for virus isolation; samples with negative virus isolation should be blindly propagated for one generation before judgment;

[0058] Step S33, determination of vaccine reserve strains: further verify whether the strains screened by the cross-hemagglutination inhibition test have a protection rate of more than 80% against other strains in the same evolutionary branch based on the challenge results, and finally determine whether the preferred strain can become a vaccine reserve strain.

[0059] Step S4, using the selected strains obtained through screening to prepare an inactivated vaccine.

[0060] This step specifically includes the following sub-steps:

[0061] Step S41, preparing an antigen solution of a preferred strain of H9 subtype avian influenza virus;

[0062] Step S42, oil phase preparation, 94 parts of white oil for injection and 2 parts of aluminum stearate are mixed evenly in an oil phase preparation tank, and then 6 parts of Siben-80 are added, and after mixing, they are sterilized by high pressure for standby use;

[0063] Step S43, aqueous phase preparation, mixing the antigen liquids of multiple H9 subtype avian influenza virus preferred strains that have passed the test in equal proportions, so that the total amount of each strain of avian influenza virus in the aqueous phase before inactivation is ≥10 7.5 EID 50 / 0.1mL; take 96 parts of the mixed antigen solution, add 4 parts of sterilized Tween-80, and stir thoroughly until the Tween-80 is completely dissolved;

[0064] Step S44, emulsification, take 2 parts of the oil phase and introduce them into an emulsification tank, slowly stir and slowly add 1 part of the water phase, mix at medium speed, and then emulsify at high speed; after emulsification, take 10mL of the vaccine and add it to a centrifuge tube, centrifuge at 3000r / min for 15min, and the water phase precipitated at the bottom of the tube should not exceed 0.5mL;

[0065] Step S45, quantitatively packing, sealing with a cover, and storing at 2-8°C.

[0066] Step S5, conducting clinical trials on poultry of different breeds and in different regions to evaluate the effectiveness of the product.

[0067] This step specifically includes the following sub-steps:

[0068] Step S51, immunizing white-feathered broilers, laying hens, and yellow-feathered broilers in farms in three provinces and cities using the multivalent inactivated vaccines prepared from the reserve vaccine strains of each evolutionary branch and the control H9 subtype avian influenza vaccine, with each farm immunizing no less than 10,000 chickens;

[0069] Step S52, 7 days, 14 days, 21 days, 28 days, and 35 days after vaccination, randomly select 40 chickens from the multivalent vaccine group and the control vaccine group in each farm to collect blood and separate serum to measure HI antibodies. At the same time, pharyngeal swabs and cloacal swabs are collected from the 40 chickens, and the pharyngeal swabs and cloacal swabs of the same chicken are mixed into one tube to detect the positive rate of H9 influenza;

[0070] Step S53, analyzing the separation positive rates of HI antibodies and H9 influenza detected in the multivalent vaccine groups and the control vaccine groups of the three farms.

[0071] A specific embodiment is given below for illustration.

[0072] A total of 954 suspected H9 subtype avian influenza samples were received from Beijing, Tianjin, Hebei, Henan, Shandong, Shanxi, Fujian, Sichuan, Jiangsu, Zhejiang, Guangdong, Guangxi, Liaoning and other regions. Virus isolation and identification were carried out on these 954 suspected samples, of which 231 samples were isolated and identified as having hemagglutination. Then serological tests and RT-PCR tests were carried out on the hemagglutinative cultures for preliminary identification. 96 strains were identified as H9 subtype avian influenza virus. The 96 H9 subtype avian influenza isolates were used to prepare inactivated vaccines to immunize SPF chickens to prepare single factor serum.

[0073] Next, HA gene sequencing and genetic evolution analysis were performed on 96 H9 subtype avian influenza viruses. According to the evolution of the HA gene, cross-hemagglutination inhibition tests were carried out on each evolutionary branch. A strain with a broad cross-spectrum of positive serum against other isolated strains was selected from each evolutionary branch. The preferred strain was then used to carry out immune protection studies in SPF chickens to verify the immunogenicity of the preferred strain.

[0074] The results show that the current mutation direction of the H9 subtype avian influenza has changed from the original 9.4.2.5a branch and 9.4.2.5b branch to the 9.4.2.5c branch. At the same time, the 9.4.2.5c branch has gradually evolved into 9.4.2.5c1, 9.4.2.5c2, and 9.4.2.5c3 branches, indicating that the current main prevention and control direction of the H9 subtype avian influenza is the 9.4.2.5c branch.

[0075] Through cross-hemagglutination inhibition test, three preferred strains were selected from 9.4.2.5c1, 9.4.2.5c2 and 9.4.2.5c3 branches, namely JN21, SY24 and WF23. Through immune protection test, it was verified that the three preferred strains can effectively protect against other strains in each branch.

[0076] Next, three selected strains were used to prepare a trivalent inactivated vaccine for the H9 subtype avian influenza, and clinical trials were carried out on white-feathered broilers, laying hens, and yellow-feathered broilers in farms in Shandong, Henan, and Jiangsu provinces. The test results showed that there was no significant difference in the antibody levels between the trivalent inactivated vaccines of JN21, SY24, and WF23 and the control vaccines at each age. However, during the detection of throat swabs and cloacal swabs 21 days after immunization, the positive rate of the H9 subtype influenza virus in the control vaccine was significantly higher than that in the trivalent inactivated vaccines of JN21, SY24, and WF23. This indicates that the trivalent inactivated vaccines of JN21, SY24, and WF23 can effectively prevent the spread of H9 subtype avian influenza in large-scale farms.

[0077] Based on this method, we can continue to carry out epidemiological investigation of H9 subtype avian influenza, select dominant strains every year, and use the dominant strains to continuously update the H9 subtype avian influenza inactivated vaccine strains, thereby achieving precise prevention and control of H9 subtype avian influenza.

[0078] Avian influenza (AIV) is the abbreviation of avian influenza, which is a syndrome of infectious diseases of birds caused by avian influenza virus. According to the pathogenicity, it can be clinically divided into highly pathogenic avian influenza and low pathogenic avian influenza. After infection with low pathogenic avian influenza virus, poultry can show mild respiratory and digestive symptoms, and the mortality rate is low. The H9N2 subtype avian influenza virus is a single-stranded negative-strand RNA virus composed of 8 gene segments. Its virulence is usually low pathogenic. It exists widely and continuously infects, reducing the production capacity of poultry. In 1992, the H9N2 avian influenza virus (AIV) first broke out in Guangdong, and then spread to other regions. In 1998, it quickly spread to most provinces in China, and then became widely prevalent in poultry groups in most parts of my country. It has become the main subtype of avian influenza virus prevalent in chickens.

[0079] The basic characteristics of H9 subtype avian influenza: First, rapid mutation is a characteristic of AI virus, and antigenic drift and antigenic conversion are important immune escape mechanisms caused by the virus HA protein. Second, the protective effect of antibodies against HA protein is better than that against HA protein antibodies. The vaccine against mucosal infection and the excretion of toxins after the virus attack depends on the similarity between the HA antigen of the vaccine strain and the HA antigen of the infected virus. Third, maternal antibodies can provide partial protection against homologous HA viruses. The effectiveness of inactivated vaccine immunity in preventing clinical symptoms, mortality and reducing in vivo excretion has been fully demonstrated, but it cannot completely prevent infection through the respiratory mucosa. The main infection routes of AIV are the respiratory tract and digestive tract. Fourth, low pathogenic AI is usually limited to replication in the respiratory tract and intestines. The main reason for the death of poultry by some strains is bacterial infection after respiratory tract damage. Fifth, the ability of different hosts to produce antibodies after infection with AI is significantly different: chicken> pheasant> turkey> quail> duck. There are also large differences between different breeds of the same host. Meat ducks and broilers with short growth periods have poor immune antibody responses, while egg breeder chickens and egg breeder ducks have relatively good immune antibodies.

[0080] The H9 subtype AI series of inactivated vaccines are almost the vaccines with the most strains among my country's veterinary biological products. In 2003, the avian influenza inactivated vaccine (H9N2 subtype, SDS696 strain) developed by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences obtained the Class II new veterinary drug certificate, becoming my country's first new veterinary drug for H9 subtype AI. At present, more than 100 H9 subtype AI inactivated vaccines have been approved in my country. The H9 subtype series of inactivated vaccines involve more than 20 H9 strains, including Re-2 strain, SDS696 strain, SS strain, L strain, HP strain, SY strain, LG1 strain, YBF003 strain, F strain, S2 strain, NJ02 strain, NJ01 strain, HZ strain, JY strain, HN106 strain, WJ57 strain, etc. The large-scale use of vaccines has indeed played an important role in the prevention and control of avian influenza. Monitoring data of H9N2 subtype avian influenza in recent years show that the currently prevalent H9N2 virus has undergone significant changes in genetic evolution and antigenic analysis. This suggests that we should screen new H9N2 vaccine strains as soon as possible for the development of new vaccines to cope with the prevention and control of mutated H9N2 subtype avian influenza. Therefore, epidemiological surveys are widely carried out in different regions, influenza virus genotype evolution analysis is conducted, the mutation direction of influenza is controlled, cross-hemagglutination inhibition tests are carried out in different branches, and preferred strains with broad protection spectrum of different branches are selected. Only by providing customized strains according to the variant branches of H9 subtype avian influenza in different regions can precise prevention and control be achieved.

[0081] 1 Materials

[0082] 1.1 Sample

[0083] The samples were 954 samples of lung, larynx, trachea, liver, spleen and other tissues of diseased chickens collected from farms in Beijing, Tianjin, Hebei, Henan, Shandong, Shanxi, Fujian, Sichuan, Jiangsu, Zhejiang, Guangdong, Guangxi, Liaoning and other places.

[0084] 1.2 Breeding eggs and experimental animals

[0085] SPF eggs and SPF chickens were purchased from Beijing Boehringer Ingelheim Laboratory Animal Technology Co., Ltd.

[0086] 1.3 Positive serum

[0087] H9 subtype avian influenza virus positive serum was purchased from Harbin Weike Biotechnology Development Company; H5 and H7 subtype avian influenza virus HI positive serum was provided by Qianyuanhao Biological Co., Ltd.; chicken Newcastle disease positive serum and EDS76HI positive serum were purchased from China Veterinary Drug Administration.

[0088] 1.4 Reagents

[0089] Total RNA extraction kit, reverse transcription kit, PCR kit, DL 2000Marker, etc. were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; pMD18-T vector kit and DH5α competent cells were purchased from TaKaRa Company; gel recovery kit was purchased from Tiangen Biochemical Technology Co., Ltd.

[0090] 2 Methods

[0091] 2.1 Virus isolation

[0092] 2.1.1 Sample processing The lungs, larynx, trachea, liver, spleen, oviduct and other tissues of the diseased chickens were collected and added to an EP tube with an appropriate amount of physiological saline. The diseased tissues were fully ground using the oscillating stainless steel strain method, and centrifuged at 5000 rpm and 4°C for 10 minutes. The supernatant was filtered and sterilized for later use.

[0093] 2.1.2 Chicken embryo inoculation: Inoculate 5 10-day-old SPF chicken embryos with the supernatant via the allantoic cavity at 0.2 mL / embryo and culture in a 37°C incubator. Check the embryos once a day, discard any chicken embryos that die within 24 hours, and harvest any that die after 24 hours at any time. After 96 hours, take out all the chicken embryos, harvest the chicken embryo fluid separately, and keep a sample for the red blood cell agglutination test.

[0094] 2.2 Virus identification

[0095] 2.2.1 Serological identification The hemagglutination-positive samples were prepared into 4HAU antigen, and serological identification was carried out by performing hemagglutination inhibition test using Newcastle disease HI positive serum, EDS76HI positive serum, avian influenza H9 subtype specific antiserum, H5, H7 subtype specific antiserum, and SPF chicken serum.

[0096] 2.2.2 RT-PCR test typing identification

[0097] Take the hemagglutination-positive sample, extract RNA with EasyPure Viral DNA / RNA K, and reverse transcribe it into cDNA. Use H9N2 subtype typing identification primers for PCR identification. After the PCR product is confirmed to be of correct size by electrophoresis, it is sent to a sequencing company for nucleotide sequence determination. Use DNA STAR software to splice the sequencing results. Obtain the HA gene sequence of a representative H9N2 subtype avian influenza virus from Gen Bank, and the HA gene sequence of the vaccine strain used in some domestic H9 subtype avian influenza inactivated vaccine products, and use the software for genetic evolution analysis.

[0098] Table 1 Primers for avian influenza H9 subtype typing and identification

[0099]

[0100] 2.3 Study on the immune efficacy of candidate strains

[0101] 2.3.1 Cross HI test

[0102] The virus without exogenous virus contamination was used to prepare antigens by conventional methods and emulsified with oil emulsion inactivated vaccine. 21-day-old SPF chickens were immunized with 0.3 mL of each vaccine, and 5 chickens were immunized with each vaccine. Five SPF chickens were not immunized as controls and raised under the same conditions. 21 days after immunization, blood was collected from 5 chickens in each immunization group and the control chickens under the same conditions, and serum was separated. The inactivated antigen of the virus liquid of each evolutionary branch was used as the hemagglutination inhibition test antigen to carry out the cross hemagglutination inhibition test.

[0103] 2.3.2 Virus attack and virus isolation

[0104] The selected strains with broad serum protection spectrum of each evolutionary branch were screened, and the prepared inactivated vaccine was used to immunize 21-day-old SPF chickens, 0.3 mL per chicken. 21 days after immunization, the other strains of each evolutionary branch were used for challenge, 10 chickens / group. The challenge method was: the virus liquid was diluted 10 times with PBS (0.1 mol / L, pH 7.0-7.4), and the challenge was performed by intravenous injection, 0.2 mL / chicken, and the challenge dose for each chicken was not less than 10 6.0 EID 50 On the fifth day after the virus attack, larynx and cloaca swabs were collected, and the two swabs from the same chicken were mixed as one sample and inoculated into 10-day-old SPF chicken embryos for virus isolation. Samples with negative virus isolation should be blindly propagated for one generation before judgment.

[0105] 3 Results

[0106] 3.1 Identification

[0107] 3.1.1 Serological identification

[0108] From 2020 to 2024, serological identification will be carried out every year by performing red blood cell agglutination inhibition tests on each hemagglutination-positive chicken embryo fluid using Newcastle disease HI positive serum, EDS76HI positive serum, avian influenza H9 subtype specific antiserum, H5, H7 subtype specific antiserum, and SPF chicken serum. In 2020, 22 out of 53 hemagglutinating samples were positive only with H9 subtype-specific antiserum, in 2021, 14 out of 35 hemagglutinating samples were positive only with H9 subtype-specific antiserum, in 2022, 16 out of 41 hemagglutinating samples were positive only with H9 subtype-specific antiserum, in 2023, 31 out of 68 hemagglutinating samples were positive only with H9 subtype-specific antiserum, in 2024, 13 out of 34 hemagglutinating samples were positive only with H9 subtype-specific antiserum, and the rest were negative, indicating that the isolated strain may be the H9 subtype avian influenza virus.

[0109] Table 2HI results of 22 hemagglutination-positive samples in 2020 using different positive sera

[0110]

[0111]

[0112] Note: “+” means positive reaction; “-” means negative reaction.

[0113] Table 3 HI results of 14 hemagglutination-positive samples in 2021 using different positive sera

[0114] sample H9 serum ND serum EDS serum H5 serum H7 serum SPF chicken serum AQ21 + - - - - - AQIU21 + - - - - - AY21 + - - - - - HB21 + - - - - - HZHOU21 + - - - - - HZ21 + - - - - - JM21 + - - - - - JN21 + - - - - - LX21 + - - - - - LC21 + - - - - - NJ21 + - - - - - NX21 + - - - - - QY21 + - - - - - SG21 + - - - - -

[0115] Note: “+” means positive reaction; “-” means negative reaction.

[0116] Table 4 HI results of 16 hemagglutination-positive samples in 2022 using different positive sera

[0117]

[0118]

[0119] Note: “+” means positive reaction; “-” means negative reaction.

[0120] Table 5 HI results of 31 hemagglutination-positive samples in 2023 using different positive sera

[0121]

[0122]

[0123] Note: “+” means positive reaction; “-” means negative reaction.

[0124] Table 6 HI results of 13 hemagglutination positive samples in 2024 using different positive sera

[0125] sample H9 serum ND serum EDS serum H5 serum H7 serum SPF chicken serum BJ24 + - - - - - CZ24 + - - - - - CZHOU24 + - - - - - DY24 + - - - - - DYING24 + - - - - - JN24 + - - - - - LY24 + - - - - - SQ24 + - - - - - SY24 + - - - - - TA24 + - - - - - YZ24 + - - - - - ZQ24 + - - - - - ZB24 + - - - - -

[0126] Note: “+” means positive reaction; “-” means negative reaction.

[0127] 3.3 Identification of viral HA gene

[0128] 3.3.1 Virus RT-PCR test results

[0129] In 2020, PCR identification was performed on 22 strains that were serologically identified as possible H9 subtype avian influenza. The results showed that the two pairs of specific primers used amplified bands of approximately 1101bp and 1038bp in size, respectively, which was consistent with expectations.

[0130] See also Figure 2 , lanes 1 to 8 on the left are the products of amplification of the H9 subtype HA1 fragment, and lane 9 is a positive control. Lanes 1 to 8 on the right are the results of amplification of the H9 subtype HA2 fragment, and lane 9 is a positive control.

[0131] In 2021, PCR identification was conducted on 14 strains that were serologically identified as possible H9 subtype avian influenza. The results showed that the two pairs of specific primers used amplified bands of approximately 1101bp and 1038bp in size, respectively, which was consistent with expectations.

[0132] In 2022, PCR identification was conducted on 16 strains that were serologically identified as possible H9 subtype avian influenza. The results showed that the two pairs of specific primers used amplified bands of approximately 1101bp and 1038bp in size, respectively, which was consistent with expectations.

[0133] In 2023, PCR identification was conducted on 31 strains that were serologically identified as possible H9 subtype avian influenza. The results showed that the two pairs of specific primers used amplified bands of approximately 1101bp and 1038bp in size, respectively, which was consistent with expectations.

[0134] In 2024, PCR identification was performed on 13 strains that were serologically identified as possible H9 subtype avian influenza. The results showed that the two pairs of specific primers used amplified bands of approximately 1101bp and 1038bp in size, respectively, which was consistent with expectations.

[0135] 3.3.2 Genetic evolution analysis of viral HA gene

[0136] The software was used to perform genetic evolution analysis on the HA genes of the isolates, vaccine strains, and reference strains. The results are shown in Figure 3 . In 2020, a total of 22 strains of H9 subtype avian influenza were isolated, in 2021, a total of 14 strains of H9 subtype avian influenza were isolated, in 2022, a total of 16 strains of H9 subtype avian influenza were isolated, in 2023, a total of 31 strains of H9 subtype avian influenza were isolated, and in 2024, a total of 13 strains of H9 subtype avian influenza were isolated.

[0137] See also Figure 2, from the evolutionary tree, we can see: the variation of H9 subtype avian influenza, 22 strains of H9 subtype avian influenza isolated in 2020, of which 6 strains of H9 subtype avian influenza belong to the 9.4.2.5a branch, 10 strains belong to the 9.4.2.5b branch, and 6 strains belong to the 9.4.2.5c branch. 14 strains of H9 subtype avian influenza isolated in 2021, of which 3 strains of H9 subtype avian influenza belong to the 9.4.2.5a branch, 3 strains belong to the 9.4.2.5b branch, and 8 strains belong to the 9.4.2.5c branch. 16 strains of H9 subtype avian influenza isolated in 2022 Among them, 2 strains of H9 subtype avian influenza belong to the 9.4.2.5a branch, 2 strains belong to the 9.4.2.5b branch, and 12 strains belong to the 9.4.2.5c branch. Among the 31 strains of H9 subtype avian influenza isolated in 2023, 1 strain of H9 subtype avian influenza belongs to the 9.4.2.5b branch, and 30 strains belong to the 9.4.2.5c branch. Among the 13 strains of H9 subtype avian influenza isolated in 2024, 1 strain of H9 subtype avian influenza belongs to the 9.4.2.5a branch, 1 strain belongs to the 9.4.2.5b branch, and 11 strains belong to the 9.4.2.5c branch. From the above analysis, it can be seen that the mutation direction of H9 subtype avian influenza has changed from being dominated by 9.4.2.5a and 9.4.2.5b branches in 2020 and 2021 to being dominated by 9.4.2.5c branches in 2022, 2023, and 2024, and the 9.4.2.5c branch is further divided into three branches: 9.4.2.5c1, 9.4.2.5c2, and 9.4.2.5c3. From the mutation direction of H9 subtype avian influenza isolates, it can be seen that the prevention and control direction of H9 subtype avian influenza in the future will focus on the prevention and control of 9.4.2.5c branch epidemic strains, in order to effectively solve the prevention and control problems of H9 subtype avian influenza.

[0138] 3.2 Study on the immune efficacy of candidate strains

[0139] 19 strains of H9 subtype avian influenza virus from branch 9.4.2.5c1, 16 strains from branch 9.4.2.5c2, and 31 strains from branch 9.4.2.5c3 were inactivated with β-propiolactone and prepared into an oil-emulsion inactivated vaccine. The vaccine was inoculated into 4-week-old SPF chickens through multiple injections with 1 mL / feather. Then, 2 and 3 immunizations were performed at intervals of 2 weeks with 0.5 mL / feather respectively. Blood was collected 21 days after the three immunizations and serum was separated. The inactivated antigens of each strain of each evolutionary branch were used as antigens for the hemagglutination inhibition test, and cross-hemagglutination inhibition tests were carried out with the positive sera of each evolutionary branch.

[0140] From the test results, it can be seen that among the 19 strains of the 9.4.2.5c1 branch, the positive sera prepared from different strains of the c1 branch were tested using different detection antigens of the c1 branch, and the positive serum of the JN21 strain had the smallest discreteness. Among the 16 strains of the 9.4.2.5c2 branch, the positive sera prepared from different strains of the c2 branch were tested using different detection antigens of the c2 branch, and the positive serum of the SY24 strain had the smallest discreteness. Among the 32 strains of the 9.4.2.5c3 branch, the positive sera prepared from different strains of the c3 branch were tested using different detection antigens of the c3 branch, and the positive serum of the WF23 strain had the smallest discreteness. This shows that the positive sera prepared from the antigens of the JN21 strain of the c1 branch, the SY24 strain of the c2 branch, and the WF23 strain of the c3 branch can better combine with the H9N2 avian influenza strains isolated in recent years, and have a greater chance of being candidate strains.

[0141] Table 79.4.2.5c1 branch 19 isolates cross HI test results

[0142]

[0143] Table 89.4.2.5C2 branch 16 isolates cross HI test results

[0144]

[0145] Table 9-19.4.2.5 Results of cross HI test of 32 isolates in branch c3

[0146]

[0147] Table 9-29.4.2.5 Results of cross-HI test of 32 isolates in branch c3

[0148]

[0149] 3.2.2 Virus isolation results after challenge

[0150] Table 10 The protective effect of trivalent inactivated vaccines of JN21, SY24 and WF23 against 19 strains of 9.4.2.5c1 branch H9 virus strains

[0151]

[0152]

[0153] On the 5th day after the challenge, cotton swabs were collected and inoculated into chicken embryos for virus isolation, and the results are shown in Table 10. The virus isolation results showed that the JN21 strain could produce ideal protection (8 / 10 to 10 / 10 protection) against both homologous and heterologous strains.

[0154] Table 11 The protective effect of trivalent inactivated vaccines of JN21, SY24 and WF23 against 16 strains of 9.4.2.5c2 branch H9 virus strains

[0155]

[0156]

[0157] On the 5th day after the challenge, cotton swabs were collected and inoculated into chicken embryos for virus isolation, and the results are shown in Table 11. The virus isolation results showed that the SY24 strain could produce ideal protection (8 / 10 to 10 / 10 protection) against both homologous and heterologous strains.

[0158] Table 12 The protective effect of trivalent inactivated vaccines of JN21, SY24 and WF23 against 32 strains of 9.4.2.5c3 branch H9 virus strains

[0159]

[0160]

[0161] On the 5th day after the challenge, cotton swabs were collected and inoculated into chicken embryos for virus isolation. The results are shown in Table 12. The virus isolation results showed that the WF23 strain could produce ideal protection (8 / 10 to 10 / 10 protection) against both homologous and heterologous strains.

[0162] 3.2.3 Clinical trials

[0163] Table 13 Verification of the efficacy of clinical trials of vaccines in farms in Liaocheng, Shandong

[0164]

[0165] Note: The immunization age is 7-day-old commercial white-feathered broilers.

[0166] Table 14 Verification of the effectiveness of clinical trials of vaccines in farms in Nanyang, Henan

[0167]

[0168] Note: The immunization age is 7-day-old commercial laying hens.

[0169] Table 15: Effect verification of vaccine clinical trial in Jianhu farm, Jiangsu

[0170]

[0171]

[0172] Note: The immunization age is 7-day-old commercial yellow-feathered broilers.

[0173] The research team conducted clinical trials on white-feathered broilers, laying hens and yellow-feathered broilers in farms in Shandong, Henan and Jiangsu provinces and cities using the trivalent inactivated vaccines of JN21, SY24 and WF23 prepared from the three preferred strains screened out through the above experiments. The results showed that there was no significant difference in antibody levels between the trivalent inactivated vaccines of JN21, SY24 and WF23 and the control vaccines at each age. However, during the detection of throat swabs and cloacal swabs 21 days after immunization, the positive rate of H9 subtype influenza virus in the control vaccine was significantly higher than that in the trivalent inactivated vaccines of JN21, SY24 and WF23. This indicates that the trivalent inactivated vaccines of JN21, SY24 and WF23 can effectively prevent the spread of H9 subtype avian influenza in large-scale farms.

Claims

1. A method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease, characterized in that: The following steps are included: Step S1, using separation and identification, gene sequencing and genotyping methods to process a large number of samples obtained over a long period and from different regions to determine the mutation direction of the virus; Step S2, performing a cross-hemagglutination inhibition test on all samples to screen out the preferred strains with a broad protection spectrum; Step S3, using the gold standard immune challenge method for immunogenicity evaluation, a challenge test is conducted on the full sample size to verify the protective effectiveness of the screened strain against other strains in the same branch; Step S4, using the selected strains obtained through screening to prepare an inactivated vaccine; Step S5, conducting clinical trials on poultry of different breeds and in different regions to evaluate the effectiveness of the product.

2. The method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease as claimed in claim 1, characterized in that: Step S1 includes the following steps, Step S11, sample processing: collect tissues such as lung, larynx, trachea, liver, spleen, and oviduct of the diseased chicken, add them into an EP tube with an appropriate amount of physiological saline, fully grind the diseased tissue using a vibrating stainless steel strain method, centrifuge at 5000 rpm and 4°C for 10 minutes, and filter and sterilize the supernatant for later use; Step S12, chicken embryo inoculation: the supernatant is inoculated into 5 10-day-old SPF chicken embryos through the allantoic cavity, 0.2 mL / embryo, and cultured in a 37°C incubator, and the embryos are inspected once a day, and the chicken embryos that die within 24 hours are discarded, and the chicken embryos that die after 24 hours are harvested at any time. After 96 hours, all chicken embryos are taken out, and the chicken embryo fluid is harvested respectively, and a sample is retained for a hemagglutination test; Step S13, serological identification: taking a hemagglutination-positive sample to prepare 4HAU antigen, and conducting a hemagglutination inhibition test with chicken Newcastle disease HI positive serum, EDS76HI positive serum, avian influenza H9 subtype specific antiserum, H5, H7 subtype specific antiserum, and SPF chicken serum to carry out serological identification; Step S14, RT-PCR test typing identification: take a hemagglutination positive sample, extract RNA with EasyPure Viral DNA / RNA K, and reverse transcribe it into cDNA; use H9N2 subtype typing identification primers for PCR identification; after the PCR product is confirmed to be of correct size by electrophoresis, it is sent to a sequencing company for nucleotide sequence determination; the sequencing results are spliced ​​using DNA STAR software; the HA gene sequence of a representative H9N2 subtype avian influenza virus and the HA gene sequence of the vaccine strain used in some domestic H9 subtype avian influenza inactivated vaccine products are obtained from GenBank, and the genetic evolution analysis is performed using software.

3. The method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease as claimed in claim 2, characterized in that: Step S2 The following steps are included: Step S21, preparing single factor serum: taking antigens without exogenous virus contamination and preparing antigens by conventional methods and emulsifying oil emulsion inactivated vaccine, immunizing 21-day-old SPF chickens with a dose of 0.3 mL per chicken, immunizing 5 chickens with the vaccine corresponding to each strain; setting up 5 SPF chickens that were not immunized as controls and raised under the same conditions; 21 days after immunization, blood was collected from 5 chickens in each immunization group and 5 chickens in the control group under the same conditions, and serum was separated; Step S22, cross hemagglutination inhibition test: using the inactivated antigen of each evolutionary branch virus liquid as the hemagglutination inhibition test antigen, and conducting a cross hemagglutination inhibition test with the prepared single factor serum of each strain; Step S23, screening of preferred strains: based on the results of the cross-hemagglutination inhibition test, the strain with the smallest dispersion between the detection antigen prepared from each strain and the single factor serum HI is selected as the preferred strain for each evolved molecule.

4. The method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease as claimed in claim 3, characterized in that: Step S3 includes the following steps, Step S31, preparation of inactivated vaccine of preferred strains and immunization: screening the preferred strains with broad serum protection spectrum of each evolutionary branch, and using the prepared inactivated vaccine to immunize 21-day-old SPF chickens, 0.3 mL per chicken; Step S32, challenge test for each evolutionary branch: 21 days after vaccination, use other strains of each evolutionary branch for challenge, 10 chickens / group; the challenge method is: dilute the virus solution 10 times with PBS, and challenge by intravenous injection, 0.2 mL / chicken, and the challenge dose for each chicken is not less than 10 6.0 EID 50 ; On the 5th day after the virus attack, collect laryngeal and cloaca cotton swabs, mix the two cotton swabs collected from the same chicken as a sample, and inoculate into 10-day-old SPF chicken embryos for virus isolation; samples with negative virus isolation should be blindly propagated for one generation before judgment; Step S33, determination of vaccine reserve strains: further verify whether the strains screened by the cross-hemagglutination inhibition test have a protection rate of more than 80% against other strains in the same evolutionary branch based on the challenge results, and finally determine whether the preferred strain can become a vaccine reserve strain.

5. The method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease as claimed in claim 4, characterized in that: Step S4 The following steps are included: Step S41, preparing an antigen solution of a preferred strain of H9 subtype avian influenza virus; Step S42, oil phase preparation, 94 parts of white oil for injection and 2 parts of aluminum stearate are mixed evenly in an oil phase preparation tank, and then 6 parts of Siben-80 are added, and after mixing, they are sterilized by high pressure for standby use; Step S43, aqueous phase preparation, mixing the antigen liquids of multiple H9 subtype avian influenza virus preferred strains that have passed the test in equal proportions, so that the total amount of each strain of avian influenza virus in the aqueous phase before inactivation is ≥10 7.5 EID 50 / 0.1mL; take 96 parts of the mixed antigen solution, add 4 parts of sterilized Tween-80, and stir thoroughly until the Tween-80 is completely dissolved; Step S44, emulsification, take 2 parts of the oil phase and introduce them into an emulsification tank, slowly stir and slowly add 1 part of the water phase, mix at medium speed, and then emulsify at high speed; after emulsification, take 10mL of the vaccine and add it to a centrifuge tube, centrifuge at 3000r / min for 15min, and the water phase precipitated at the bottom of the tube should not exceed 0.5mL; Step S45, quantitatively packing, sealing with a cover, and storing at 2-8°C.

6. The method for preparing a multivalent inactivated vaccine for preventing H9 subtype avian influenza virus disease as claimed in claim 5, characterized in that: Step S5 The following steps are included: Step S51, immunizing white-feathered broilers, laying hens, and yellow-feathered broilers in farms in three provinces and cities using the multivalent inactivated vaccines prepared from the reserve vaccine strains of each evolutionary branch and the control H9 subtype avian influenza vaccine, with each farm immunizing no less than 10,000 chickens; Step S52, 7 days, 14 days, 21 days, 28 days, and 35 days after vaccination, randomly select 40 chickens from the multivalent vaccine group and the control vaccine group in each farm to collect blood and separate serum to measure HI antibodies. At the same time, pharyngeal swabs and cloacal swabs are collected from the 40 chickens, and the pharyngeal swabs and cloacal swabs of the same chicken are mixed into one tube to detect the positive rate of H9 influenza; Step S53, analyzing the separation positive rates of HI antibodies and H9 influenza detected in the multivalent vaccine groups and the control vaccine groups of the three farms.

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