H9N2 subtype avian influenza virus bivalent inactivated vaccine and preparation method thereof

Abstract

The invention discloses an H9N2 subtype avian influenza virus, the H9N2 subtype avian influenza virus is A / chick / Guangxi / C1228 / 2015 (H9N2), and the preservation number of the H9N2 subtype avian influenza virus is CGMCC (China General Microbiological Culture Collection Center) No.45222; or the H9N2 subtype avian influenza virus is A / quil / Guangxi / 210Q33 / 2015 (H9N2), and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 46091. The bivalent inactivated vaccine of CK / GX / C1228 / 15 with better antigenicity and the strain QL / GX / 210Q33 / 15 which has better antigenicity and is separated from quails in the live poultry market has a better protection effect on two different branches of H9N2 subtype avian influenza viruses, the lowest immune dose (0.3 mL) can reach the protection rate of 80% or above, and the generated effective protection antibody can last to 24W.

Description

Technical Field

[0001] The present invention relates to the field of vaccine preparation, and particularly to a bivalent inactivated vaccine against H9N2 subtype avian influenza virus and a preparation method thereof. Background Art

[0002] Avian influenza (AI) is an avian infectious disease caused by avian influenza virus A (AIV), which affects poultry and wild birds. The genome of influenza A virus consists of eight segments, including PB2, PB1, PA, HA, NP, NA, M, and NS. According to the antigenicity of hemagglutinin (HA) and neuraminidase (NA) on the surface of AIV, AIV can be divided into multiple subtypes. Currently, there are eighteen known HA subtypes and eleven NA subtypes. In addition, according to the pathogenicity to chickens, AIV can be further divided into highly pathogenic AIV (HPAIV) and low pathogenic AIV (LPAIV).

[0003] Low pathogenic H9N2 subtype avian influenza virus widely exists in poultry populations in many countries and shows endemic prevalence. Although poultry will not die in large numbers after being infected with H9N2 subtype avian influenza virus, this virus will cause consequences such as a decrease in egg production and a weakening of the body's immunity in poultry, and it is easy to be co-infected with other pathogens, thereby increasing the mortality rate and bringing significant economic losses to the poultry industry. In addition, the H9N2 subtype avian influenza virus has strong transmission ability and a wide range, posing challenges to prevention and control. The host of H9N2 subtype AIV is also very wide, and this subtype of AIV has also been found in other animals such as ducks, quails, chickens, pigeons, egrets, and pigs. Currently, the most effective preventive measure is still vaccination. However, due to the continuous mutation of the H9N2 subtype AIV antigen, existing vaccines can no longer provide sufficient immune protection. Our laboratory has long monitored low pathogenic avian influenza virus in live poultry markets and wild bird markets in Guangxi, and a large number of H9N2 subtype avian influenza viruses have been found during the monitoring process. Therefore, high attention still needs to be paid to the prevention and control of avian influenza epidemics. Most of the H9 subtype AIV in the live poultry market in Guangxi belongs to Y280-Like, and most of the H9 subtype AIV in the wild bird market belongs to G1-Like. In recent years, a small amount of H9 subtype AIV strains belonging to G1-Like have also been monitored in chickens and ducks in the live poultry market. By comparing the HA genes of H9N2 subtype avian influenza viruses in different years, it is found that the similarity in recent years is significantly lower and the difference is significantly increased compared with the past. By selecting H9N2 subtype avian influenza viruses from different years in Guangxi to prepare SPF chicken hyperimmune sera and conducting cross-HI antibody titer experiments, it is also found that the antigen differences between them are relatively large.

[0004] During the past 20 years of genetic evolution, obvious antigenic drift has occurred in H9N2 subtype AIV. Three commercial H9N2 vaccine strains were isolated in the 1990s, and clinical data showed that none of these three vaccines could effectively protect poultry from the current prevalent strains. Currently, in China, inactivated vaccines against H9N2 subtype belonging to two antigenic groups of Y280-Like branch and G1-Like branch are still blank. Therefore, the practical application prospect of this research is extremely broad, and it has important research value for the prevention and control of the spread of avian influenza virus. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a bivalent inactivated vaccine against H9N2 subtype avian influenza virus and its preparation method, aiming to conduct in-depth analysis on the prevalent strains in Guangxi region isolated from 2000 to 2020. Through comprehensive epidemiological analysis, antigenicity analysis and immunogenicity analysis, potential vaccine strains are screened out. Exhaustive immunogenicity verification is carried out on the screened vaccine strains to improve the antigenic matching degree between the vaccine strains and the prevalent strains, thereby enhancing the effective immune effect of H9N2 subtype avian influenza vaccine.

[0006] To achieve the above purpose, the technical solutions provided by the present invention are as follows:

[0007] A kind of H9N2 subtype avian influenza virus, the H9N2 subtype avian influenza virus is A / chicken / Guangxi / C1228 / 2015(H9N2) (abbreviation: CK / GX / C1228 / 15), and the preservation number is CGMCC No.45222; or the H9N2 subtype avian influenza virus is A / quail / Guangxi / 210Q33 / 2015(H9N2) (abbreviation: QL / GX / 210Q33 / 15), and the preservation number is CGMCC No.46091.

[0008] Furthermore, the application of the H9N2 subtype avian influenza virus in the preparation of drugs for preventing and controlling avian influenza.

[0009] Furthermore, the drug is a vaccine, and the vaccine is an inactivated vaccine.

[0010] A bivalent inactivated vaccine against H9N2 subtype avian influenza virus, the vaccine is prepared by inactivating the H9N2 subtype avian influenza virus A / chicken / Guangxi / C1228 / 2015(H9N2) with the preservation number of CGMCC No.45222 and A / quail / Guangxi / 210Q33 / 2015(H9N2) with the preservation number of CGMCC No.46091 as antigens.

[0011] A method for preparing a bivalent inactivated vaccine of H9N2 subtype avian influenza virus comprises the following steps:

[0012] S1 inactivated vaccine: A / chicken / Guangxi / C1228 / 2015(H9N2) and A / quail / Guangxi / 210Q33 / 2015(H9N2) H9N2 virus liquid were inactivated with BPL at a final concentration of 0.05% at 20°C for 16 h;

[0013] S2: Prepare the oil phase: Mix 94 parts of white oil for injection and 6 parts of Siben-80 evenly according to the volume ratio, sterilize and set aside;

[0014] S3: Prepare the aqueous phase: according to the volume ratio, take 48.5 parts of the inactivated C1228 strain and 210Q33 strain stock solutions, mix them, add 3 parts of sterilized Tween-80 and stir thoroughly until dissolved;

[0015] S4 emulsification: Emulsify at a volume ratio of 1:2 between the water phase and the oil phase, slowly add the water phase to the oil phase, pre-emulsify for 1 minute, gradually add the virus liquid to the oil phase during the emulsification process, and then emulsify at high speed for 5 minutes. After the emulsification process is completed, you can test its stability by dropping a few drops of inactivated vaccine on the water surface. If the vaccine does not disperse and presents a good oil-in-water state, it means that its emulsification is good; obtain a well-emulsified H9N2 subtype AIV bivalent inactivated vaccine, seal it, mark it, and finally store it in a 4°C refrigerator.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The bivalent inactivated vaccine of the present invention, which uses CK / GX / C1228 / 15 with better antigenicity of the h9.4.2.5 branch and QL / GX / 210Q33 / 15 with better antigenicity of the h9.4.1 branch and isolated from quails in the live poultry market, has better protection effect on two different branches of H9N2 subtype avian influenza virus, and the lowest immunization dose (0.3 mL) can achieve a protection rate of more than 80%, and the generated effective protective antibodies can last up to 24 weeks.

[0018] Depository Information

[0019] The H9N2 subtype avian influenza virus A / chicken / Guangxi / C1228 / 2015 (H9N2) was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on July 4, 2022, with the deposit number CGMCC No.45222.

[0020] The avian influenza virus A / quail / Guangxi / 210Q33 / 2015 (H9N2) of the H9N2 subtype was deposited with the China General Microbiological Culture Collection Center (abbreviated as CGMCC) on July 22, 2024, and the deposit number is CGMCC No. 46091. Description of the Drawings

[0021] Figure 1 It is the cytopathic effect produced by the virus after infecting cells, and low melting point agarose is used to fix the diseased cells; healthy control (left), plaque of H9N2 subtype AIV strain C1228 on MDCK cells (right).

[0022] Figure 2 It is the phylogenetic tree of the HA gene of some H9N2 subtype avian influenza viruses in Guangxi from 2000 to 2020.

[0023] Figure 3 It is the protection rate of inactivated vaccines of strains C1228, 210Q33 and SS.

[0024] Figure 4 It is the antibody titer of inactivated vaccines of strains C1228, 210Q33 and SS in SPF chickens 21 days after immunization.

[0025] Figure 5 It is the comparison of HI antibody titers in SPF chickens 21 days after immunization with different immunization doses. Detailed Embodiments

[0026] The following describes the detailed embodiments in conjunction with the drawings, but it should be understood that the protection scope of the present invention is not limited by the detailed embodiments. The raw materials and reagents used in the examples are all commercially available unless otherwise specified.

[0027] SPF chicken embryos were purchased from Beijing Boehringer Ingelheim Vetmedica Biotechnology Co., Ltd., SPF chicken embryos were hatched by themselves, and SPF chickens were raised in a negative pressure SPF chicken isolator.

[0028] The inactivated H9 subtype avian influenza vaccine (strain SS) and its detection antigen (strain SS, HA titer greater than or equal to 8log2) were purchased from Guangzhou South China Agricultural University Biopharmaceutical Co., Ltd. The sequences of other reference strains were derived from the GenBank database. The detailed information of all strains is shown in Table 3.

[0029] The viral RNA extraction kit, gel purification and recovery kit, and DNA Marker were all purchased from TransGen Biotech Co., Ltd. Reverse Transcriptase M-MLV, 6× Loading Buffer, PrimeSTAR Max DNA Polymerase (high-fidelity enzyme), and Ex Taq DNA Polymerase were purchased from Takara Company. Tween-80 was purchased from Shanghai High Dimension Chemical Co., Ltd.; Span-80 was purchased from Shanghai Sangon Biotech Co., Ltd.; white oil was purchased from Hangzhou Refinery of Sinopec Group; β-propiolactone (BPL) virus inactivator was purchased from Bupps (Hong Kong) Trading Co., Ltd. The ATS high-pressure homogenizer was purchased from Shanghai ATS Industrial Systems Co., Ltd.; the negative-pressure SPF chicken isolator was purchased from Jiangsu Feng's Animal Equipment Co., Ltd. The disposable blood collection tubes for experimental animals were products of Kangweishi Company.

[0030] The positive serum of H9N2 subtype AIV for hemagglutinin inhibition (HI) test was a product of Harbin Veterinary Research Institute, China. Positive sera of other subtypes of AIV were prepared and stored by our laboratory; the 1% SPF chicken red blood cell suspension was prepared by drawing blood in our laboratory.

[0031] The ordinary PCR instrument was purchased from BIO-RAD of the United States, the gel imaging system was purchased from GENE Company, the constant temperature water bath was purchased from Changzhou Tianrui Instrument Co., Ltd., and the centrifuge was purchased from Beckman Coulter. The 37°C biochemical incubator was purchased from Shaoguan Taihong Medical Devices Co., Ltd. The chicken embryo incubator was purchased from Shandong Taichang Incubation Equipment Co., Ltd. The -30°C refrigerator and -70°C ultra-low temperature refrigerator were purchased from Qingdao Haier Biomedical Co., Ltd.

[0032] Example 1

[0033] 1.1 Virus isolation and purification

[0034] During the period from 2000 to 2020, throat and cloacal swab samples of live poultry and wild birds were collected from cities such as Nanning, Liuzhou, Guilin, Beihai, and Fangchenggang in Guangxi. These samples were immediately immersed in a quadruple-antibody solution (PBS containing penicillin (concentration: 8000 units / mL), streptomycin sulfate (concentration: 10000 units / mL), gentamicin (concentration: 0.5 mg / mL), and nystatin (concentration: 0.5 mg / mL)) for about 4 h to ensure sufficient sterilization. Then, the swabs were repeatedly squeezed and washed, and the supernatant of the swabs was obtained by centrifugation at 6000 r / min for 5 min.

[0035] After sample treatment, inoculate the allantoic cavity of 10-day-old SPF chicken embryos, inoculating 0.2 mL per embryo, incubate at 37°C, discard the chicken embryos that died within 24 hours, after culturing for 72 hours, collect the allantoic fluid of the chicken embryos, conduct a hemagglutination test, and determine the serum subtype of the isolate by performing HA-HI tests with influenza standard typing sera after preparing four-unit antigens. A total of 132 H9 subtype AIV strains were isolated.

[0036] Refer to the primers designed by Hoffman for amplifying influenza virus HA and NA genes to conduct RT-PCR tests for assistance in identification. The specific operating procedures are carried out according to the international standard methods of the World Organization for Animal Health. For the viruses with mixed infections, use the serum neutralization method and plaque purification method to purify the viruses. Aliquot the identified viruses and store them in a -70°C refrigerator for standby.

[0037] 1.2. PCR amplification and sequence determination of virus-specific fragments

[0038] Use the full-length amplification primers designed by Hoffman for amplifying each gene fragment of avian influenza virus to amplify each gene of avian influenza virus. Conduct gel electrophoresis on the PCR products, cut and recover the target fragments, ligate them with the pEASY-Blunt vector of TransGen Biotech Co., Ltd., and select positive clones to send to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing. The specific information of the amplification primers is shown in Table 1:

[0039] Table 1 List of full-length amplification primers for each gene fragment of avian influenza virus

[0040]

[0041]

[0042] 1.3. HA gene sequence analysis

[0043] Use DNAStar software to splice and proofread the sequencing return results, and compare them with the HA gene sequences of representative strains of H9N2 subtype AIV downloaded from GenBank. Use MEGA6.0 software to draw the phylogenetic tree of its HA gene, and the results are as Figure 2 shown.

[0044] Example 2

[0045] Antigenicity analysis of H9N2 subtype AIV isolated strains and screening of vaccine strains

[0046] Screening of representative strains for antigen analysis

[0047] In Example 1.1, the HA gene of H9 subtype AIV isolated during the period from 2000 to 2020 was compared with the commonly used vaccine strains in China: A / chicken / Guangdong / SS / 94(H9N2) (SS strain), A / chicken / Guangxi / 10 / 99(H9N2) (Re-2 strain), A / Chicken / Shanghai / F / 98(H9N2) (F strain), A / chicken / Shandong / 6 / 96(H9N2) (SD696 strain), A / chicken / Jiangsu / 1 / 1999(H9N2) (JY strain), A / chicken / Henan / 1 / 2001(H9N2) (HL strain), and was also compared with the representative strains of each branch of H9 subtype AIV at home and abroad. An evolutionary tree was established. Considering various aspects such as the different isolation times, locations, hosts, genetic evolution analysis, epidemic situation, and differences from the vaccine strain branches, 15 purified H9 subtype AIV isolates were selected, namely: NN1(2011), 120D25(2012), 35B15(2013), CX13(2013), LF2(2014), 92B7(2014), Fcg01C3(2014), 210Q33(2015), C227(2015), C1228(2015), C228(2016), 160B8(2016), 241B5(2018), CWM(2019), Lz071D5(2020). Among them, a strain of H9N2 subtype avian influenza virus C1228(2015) from Guangxi was named A / chicken / Guangxi / C1228 / 2015(H9N2), and the deposit number was CGMCC No.45222. Another strain of H9N2 subtype avian influenza virus 210Q33(2015) also from Guangxi was named A / quail / Guangxi / 210Q33 / 2015(H9N2), and the deposit number was CGMCC No.46091. Both were deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit dates were July 4, 2022 and July 22, 2024 respectively. The sequences of strain C1228(2015) and strain 210Q33(2015) have been uploaded to the GenBank database. The accession number of C1228(2015) is KX185883-KX185890, and the accession number of 210Q33(2015) is MN093962-MN093969, which can be queried and downloaded on the official website of the National Center for Biotechnology Information (NCBI).

[0048] Example 3

[0049] Determination of 50% embryo infectious dose (EID 50 )

[0050] The virus was serially diluted 10-fold with sterile PBS. Dilutions between 10 -5 and 10 -10 were selected. Five 10-day-old SPF chicken embryos were inoculated with each dilution at a dose of 0.2 mL per embryo and incubated at 37°C. The embryos were examined daily, and those that died within 24 h were discarded. Allantoic fluid was collected after 72 h of incubation, and the hemagglutination activity of the allantoic fluid was measured. The EID 50 (50%embryo infective dose) was calculated using the Reed-Muench method. The results are shown in Table 2.

[0051] Example 4

[0052] Preparation of inactivated oil emulsion vaccine against H9N2 subtype AIV

[0053] 4.1 Inactivation of virus solution

[0054] Fifteen selected H9N2 subtype AIV isolates were serially diluted 10 -4 times with sterile PBS and inoculated into the allantoic cavities of 9- to 10-day-old SPF chicken embryos at a dose of 0.2 mL per embryo. The embryos were incubated at 35°C for 96 h, and then the pooled allantoic fluid, including that from both dead and surviving embryos, was collected and its HA titer was measured.

[0055] BPL virus inactivator was mixed into the above allantoic fluid at a ratio of 0.05% and added dropwise with gentle shaking to mix evenly. Then, the mixture was placed in a constant temperature shaker and inactivated at 20°C at a rotation speed of 60 r / min for 16 h. After that, it was stored at 4°C for later use. The HA titer was measured again, and it was preferred that the titer only decreased by 1log2 - 2log2. Finally, the inactivated allantoic fluid stock solution was inoculated into the allantoic cavities of SPF chicken embryos. After two blind passages, no virus proliferation was observed, indicating that the virus had been completely inactivated.

[0056] 4.2 Preparation of inactivated oil emulsion vaccine against H9N2 subtype AIV

[0057] Preparation of inactivated vaccine adjuvant: For the oil phase preparation, white oil for injection was used as the main component. 94 parts of white oil were taken according to the weight ratio, 6 parts of Span-80 were added, and they were fully mixed evenly and then sterilized for later use. For the aqueous phase preparation, the inactivated strain antigen solution was used as the main body, 48.5 parts were taken according to the weight ratio, and 3 parts of sterilized Tween-80 were added and stirred until the Tween-80 was completely dissolved.

[0058] During the emulsification process, the aqueous phase was slowly added to the prepared oil phase according to a volume ratio of 1:2 of the aqueous phase to the oil phase. The pre-emulsification treatment was carried out for 1 min using an emulsifier, and then the virus solution was gradually introduced into the oil phase. Subsequently, high-speed shear emulsification was carried out for 5 min using the emulsifier to complete the emulsification, and the inactivated oil emulsion vaccine of H9N2 subtype AIV (i.e., the inactivated vaccine) was obtained. After the emulsification was completed, the quality of the vaccine could be detected by dropping a few drops of the prepared inactivated vaccine onto the water surface. If the vaccine did not spread, it indicated that it had good water-in-oil characteristics.

[0059] Verify the stability of water-in-oil: Pipette 10 mL of the inactivated vaccine obtained in 4.1 into a centrifuge tube and carry out centrifugation at a speed of 3500 r / min for 15 min. After centrifugation, the volume of the liquid precipitated at the bottom of the tube should not exceed 0.5 mL. The vaccines that passed the quality inspection need to be capped and labeled to ensure their quality and safety.

[0060] 4.3 Preparation of monovalent immune serum against H9N2 subtype AIV

[0061] Using the inactivated oil emulsion vaccine prepared in step 4.2, 3-week-old SPF chickens were immunized by subcutaneous injection in the neck, and the vaccination dose for each chicken was 0.3 mL of the vaccine. On the 21st day after the immunization, 0.5 mL of blood samples were collected from the vein of each chicken for HI antibody titer detection. If the HI antibody titer reached a level above 8 log2, the second immunization was carried out in the same manner and dose. On the 21st day after the second immunization, venous blood samples were collected again, also 0.5 mL. The collected blood samples were allowed to stand in a sterile environment at 37°C for 1 h, and after the serum was separated, the HI antibody level was detected. For SPF chickens with serum titers meeting the requirements, 10 mL of blood samples were further collected from the wing vein or the neck vein. After the serum was allowed to separate naturally at room temperature, monovalent immune serum against H9N2 subtype AIV was obtained. It was aliquoted into small tubes in a sterile environment and stored in a -20°C refrigerator.

[0062] 4.4 Determination of HI antibody cross-reaction among different strains

[0063] Fifteen H9N2 subtype AIV isolated strains and the SS strain vaccine strain were selected. After the HA titers were uniformly determined, the preparation of 4-unit antigens was carried out. The 4-unit antigens of each virus were subjected to cross-HI tests with the 16 monovalent sera against H9N2 subtype AIV prepared in 4.3, and the inhibition titers were recorded. The antigenic difference was expressed by the difference coefficient R value, and the calculation method of its R value referred to the method in the second edition of "Animal Virology" (Yin Zhen et al., 1997). The calculation formula was as follows:

[0064] Let r1 be the ratio of the HI titer of virus 1 against serum 2 to the HI titer of virus 1 against serum 1, and r2 be the ratio of the HI titer of virus 2 against serum 1 to the HI titer of virus 2 against serum 2. When R = 1, it indicates that virus 1 and virus 2 have the same antigenicity; when 0.67 ≤ R ≤ 1.5, it indicates that there is no significant difference in antigenicity between virus 1 and virus 2; when 0.5 ≤ R ≤ 0.67, it indicates that there is a minor difference in antigenicity between virus 1 and virus 2; when R < 0.5, it indicates that there is a large difference in antigenicity between virus 1 and virus 2.

[0065] 4.5, Median Protective Dose (PD 50 ) To evaluate the effectiveness of the vaccine

[0066] In this experiment, 15 representative virus strains of H9N2 subtype AIV prepared by step 1.1 and the single-factor serum of the classical vaccine strain SS strain were used as antibody samples. These sera were diluted at intervals of 2 -1 ~2 -16 . Subsequently, an equal volume of 200 EID 50 virus diluent was added to each antibody at different dilutions. After thorough mixing, these mixtures were incubated at 37°C for 60 min. Each mixture at different gradients was inoculated into SPF chicken embryos at a dose of 0.2 mL per embryo and incubated at 35°C. If the chicken embryos showed non-specific death within 24 h, they were regarded as invalid and eliminated. After 72 h, the hemagglutination titers of each embryo were measured respectively to determine whether the chicken embryos were infected (micro-method), and the infection ratio and the corresponding dilution were recorded. The Reed-Muench method was used to calculate the serum dilution that could protect 50% of the chicken embryos from virus infection. The result of the experiment was recorded as the dilution that protected 50% of the chicken embryos. In this experiment, by fixing the virus dose and diluting the antibody, the PD 50 (Protective Dose for 50%) was calculated. The higher the dilution of the antibody, the stronger its neutralizing ability against the virus.

[0067] Example 5

[0068] Immunogenicity Efficacy and Comparative Test among Screening Strains of H9N2 Subtype AIV Vaccine

[0069] The SPF negative pressure isolator used for raising chickens was cleaned, and after cleaning, it was fumigated and disinfected with formaldehyde and potassium permanganate (ratio 2:1). While disinfecting the inside of the isolator, the external environment of the isolator was also fumigated with the same ratio of drugs. After 24 h of fumigation, the ventilation system was started to exhaust the waste gas and introduce fresh filtered air. Subsequently, SPF chickens were allocated into the isolator.

[0070] The specific allocation method is as follows: SPF chickens at 3 weeks of age are randomly divided into three groups, with 10 chickens in each group. Each group of chickens wears leg rings of different colors and serial numbers for identification. Each group of chickens will receive subcutaneous injection in the neck. The injection substances for the three groups of chickens are inactivated vaccines of strain C1228, strain 210Q33, and classical SS strain respectively, and the immunization dose is 0.3 mL per chicken. At the same time, two control groups are set up, with 5 chickens in each group. The SPF chickens in the control groups will receive the same dose of oil adjuvant as a blank control.

[0071] On the 21st day after immunization, venous blood will be collected from all SPF chickens and serum will be separated, and HI test will be carried out to determine the serum antibody titer. Subsequently, each group of chickens will be evenly divided into two small subgroups again. Each small subgroup will be inoculated with strain C1228 and strain 210Q33 by wing vein injection respectively, and each chicken will be inoculated with 0.2 mL (containing 2×10 7.5 EID 50 ). The control groups will be inoculated with an equal amount of PBS. On the 5th day after challenge, cloacal swabs of the experimental chickens will be collected and these swabs will be placed in a sterilized PBS (pH 7.4) solution containing four antibiotics. After washing the cotton swabs, each swab sample will be inoculated into 10-day-old SPF chicken embryos through the allantoic cavity, with two chicken embryos inoculated for each sample and 0.2 mL inoculated for each chicken embryo. Dead embryos will be eliminated within 24 hours after inoculation, and allantoic fluid will be collected after 72 hours. Finally, virus isolation will be determined by HA test.

[0072] Example 6

[0073] Study on the Immunogenicity of Bivalent Inactivated Vaccine (Strain C1228 + Strain 210Q33) against H9N2 Subtype AIV

[0074] 6.1 Preparation of Bivalent Inactivated Vaccine (Strain C1228 + Strain 210Q33) against H9N2 Subtype AIV

[0075] The virus solutions of strain C1228 and strain 210Q33 are inactivated with BPL at a final concentration of 0.05% at 20°C for 16 h. When preparing the inactivated vaccine adjuvant, first prepare the oil phase by mixing 94 parts of white oil for injection and 6 parts of Span-80 evenly, and sterilize it for later use. Then prepare the aqueous phase by mixing 48.5 parts of the inactivated original solutions of strain C1228 and strain 210Q33 respectively, adding 3 parts of sterilized Tween-80 and stirring well until dissolved (the above parts are all volume fractions).

[0076] Emulsify according to the volume ratio of water phase to oil phase of 1:2. Slowly add the water phase to the oil phase and pre-emulsify for 1 min using an emulsifier. During the emulsification process, gradually add the virus solution to the oil phase, and then perform high-speed shear emulsification for 5 min. After completing the emulsification process, the stability of the inactivated vaccine can be detected by dropping a few drops of the inactivated vaccine on the water surface. If the vaccine does not disperse and shows a good water-in-oil state, it indicates that the emulsification is good.

[0077] Next, take out 10 mL of the inactivated vaccine and add it to a centrifuge tube. Perform centrifugation for 15 min at a set speed of 3500 revolutions per minute. The volume of the water phase precipitated at the bottom of the tube after centrifugation should not exceed 0.5 mL. The vaccines that meet the requirements after testing are quantitatively sub-packaged to obtain a well-emulsified bivalent inactivated vaccine of H9N2 subtype AIV, sealed and marked, and finally stored in a 4°C refrigerator.

[0078] 6.2 Minimum immunization dose test of bivalent inactivated vaccine of H9N2 subtype AIV (strain C1228 + strain 210Q33)

[0079] Select 21 SPF chickens and randomly divide them into four groups, namely groups A, B, C, and D, with 3 chickens in each group. For these four groups of chickens, bivalent inactivated vaccine will be inoculated subcutaneously in the neck, and the inoculation doses are 0.1 mL, 0.2 mL, 0.3 mL, and 0.4 mL respectively. In addition, select another 12 SPF chickens and randomly divide them into four groups, namely groups E, F, G, and H, with 3 chickens in each group as well. These chickens will be used as the control group and injected with 0.1 mL, 0.2 mL, 0.3 mL, and 0.4 mL of blank oil adjuvant respectively. On the 21st day after immunization, collect blood from all groups and separate the serum. Subsequently, use the four-unit virus solution of strains C1228 and 210Q33 to detect the antigen, and determine the antibody titer of the serum through the HI test.

[0080] 6.3 Comparative test on the immune efficacy of bivalent inactivated vaccine of H9N2 subtype AIV (strain C1228 + strain 210Q33) and single vaccine

[0081] Select 40 SPF chickens and evenly divide them into groups A, B, C, and D. Among them, 10 chickens in group A are inoculated subcutaneously in the neck with bivalent inactivated vaccine of H9N2 subtype AIV (strain C1228 + strain 210Q33), 10 chickens in group B are inoculated subcutaneously in the neck with inactivated vaccine of strain C1228, 10 chickens in group C are inoculated subcutaneously in the neck with inactivated vaccine of strain 210Q33, and 10 chickens in group D are inoculated with an equal amount of oil adjuvant as the blank control group. The immunization dose for each chicken is 0.3 mL.

[0082] On the 21st day after vaccination, blood samples of each group were collected to isolate serum. The HI antibody titer was detected by a four-unit antigen assay against strains C1228 and 210Q33. Meanwhile, SPF chickens of each group and SPF chickens of the control group, with 10 chickens in each group evenly divided into two small subgroups, were inoculated with strains C1228 and 210Q33 via the wing vein, and the inoculation dose for each chicken was 0.2 mL (containing 2×10 7.5 EID 50 ). On the 5th day after challenge, cloacal swab samples of the chickens were collected. Each sample was placed in a sterilized PBS solution tube containing four antibiotics and thoroughly washed by shaking. Thereafter, each sample was inoculated into 10-day-old SPF chicken embryos, and those that died within 24 hours were discarded. After culturing for 72 hours, allantoic fluid was collected and the HA titer was detected to confirm whether the virus was contained in the sample.

[0083] 6.4. Monitoring of antibody levels after immunization with bivalent inactivated vaccine against H9N2 subtype AIV (strain C1228 + strain 210Q33)

[0084] Three 21-day-old SPF chickens were subcutaneously inoculated with the bivalent inactivated vaccine against H9N2 subtype AIV (strain C1228 + strain 210Q33) via the neck, with 0.3 mL for each chicken. Another three SPF chickens of the same age were injected with an equal amount of oil adjuvant in the same way as a control. Blood samples were collected from one group of immunized chickens and one group of control chickens at 1W, 2W, 3W, 4W, 8W, 12W, 16W, 20W, and 24W after immunization to isolate serum and detect the HI titer.

[0085] Results

[0086] 1. Plaque formation and virus purification of H9N2 subtype avian influenza virus from different sources

[0087] When purifying the first-generation virus with MDCK cells (the final concentration of TPCK-trypsin in the first layer of nutrient agar was 2 μg / mL), the plaque appearance time varied from 48 to 72 hours, and the plaque diameters were different (as Figure 1 shown). Independent and dominant plaques were selected for subsequent purification. After three generations of purification, the virus was propagated on SPF chicken embryos for one more generation, the embryos were harvested, the virus was subpackaged, and stored at -70°C for standby.

[0088] 2. Genetic evolution analysis of the HA gene of H9N2 subtype AIV

[0089] The MEGA6.0 software was used to perform genetic evolution analysis on the HA genes of some H9N2 subtype avian influenza viruses isolated from Guangxi region from 2000 to 2020 and draw an evolutionary tree. This HA gene evolutionary tree was further optimized using the Evolview website (as Figure 2As shown (in the figure). It can be seen from the phylogenetic tree that after nearly 20 years of prevalence, the HA gene of the H9N2 subtype avian influenza virus in Guangxi region has undergone relatively large mutations, forming five different branches: I, II, III, IV, and V. By comparing 132 isolates with the commonly used vaccine strains in China and the representative strains of H9 subtype AIV, the phylogenetic tree as shown in Figure 2 was constructed. The specific details of the virus strains are shown in Table 3.

[0090] Analysis of the phylogenetic tree data shows that: among the isolates, 49 belong to branch I, 28 belong to branch II, 16 belong to branch III, and another 37 isolates belong to G1-Like. It is worth noting that G1-Like strains have been isolated from poultry such as pigeons and quails in Guangxi region, showing a certain prevalence trend. The isolates of branch III show relatively high homology with the vaccine strain A / chicken / Henan / 1 / 2001 (H9N2) (HL strain); while the isolates are mainly concentrated in branch I, branch II, and branch III, widely prevalent in Guangxi region, and there is an obvious genetic distance from the existing vaccine strains.

[0091] It is thus speculated that during the 20-year prevalence process of the H9N2 subtype avian influenza virus, antigenic drift may have occurred, and the antigenic differences among the strains may have been relatively large, and the existing vaccine strains may not be able to provide sufficient protection. Therefore, the present invention focuses on the antigenicity analysis of the strains of branch I, branch II, branch III of the Y280-Like lineage and branch V belonging to the G1-Like lineage. For in-depth study, the commercially available vaccine A / chicken / Guangdong / SS / 94 (H9N2) (i.e., SS strain) most widely used in Guangxi region was selected for antigenic difference comparison experiments with 15 representative isolates. The strain backgrounds of the isolates and the commercially available vaccine are shown in detail in Table 2.

[0092] Table 2 Strains selected for antigenicity analysis test

[0093]

[0094]

[0095] “-” indicates that the corresponding strain did not measure EID50

[0096] Table 3 Information of H9N2 subtype AIV strains

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] 3. Antigenicity analysis of H9N2 subtype AIV isolates and screening of vaccine strains 3.1 Genotype and EID of HA gene of H9N2 subtype AIV strains 50

[0103] Determine the EID of each H9N2 subtype AIV isolate 50 for antigenicity analysis of each H9N2 subtype AIV isolate and further virus challenge protection experiments. The number of infected chicken embryos was judged according to hemagglutination activity, and the EID was calculated according to the Reed-Muench method 50 . The results showed the EID of each strain 50 was between 10 -7 and 10 -8.5 . The obtained results are shown in Table 2 in detail

[0104] The strains used to prepare the hyperimmune serum of SPF chickens in the present invention are representative strains of different branches and different years in Guangxi region from 2000 to 2020, namely: NN1(2011), 120D25(2012), 35B15(2013), CX13(2013), LF2(2014), 92B7(2014), Fcg01C3(2014), 210Q33(2015), C227(2015), C1228(2015), C228(2016), 160B8(2016), 241B5(2018), CWM(2019), Lz071D5(2020). The basic information of the strains is shown in Table 2. Hyperimmune sera of SPF chickens against 15 H9N2 subtype avian influenza viruses and vaccine strain SS were successfully prepared. The hemagglutination inhibition test showed that the titers of the hyperimmune sera all reached 2 10 or above

[0105] Antibody cross-reaction determination

[0106] Genetic evolution analysis shows that the H9N2 subtype AIV isolates in the present invention are constantly evolving. In order to screen for vaccines suitable for the current epidemic environment, it is necessary to first understand the antigen variation of the isolates in this study for antigen difference analysis. According to the isolation time and evolutionary relationship, monovalent antisera were prepared for the above 15 H9N2 subtype AIV isolates and the classical SS vaccine strain. The HI test and R value results show that there are certain differences in the HI titers of the monovalent antisera of each isolate against homologous and heterologous virus antigens. In this experiment, there are significant antigenicity differences between the epidemic strains in the h9.4.2 and h9.4.1 branches. The antigenicity differences between the vaccine strain and the isolates in h9.4.2 and h9.4.1 are also obvious, which is consistent with the results of the genetic evolutionary tree analysis. It is worth noting that the antigenicity difference between the isolates from the h9.4.1 branch and the vaccine strain is the most significant. Since the significant antigenicity difference may affect the effectiveness of the vaccine, this indicates that the currently used vaccine strain may not provide sufficient protection.

[0107] The HA titers of the antigens of 15 H9 subtype AIV and the SS strain vaccine were measured, and 4-unit antigens of 16 strains were prepared according to the method described above. The HI antibody titers of each SPF chicken were measured respectively, and 3 replicates were made to calculate the average value. The HI antibody titers of the sera of 3 SPF chickens in each experimental group against each antigen were statistically analyzed and the average value was calculated to obtain the reciprocal HI values between the isolates and the vaccine strain. According to the results of the HI reciprocal test, the antigen-related value R between each isolate and the vaccine strain was calculated, and the results are shown in Table 4.

[0108] According to the R value, there are significant antigenicity differences between the strains in the h9.4.1 branch and the h9.4.2 branch. Branch Ⅴ belongs to the h9.4.1 branch, and the antigen differences between it and the strains of other branches are too large, indicating that the vaccine designed for the strains in the h9.4.2 branch cannot provide good and comprehensive prevention and control for the currently prevalent strains. It is necessary to screen for suitable vaccine strains for the strains belonging to the two lineages of h9.4.2 and h9.4.1.

[0109] Table 4 Antigen correlation coefficient R values between strains

[0110]

[0111]

[0112] 3.3 Results of chicken embryo neutralization test

[0113] To further verify the antigenicity differences between different strains and screen for suitable vaccine strains, chicken embryo neutralization tests were carried out. The main focus was on the binding and neutralization ability between different sera and different strains, and the highest dilution multiple of the serum that could protect 50% of the chicken embryos from infection was used as the 50% neutralization titer (PD50 )。

[0114] The chicken embryo neutralization and cross - test showed that there were certain differences in the neutralization titers of 15 single - factor antisera against homologous and heterologous viruses. The test showed that there were obvious differences in the PD 50 values between the epidemic strains in the h9.4.2 and h9.4.1 branches. There were also obvious differences in the PD 50 values between the epidemic strains in the h9.4.2 branch, and according to the results of the serum cross - test, they were also clearly divided into two different antigenic groups. The cross - reaction of neutralizing antibodies was good among the strains belonging to the h9.4.2 branch, and the cross - reaction of neutralizing antibodies was also good among the strains belonging to the h9.4.1 branch. Analyzing each strain's PD 50 , it was found that the serum of strain C1228 in the h9.4.2 antigenic group had a relatively high neutralization titer against the strains in the h9.4.2 antigenic group, indicating that strain C1228 had a wide coverage of the viruses in the h9.4.2 antigenic group, and the lowest PD 50 value was 1:16; the serum of strain 210Q33 in the h9.4.1 antigenic group had a relatively high neutralization titer against the strains in the h9.4.1 antigenic group, indicating that strain 210Q33 had a wide coverage of the viruses in the h9.4.1 antigenic group, and the lowest PD 50 value was 1:16. Therefore, the development of new vaccines was further carried out for these two antigenic groups. It was suggested that strains C1228 and 210Q33 met the conditions to be candidate vaccine strains, and the results were shown in Table 5 for details.

[0115] Table 5 Chicken embryo neutralizing antibody cross - reaction (serum dilution ratio for protecting 50% of chicken embryos)

[0116]

[0117]

[0118] 3.4, Immunogenicity efficacy and comparative test among the screened strains of H9N2 subtype AIV vaccine

[0119] According to antigenicity, strains C1228 and 210Q33 were selected as candidate vaccine screening strains. In order to verify whether the candidate vaccine screening strains had good immunogenicity and clinical significance, the present invention designed an immunoprotection test. After 21 days of immunization, the antibody levels of each group were examined. The three vaccine groups used their own strains as HI diagnostic antigens, and the HI titers all reached above 10log2 ( Figure 4 ), and compared with the HI diagnostic antigens of non - self strains, all showed extremely significant differences (P < 0.0001), as shown in Table 7 for details.

[0120] The results of the virulence protection test showed that the vaccination protection rate of the C1228 vaccine group against the C1228 strain was 100%, and the vaccination protection effect against the 210Q33 strain was 40%. The vaccination protection rate of the 210Q33 vaccine group against the 210Q33 strain was 100%, while the vaccination protection rate against the C1228 strain was only 10%. The vaccination protection rates of the SS vaccine group against the C1228 strain and the 210Q33 strain were 40% and 0 respectively. Based on comprehensive data analysis, each vaccine group had a good vaccination protection effect against its own strain, reaching 100%, while the vaccination protection effect against heterologous strains was between 0 and 40% (Table 6 and Figure 3 ). The results showed that a vaccine against a single branch strain could not effectively protect chickens from infection by most h9.4.2 or h9.4.1 epidemic strains at the same time. Based on the experimental results, it was inferred that the combined action of the C1228 strain and 210Q33 strain vaccines could provide better defense against current epidemic strains than a single branch vaccine. The experiment proved that the currently used SS strain vaccine could no longer effectively protect chickens from infection by h9.4.2 or h9.4.1 branch epidemic strains.

[0121] Table 6 Vaccination protection status of SPF chickens after immunization

[0122]

[0123]

[0124] 3.5. Exploration of the immunization efficacy of the bivalent inactivated vaccine (C1228 strain + 210Q33 strain) against H9N2 subtype AIV

[0125] 3.5.1. Minimum immunization dose test of the bivalent inactivated vaccine (C1228 strain + 210Q33 strain) against H9N2 subtype AIV

[0126] To more effectively prevent and control the currently prevalent H9N2 subtype AIV, a bivalent inactivated vaccine (C1228 strain + 210Q33 strain) was designed. The test results showed that the bivalent inactivated vaccine (C1228 strain + 210Q33 strain) of H9N2 subtype AIV was used to immunize three-week-old SPF chickens for 21 days at immunization doses of 0.1 mL, 0.2 mL, 0.3 mL, and 0.4 mL. As the immunization dose increased, the HI antibody titer also increased accordingly. When the immunization dose was 0.1 mL, the average HI antibody titer was 5 log2. When the immunization dose was 0.2 mL, the average HI antibody titer was 7 log2. When the immunization dose was increased to 0.3 mL, the average HI antibody titer could reach more than 10 log2, and there was no significant difference in the HI antibody titer between the 0.3 mL group and the 0.4 mL group. The antibody titers of the 0.3 mL group and the 0.4 mL group were both higher than those of the 0.2 mL group (see details in Figure 5 ).

[0127] 3.5.2、Comparison Test on the Immune Efficacy between the Bivalent Inactivated Vaccine (Strain C1228 + Strain 210Q33) of Subtype H9N2 AIV and the Monovalent Vaccine

[0128] The test results showed that after immunizing SPF chickens with the bivalent inactivated vaccine (Strain C1228 + Strain 210Q33) of subtype H9N2 AIV for 21 days, when detecting the bivalent inactivated vaccine (Strain C1228 + Strain 210Q33) and the inactivated vaccine of Strain C1228 with the four-unit antigen of Strain C1228, the antibody titer reached above 10log2, and there was no difference between the two groups of values (P>0.05), indicating that the difference in antibody titers between the two groups was not significant. When detecting the bivalent inactivated vaccine (Strain C1228 + Strain 210Q33) and the inactivated vaccine of Strain 210Q33 with the diagnostic antigen of Strain 210Q33, the antibody level could also reach above 10log2, and there was no difference between the two groups of values (P>0.05), indicating that the difference in antibody titers between the two groups was not significant. Cloacal swabs were collected for virus isolation 5 days after virus challenge. Most of the virus isolations from the test chickens were negative for avian influenza, while all the virus isolations from the control chickens were positive for avian influenza. All the chickens in the control group and the test group were healthy. According to the analysis of the virus isolation results, the protection rates of the bivalent inactivated vaccine (Strain C1228 + Strain 210Q33) against the challenge of Strain C1228 or Strain 210Q33 both reached over 80%. The protection rate of the Strain C1228 vaccine group against the challenge of Strain C1228 also reached 100%, but the protection rate against the challenge of Strain 210Q33 was only 40%; the protection rate of the Strain 210Q33 vaccine group against the challenge of Strain 210Q33 reached 100%, but the protection rate against the challenge of Strain C1228 was only 20%. Therefore, compared with the monovalent vaccine, the bivalent inactivated vaccine (Strain C1228 + Strain 210Q33) can bring better protection effects to the chicken flock and enable it to more effectively resist the threats of viruses from different branches. The data are shown in Table 7 for details.

[0129] Table 7 HI Antibody Conditions of SPF Chickens Immunized with the Bivalent Inactivated Vaccine (Strain C1228 + Strain 210Q33) and the Corresponding Monovalent Vaccine

[0130]

[0131] 3.6、Monitoring of Antibody Levels after Immunization with the Bivalent Inactivated Vaccine (Strain C1228 + Strain 210Q33) of Subtype H9N2 AIV

[0132] The test results showed that when the inactivated bivalent vaccine was inoculated into SPF chickens at 21 days old, antibodies could be detected at the 1st week after immunization, but the average HI antibody titer was relatively low, only 3log2 - 4log2, which was not sufficient to protect the chicken flock well from the infection of H9N2 subtype AIV. However, the average antibody titer increased by 8log2 (when the detected antigen was C1228) and 7log2 (when the detected antigen was 210Q33) at the 2nd week after immunization. By the 3rd week, the average HI antibody titers corresponding to the test antigens of C1228 strain and 210Q33 strain reached above 9log2. By the 4th week after immunization, the average HI antibody titers of each test group reached the peak, all reaching above 10log2, and the high-level HI antibody titers continued until the 24th week after immunization when there was a slight downward trend. The results are shown in Table 8 for details.

[0133] Table 8 Changes in antibody titers of SPF chickens immunized with inactivated bivalent vaccine against H9N2 subtype AIV

[0134]

[0135] From the above results, it can be seen that the antigenicity analysis of the present invention shows that there are great differences in the antigens of viruses in the h9.4.2 branch and h9.4.1 branch, and C1228 from the h9.4.2 branch and 210Q33 strain from the h9.4.1 branch have the potential to prevent the infection of current prevalent strains.

[0136] The results of the immunoprotection test showed that the original classical SS strain vaccine could not provide complete protection against the current prevalent strains. It could only provide a 40% protection rate against the C1228 strain and a 0% protection rate against the 210Q33 strain. Although the individual C1228 strain and 210Q33 strain had a slightly improved protective effect against some current prevalent strains compared with the SS strain, the inactivated bivalent vaccine prepared with these two strains could provide a more comprehensive protective effect against the strains in the Y280-Like and G1-Like branches.

[0137] The protection rate of the bivalent vaccine (C1228 strain + 210Q33 strain) against the current prevalent strains reached more than 80%. The minimum immunoprotective dose was 0.3 mL, and the effective immunoprotective period was 24W.

[0138] The foregoing description of specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the present invention to the precise forms disclosed, and obviously, many changes and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical applications, so that those skilled in the art can implement and utilize various different exemplary embodiments of the present invention as well as various different selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. An H9N2 subtype avian influenza virus, characterized in that: The H9N2 subtype avian influenza virus is A / chicken / Guangxi / C1228 / 2015 (H9N2), with a preservation number of CGMCC No.45222; or the H9N2 subtype avian influenza virus is A / quail / Guangxi / 210Q33 / 2015 (H9N2), with a preservation number of CGMCC No.46091.

2. Use of the H9N2 subtype avian influenza virus as claimed in claim 1 in the preparation of drugs for preventing and controlling avian influenza.

3. The use of the H9N2 subtype avian influenza virus according to claim 2 in the preparation of a drug for preventing and controlling avian influenza, characterized in that: The medicine is a vaccine, and the vaccine is an inactivated vaccine.

4. A bivalent inactivated vaccine for H9N2 subtype avian influenza virus, characterized in that: The vaccine is prepared by inactivating the H9N2 subtype avian influenza virus A / chicken / Guangxi / C1228 / 2015 (H9N2) with a preservation number of CGMCC No. 45222 and the H9N2 subtype avian influenza virus A / quail / Guangxi / 210Q33 / 2015 (H9N2) with a preservation number of CGMCC No. 46091 as antigens.

5. A method for preparing a bivalent inactivated vaccine of H9N2 subtype avian influenza virus as claimed in claim 4, characterized in that: The following steps are included: S1 inactivated vaccine: The H9N2 subtype avian influenza virus A / chicken / Guangxi / C1228 / 2015(H9N2) with a deposit number of CGMCC No.45222 and the H9N2 subtype avian influenza virus A / quail / Guangxi / 210Q33 / 2015(H9N2) with a deposit number of CGMCC No.46091 were inactivated with BPL at a final concentration of 0.05% respectively; S2: Prepare the oil phase: Mix 94 parts of white oil and 6 parts of Siben-80 evenly according to the volume ratio, sterilize and set aside; S3: Prepare the aqueous phase: according to the volume ratio, take 48.5 parts of the inactivated C1228 strain and 210Q33 strain stock solutions, mix them, add 3 parts of sterilized Tween-80 and stir thoroughly until dissolved; S4 emulsification: Emulsify according to the volume ratio of water phase to oil phase 1:2, slowly add the water phase into the oil phase, pre-emulsify for 1 minute, during the emulsification process, gradually add the virus liquid into the oil phase, and then emulsify at high speed for 5 minutes to obtain a well-emulsified H9N2 subtype AIV bivalent inactivated vaccine.

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