2.3. 4.4b branch H5 subtype avian influenza virus strain and application thereof

Abstract

The invention discloses a 2.3. 4.4b branch H5 subtype avian influenza virus strain and an application thereof. The 2.3. 4.4b branch H5 subtype avian influenza virus strain is an A type avian influenza virus A / chick / Shenyang / 12301 / 2024, is preserved in the China General Microbiological Culture Collection Center (CGMCC), has a preservation number of CGMCC No.46299, and is preserved for December 12, 2024, and the strain has a preservation number of CGMCC No.46299, a preservation number of CGMCC No.46299, a preservation number of CGMCC No.46299 and a preservation number of CGMCC No.46299. According to the invention, 2.3. 4.4b branch H5 subtype AIV with good cross immunoreaction is searched as a vaccine candidate strain to form a whole virus inactivated vaccine, so as to effectively prevent and control the infection of the latest epidemic H5 subtype AIV to poultry, and further eliminate the potential threat of influenza virus to public health safety in poultry breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of animal vaccines, and in particular to a 2.3.4.4b branch H5 subtype avian influenza virus strain and application thereof. Background Art

[0002] Since 2021, a new H5 subtype AIV of the 2.3.4.4b evolutionary branch has spread worldwide, causing huge economic losses to the global poultry industry, and its sporadic infections in wild carnivores, mink farms and marine mammals have increased significantly.

[0003] Currently, the most widely used vaccine strain for the H5 subtype of the 2.3.4.4b evolutionary branch is H5-Re14-WS / SX / 4-1 / 2020-(2.3.4.4b), which was selected from the H5 subtype strain prevalent in China in 2020. The continuous genetic evolution of the virus may lead to a poor antigenic reaction between the vaccine strain that has not been updated for a long time and the newly isolated virus. According to literature reports (doi: 10.1016 / S2666-5247(22)00148-3), the vaccine strain has a certain difference in antigenicity with the strain currently prevalent in China. In addition, migratory birds bring foreign epidemic strains into China through migration, which further evolve into new epidemic strains, making the protection effect of the previously used inactivated vaccine worse. The above studies show that the currently prevalent 2.3.4.4b lineage H5 subtype virus has undergone a certain degree of genetic evolution and poses a potential threat to human public health security. Given this, a new vaccine strain is urgently needed to address this evolving threat. Summary of the invention

[0004] The purpose of the present invention is to provide a 2.3.4.4b branch H5 subtype avian influenza virus strain and its application. The present invention seeks a 2.3.4.4b branch H5 subtype AIV with good cross-immune reaction as a vaccine candidate strain to complete the virus inactivated vaccine, so as to effectively prevent and control the infection of the recently popular H5 subtype AIV to poultry, thereby eliminating the potential threat of influenza virus in poultry farming to public health safety.

[0005] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0006] The first aspect of the present invention provides a 2.3.4.4b branch H5 subtype avian influenza virus strain, wherein the 2.3.4.4b branch H5 subtype avian influenza virus strain is a type A avian influenza virus A / chicken / Shenyang / 12301 / 2024, which is deposited in the General Microbiological Center of the China Culture Collection Administration, with a deposit number of CGMCC No.46299 and a deposit time of December 12, 2024.

[0007] The second aspect of the present invention provides a use of the above-mentioned 2.3.4.4b branch H5 subtype avian influenza virus strain in the preparation of vaccines or biological materials.

[0008] Preferably, the biological material is any one of (1) to (4);

[0009] (1) Chicken embryo allantoic fluid containing the 2.3.4.4b clade H5 subtype avian influenza virus strain of claim 1;

[0010] (2) cells containing the 2.3.4.4b clade H5 subtype avian influenza virus strain of claim 1;

[0011] (3) the genomic nucleic acid of the 2.3.4.4b clade H5 subtype avian influenza virus strain according to claim 1;

[0012] (4) Recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector, engineered bacteria or cell line containing the genomic nucleic acid of the 2.3.4.4b branch H5 subtype avian influenza virus strain as described in claim 1.

[0013] The third aspect of the present invention provides a 2.3.4.4b branch H5 subtype avian influenza vaccine, which comprises an immunizing amount of the 2.3.4.4b branch H5 subtype avian influenza virus strain antigen.

[0014] Preferably, the 2.3.4.4b branch H5 subtype avian influenza virus strain antigen is an inactivated 2.3.4.4b branch H5 subtype avian influenza virus strain.

[0015] Preferably, the 2.3.4.4b branch H5 subtype avian influenza vaccine also includes an oil-emulsion adjuvant.

[0016] Preferably, the antigen content of the 2.3.4.4b branch H5 subtype avian influenza virus strain is 10 8.5 EID 50 / mL.

[0017] The fourth aspect of the present invention provides a method for preparing the above-mentioned 2.3.4.4b branch H5 subtype avian influenza vaccine, the preparation method comprising the following steps:

[0018] (a) The hemagglutination titer is 27 and the virus titer is 10 8.5 EID 50 / mL of the 2.3.4.4b branch H5 subtype avian influenza virus strain chicken embryo allantoic fluid is inactivated with formaldehyde solution to obtain a 2.3.4.4b branch H5 subtype avian influenza virus strain antigen solution;

[0019] (b) Mixing and emulsifying the 2.3.4.4b clade H5 subtype avian influenza virus strain antigen solution and the oil-emulsion adjuvant to obtain the 2.3.4.4b clade H5 subtype avian influenza vaccine.

[0020] Preferably, the volume ratio of the chicken embryo allantoic fluid of the 2.3.4.4b branch H5 subtype avian influenza virus strain to the formaldehyde solution is (800-1200):1.

[0021] Preferably, the concentration of the formaldehyde solution is 37% to 40%.

[0022] Preferably, the volume ratio of the 2.3.4.4b branch H5 subtype avian influenza virus strain antigen solution and the oil-emulsion adjuvant is (1.5-2.3):1.

[0023] Compared with the prior art, the beneficial effects of the present invention include at least:

[0024] The premise for the application of inactivated vaccines is that the production and stability of the virus must be guaranteed. At the same time, the effective immune protection of inactivated vaccines against the 2.3.4.4b branch H5 subtype AIV currently prevalent in China is also an important part of vaccine evaluation. To this end, the present invention evaluates the stability of the virus by continuous passage in chicken embryos, and evaluates its immune efficacy by immune chicken protection tests; the study found that the 2.3.4.4b branch H5 subtype avian influenza virus strain with good cross-immune reaction (type A avian influenza virus A / chicken / Shenyang / 12301 / 2024, referred to as SY / 01 / 24 strain) isolated by the present invention is a representative strain of the current 2.3.4.4b branch H5 subtype AIV. The virus can still maintain a stable hemagglutination titer when it is continuously passed in chicken embryos for 15 generations, and the hemagglutination titer is stable at 2 7 The virus titer reached 10 8.5 EID 50 / mL, indicating that the virus has good stability; the whole virus of the SY / 01 / 24 strain was inactivated and then assisted with an adjuvant to prepare a whole virus inactivated vaccine. The results of the vaccine's immune protection test on chickens confirmed that the vaccine can quickly produce resistance to 2.3.4.4b branch H5 subtype AIV infection after immunization; the research results show that the vaccine candidate strain used in the present invention can be stably propagated in chicken embryos, the strain is stable, and it is very safe and non-pathogenic to chickens; protective studies have shown that the vaccine has a good protective effect on the currently popular 2.3.4.4b branch H5 subtype AIV in China; the vaccine can also quickly (2 weeks after one immunization) produce immune protection efficacy, laying a foundation for the prevention and control of influenza viruses in chickens. DETAILED DESCRIPTION

[0025] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0026] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0027] The embodiment of the present invention provides a 2.3.4.4b branch H5 subtype avian influenza virus strain, wherein the 2.3.4.4b branch H5 subtype avian influenza virus strain is a type A avian influenza virus A / chicken / Shenyang / 12301 / 2024, which is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.46299 and a deposit time of December 12, 2024.

[0028] Another embodiment of the present invention provides a use of the above-mentioned 2.3.4.4b branch H5 subtype avian influenza virus strain in the preparation of vaccines or biological materials.

[0029] In one embodiment, the biomaterial is any one of (1) to (4);

[0030] (1) Chicken embryo allantoic fluid containing the 2.3.4.4b clade H5 subtype avian influenza virus strain of claim 1;

[0031] (2) cells containing the 2.3.4.4b clade H5 subtype avian influenza virus strain of claim 1;

[0032] (3) the genomic nucleic acid of the 2.3.4.4b clade H5 subtype avian influenza virus strain according to claim 1;

[0033] (4) Recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector, engineered bacteria or cell line containing the genomic nucleic acid of the 2.3.4.4b branch H5 subtype avian influenza virus strain as described in claim 1.

[0034] Another embodiment of the present invention provides a 2.3.4.4b branch H5 subtype avian influenza vaccine, which includes an immunizing amount of the 2.3.4.4b branch H5 subtype avian influenza virus strain antigen.

[0035] In one embodiment, the 2.3.4.4b clade H5 subtype avian influenza virus strain antigen is an inactivated 2.3.4.4b clade H5 subtype avian influenza virus strain.

[0036] In one embodiment, the 2.3.4.4b clade H5 subtype avian influenza vaccine further comprises an oil-emulsion adjuvant.

[0037] In one embodiment, the antigen content of the 2.3.4.4b clade H5 subtype avian influenza virus strain is 10 8.5 EID 50 / mL.

[0038] Another embodiment of the present invention provides a method for preparing the above-mentioned 2.3.4.4b branch H5 subtype avian influenza vaccine, the preparation method comprising the following steps:

[0039] (a) inactivating the chicken embryo allantoic fluid of the 2.3.4.4b branch H5 subtype avian influenza virus strain having a hemagglutination titer of 27 and a virus titer of 108.5 EID50 / mL with a formaldehyde solution to obtain a 2.3.4.4b branch H5 subtype avian influenza virus strain antigen solution;

[0040] (b) Mixing and emulsifying the 2.3.4.4b clade H5 subtype avian influenza virus strain antigen solution and the oil-emulsion adjuvant to obtain the 2.3.4.4b clade H5 subtype avian influenza vaccine.

[0041] In one embodiment, the volume ratio of the chicken embryo allantoic fluid of the 2.3.4.4b branch H5 subtype avian influenza virus strain to the formaldehyde solution is any value in the range of (800-1200):1, for example, 800:1, 1000:1 or 1200:1.

[0042] In one embodiment, the concentration of the formaldehyde solution is any value between 37% and 40%.

[0043] In one embodiment, the volume ratio of the 2.3.4.4b branch H5 subtype avian influenza virus strain antigen solution and the oil-emulsion adjuvant can be any value in the range of (1.5-2.3):1, for example, 1.5:1, 2.0:1 or 2.3:1.

[0044] The technical solution of the present invention is further described in detail below through specific embodiments.

[0045] The experimental methods used are as follows:

[0046] Hemagglutination (HA) test, as follows:

[0047] Use a pipette to add 25μl of saline to each well of the 96-well microhemagglutination plate, add 25μl of the virus antigen to be tested to the first well of each row, pipette 3-4 times to mix, then pipette 25μl to the second well, dilute it in turn to the 11th well and discard 25μl. No antigen is added to the 12th well as a negative control well. After multiple dilutions, pipette 25μl of 1% chicken red blood cell suspension and add it to each well from the highest dilution to the lowest dilution. After shaking and mixing, let it stand at room temperature for 30 minutes and observe the results. Tilt the reaction plate, and the red blood cells that sink to the bottom of the well flow linearly along the inclined surface downward as precipitation, indicating that the red blood cells are not or not completely agglutinated by the virus; if the red blood cells at the bottom of the well flatten the bottom of the well and agglutinate into a uniform thin layer, the red blood cells do not flow after tilting, indicating that the red blood cells are agglutinated by the virus. Method for determining the virus hemagglutination titer: the maximum dilution of the virus that can agglutinate 1% red blood cell suspension.

[0048] Hemagglutination inhibition (HI) test, as follows:

[0049] Prepare four units of antigen according to the virus hemagglutination titer, that is, 4 hemagglutination units of virus liquid. Divide the hemagglutination titer by 4 to get the dilution multiple of the antigen. Add physiological saline according to the dilution multiple to dilute the virus stock liquid to obtain four units of antigen. After the preparation is completed, it should be verified according to the HA test method to see if the preparation is accurate. Only after the preparation is accurate can the subsequent HI test be carried out.

[0050] Specific method of HI test: Use a pipette to add 25μl of physiological saline to each well of the 96-well microhemagglutination plate. Pipette 25μl of the serum to be tested and place it in the first well. Mix it by blowing 3 to 4 times and then pipette 25μl into the second well. Dilute it to the 11th well in turn, discard 25μl, and set the last well as a negative control without dilution. Add 25μl of the prepared four-unit antigen to each well from the highest dilution of serum to the lowest dilution, shake and mix, and let it stand at room temperature for 30 minutes. Pipette 25μl of 1% red blood cell suspension from the highest dilution to the lowest dilution, shake and mix, and let it stand at room temperature for 30 minutes to observe the results. The maximum dilution that completely inhibits red blood cell agglutination is the hemagglutination inhibition test titer of the serum, that is, the hemagglutination inhibition titer.

[0051] Example 1

[0052] This example is the isolation and identification of the 2.3.4.4b branch H5 subtype avian influenza virus strain (SY / 01 / 24 strain):

[0053] The 2.3.4.4b branch H5 subtype avian influenza virus strain in the present invention was isolated from chickens in a certain area of ​​Shenyang City, Liaoning Province, and was identified as type A avian influenza virus by the Animal Influenza Laboratory of China Agricultural University. The isolated strain SY / 01 / 24 was named A / chicken / Shenyang / 12301 / 2024 strain, and the classification name was: type A avian influenza virus.

[0054] The isolation and identification process of the strain is as follows:

[0055] (1) Sample processing

[0056] Add 1 ml of double-antibody PBS solution (10,000 UL / ml) to each swab sample and vortex to mix for 30 seconds; store the treated samples in a 4°C refrigerator overnight and inoculate 9-11 day old chicken embryos the next day.

[0057] (2) Chicken embryo inoculation and allantoic fluid harvesting

[0058] The treated samples were inoculated into 9-11 day old chicken embryos by the method of allantoic cavity inoculation. Two chicken embryos were inoculated per sample, and 0.5 ml was inoculated per embryo. After inoculation, the embryos were sealed with paraffin and placed in a 35°C incubator for 48-72 hours. The growth of the chicken embryos was checked every 12 hours. If dead embryos were found within 24 hours, they should be discarded in time. After 48-72 hours, the chicken embryos were placed in a 4°C refrigerator overnight. When harvesting chicken embryos, sterile tweezers were used to tear the eggshell of the chicken embryo air chamber, pierce the chorioallantoic membrane where there are no large blood vessels, and aspirate 25 μl of allantoic fluid. The corresponding chicken embryo allantoic fluid was collected in a cryopreservation tube and stored in a -80°C refrigerator.

[0059] The universal primer for reverse transcription of AIVs is Unit 12. The primer sequence for identification amplification of the HA gene fragment of H5 AIVs is:

[0060] HA-F: 5'-TGGAAAGTRTAARAAACGGAACGT-3', SEQ IDNO: 1;

[0061] HA-R: 5'-TGCTAGGGARCTCGCCACTG-3', SEQ ID NO: 2.

[0062] The storage concentration of the primer is 100 μM, the working concentration is 20 μM, and it is stored in a -20°C refrigerator for future use.

[0063] Viral RNA was extracted according to the instructions of RaPure Total RNA Mini Kit.

[0064] The reaction system for reverse transcription is shown in Table 1: Table 1

[0065] Add the components in Table 1 in sequence, mix well and centrifuge immediately. Perform reverse transcription in a 37°C water bath for 1 hour. The cDNA product obtained is the template for PCR amplification reaction.

[0066] The PCR reaction system is shown in Table 2: Table 2

[0067] Add the ingredients in Table 2 in sequence, mix well and centrifuge instantly.

[0068] PCR reaction conditions: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min 30 s, 35 cycles, 72°C final extension for 10 min, 4°C ∞;

[0069] After the PCR amplification reaction, 6 µL of PCR product was subjected to 1% agarose gel electrophoresis for identification, and PCR identification of the isolated strains was positive.

[0070] Based on the above identification results, the A / chicken / Shenyang / 12301 / 2024 strain was determined to be the H5N6 subtype of avian influenza virus type A.

[0071] The avian influenza virus A / chicken / Shenyang / 12301 / 2024A / chicken / Shenyang / 12301 / 2024 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 12, 2024, with the deposit number CGMCC No. 46299. Its scientific description: 2.3.4.4b branch H5 subtype avian influenza virus; reference biological material (strain): A / chicken / Shenyang / 12301 / 2024.

[0072] Example 2

[0073] This example is the preparation of 2.3.4.4b branch H5 subtype AIV inactivated vaccine:

[0074] 1. Screening of vaccine virus strains

[0075] Test vaccine virus strains: (1) A 2.3.4.4b branch H5 subtype AIV, namely A / chicken / Shenyang / 12301 / 2024 (Ck / SY / 01 / 24) isolated in Example 1. (2) Six strains of 2.3.4.4b branch H5 subtype AIV currently preserved in our laboratory, namely A / duck / Hainan-wanning / 10 / 2024 (DK / HN / 10 / 24), A / chicken / Shenzhen / 15 / 2023 (CK / SZ / 15 / 23), A / chicken / Shandong / 24 / 2024 (Ck / SD / 24 / 24), A / chicken / Shenyang / 39 / 2023 (CK / SY / 23), A / wildbird / Zhejiang / T12 / 2024 (WB / ZJ / 24), and H5-Re14-WS / SX / 4-1 / 2020-(2.3.4.4b) (WS / SX / 4-1 / 20).

[0076] The 10-fold diluted sera of the seven test strains were diluted to the corresponding multiples until the blood inhibition titer reacting with the self-antigen was 1280, and the isolated strains and antigenic analysis strains were used to perform hemagglutination inhibition (HI) tests with antisera to select vaccine candidate strains.

[0077] Table 3 Selection of candidate strains by HI test

[0078] From Table 3, we can see that:

[0079] The 2.3.4.4b branch H5 subtype AIV recently isolated and preserved in the laboratory has a large difference in antigenic reaction with the currently used vaccine strains; the present invention selects A / chicken / Shenyang / 12301 / 2024 (Ck / SY / 01 / 24) which has a better antigenic reaction with other isolated 2.3.4.4b branch H5 subtype strains among the isolated strains, that is, the A type 2.3.4.4b branch avian influenza virus A / chicken / Shenyang / 12301 / 2024 (Ck / SY / 01 / 24) CGMCC No.46299 isolated in Example 1 as a vaccine candidate strain.

[0080] 2. Virus hemagglutination titer determination

[0081] The chicken embryo allantoic fluid of influenza virus Ck / SY / 01 / 24 strain CGMCC No.46299 of clade 2.3.4.4b H5 subtype was used for 10 -1 ~10 -3The allantoic fluid of 9-11 day old SPF chicken embryos was inoculated with 0.2 mL / embryo through the allantoic cavity and incubated at 37°C. The allantoic fluid of 48-96 h old chicken embryos was collected aseptically and its hemagglutination titer was determined by 1% chicken red blood cell hemagglutination test (HA). The allantoic fluid of chicken embryos with higher hemagglutination titer was collected and mixed and diluted 10% with sterile saline. -1 ~10 -3 Then continue to inoculate the allantoic cavity of 10-day-old SPF chicken embryos. Repeated passage until the hemagglutination titer of the harvested virus allantoic fluid is stable. After SPF chicken embryo expansion culture, collect 72h to 96h chicken embryo allantoic fluid, mix and measure its hemagglutination titer, and use it as vaccine seed virus.

[0082] It was determined that the 2.3.4.4b branch H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024CGMCC No.46299 could maintain a stable hemagglutination titer when it was continuously propagated in chicken embryos for 15 generations, and the hemagglutination titer was stable at 2 7 .

[0083] 3. Virus EID 50 Determination

[0084] (1) Preparation of chicken embryos: Place 9-11 day old chicken embryos in a dark room and observe them with an egg candling device. Mark the air chamber of each chicken embryo, avoiding marking large blood vessels.

[0085] (2) Virus dilution: Take out the 2.3.4.4b branch H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No.46299 from the -80℃ refrigerator and thaw naturally on ice. Prepare 10 1.5 mL EP tubes and add 900 μL of double-antibody PBS to each tube. Add 100 μL of virus stock solution to the first tube, mix thoroughly and then aspirate 100 μL to the second tube, dilute to the tenth tube in sequence, mark well, and store on ice.

[0086] (3) Inoculate the virus: Disinfect the marked chicken embryos with 75% alcohol, sterilize the puncher and punch holes above the air chamber and at the ligand. Insert a 1mL syringe 0.5cm along the hole in the embryo, inject 0.1mL of virus solution, inoculate three chicken embryos with each dilution of virus solution, and mark them. Seal with paraffin. Place in an incubator and incubate, and illuminate the eggs daily.

[0087] (4) Harvesting chicken embryos: 48-72 hours after infection, place the chicken embryos in a 4℃ refrigerator for 1 hour to prevent bleeding. After disinfecting the air chamber with 75% alcohol, open it with tweezers and draw 25μL of allantoic fluid into a 96-well hemagglutination plate for a hemagglutination test and record the results.

[0088] According to the Reed-Muench method, the EID 50 ), the correction factor for 10-fold serial dilution is 1.

[0089] logEID 50 = logarithm of virus dilution with infection rate higher than 50% + logarithm of corresponding distance ratio × dilution factor;

[0090] Corresponding distance ratio = (positive percentage above 50% - 50%) / (positive percentage above 50% - positive percentage below 50%) × correction factor;

[0091] It was determined that the virus titer of 2.3.4.4b branch H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024CGMCC No.46299 remained stable at 10 after 15 generations of continuous propagation in chicken embryos. 8.5 EID 50 / mL.

[0092] 4. Preparation of inactivated vaccines

[0093] (1) Preparation of vaccine antigens;

[0094] Preparation method of vaccine antigen: 7 , the virus titer is 10 8.5 EID 50 / mL of 2.3.4.4b branch H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No.46299 chicken embryo allantoic fluid analytically pure formaldehyde solution for inactivation, during inactivation, the volume ratio of the 2.3.4.4b branch H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 chicken embryo allantoic fluid and the formaldehyde solution is 1000:1, to obtain vaccine antigens;

[0095] (2) Vaccine preparation: Refer to the "Regulations for Veterinary Biological Products" and mix the vaccine antigen prepared above (the virus content of 2.3.4.4b branch H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No.46299 is 10 8.5 EID 50 / mL) and oil-emulsion adjuvant in a volume ratio of 1.5:1 and emulsified by conventional methods to obtain 2.3.4.4b branch H5 subtype avian influenza virus inactivated vaccine.

[0096] Example 3

[0097] This example is a protective evaluation of the inactivated vaccine of subtype AIV of branch 2.3.4.4b H5

[0098] Immune challenge protection experiment: divided into immunization group and control group (9 2-week-old chickens in each group, and numbered). The immunization group was intravenously injected with 0.3 mL of the 2.3.4.4b branch H5 subtype avian influenza virus inactivated vaccine prepared in Example 2; the control group was not vaccinated.

[0099] Six chickens (immune group) at 2 weeks after immunization and six non-immune chickens (control group) at the same age were inoculated with 10 6 EID 50 / ml (0.2ml) of different viruses (A / chicken / Shenyang / 12301 / 2024 (Ck / SY / 01 / 24) isolated in Example 1).

[0100] 24 hours after infection, the remaining 3 chickens in the immunized group and the control group were placed in the corresponding infected group. Oral swabs and cloacal swabs were collected from the infected chickens and the cohabiting chickens 3, 5, and 7 days after Ck / SY / 01 / 24 virus inoculation, and the virus titers in the swabs were measured (EID 50 / mL), the measurement results are shown in Table 4.

[0101] Table 4 Evaluation of the protective effect of inactivated vaccines of subtype H5 AIV in clade 2.3.4.4b

[0102] From Table 4, we can see that:

[0103] The chickens in the immunized group did not excrete toxins after virus infection, and the chickens that were put in later 2 weeks after the first immunization were not infected with the Ck / SY / 01 / 24 virus. The chickens in the control group excreted toxins after virus infection, and the non-immunized chickens of the same age that were put in later were infected with the Ck / SY / 01 / 24 virus. It is proved that the 2.3.4.4b branch H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No.46299 of the present invention can quickly (2 weeks after a single immunization) produce resistance to 2.3.4.4b branch H5 subtype virus infection after immunization as an inactivated vaccine, and has the characteristics of high safety and good immune effect, which lays a foundation for the prevention and treatment of 2.3.4.4b branch H5 subtype avian influenza virus.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A 2.3.4.4b clade H5 subtype avian influenza virus strain, characterized in that: The 2.3.4.4b branch H5 subtype avian influenza virus strain is type A avian influenza virus A / chicken / Shenyang / 12301 / 2024, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.46299 and the deposit time being December 12, 2024.

2. Use of the 2.3.4.4b branch H5 subtype avian influenza virus strain according to claim 1 in the preparation of vaccines or biological materials.

3. The use according to claim 2, characterized in that: The biological material is any one of (1) to (4); (1) Chicken embryo allantoic fluid containing the 2.3.4.4b clade H5 subtype avian influenza virus strain of claim 1; (2) cells containing the 2.3.4.4b clade H5 subtype avian influenza virus strain of claim 1; (3) the genomic nucleic acid of the 2.3.4.4b clade H5 subtype avian influenza virus strain according to claim 1; (4) Recombinant DNA, expression cassette, transposon, plasmid vector, phage vector, viral vector, engineered bacteria or cell line containing the genomic nucleic acid of the 2.3.4.4b branch H5 subtype avian influenza virus strain as described in claim 1.

4. A 2.3.4.4b clade H5 subtype avian influenza vaccine, characterized in that: The 2.3.4.4b branch H5 subtype avian influenza vaccine comprises an immunizing amount of the 2.3.4.4b branch H5 subtype avian influenza virus strain antigen according to claim 1.

5. The 2.3.4.4b clade H5 subtype avian influenza vaccine according to claim 4, characterized in that: The 2.3.4.4b branch H5 subtype avian influenza virus strain antigen is the inactivated 2.3.4.4b branch H5 subtype avian influenza virus strain.

6. The 2.3.4.4b clade H5 subtype avian influenza vaccine according to claim 4, characterized in that: The 2.3.4.4b branch H5 subtype avian influenza vaccine also includes an oil-emulsion adjuvant.

7. The 2.3.4.4b clade H5 subtype avian influenza vaccine according to claim 4, characterized in that: The antigen content of the 2.3.4.4b branch H5 subtype avian influenza virus strain is 10 8.5 EID 50 / mL.

8. The method for preparing the 2.3.4.4b clade H5 subtype avian influenza vaccine according to any one of claims 4 to 7, characterized in that: The preparation method comprises the following steps: (a) The hemagglutination titer is 27 and the virus titer is 10 8.5 EID 50 / mL of chicken embryo allantoic fluid of the 2.3.4.4b branch H5 subtype avian influenza virus strain of claim 1 is inactivated with a formaldehyde solution to obtain a 2.3.4.4b branch H5 subtype avian influenza virus strain antigen solution; (b) Mixing and emulsifying the 2.3.4.4b clade H5 subtype avian influenza virus strain antigen solution and the oil-emulsion adjuvant to obtain the 2.3.4.4b clade H5 subtype avian influenza vaccine.

9. The preparation method according to claim 8, characterized in that: The volume ratio of the chicken embryo allantoic fluid of the 2.3.4.4b branch H5 subtype avian influenza virus strain to the formaldehyde solution is (800-1200):

1.

10. The preparation method according to claim 8, characterized in that: The volume ratio of the 2.3.4.4b branch H5 subtype avian influenza virus strain antigen solution and the oil emulsion adjuvant is (1.5-2.3):1.

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