A 2.3.4.4b-branch H5 subtype avian influenza virus strain and its application

Abstract

The present invention discloses a 2.3.4.4b clade H5 subtype avian influenza virus strain and its application. The 2.3.4.4b clade H5 subtype avian influenza virus strain is avian influenza virus A / chicken / Shenyang / 12301 / 2024, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 46299 and the deposit date of December 12, 2024. By searching for a 2.3.4.4b clade H5 subtype AIV with good cross-immune reaction as a vaccine candidate strain and preparing a whole virus inactivated vaccine, the present invention aims to effectively prevent and control the infection of recently prevalent H5 subtype AIV in poultry, and further eliminate the potential threat of influenza virus in poultry farming to public health safety.

Description

Technical Field

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

[0002] Since 2021, a novel 2.3.4.4b-evolved branch of H5 subtype AIV has been prevalent globally, causing huge economic losses to the global poultry industry, and its accidental infections in wild carnivores, mink farms and marine mammals have increased significantly.

[0003] Currently, the vaccine strain with a relatively wide application range for the 2.3.4.4b-evolved branch of H5 subtype is H5-Re14-WS / SX / 4-1 / 2020-(2.3.4.4b), which is selected from the H5 subtype virus strains 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 the literature report (doi:10.1016 / S2666-5247(22)00148-3), there have been certain differences in antigenicity between the vaccine strain and the currently prevalent virus strains in China. Moreover, migratory birds bring foreign prevalent virus strains into the country through migration and further evolve into new prevalent virus strains, making the protective effect of the previously applied inactivated vaccine worse. The above research shows that the currently prevalent 2.3.4.4b-branch H5 subtype virus has undergone a certain degree of genetic evolution and variation and poses a potential threat to human public health safety. In view of this, there is an urgent need for a new vaccine strain to cope with this continuously 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 finds a 2.3.4.4b-branch H5 subtype AIV with good cross-immune reaction as a vaccine candidate strain to prepare a whole-virus inactivated vaccine, in order to effectively prevent and control the infection of recently prevalent H5 subtype AIV in poultry, and further eliminate the potential threat of influenza virus in poultry farming to public health safety.

[0005] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0006] The first aspect of the present invention provides a 2.3.4.4b-branch H5 subtype avian influenza virus strain, and the 2.3.4.4b-branch H5 subtype avian influenza virus strain is avian influenza virus A / chicken / Shenyang / 12301 / 2024, which is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No. 46299, and the preservation time is December 12, 2024.

[0007] The second aspect of the present invention provides an application 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-branch H5 subtype avian influenza virus strain;

[0010] (2) Cells containing the 2.3.4.4b-branch H5 subtype avian influenza virus strain;

[0011] (3) Genomic nucleic acid of the 2.3.4.4b-branch H5 subtype avian influenza virus strain;

[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.

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

[0014] Preferably, the antigen of the 2.3.4.4b-branch H5 subtype avian influenza virus strain is the 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 further comprises an oil emulsion adjuvant.

[0016] Preferably, the content of the antigen 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 preparation method of the above-mentioned 2.3.4.4b-branch H5 subtype avian influenza vaccine, and the preparation method comprises the following steps:

[0018] (a) Inactivating the chicken embryo allantoic fluid of the 2.3.4.4b-branch H5 subtype avian influenza virus strain with a hemagglutination titer of 2 7 and a virus titer of 10 8.5 EID 50 / mL with a formaldehyde solution to obtain an antigen solution of the 2.3.4.4b-branch H5 subtype avian influenza virus strain;

[0019] (b) Mix and emulsify the antigen solution of the 2.3.4.4b-branch H5 subtype avian influenza virus strain and the oil adjuvant to obtain the 2.3.4.4b-branch H5 subtype avian influenza vaccine.

[0020] Preferably, the volume ratio of the allantoic fluid of chicken embryos 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%-40%.

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

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

[0024] The prerequisite for the application of inactivated vaccines is to ensure the virus yield and stability. At the same time, the effective immune protection efficacy of inactivated vaccines against the currently prevalent 2.3.4.4b-branch H5 subtype AIV in China is also an important part of vaccine evaluation. For this reason, the present invention evaluates the virus stability by continuous passage in chicken embryos and evaluates its immune efficacy through an immunized chicken challenge protection test; it is found that using the 2.3.4.4b-branch H5 subtype avian influenza virus strain with good cross-immune reaction isolated in the present invention (avian influenza virus A / chicken / Shenyang / 12301 / 2024, abbreviated as SY / 01 / 24 strain) as the representative strain of the currently prevalent 2.3.4.4b-branch H5 subtype AIV, the virus can still maintain a stable hemagglutination titer when continuously passaged in chicken embryos up to 15 generations, and the hemagglutination titer is stable at 2 7 , and the virus titer is measured to reach 10 8.5 EID 50 / mL, indicating that the virus has good stability; after inactivating the whole virus of the SY / 01 / 24 strain and supplementing it with an adjuvant to make a whole virus inactivated vaccine, the results of the immunized chicken challenge protection test of the vaccine confirm that the vaccine can rapidly generate resistance to the infection of the 2.3.4.4b-branch H5 subtype AIV after immunization; the research results show that the vaccine candidate strain used in the present invention can stably reproduce in chicken embryos, the strain is stable, and it has high safety and is not pathogenic to chickens; the protective research shows that the vaccine has a good protective effect on the currently prevalent 2.3.4.4b-branch H5 subtype AIV in China; the vaccine can also rapidly (2 weeks after a single immunization) generate immune protection efficacy, laying a foundation for the prevention and control of influenza virus in chickens. Specific embodiments

[0025] The embodiments of the technical solution of the present invention will be described in detail below in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, so they are 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 ordinary meaning 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, and the 2.3.4.4b-branch H5 subtype avian influenza virus strain is avian influenza virus A / chicken / Shenyang / 12301 / 2024, which is preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No. 46299, and the preservation time is December 12, 2024.

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

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

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

[0031] (2) Cells containing the 2.3.4.4b-branch H5 subtype avian influenza virus strain;

[0032] (3) The genomic nucleic acid of the 2.3.4.4b-branch H5 subtype avian influenza virus strain;

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

[0034] Another embodiment of the present invention provides a 2.3.4.4b-branch H5 subtype avian influenza vaccine, and the 2.3.4.4b-branch H5 subtype avian influenza vaccine 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-branch H5 subtype avian influenza virus strain antigen is the inactivated 2.3.4.4b-branch H5 subtype avian influenza virus strain.

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

[0037] In one embodiment, the antigen content of the 2.3.4.4b lineage 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 lineage H5 subtype avian influenza vaccine, and the preparation method comprises the following steps:

[0039] (a) Inactivating the allantoic fluid of chicken embryos of the 2.3.4.4b lineage H5 subtype avian influenza virus strain with a hemagglutination titer of 2 7 and a virus titer of 108.5 EID50 / mL with a formaldehyde solution to obtain a 2.3.4.4b lineage H5 subtype avian influenza virus strain antigen solution;

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

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

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

[0043] In one embodiment, the volume ratio of the 2.3.4.4b lineage H5 subtype avian influenza virus strain antigen solution to the oil adjuvant can be any value in (1.5 - 2.3):1, such as 1.5:1, 2.0:1 or 2.3:1.

[0044] The technical solutions of the present invention are further described in detail below through specific examples.

[0045] The experimental methods adopted below are as follows:

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

[0047] Add 25 μl of normal saline to each well of a 96-well microtiter hemagglutination plate using a pipette. Add 25 μl of the virus antigen to be tested to the first well of each row. After pipetting up and down 3 - 4 times to mix evenly, aspirate 25 μl and add it to the second well. Perform serial dilutions in this way until the 11th well, then discard 25 μl. Do not add antigen to the 12th well, which serves as the negative control well. After serial dilution, aspirate 25 μl of 1% chicken red blood cell suspension and add it to each well in sequence from the highest dilution to the lowest dilution. After shaking and mixing evenly, let it stand at room temperature for 30 min and then observe the results. Tilt the reaction plate. If the red blood cells that sink to the bottom of the well flow downward in a linear shape along the inclined surface, it is precipitation, indicating that the red blood cells are not or incompletely agglutinated by the virus; if the red blood cells at the bottom of the well spread flat on the bottom of the well, form a uniform thin layer, and do not flow after tilting, it indicates that the red blood cells are agglutinated by the virus. The 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 is as follows:

[0049] Prepare four-unit antigen according to the virus hemagglutination titer, that is, a virus solution with 4 hemagglutination units. Divide the hemagglutination titer by 4 to obtain the dilution factor of the antigen. Dilute the virus stock solution with normal saline according to the dilution factor to obtain the four-unit antigen. After preparation, verify whether the preparation is accurate according to the HA test method. Only after accurate preparation can the subsequent HI test be carried out.

[0050] The specific method of the HI test: Add 25 μl of normal saline to each well of a 96-well microtiter hemagglutination plate using a pipette. Aspirate 25 μl of the serum to be tested and place it in the first well. After pipetting up and down 3 - 4 times to mix evenly, aspirate 25 μl and transfer it to the second well. Perform serial dilutions in this way until the 11th well, then discard 25 μl. The last well is set as the negative control and is not diluted. Add 25 μl of the prepared four-unit antigen to each well in sequence from the highest dilution to the lowest dilution of the serum. After shaking and mixing evenly, let it stand at room temperature for 30 min. Aspirate 25 μl of 1% red blood cell suspension and add it to each well in sequence from the highest dilution to the lowest dilution. After shaking and mixing evenly, let it stand at room temperature for 30 min and then observe the results. The maximum dilution that completely inhibits red blood cell agglutination is the hemagglutination inhibition test titer of this serum, that is, the hemagglutination inhibition titer.

[0051] Example 1

[0052] This example is about the isolation and identification of a 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 an avian influenza virus type A by the Animal Influenza Laboratory of China Agricultural University. The isolated strain SY / 01 / 24 was named A / chicken / Shenyang / 12301 / 2024, and the classification name is: avian influenza virus type A.

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

[0055] (1) Sample treatment

[0056] Add 1 ml of double-antibody PBS solution containing (10000 U / L / ml) to each swab sample, vortex and mix for 30 s; place the treated sample in a 4°C refrigerator and store overnight, and inoculate 9-11-day-old chicken embryos the next day.

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

[0058] Inoculate the treated samples into 9-11-day-old chicken embryos by the allantoic cavity inoculation method, inoculate 2 chicken embryos for each sample, inoculate 0.5 ml for each embryo, seal with paraffin after inoculation, and place in a 35°C incubator for 48-72 h. Check the growth of chicken embryos every 12 h. If dead embryos are found within 24 h, they should be discarded in time. After 48-72 h, place the chicken embryos in a 4°C refrigerator overnight. When harvesting chicken embryos, use sterile forceps to tear the eggshell of the chicken embryo air chamber, pierce at the place where there are no large blood vessels on the chorioallantoic membrane, aspirate 25 μl of allantoic fluid, collect the corresponding chicken embryo allantoic fluid into a cryopreservation tube, and store it in an -80°C refrigerator.

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

[0060] HA-F: 5’-TGGAAAGTRTAARAAACGGAACGT-3’, SEQ ID NO: 1;

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

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

[0063] Extract the viral RNA according to the instructions of the RaPure Total RNA Mini Kit RNA extraction kit.

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

[0065] Table 1

[0066]

[0067]

[0068] Add each component in Table 1 in sequence. After mixing, centrifuge immediately. React by water bath at 37 °C for reverse transcription for 1 h. The cDNA product thus obtained is the template for the PCR amplification reaction.

[0069] The PCR reaction system is shown in Table 2:

[0070] Table 2

[0071] <![CDATA[dd H 2 O]]> 9.5μL Forward primer (20uM) 0.5μL Reverse primer (20uM) 0.5μL cDNA 2μL 2×EasyTaq PCR Supermix 12.5μL Total volume 25μL

[0072] Add each component in Table 2 in sequence. After mixing, centrifuge immediately.

[0073] PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min 30 s, 35 cycles, final extension at 72 °C for 10 min, 4 °C ∞;

[0074] After the PCR amplification reaction, take 6 μL of the PCR product for identification by 1% agarose gel electrophoresis. The PCR identification of the isolated strain is all positive.

[0075] In view of the above identification results, it is determined that the strain A / chicken / Shenyang / 12301 / 2024 is an H5N6 subtype of avian influenza virus A.

[0076] On December 12, 2024, the avian influenza virus A / chicken / Shenyang / 12301 / 2024 was deposited with the China General Microbiological Culture Collection Center, and the deposit number is CGMCC No. 46299. Its scientific description: an H5 subtype avian influenza virus of the 2.3.4.4b lineage; the biological material (strain) referred to: A / chicken / Shenyang / 12301 / 2024.

[0077] Example 2

[0078] This example is for the preparation of an inactivated vaccine against H5 subtype AIV of the 2.3.4.4b lineage:

[0079] 1. Screening of vaccine virus strains

[0080] Vaccine virus strains for testing: (1) One H5 subtype AIV of the 2.3.4.4b lineage, namely A / chicken / Shenyang / 12301 / 2024 (Ck / SY / 01 / 24) isolated in Example 1. (2) Six H5 subtype AIVs of the 2.3.4.4b lineage previously preserved in this 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), H5-Re14-WS / SX / 4-1 / 2020-(2.3.4.4b) (WS / SX / 4-1 / 20).

[0081] The sera of the seven test strains diluted 10-fold were respectively diluted to a hemagglutination inhibition (HI) titer of 1280 for reacting with their own antigens, and the hemagglutination inhibition (HI) test was performed using the isolated strain and antigenic analysis strains with the antiserum to select vaccine candidate strains.

[0082] Table 3 Selection of candidate strains by HI test

[0083]

[0084] As can be seen from Table 3:

[0085] The antigenic reaction of the recently isolated and preserved H5 subtype AIV of the 2.3.4.4b lineage with the currently used vaccine strain is quite different; the present invention selects A / chicken / Shenyang / 12301 / 2024 (Ck / SY / 01 / 24) with a better antigenic reaction among the isolated strains with other isolated H5 subtype strains of the 2.3.4.4b lineage, that is, the avian influenza virus of the 2.3.4.4b lineage A / chicken / Shenyang / 12301 / 2024 (Ck / SY / 01 / 24) CGMCC No. 46299 isolated in Example 1 as the vaccine candidate strain.

[0086] 2. Determination of virus hemagglutination titer

[0087] The allantoic fluid of chicken embryos of the Ck / SY / 01 / 24 strain CGMCC No. 46299 of the H5 subtype influenza virus of the 2.3.4.4b lineage was made into 10 -1 ~10 -3Dilute, inoculate 9- to 11-day-old SPF chicken embryos via the allantoic cavity, with an inoculation volume of 0.2 mL / embryo, and incubate at 37°C. Sterilely collect the allantoic fluid of chicken embryos at 48 h to 96 h, and measure its hemagglutination titer using 1% chicken red blood cell hemagglutination assay (HA). Mix the allantoic fluid of chicken embryos with a higher hemagglutination titer, and dilute it 10 -1 to 10 -3 times with sterile normal saline, and then continue to inoculate the allantoic cavity of 10-day-old SPF chicken embryos. Passage repeatedly until the hemagglutination titer of the harvested viral allantoic fluid is stable. After large-scale culture in SPF chicken embryos, collect the allantoic fluid of chicken embryos at 72 h to 96 h, mix it, and measure its hemagglutination titer as the vaccine seed virus.

[0088] It was determined that the 2.3.4.4b clade H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No. 46299 could still maintain a stable hemagglutination titer when continuously passaged in chicken embryos up to 15 passages, and the hemagglutination titer was stable at 2 7 .

[0089] 3. Virus EID 50 Determination

[0090] (1) Preparation of chicken embryos: Place 9- to 11-day-old chicken embryos in a dark room, observe with an egg candler, mark the air chamber of each chicken embryo, and mark it avoiding large blood vessels;

[0091] (2) Serial dilution of the virus: Take out the 2.3.4.4b clade H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No. 46299 from the -80°C refrigerator and let it melt 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 the virus stock solution to the first tube, mix well, and then aspirate 100 μL and transfer it to the second tube, and dilute it sequentially to the tenth tube, make marks, and store it on ice.

[0092] (3) Inoculation of the virus; Disinfect the marked chicken embryos with 75% alcohol, disinfect the puncher, and punch holes above the air chamber and at the ligand. Insert a 1-mL syringe 0.5 cm along the small hole in the embryo body and inject 0.1 mL of the virus solution. Inoculate three chicken embryos with the virus solution of each dilution, make marks. Seal with paraffin. Place it in an incubator for incubation and candling every day.

[0093] (4) Harvest of chicken embryos: 48 - 72 h after virus inoculation, place the chicken embryos in a 4°C refrigerator for 1 h to prevent chicken embryo bleeding. After disinfecting the air chamber with 75% alcohol, open it with forceps, aspirate 25 μL of the allantoic fluid into a 96-well hemagglutination plate, perform a hemagglutination test, and record the results.

[0094] According to the Reed-Muench formula, calculate the median infective dose (EID50 ) For the 10-fold serial dilution, the correction factor is 1.

[0095] logEID 50 = logarithm of virus dilution at infection rate higher than 50% + logarithm of dilution coefficient × corresponding distance ratio;

[0096] Corresponding distance ratio = (positive percentage higher than 50% - 50%) / (positive percentage higher than 50% - positive percentage lower than 50%) × correction factor;

[0097] It was determined that when the 2.3.4.4b lineage H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No. 46299 was continuously passaged in chicken embryos to the 15th generation, the virus titer could still be stabilized at 10 8.5 EID 50 / mL.

[0098] 4. Preparation of inactivated vaccine

[0099] (1) Preparation of vaccine antigen;

[0100] Method for preparing vaccine antigen: Inactivate the allantoic fluid of chicken embryos of the 2.3.4.4b lineage H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No. 46299 with a hemagglutination titer of 2 7 and a virus titer of 10 8.5 EID 50 / mL with analytical pure formaldehyde solution. During inactivation, the volume ratio of the allantoic fluid of chicken embryos of the 2.3.4.4b lineage H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 strain to the formaldehyde solution is 1000:1, and the vaccine antigen is obtained;

[0101] (2) Preparation of the vaccine Refer to the method of "Regulations for Veterinary Biological Products", mix the above-prepared vaccine antigen (where the virus content of the 2.3.4.4b lineage H5 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024 CGMCC No. 46299 is 10 8.5 EID 50 / mL) and oil adjuvant in a volume ratio of 1.5:1, and emulsify by the conventional method to obtain the inactivated vaccine of the 2.3.4.4b lineage H5 subtype avian influenza virus.

[0102] Example 3

[0103] This example is for the protective evaluation of the inactivated vaccine of the 2.3.4.4b lineage H5 subtype AIV

[0104] Immune challenge protection experiment: Divided into an immune group and a control group (9 two-week-old chickens in each group, and they were numbered). Chickens in the immune group were intravenously injected with 0.3 mL of the inactivated vaccine of H5 subtype avian influenza virus of the 2.3.4.4b branch prepared in Example 2; chickens in the control group were not vaccinated.

[0105] Six chickens from the immunized chickens (immune group) two weeks after immunization and six chickens of the same age as non-immunized chickens (control group) were inoculated by intranasal and ocular instillation with 10 6 EID 50 / ml (0.2 ml) of different viruses (A / chicken / Shenyang / 12301 / 2024 (Ck / SY / 01 / 24) isolated in Example 1).

[0106] Twenty-four hours after infection, the remaining 3 chickens in the immune group and the control group were respectively placed in the corresponding infected groups for cohabitation. Oral swabs and cloacal swabs of the infected chickens and cohabiting chickens were collected at 3 days, 5 days, and 7 days after inoculation with the Ck / SY / 01 / 24 virus, and the virus titer (EID 50 / mL) in the swabs was measured, and the measurement results are shown in Table 4.

[0107] Table 4 Protective evaluation of the inactivated vaccine of H5 subtype AIV of the 2.3.4.4b branch

[0108]

[0109]

[0110] As can be seen from Table 4:

[0111] The chickens in the immune group did not excrete the virus after virus infection, and the chickens two weeks after a single immunization put in later were not infected with the Ck / SY / 01 / 24 virus. While the chickens in the control group excreted the virus after virus infection, and the non-immunized chickens of the same age put in later were infected with the Ck / SY / 01 / 24 virus. It is proved that the H5 subtype avian influenza virus of the 2.3.4.4b branch of the present invention, A / chicken / Shenyang / 12301 / 2024 CGMCC No. 46299, can rapidly (two weeks after a single immunization) produce resistance to the infection of the H5 subtype virus of the 2.3.4.4b branch after being immunized as an inactivated vaccine, and has the characteristics of high safety and good immune effect, laying a foundation for the prevention and control of the H5 subtype avian influenza virus of the 2.3.4.4b branch.

[0112] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered by the scope of the claims and the description 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 the H5N6 subtype avian influenza virus A / chicken / Shenyang / 12301 / 2024, which is deposited in the General Microbiology Center of the China 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 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) A recombinant DNA containing the genomic nucleic acid of the 2.3.4.4b clade 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; 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.

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 vaccine also includes an oil-emulsion adjuvant.

6. 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.

7. The method for preparing the 2.3.4.4b clade H5 subtype avian influenza vaccine according to any one of claims 4 to 6, characterized in that: The preparation method comprises the following steps: (a) Set the hemagglutination titer to 2 7 , 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.

8. The preparation method according to claim 7, 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.

9. The preparation method according to claim 7, 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.

Images