A strain of Akabane disease virus and its application

Abstract

The present invention relates to the technical field of veterinary biological products, and in particular to an Akabane disease virus strain and its application. The Akabane disease virus strain is Akabane virus AKAV / JL / 2022, and its deposit number is CGMCC No.45375. The present invention provides an Akabane disease virus strain with good passage stability, strong pathogenicity, good immunogenicity, and the ability to induce the body to produce high-titer neutralizing antibodies. After being prepared into a vaccine, it can target the prevalent Akabane disease, provide important medical value for the prevention, control and treatment of Akabane disease in pastoral areas, and has far-reaching and significant practical application significance.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary biological products, and in particular to an Akabane disease virus strain and an application thereof. Background Art

[0002] Akabane disease (AKABANES) is a viral infectious disease caused by Akabane disease virus (AKAV), also known as Akabane disease. The virus is mainly transmitted mechanically by blood-sucking insects (such as mosquitoes and Culicoides), and the Akabane virus can also be transmitted vertically through the mother. Generally speaking, AKAV mainly causes reproductive disorders and congenital malformations in ruminants such as cattle and sheep. It causes premature birth, miscarriage and stillbirth in pregnant female livestock, and symptoms such as joint curvature, abnormal body shape and brain defects in aborted fetuses. The virus infects adult bulls, sheep or non-pregnant cows and sheep, and often manifests as latent infection. The infected animals are carriers of the virus but have no obvious clinical symptoms. Akabane disease affects fetal development. In the early stages of infection, the younger the gestational age, the more likely it is to have a miscarriage, and the older the gestational age, the more likely it is to have premature birth. In the middle stages of infection, cows often experience dystocia, which is caused by the bending, stiffness, and deformity of the calf's limbs. Even calves born naturally have difficulty standing on their own. In late pregnancy, calves born from infected cows are smart and alert, but unable to stand, and have ataxia and muscle atrophy. When sheep are infected, joint bending and neurological symptoms usually appear at the same time.

[0003] Akabane virus belongs to the genus Orthobunya virus of the family Peribunyaviridae in the order Bunyavirales. Viruses are mostly transmitted by arthropods, and some of them pose a major threat to human health. However, there are no reports of Akabane virus infecting humans. The AKAV genome is a single-stranded negative-strand RNA composed of three RNA segments: L, M, and S. Among the three RNA segments, the M segment encoding the glycoprotein that induces neutralizing antibodies and hemagglutination inhibition antibodies is the most variable among isolates, and the S segment encoding the nucleocapsid protein and non-structural protein is highly conserved. Current studies have shown that AKAV has only one serotype. Based on the S segment sequence, AKAV isolates are divided into five genotypes (IV), of which the type I genotype can be divided into subgroups Ia and Ib, with Ia being more common. AKAV virus particles are spherical, with a diameter of 90 to 100 nm, and have an envelope and fibers.

[0004] There is no effective treatment for Akabane disease, because calves are born with deformed limbs and unstable standing, and there is no way to treat it. Calves that can drink milk generally do not die, but their growth rate is relatively slow, which consumes a lot of manpower and material resources and has low economic value. If the calf's symptoms are more serious and there are signs of difficulty in swallowing, most of them will die in the end. Therefore, once cattle and sheep are infected, most of the infected animals are killed. On the other hand, since Akabane virus infection in adult male animals and non-pregnant female animals mostly manifests as latent infection without obvious clinical symptoms, coupled with possible virus mutations, latent infection and virus mutations determine the difficulty of prevention and control of Akabane disease. Therefore, the development of an effective Akabane disease vaccine is of great significance for the prevention and control of Akabane disease. Summary of the invention

[0005] In a first aspect, the present invention provides an Akabane disease virus strain, which is Akabane virus AKAV / JL / 2022, and its preservation number is CGMCC No.45375.

[0006] This strain is an epidemic strain of Akabane disease virus obtained by purification and isolation of plaque cloning. It can be stably propagated in cells, has strong pathogenicity and good immunogenicity, and can induce the body to produce high-titer neutralizing antibodies, providing a basis for effective vaccine preparation and prevention and control of Akabane disease.

[0007] The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 25, 2022. The address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Classification Name: Akabane Virus, and the deposit number is CGMCC No.45375.

[0008] The primary strain of Akabane disease virus of the present invention is separated from the afterbirth tissue collected from Akabane disease positive cattle in Jilin.

[0009] The method for isolating the AKAV / JL / 2022 strain of the present invention comprises the following steps:

[0010] (1) Disease material treatment: The afterbirth of aborted cows with Akabane disease symptoms was taken and made into a suspension with DMEM, ground, frozen and thawed, and then centrifuged. The supernatant was filtered and double-antibody was added to obtain a virus suspension;

[0011] (2) Isolation and screening: Take the MDBK adherent cells that have been spread over a monolayer, discard the original culture medium, add the virus suspension obtained above, place in a 37°C, 5% CO2 incubator for adsorption, discard the inoculum, and add DMEM medium containing penicillin and streptomycin for culture. Observe the cells daily to see if they produce CPE. If there is no lesion, collect the culture medium on the 5th day, freeze and thaw three times, and continue to subculture. If the cells produce lesions, collect the virus liquid to obtain the primary virus strain;

[0012] The criteria for judging cell lesions are as follows: the degree of cell lesions is above 80%. In the early stage of lesions, typical cell enlargement and rounding can be seen, and the refractive index increases, and then local cells begin to fuse; in the late stage, cells begin to fuse over a large area, break up, and finally the fused cells necrotize and fall off;

[0013] (3) Plaque clone purification: The F1 strain virus solution was diluted 10-fold to 10 in DMEM. -1 ~10 -5 After that, the culture medium in the six-well plate of MDBK cells grown into a dense monolayer was discarded, and after washing twice with the maintenance solution, 400ul of the above-mentioned virus solution of different dilution multiples was added to each well, and the plate was placed in an incubator for adsorption for 1 hour, and then the liquid was discarded, and a first layer of nutrient agar with a thickness of about 3mm was spread. The nutrient agar was 2% low melting point agarose and 2×DMEM mixed in equal volumes, and then allowed to stand at room temperature for 30 minutes, and then inverted and cultured at 37°C, 5% CO2 for 3 days. When the cells showed pathological changes, a second layer of nutrient agar was spread, and then allowed to stand at room temperature for 30 minutes, and then inverted and cultured at 37°C, 5% CO2 in the dark, and the growth of plaques was observed and recorded every day;

[0014] After plaques are formed, small and isolated plaques in the cell wells are selected, aspirated with a 10μl pipette tip, and placed together with agarose in 1.0ml of serum-free DMEM. Repeated freezing and thawing three times to fully release the virus, inoculate new blank MDBK cells for virus proliferation, and repeat cloning three times. Plaques are randomly selected for subculture, and strains with stable subculture and high virus content are selected.

[0015] The primary Akabane disease virus strain of the present invention produces obvious lesions in the first generation of visible cells. The S gene of the isolated AKAV / JL / 2022 strain was sequenced and identified by RT-PCR. It was identified that the strain was different from the two strains of CH-01 (accession number: MW194117.1) and GXDH01 (accession number: MH174977.1) of genotype Ia subtype published in China in 2020, and had the highest homology of 98.5% with the Israeli AKAV / S / ISR / -170 / 18 (accession number: MW822048.1) published in 2018, both of which were genotype Ib subtype.

[0016] During the plaque cloning and purification process, the present invention screened a large number of plaque samples for passage, and observed the degree of pathological changes in cells infected by different strains during the passage process. It was found that some strains could not be passaged for a long time, and their passage stability was poor, while the degree of pathological changes in cells infected by other strains was poor, and their virulence was weak. After a large number of screenings, only two strains with both good passage stability and high virulence were found. The virus content of the two passaged strains was determined respectively, and the strain with the highest virus content was selected. The preserved strain AKAV / JL / 2022 with strong pathogenicity and good immunogenicity of the present invention was obtained through immunological test verification.

[0017] In a second aspect, the present invention provides the use of the above-mentioned Akabane disease virus strain in the preparation of a drug or reagent, wherein the drug or reagent is used to treat, prevent or diagnose Akabane disease.

[0018] In a third aspect, the present invention provides a product containing the Akabane disease virus strain; the product is any one selected from a medicine, a feed or a feed additive.

[0019] In a fourth aspect, the present invention provides a vaccine comprising the Akabane disease virus strain or a culture thereof.

[0020] As a preferred embodiment of the present invention, the vaccine is an inactivated vaccine.

[0021] As a preferred embodiment of the present invention, the vaccine is a veterinary vaccine.

[0022] As a preferred embodiment of the present invention, the titer of the Akabane disease virus strain or its culture in the vaccine is 10 6.0 TCID 50 / mL or above.

[0023] As a preferred embodiment of the present invention, after vaccination with the vaccine, the anti-Akabane virus neutralizing antibody titer level is not less than 1:128.

[0024] As a preferred embodiment of the present invention, the vaccine further comprises an adjuvant.

[0025] Preferably, the adjuvant is ISA 201VG adjuvant or oil adjuvant.

[0026] In a fifth aspect, the present invention provides a method for preparing a vaccine in any of the above embodiments, comprising:

[0027] The Akabane disease virus strain is inoculated into cells for cultivation to obtain a virus liquid culture; the virus liquid culture is then inactivated and mixed with an adjuvant to obtain the product after emulsification.

[0028] Preferably, MDBK cells are used for virus culture.

[0029] Preferably, the virus culture fluid is harvested when the cell pathological effect reaches 80-90%.

[0030] Preferably, β-propiolactone is used for inactivation, more preferably 0.25‰ β-propiolactone.

[0031] Preferably, the step of inactivation comprises:

[0032] After the virus liquid culture is mixed with β-propiolactone, it is inactivated at 80-100 r / min and 0-8° C., and then placed in a water bath at 35-40° C. for hydrolysis.

[0033] Preferably, the inactivation is performed for more than 24 hours; and / or the hydrolysis is performed for more than 24 hours.

[0034] Preferably, the inactivated virus liquid is mixed with the adjuvant in a weight ratio of 1:0.5-1.5 and emulsified at 30-33°C to obtain the preparation.

[0035] Preferably, the emulsification time is more than 30 minutes.

[0036] In a sixth aspect, the present invention provides the use of the vaccine or the method for preparing the vaccine in any of the above embodiments in the preparation of drugs or reagents for diagnosing, preventing, or treating Akabane disease.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention provides an Akabane disease virus strain with good passage stability, strong pathogenicity, good immunogenicity, and the ability to induce the body to produce high-titer neutralizing antibodies. After being prepared into a vaccine, it can specifically prevent Akabane disease, provide important medical value for the prevention, control and treatment of Akabane disease in pastoral areas, and has far-reaching and significant practical application significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : This is an optical microscope photo of MDBK cells infected with the primary strain of AKAV / JL / 2022 provided by the present invention; A is a negative control; B is MDBK cells infected with the primary strain of AKAV / JL / 2022.

[0040] Figure 2 This is the F10 growth curve of the AKAV / JL / 2022 strain provided by the present invention.

[0041] Figure 3 This is an electron microscope photo of the AKAV / JL / 2022 strain provided by the present invention.

[0042] Figure 4 This is an infographic of the existing Akabane virus strains.

[0043] Figure 5 This is the result of homology analysis of S gene sequencing of AKAV / JL / 2022 strain.

[0044] Figure 6 This is the result of the evolutionary tree analysis of the S gene sequencing system of the AKAV / JL / 2022 strain. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0047] The methods for obtaining various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0048] Example 1 Strain Isolation and Identification

[0049] 1.1 Cells and Pathological Materials

[0050] MDBK cells were obtained through commercial channels, and the pathological materials were collected from the afterbirths of aborted cows in positive cattle farms in Jilin Province in 2022.

[0051] 1.2 Treatment of diseased materials

[0052] Before inoculating cells, the placenta of aborted cows that were positive for AKAV by RT-PCR was made into a suspension with DMEM at a volume ratio of 1:5, ground with a grinder, frozen and thawed once at -80°C, centrifuged at 12000r / min for 30min at 4°C, and the supernatant was filtered with a 0.22μm filter to obtain a virus suspension, added with double antibodies (100μg streptomycin / ml, 100U penicillin / ml) and placed in a -4°C refrigerator for storage. When isolating the virus, the placenta suspension was quickly frozen at -80°C, which accelerated the release of the virus from the placenta while reducing the loss of live virus during the freezing process, thereby increasing the probability of successful virus isolation.

[0053] 1.3 Virus isolation and screening

[0054] Select a monolayer of MDBK adherent cells (25 cm 2 ), discard the original culture medium, wash the cells once with PBS, add 1 ml of the above-obtained virus suspension, place in a 37°C, 5% CO2 incubator for adsorption for 1 hour, discard the inoculum, add 10 ml of DMEM medium containing double antibodies (100 μg streptomycin / ml, 100 U penicillin / ml) for culture. Observe the cells daily to see if they produce CPE. If there is no lesion, collect the culture medium on the 5th day, freeze and thaw 3 times, and continue blind transmission.

[0055] Results The isolated AKAV / JL / 2022 primary strain produced obvious pathological changes in cells in the first generation. Figure 1 As shown, in the early stage of the lesion, typical cell enlargement and rounding can be seen, and the refractive index increases, and then local cells begin to fuse; in the late stage, cells begin to fuse in a large area, break, and finally the fused cells necrotize and fall off. When the cell lesion rate reaches more than 80%, freeze and thaw three times, harvest the virus liquid, which is the F1 generation virus, and subculture the F1 generation virus on MDBK adherent cells for acclimation culture.

[0056] 1.4 Plaque purification of viruses

[0057] (1) The Akabane virus F1 virus solution was diluted 10-fold to 10 -1 ~10 -5 After that, the culture medium in the six-well plate of MDBK cells grown into a dense monolayer was discarded, and after washing twice with the maintenance solution, 400ul of the above-mentioned virus solution of different dilution multiples was added to each well, and the plate was placed in an incubator for adsorption for 1 hour, and then the liquid was discarded, and a first layer of nutrient agar with a thickness of about 3mm was added. The nutrient agar refers to 2% low melting point agarose and 2×DMEM mixed in equal volumes, and then allowed to stand at room temperature for 30 minutes, and then inverted in a CO2 incubator at 37°C and 5% CO2 for 3 days. When the cells showed pathological changes, a second layer of nutrient agar was added, and then allowed to stand at room temperature for 30 minutes, and then inverted in a dark environment at 37°C and 5% CO2, and the growth of plaques was observed and recorded every day;

[0058] (2) After the plaques are formed, select small and isolated plaques in the cell wells, aspirate them with a 10μl pipette tip, and place them together with agarose in 1.0ml of serum-free DMEM. Repeat freezing and thawing 3 times to fully release the virus, inoculate new blank MDBK cells, and multiply the virus. Repeat the cloning three times, and randomly select a large number of plaques of the fourth-generation clones for subculture. At the same time, observe the degree of pathological changes in cells infected with different strains during the subculture process. The results show that some strains cannot be subcultured for a long time, and the degree of pathological changes in cells infected by other strains is poor. After screening, only two strains that can be subcultured through MDBK cells for a long time were found. After continuous subculture to the 20th generation, the virus content of the subculture was measured respectively, and the strain with the highest virus content was selected, which is the purified Akabane virus AKAV / JL / 2022.

[0059] 1.5 Determination of virus titer

[0060] Take a 96-well cell culture plate of MDBK cells that have grown into a well-contaminated monolayer, discard the cell culture medium, and wash twice with PBS. Dilute the virus solution of the serial passages in serum-free DMEM medium 10 times in series, and dilute 10 -2 -10 -6 The diluted virus solution was inoculated into 96-well cell culture plates, and 4 wells were inoculated for each dilution. Positive and negative controls were set up at the same time. The culture plates were placed in a 37°C, 5% CO2 incubator for 3-5 days, and CPE was observed and recorded daily. The virus titer was calculated according to the Reed-Muench method. The experimental results showed that the virus titers of the 3rd, 4th, 5th, 7th, 10th, 12th, 15th, 17th, and 20th generations of two consecutive passages were 10 4.5 TCID 50 / mL, 10 5.5 TCID 50 / mL, 10 6.0 TCID 50 / mL, 10 5.5 TCID 50 / mL, 10 6.0 TCID 50 / mL, 10 5.5 TCID 50 / mL, 10 6.0 TCID 50 / mL, 10 6.0 TCID 50 / mL, 10 5.75 TCID 50 / mL and 10 4.5 TCID 50 / mL, 10 4.25 TCID50 / mL, 10 4.75 TCID 50 / mL, 10 5 TCID 50 / mL, 10 4.0 TCID 50 / mL, 10 5 TCID 50 / mL, 10 4.5 TCID 50 / mL, 10 4.75 TCID 50 / mL, 10 4.55 TCID 50 / mL. The results showed that one of the Akabane virus plaque strains, AKAV / JL / 2022, could be stably propagated on MDBK cells, and a high-titer virus liquid could be obtained, which could be used as a candidate strain for Akabane disease vaccine.

[0061] 1.6 Determination of virus growth curve

[0062] In order to study the growth characteristics of the virus, AKAV / JL / 2022 virus was inoculated into MDBK cells to draw a growth curve. The separated F10 virus liquid was inoculated into MDBK cells at 1% of the virus amount. After adsorption for 1 h in a 37°C, 5% CO2 incubator, the inoculum was discarded, and the maintenance liquid was added to continue the culture. Samples were taken every 8 hours until 48 h after inoculation. The virus liquid was collected at different time points to determine the TCID 50 , based on the test results, the growth curve of the virus on MDBK cells was drawn, such as Figure 2 .

[0063] 1.7 Electron microscopy observation

[0064] Take Akabane virus AKAV / JL / 2022 virus solution, centrifuge at 10000r / min for 30 minutes, take the supernatant and centrifuge at 40000r / min for 5 hours, discard the supernatant, dissolve the precipitate with 0.5ml deionized water, negatively stain with 2% sodium phosphotungstate for 30 minutes, dry naturally at room temperature, and observe under transmission electron microscope. Figure 3 As shown, the isolate is approximately round, with a size of 100 nm, a capsule and fibrils, which is consistent with the characteristics of Akabane virus.

[0065] 1.8 PCR identification and sequence analysis

[0066] This experiment used specific detection primers from the "Genetic analysis of Akabane virus isolates from cattle in Korea" published in Veterinary Microbiology in 2010 by the National Veterinary Research and Quarantine Service in Anyang, Gyeonggi Island, South Korea. The primers were designed based on the gene sequence encoding the Akabane virus S protein, and the target gene fragment size was 794bp.

[0067] The above specific detection primers were used to perform PCR identification on the isolated AKAV / JL / 2022 virus strain. The electrophoresis results of the PCR identification of the virus strain clearly showed that the obtained PCR product was a target band with a gene fragment size of about 794 bp. The product was sent to Shanghai Bioengineering Company for sequencing and the results were spliced ​​with the 13 Akabane virus gene sequences published in the GenBank database (such as Figure 4 ) to compare homology and draw a phylogenetic tree, such as Figure 5 , Figure 6 As shown. The results showed that the isolate AKAV / JL / 2022 had the highest homology with AKAV / S / ISR / -170 / 18 (accession number: MW822048.1) at 98.5%, 97.4% with AKAV / S / ISR / -01 (accession number: AY378155.1), 94.7% and 95.3% with two genotype 1a subtype CH-01 (accession number: MW194117.1) and GXDH01 (accession number: MH174977.1), respectively. The homology with two genotype 1a subtype NT-14 (accession number: AF529883.1) and Iriki (accession number: AB297855.1) was 96.3% and 96.4%, respectively. The homology with Okayama2004 (AB289320) and Obe-1 (AF034942.1) of genotype II is 94.4% and 94.9% respectively. The homology with 93FMX (FJ498797) and AK7 (FJ498795.1) of genotype II is 95.3% and 95.2% respectively. The homology with B8935 (MH734940.1) of genotype III is 92.2%, and the lowest homology with MP496 (AB297885.1) of genotype IV is 83.9%. The genetic evolution tree also shows that it is in the same branch with AKAV / S / ISR / -170 / 18 (accession number: MW822048.1) and AKAV / S / ISR / -01 (accession number: AY378155.1), indicating that the isolated strain is of genotype Ib.

[0068] 1.9 virus challenge experiment in suckling mice

[0069] The 5th, 10th and 15th generations (10 6 TCID 50 After centrifugation at 10000 rpm for 15 minutes, the virus solution was serially diluted 10-fold using serum-free DMEM medium. -7 The virus solution was inoculated into 1-3 day old suckling mice at each dilution, 4 mice were inoculated at each dilution, and 10ul was inoculated intracranially. Empty DMEM was set as a negative control. The death of suckling mice was observed every day for 14 days. The results showed that from the third day, suckling mice showed neurological symptoms, convulsions, stiff limbs, and died one after another. After 14 days, the median lethal dose was calculated according to the Reed-Muench method based on the death situation as shown in Table 1. The median lethal dose of F5, F10, and F15 generations was 10 6 LD 50 / ml, 10 6 LD 50 / ml, 10 5.83 LD 50 / ml.

[0070] Table 1 Death of suckling mice challenged with AKAV / JL / 2022 virus

[0071]

[0072] Under sterile conditions, brain tissues of dead suckling mice after inoculation with viruses of different generations and dilutions were taken for PCR testing. The results showed that all were PCR positive, indicating that the AKAV / JL / 2022 isolate has strong virulence. After 15 generations of passaging in MDBK cells, the virulence changed little. It can be used as a test strain for Akabane disease vaccine and for the preparation of other diagnostic reagents.

[0073] Example 2 Preparation of inactivated virus vaccine

[0074] 2.1 Preparation of Akabane virus seed

[0075] Take the MDBK adherent cells grown to a good monolayer, discard the original culture medium, add the AKAV / JL / 2022 strain F9 virus solution at a ratio of 0.5%-2% (v / v), and culture at 37°C. When the cell lesion rate is ≧80%, harvest the virus solution and freeze-thaw it three times to obtain the basic virus. This basic virus seed is serially diluted 10 times with DMEM culture medium, and 10 2 -10 6A total of 5 dilutions were used to inoculate the MDBK cells grown into a monolayer in a 96-well plate. Each dilution was inoculated into 4 wells, and 100ul of dilution solution was added to each well, followed by 100ul of DMEM. Negative and positive controls were also set up. The cells were cultured in a 37°C, 5% CO2 incubator for 5 days, and the cell lesions were observed under a microscope. The TCID was calculated according to the Reed-Muench method. 50 . It is calculated that the virus content of this strain is not less than 10 6 TCID 50 / ml.

[0076] 2.2 Purity test of virus solution for seedling preparation

[0077] The test was carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia", and the results showed that the basic toxin species was not contaminated with bacteria, mycoplasma and exogenous viruses.

[0078] 2.3 Inactivation of virus solution for vaccine preparation

[0079] Use 0.25‰ of β-propiolactone to inactivate the virus at 4℃ for 24h on a shaker with a speed of 80-100r / min, and inactivate it again at room temperature for 24 hours to hydrolyze the residual β-propiolactone to obtain an inactivated virus solution. After diluting the inactivated virus solution with DMEM culture medium, inoculate it into well-growing MDBK adherent cells at a ratio of 10% (V / V) of the culture medium, and culture it in a 37℃, 5% CO2 incubator for 5 days, observe it day by day, freeze and thaw the culture at -80℃ for 3 times, and blindly propagate it for 3 generations according to the above method, and observe the CPE of each generation of culture. The results showed that no CPE was produced, and it was determined that the inactivation was complete.

[0080] 2.4 Vaccine preparation

[0081] (1) Preparation of aqueous phase: Heat the inactivated virus solution to 31±1°C for later use.

[0082] (2) Preparation of oil phase: Use oil adjuvant and preheat to 31±1°C for use.

[0083] (3) Emulsification: The oil phase and the water phase are mixed in a mass ratio of adjuvant: water phase = 1:1. The specific operation is: feed the adjuvant into the emulsification tank, and under stirring conditions, slowly feed the water phase into the emulsification tank under positive pressure, stir at 30-33°C for 30 minutes until the water phase and the adjuvant are fully mixed and emulsified into a two-phase oil emulsion vaccine. Then, quantitatively package, seal with a cap, label, and store at 2-8°C.

[0084] 2.5 Vaccine Product Testing

[0085] (1) Appearance: Milky white slightly viscous emulsion.

[0086] (2) Dosage form: water-in-oil-in-water type.

[0087] (3) Stability: Pipette 10 ml of the vaccine into a centrifuge tube and centrifuge at 3000 r / min for 15 minutes. No more than 0.5 ml of water phase should be precipitated at the bottom of the tube.

[0088] (4) Viscosity: Tested in accordance with the current Appendix of the Chinese Pharmacopoeia of Veterinary Medicine and in compliance with the regulations.

[0089] (5) Filling quantity inspection: Inspection shall be carried out in accordance with the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine and shall comply with the regulations.

[0090] (6) Sterility test: Test according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine, sterile growth.

[0091] (7) Safety inspection:

[0092] Two guinea pigs weighing 350-450g were injected subcutaneously with 2ml of the vaccine, and five mice weighing 18-22g were injected subcutaneously with 0.5ml of the vaccine. The animals were observed daily for 7 days, and no deaths or obvious local or systemic adverse reactions due to the injection of the vaccine occurred.

[0093] 2.6 Neutralizing antibody titer detection

[0094] Eight healthy female guinea pigs weighing 350-450g (Akabane virus neutralizing antibody titer not higher than 1:4) were selected and randomly divided into two groups. The first group of four guinea pigs were injected with 1.0ml of vaccine into the muscle of each leg as the immunization group; the second group of four guinea pigs were injected with 1.0ml of DMEM into each leg as the control group; 21 days later, the immunization was strengthened in the same way, and 21 days after the second immunization, the heart blood was collected, the serum was separated, and the titer level of Akabane virus neutralizing antibody was determined. The results are shown in Table 2. The results show that the titer level of anti-Akabane virus neutralizing antibody in the serum of the four guinea pigs in the immunization group was not less than 1:128, while the titer of neutralizing antibody in the control group was not higher than 1:2.

[0095] Table 2 Guinea pig serum neutralizing antibody titer test results

[0096]

[0097]

[0098] The above results show that after immunizing guinea pigs with the inactivated vaccine prepared using the Akabane disease virus strain provided by the present invention, the guinea pigs can be induced to produce high-titer anti-Akabane disease neutralizing antibodies (≥128). Therefore, the inactivated vaccine provided by the present invention can effectively protect guinea pigs from infection with Akabane disease virus for a long time.

[0099] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Akabane disease virus, characterized in that: It is Akabane virus AKAV / JL / 2022, and its preservation number is CGMCC No.45375.

2. Use of the Akabane disease virus strain according to claim 1 in the preparation of a drug or reagent, wherein the drug or reagent is used to treat, prevent or diagnose Akabane disease.

3. A product, characterized in that: The product comprises the Akabane disease virus strain according to claim 1; the product is any one selected from medicine, feed or feed additive.

4. A vaccine, characterized in that It contains the Akabane disease virus strain or its culture as described in claim 1.

5. The vaccine according to claim 4, characterized in that The vaccine is an inactivated vaccine.

6. The vaccine according to claim 4 or 5, characterized in that The titer of the Akabane disease virus strain or its culture is 10 6.0 TCID 50 / mL or above.

7. The vaccine according to claim 4 or 5, characterized in that After vaccination with the vaccine, the anti-Akabane virus neutralizing antibody titer level is not less than 1:

128.

8. The vaccine according to claim 6, characterized in that After vaccination with the vaccine, the anti-Akabane virus neutralizing antibody titer level is not less than 1:

128.

9. The vaccine according to claim 4, 5 or 8, characterized in that The vaccine also includes an adjuvant.

10. The vaccine according to claim 6, characterized in that The vaccine also includes an adjuvant.

11. The vaccine according to claim 7, characterized in that The vaccine also includes an adjuvant.

12. The method for preparing the vaccine according to any one of claims 9 to 11, characterized in that: include: The Akabane disease virus strain of claim 1 is inoculated into cells for cultivation to obtain a virus liquid culture; the virus liquid culture is then inactivated and mixed with an adjuvant, and then emulsified to obtain the product.

13. Use of the vaccine according to any one of claims 4 to 11, or the method for preparing the vaccine according to claim 12, in preparing a drug or reagent for diagnosing, preventing, or treating Akabane disease.

Images