CN118325848A - Akabane virus strain and application thereof - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
The invention relates to the technical field of biological products for animals, in particular to an akabane virus strain and application thereof. The akabane virus strain is akabane virus AKAV/JL/2022, and the preservation number is CGMCC No.45375. The akabane virus strain provided by the invention has the advantages of good passage stability, strong pathogenicity and good immunogenicity, can induce organisms to generate high-titer neutralizing antibodies, can be used for treating epidemic akabane after being prepared into vaccines, provides important medical value for prevention, control and treatment of akabane in pasture areas, and has profound and great practical application significance.
Description
Technical Field
The invention relates to the technical field of biological products for animals, in particular to an akabane virus strain and application thereof.
Background
Akabane (Akabane disease) is a viral infectious disease caused by akabane virus (Akabane disease virus, AKAV), also known as acariasis. The virus is mainly transmitted mechanically by blood sucking insect (such as mosquito and culicoides) medium, and the akabane virus can also be transmitted vertically by matrix. Generally, AKAV mainly causes ruminant reproductive disorders and congenital malformations such as cattle, sheep and the like. Leading the sick pregnant female animals to have the symptoms of premature birth, abortion, stillbirth, abortion fetal joint bending, abnormal body shape, cerebral defect and the like. The virus infects adult bulls, sheep or infects non-pregnant cows and sheep, often appearing as a recessive infection, with the infected animals carrying the virus but without obvious clinical symptoms. Akabane affects fetal development, and in early stage of infection, abortion is more likely to occur when the gestational age is smaller, and premature birth is more likely to occur when the gestational age is larger; calves in the middle of infection often suffer from dystocia due to bending, stiffness and deformity of the joints of the limbs of the calves, and even calves with parturition are difficult to stand by themselves; infected cows in the late gestation period, calves born are clever and alert, but cannot stand, and ataxia and muscular atrophy are caused. When sheep are infected, joint bending and neurological symptoms generally occur simultaneously.
Akabane virus belongs to the order bunyaviridae (Bunya virales) of the genus bunyaviridae (Peribunya viridae) of the genus bunyaviridae (Orthobunya virus). Viruses are transmitted by arthropods, some of which pose a major threat to human health. However, no report on akabane virus infection is currently available. AKAV the genome is a single-stranded negative strand RNA, consisting of L, M, S RNA fragments. Of the 3 RNA fragments, the M fragment encoding glycoprotein that induces neutralizing antibodies and hemagglutination inhibition antibodies is the most variable in the isolate, and the S fragment encoding nucleocapsid protein and non-structural protein is highly conserved. Current studies indicate that AKAV have only 1 serotype. Based on the S fragment sequence, AKAV isolates were divided into 5 gene groups (I-V), where type I gene groups in turn divided the subgroups Ia and Ib, ia being more common. AKAV virus particles are spherical, have a diameter of 90-100 nm, and comprise a capsule and a fiber.
Akabane has no effective treatment scheme, because calf is malformed in limbs and unstable in standing when born, and no treatment is available at all. Calves which can eat milk generally cannot die, but the growth speed is slower, a large amount of manpower and material resources are consumed, and the economic value is low. If the calf symptoms are severe, dysphagia manifests itself, and eventually dies. Therefore, once cattle and sheep are infected, the animals infected are usually killed. On the other hand, since adult male animals and non-pregnant female animals infected by akabane virus are often shown as recessive infections, no obvious clinical symptoms are caused, and the prevention and control difficulty of akabane disease is determined by recessive infections and virus variations possibly existing. Therefore, the developed effective akabane vaccine has great significance for preventing and treating akabane disease.
Disclosure of Invention
In a first aspect, the invention provides an akabane virus strain which is akabane virus AKAV/JL/2022 and has a preservation number of CGMCC No.45375.
The strain is a akabane virus epidemic strain obtained by plaque clone purification, separation and culture, can be stably passaged in cells, has strong pathogenicity and good immunogenicity, can induce organisms to generate high-titer neutralizing antibodies, and provides a foundation for effective vaccine preparation and akabane prevention and control.
The strain is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms at 2022, 12 and 25. Deposit unit address: the institute of microorganisms at national academy of sciences of China, national academy of sciences, no. 1, north Star West way, no. 3, chat.Chao, beijing, city; postal code: 100101, classification naming: the akabane virus has a preservation number of CGMCC No.45375.
The primary strain of akabane virus of the invention is isolated from fetal membranes collected from akabane positive cattle.
The method for separating AKAV/JL/2022 strain comprises the following steps:
(1) And (3) treating the disease materials: preparing the placenta of aborted cattle with akabane symptom into suspension by using DMEM, grinding, freeze thawing, centrifuging, filtering the supernatant, and adding double-antibody virus suspension;
(2) And (3) separating and screening: taking MDBK adherent cells fully paved with a monolayer, discarding the original culture solution, adding the obtained virus suspension, placing the virus suspension in a 37 ℃ and 5% CO 2 incubator to adsorb the discarded inoculum, and adding a DMEM culture medium containing the green and the streptomycin for culture. Observing whether the cells generate pathological CPE or not every day, if no pathological changes exist, collecting culture solution for 3 times after freezing and thawing on the 5 th day, and continuing to subculture, and if the cells generate pathological changes, collecting the virus solution to obtain primary virus strains;
Wherein, the judgment standard of the pathological changes of the cells is as follows: the cytopathic effect is more than 80%, typical cell enlargement and rounding phenomena are seen in early stage of cytopathic effect, the refraction is enhanced, and then local cells begin to fuse; late cells begin to appear as large-area fusion, disruption, and finally fused cell necrosis and shedding;
(3) Plaque clone purification: f1 strain virus liquid is subjected to 10-time gradient dilution to 10 -1～10-5 times by using DMEM, then culture liquid in a six-hole plate of MDBK cells growing into a compact single layer is discarded, after the culture liquid is washed for 2 times by using maintenance liquid, 400ul of virus liquid with different dilution factors is added into each hole, the virus liquid is placed into an incubator to be adsorbed for 1 hour, the liquid is discarded, a first layer of nutrient agar with the thickness of about 3mm is paved, the nutrient agar is uniformly mixed with 2% low-melting-point agarose and 2 XDMEM in equal volume, after standing at room temperature for 30min, after the culture is performed for 3 days in an inversion mode under the condition of 37 ℃ and 5% CO 2, a second layer of nutrient agar is paved when the cells are diseased, after standing at room temperature for 30min, the culture is performed in an inversion mode under the condition of 5% CO 2, and the growth condition of plaques is observed and recorded every day;
After plaque is formed, small and isolated plaques in the cells are selected, the small and isolated plaques are sucked out by a 10 mu l gun head, the small and isolated plaques are placed into 1.0ml of serum-free DMEM together with agarose, the freeze thawing is repeated for 3 times, the virus is fully released, new blank MDBK cells are inoculated for virus proliferation, the cloning is repeated for three times, the plaques are randomly selected for subculture, and strains with stable passage and high virus content are selected.
The primary akabane virus strain of the present invention produces a distinct lesion in the first-generation visible cells. The S gene of the AKAV/JL/2022 strain is separated by RT-PCR method, and the identified strain is different from CH-01 (accession number: MW 194117.1) and GXDH (accession number: MH 174977.1) which are published in China 2020 and have the homology of 98.5% at most with AKAV/S/ISR/-170/18 (accession number: MW 822048.1) of israel published in 2018, and is the subtype of gene Ib.
In the plaque clone purification process, a large number of plaque samples are screened for passage, meanwhile, the pathological change degree of cells infected by different strains is observed in the passage process, and the result shows that one part of strains cannot be passed for a long time, the passage stability is poor, the pathological change degree of cells infected by the other part of strains is poor, and the toxicity is weak. After a large number of screening, only two strains with good passage stability and high virulence are found, the virus content of the two passage strains is respectively measured, one strain with the highest virus content is selected, and the preserved strain AKAV/JL/2022 with strong pathogenicity and good immunogenicity is obtained through immune test verification.
In a second aspect, the present invention provides the use of an akabane virus strain as described above in the manufacture of a medicament or agent for the treatment, prophylaxis or diagnosis of akabane.
In a third aspect, the present invention provides a product comprising said akabane virus strain; the product is any one selected from medicines, feeds or feed additives.
In a fourth aspect, the present invention provides a vaccine comprising said akabane virus strain or a culture thereof.
As a preferred embodiment of the invention, the vaccine is an inactivated vaccine.
As a preferred embodiment of the invention, the vaccine is a veterinary vaccine.
As a preferred embodiment of the present invention, the titer of the akabane virus strain or a culture thereof in the vaccine is 10 6.0TCID50/mL or more.
As a preferred embodiment of the present invention, the neutralizing antibody titer level against akabane virus after the vaccination is not less than 1:128.
As a preferred embodiment of the invention, an adjuvant is also included in the vaccine.
Preferably, the adjuvant is ISA 201VG adjuvant or oil adjuvant.
In a fifth aspect, the invention provides a method of preparing a vaccine according to any one of the embodiments above, comprising:
inoculating the akabane virus strain into cells for culturing to prepare a virus liquid culture; and mixing the virus liquid culture after inactivation with an adjuvant, and emulsifying to obtain the virus liquid.
Preferably, the virus culture is performed using MDBK cells.
Preferably, the virus liquid culture is harvested when the culture reaches 80-90% of cytopathy.
Preferably, beta-propiolactone is used for inactivation, more preferably 0.25% beta-propiolactone.
Preferably, the step of inactivating comprises:
Mixing the virus liquid culture with beta-propiolactone, inactivating at 80-100 r/min and 0-8 deg.c, and hydrolyzing in 35-40 deg.c water bath.
Preferably, the inactivation is performed for more than 24 hours; and/or hydrolyzing for more than 24 hours.
Preferably, the weight ratio of the inactivated virus liquid to the adjuvant is 1: mixing 0.5-1.5, emulsifying at 30-33 deg.C.
Preferably, the time of emulsification is 30min or more.
In a sixth aspect, the invention provides the use of a vaccine or a method of preparing a vaccine according to any of the embodiments described above, in the manufacture of a medicament or agent for diagnosing, preventing, or treating akabane.
Compared with the prior art, the invention has the beneficial effects that:
The akabane virus strain provided by the invention has the advantages of good passage stability, strong pathogenicity and good immunogenicity, can induce organisms to generate high-titer neutralizing antibodies, can specifically prevent akabane after being prepared into vaccines, provides important medical value for prevention, control and treatment of akabane in pasture areas, and has profound and important practical application significance.
Drawings
FIG. 1 is an optical micrograph of a AKAV/JL/2022 primary strain provided by the invention infecting MDBK cells; a is a negative control; b is AKAV/JL/2022 primary strain infected MDBK cells.
FIG. 2 is a plot of the F10 generation growth of AKAV/JL/2022 strain provided by the present invention.
FIG. 3 is a AKAV/JL/2022 strain electron micrograph provided by the invention.
FIG. 4 is a diagram showing strain information of the conventional akabane virus.
FIG. 5 is a diagram showing the results of a AKAV/JL/2022 strain S gene sequencing homology analysis.
FIG. 6 is a diagram showing the results of a phylogenetic tree analysis of AKAV/JL/2022 strain S gene sequencing.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The methods used in the examples below are conventional methods unless otherwise specified, and specific steps can be found in: molecular cloning experiment guide "("Molecular Cloning:A Laboratory Manual"Sambrook,J.,Russell,DavidW.,Molecular Cloning:A Laboratory Manual,3rd edition,2001,NY,Cold SpringHarbor).
The various biomaterials described in the examples were obtained by merely providing an experimental route for achieving the objectives of the specific disclosure and should not be construed as limiting the source of biomaterials of the present invention. In fact, the source of the biological material used is broad, and any biological material that is available without violating law and ethics may be used instead as suggested in the examples.
EXAMPLE 1 isolation and identification of strains
1.1 Cells, disease Material
MDBK cells were obtained by commercial means and the disease was harvested in 2022 from the placenta of Ji Linsheng-positive cattle aborted in the cattle farm.
1.2 Treatment of disease Material
Before inoculating cells, the placenta of the aborted cattle, which is detected to be AKAV positive by RT-PCR, is treated with DMEM according to a volume ratio of 1:5 preparing a suspension, grinding by a grinder, freezing and thawing once at-80 ℃, centrifuging for 30min at-4 ℃ at 12000r/min, filtering the supernatant by a filter with the thickness of 0.22 mu m to obtain a virus suspension, adding the double antibody (100 mu g streptomycin/ml and 100U penicillin/ml), and refrigerating in a refrigerator with the temperature of-4 ℃ for later use. When separating viruses, the placenta suspension is quickly frozen at-80 ℃, so that the loss of live viruses in the freezing process is reduced while the release of the viruses from the placenta is quickened, and the probability of successful virus separation is improved.
1.3 Virus isolation screening
Selecting MDBK adherent cells (25 cm 2) fully paved with a monolayer, discarding the original culture solution, washing the cells once by PBS, adding 1ml of the obtained virus suspension, placing the virus suspension in a 37 ℃ incubator with 5% CO 2 for adsorption for 1h, discarding the inoculum, and adding 10ml of DMEM culture medium containing double antibodies (100 mug streptomycin/ml and 100U penicillin/ml) for culture. The cells were observed daily for the presence of diseased CPE, and if no disease occurred, the culture medium was collected 3 times after freeze thawing on day 5 and continued blind transfer.
Results the AKAV/JL/2022 primary strain isolated produced a clear lesion in the first generation of visible cells. As shown in FIG. 1, typical cell enlargement and rounding phenomena can be seen in early stage of lesions, the refraction is enhanced, and then local cells begin to fuse; late cells begin to appear as large-area fusion, disruption, and finally necrosis and shedding of the fused cells. When cytopathy reaches more than 80%, freezing and thawing for 3 times, collecting virus liquid, namely F1 generation virus, and carrying out domestication culture on the F1 generation virus on MDBK adherent cells.
1.4 Plaque purification of viruses
(1) Performing gradient dilution on akabane virus F1 generation virus liquid by using DMEM (DMEM) for 10 times to 10 -1～10-5 times, discarding culture solution in a six-hole plate of MDBK cells growing into a compact single layer, washing 2 times by using a maintenance solution, adding 400ul of virus liquid with different dilution factors into each hole, placing the mixture into an incubator to absorb the virus liquid for 1 hour, discarding the liquid, adding a first layer of nutrient agar with the thickness of about 3mm, uniformly mixing 2% low-melting-point agarose with 2 XDMEM in equal volume, standing at room temperature for 30min, then inversely culturing the mixture in a CO 2 incubator at 37 ℃ under the condition of 5% CO 2, adding a second layer of nutrient agar when lesions appear on the cells, standing at room temperature for 30min, inversely culturing the cells at 37 ℃ under the condition of 5% CO 2 in a dark place, and observing and recording the growth condition of plaques every day;
(2) After plaque is formed, small and isolated plaques in the cells are selected, the small and isolated plaques are sucked out by a 10 mu l gun head, the small and isolated plaques are placed into 1.0ml of serum-free DMEM together with agarose, the repeated freeze thawing is carried out for 3 times, viruses are fully released, new blank MDBK cells are inoculated for virus proliferation, the repeated cloning is carried out for three times, a large number of plaques of fourth-generation clones are randomly selected for subculture, meanwhile, the pathological changes of cells infected by different strains are observed in the passaging process, and as a result, the fact that one part of strains cannot be passaged for a long time and the pathological changes of cells infected by the other part of strains are poor is found. After screening, only two strains capable of long-term passage through MDBK cells are found, after continuous passage to the 20 th generation, the virus content of the passage is respectively measured, and one strain with the highest virus content is selected, namely the purified akabane virus AKAV/JL/2022.
1.5 Determination of viral titers
The cell culture medium was discarded from the 96-well cell culture plate of MDBK cells grown to well confluency, and washed twice with PBS. Serial passage of virus fluid was serially diluted 10-fold with serum-free DMEM medium, 10 -2-10-6 dilutions of virus fluid were inoculated into 96-well cell culture plates, each dilution was inoculated into 4 wells, and positive and negative controls were established simultaneously. The plates were incubated at 37℃in a 5% CO 2 incubator for 3-5 days, CPE was observed daily and recorded. Viral titers were calculated according to the Reed-Muench method. Experimental results show that the titers of the 3 rd generation, the 4 th generation, the 5 th generation, the 7 th generation, the 10 th generation, the 12 th generation, the 15 th generation, the 17 th generation and the 20 th generation of the two serial generation of the plaque strains are 104.5TCID50/mL、105.5TCID50/mL、106.0TCID50/mL、105.5TCID50/mL、106.0TCID50/mL、105.5TCID50/mL、106.0TCID50/mL、106.0TCID50/mL、105.75TCID50/mL th generation and 104.5TCID50/mL、104.25TCID50/mL、104.75TCID50/mL、105TCID50/mL、104.0TCID50/mL、105TCID50/mL、104.5TCID50/mL、104.75TCID50/mL、104.55TCID50/mL. th generation respectively, and the results show that one of the two serial generation of the plaque strains of the akabane virus AKAV/JL/2022 can be stably passaged on MDBK cells, and virus liquid with high titer can be obtained and can be used as a candidate strain of akabane vaccine.
1.6 Determination of the Virus growth Curve
To study the growth characteristics of the virus, AKAV/JL/2022 virus was inoculated to MDBK cells to draw a growth curve, the isolated F10 virus solution was inoculated to MDBK cells at 1% of the virus amount, the inoculum was discarded after adsorption in a 5% co 2 incubator at 37 ℃ for 1h, the maintenance solution was added to continue the culture, samples were taken every 8 hours until 48h after inoculation, virus solution was collected at different time points to determine TCID 50, and the growth curve of the virus on MDBK cells was drawn according to the determination results, as shown in fig. 2.
1.7 Electron microscope observations
Taking the akabane virus AKAV/JL/2022 virus liquid, centrifuging at 10000r/min for 30min, taking supernatant fluid 40000r/min for 5 h, discarding the supernatant fluid, dissolving precipitate with 0.5ml deionized water, carrying out negative dyeing for 30min with 2% sodium phosphotungstate, naturally drying at room temperature, and observing under a transmission electron microscope. As shown in FIG. 3, the isolate is approximately round, has a morphology size of 100nm, has a capsule membrane and a fiber, and accords with the characteristics of akabane virus.
1.8PCR identification and sequence analysis
The experiment utilizes a specific detection primer in GENETIC ANALYSIS of Akabane virus isolates from cattle in Korea published by Han Guoqing Ji Dao an yang national veterinary research and quarantine agency 2010 on veterinary microorganisms, wherein the primer is designed according to a gene sequence for encoding S protein of akabane virus, and the size of a target gene fragment is 794bp.
And carrying out PCR identification on the AKAV/JL/2022 virus strain obtained by separation by utilizing the specific detection primer, and obviously obtaining a target band with the size of about 794bp of a gene fragment in the PCR product by an electrophoresis result of PCR identification on the virus strain. The product is sent to Shanghai worker company for sequencing and splicing, homology comparison is carried out on the result and 13 akabane virus gene sequences published in GenBank database (shown in figure 4), and a phylogenetic tree is drawn, as shown in figures 5 and 6. As a result, it was found that the homology of the isolate AKAV/JL/2022 to AKAV/S/ISR/-170/18 (accession number: MW 822048.1) was 98.5% at the highest, the homology to AKAV/S/ISR/-01 (accession number: AY 378155.1) was 97.4%, and the homology to CH-01 (accession number: MW 194117.1) and GXDH01 (accession number: MH 174977.1) of subtype 1a, respectively, were 94.7% and 95.3%. Homology to NT-14 (accession number: AF 529883.1) and Iriki (accession number: AB 297855.1) of the two Ia subtypes was 96.3% and 96.4%, respectively. Homology to Okayama2004 (AB 289320), obe-1 (AF 034942.1) of genotype II was 94.4% and 94.9%. Homology to 93FMX (FJ 498797), AK7 (FJ 498795.1) of genotype II was 95.3% and 95.2%. The homology with B8935 (MH 734940.1) of the gene III is 92.2%, and the homology with MP496 (AB 297885.1) of the gene IV is 83.9% at the minimum. The genetic evolutionary tree also showed the same branches as AKAV/S/ISR/-170/18 (accession number: MW 822048.1) and AKAV/S/ISR/-01 (accession number: AY 378155.1), indicating that the isolate was of subtype Ib gene.
1.9 Experiment of virus in the case of Dairy mice
The AKAV th, 10 th and 15 th generation (10 6TCID50/ml) virus solutions of AKAV th, JL/2022 isolated strains are respectively centrifuged for 15 minutes at 10000rpm, then serial dilutions of 10 times are carried out by serum-free DMEM culture medium, the original-10 -7 dilution virus solutions are respectively inoculated into 1-3 days of suckling mice, 4 dilutions are inoculated into each of the mice, 10ul of each of the mice is inoculated into the brain, and meanwhile, the empty DMEM is used as a negative control. The mice were observed daily for 14 days. The results showed that the mice developed neurological symptoms, convulsions, limb hardness and death after day 3, and the half-mortality after 14 days was 10 6LD50/ml,106LD50/ml,105.83LD50/ml according to Reed-Muench method as shown in Table 1, and the half-mortality of F5, F10, and F15 generations was 10.
TABLE 1 AKAV/JL/2022 strain case of death of challenge mice
Under the aseptic condition, the brain tissues of the dead mice inoculated with different dilutions of viruses of different generations are subjected to PCR detection, and the results show that the samples are positive in PCR, which shows that AKAV/JL/2022 isolate has strong toxicity, and the toxicity change is small after 15 generations of MDBK cells passage, so that the samples can be used as test strains of akabane vaccines and can be used for preparing other diagnostic reagents.
Example 2 preparation of viral inactivated vaccine
2.1 Preparation of akabane virus seed Virus
Taking MDBK adherent cells growing to a good monolayer, discarding the original culture solution, adding AKAV/JL/2022 strain F9 virus solution according to the proportion of 0.5% -2% (v/v), culturing at 37 ℃, when cytopathy is larger than or equal to 80%, harvesting the virus solution, freezing and thawing for 3 times to obtain basic seed virus, serial diluting the basic seed virus with DMEM culture solution 10 times, taking 10 2-106 for 5 dilutions, inoculating MDBK cells growing to a monolayer into 96-well plates, inoculating 4 wells respectively for each dilution, adding 100ul of diluent into each well, then supplementing 100ul of DMEM, and setting negative and positive controls. The cells were incubated at 37℃in a 5% CO 2 incubator for 5 days, and the cytopathic effect was observed under a microscope, and TCID 50 was calculated according to the Reed-Muench method. The virus content of the virus seed is calculated to be not less than 10 6TCID50/ml.
2.2 Purity test of virus liquid for seedling preparation
The test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the result shows that the basic virus seed has no bacterial, mycoplasma and exogenous virus pollution.
2.3 Inactivation of virus liquid for seedling preparation
Inactivating the beta-propiolactone for 24 hours at 4 ℃ on a shaking table with the rotating speed of 80-100r/min by using 0.25 per mill, and inactivating the beta-propiolactone again for 24 hours at room temperature to hydrolyze residual beta-propiolactone, thereby obtaining the inactivated virus liquid. Diluting the inactivated virus liquid with DMEM culture solution, inoculating the diluted virus liquid into MDBK adherent cells growing well according to the proportion of 10% (V/V) of the culture solution, culturing in a culture box containing 5% CO 2 at 37 ℃ for 5 days, observing day by day, freezing and thawing the culture at-80 ℃ for 3 times, carrying out blind transfer for 3 generations according to the method, and observing CPE of each culture. The results showed no CPE production and complete inactivation.
2.4 Vaccine preparation
(1) Preparation of an aqueous phase: heating the inactivated virus liquid to 31+/-1 ℃ for standby.
(2) Preparation of an oil phase: and (3) adopting an oil adjuvant, and preheating to 31+/-1 ℃ for standby.
(3) Emulsification: mixing the oil phase and the water phase at a ratio of adjuvant to water phase=1:1. The specific operation is as follows: feeding the adjuvant into an emulsifying cylinder, slowly feeding the water phase into the emulsifying cylinder under positive pressure under stirring, stirring at 30-33deg.C for 30min until the water phase and adjuvant are fully mixed, and emulsifying to obtain the biphasic oil emulsion vaccine. And then quantitatively split charging, capping, sealing, labeling, and storing at 2-8 ℃.
2.5 Vaccine product detection
(1) Appearance: milky white slightly viscous emulsion.
(2) Dosage form: water-in-oil-in-water.
(3) Stability: 10ml of the extracted vaccine is added into a centrifuge tube, and the mixture is centrifuged at 3000r/min for 15 minutes, and the water phase separated from the bottom of the tube is not more than 0.5ml.
(4) Viscosity: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and meets the regulations.
(5) And (3) checking the loading: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and meets the regulations.
(6) And (3) sterile inspection: the test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the sterile growth is carried out.
(7) And (3) safety inspection:
2 guinea pigs with the weight of 350-450 g are used for subcutaneous injection of 2ml of vaccine; 5 mice weighing 18-22 g were used, each injected subcutaneously with 0.5ml of vaccine. The results were observed for 7 days daily, and no death or obvious local reaction or systemic adverse reaction caused by vaccine injection occurred.
2.6 Neutralizing antibody titre detection
8 Healthy female guinea pigs (akabane virus neutralizing antibody titer is not higher than 1:4) with the weight of 350-450 g are selected and randomly divided into 2 groups. The first group of 4 guinea pigs was given 1.0ml of intramuscular vaccine for each leg, as an immunized group; a second group of 4 guinea pigs injected with 1.0ml DMEM per leg as a control group; immunization was enhanced in the same manner after 21 days, heart was collected 21 days after the double immunization, serum was separated, and the level of neutralizing antibody against akabane virus was measured, and the results are shown in table 2. As a result, it was found that the average neutralizing antibody titer against akabane virus in the serum of 4 guinea pigs in the immunized group was not lower than 1:128, while the neutralizing antibody titer in the control group was not higher than 1:2.
TABLE 2 results of guinea pig serum neutralizing antibody titer detection
The results show that after the akabane virus strain provided by the invention is used for preparing an inactivated vaccine to immunize guinea pigs, the high-titer neutralizing antibody (more than or equal to 128) for resisting akabane can be induced to be generated by the guinea pigs, so that the inactivated vaccine provided by the invention can effectively protect the guinea pigs from being infected by akabane viruses for a long time.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The akabane virus strain is characterized in that the akabane virus strain is akabane virus AKAV/JL/2022, and the preservation number of the akabane virus strain is CGMCC No.45375.
2. Use of the akabane virus strain of claim 1 in the preparation of a medicament or agent for the treatment, prevention or diagnosis of akabane.
3. A product comprising the akabane virus strain of claim 1; the product is any one selected from medicines, feeds or feed additives.
4. A vaccine comprising the akabane virus strain of claim 1 or a culture thereof.
5. The vaccine of claim 4, wherein the vaccine is an inactivated vaccine.
6. The vaccine of claim 4 or 5, wherein the akabane virus strain or a culture thereof has a titer of 10 6.0TCID50/mL or more.
7. The vaccine of any one of claims 4-6, wherein after vaccination the vaccine has an anti-akabane virus neutralising antibody titer level of no less than 1:128.
8. The vaccine according to any one of claims 4 to 7, further comprising an adjuvant.
9. A method of preparing a vaccine as claimed in claim 8, comprising:
inoculating the akabane virus strain of claim 1 into cells for culturing to prepare a virus liquid culture; and mixing the virus liquid culture after inactivation with an adjuvant, and emulsifying to obtain the virus liquid.
10. Use of the vaccine of any one of claims 4 to 8, or the method of preparing the vaccine of claim 9, for the preparation of a medicament or agent for diagnosing, preventing, or treating akabane disease.
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| JPH0811736B2 (en) * | 1986-08-07 | 1996-02-07 | 財団法人化学及血清療法研究所 | Cattle epidemic fever, Ibaraki disease, Akabane disease 3 types inactivated vaccine |
| KR100834014B1 (en) * | 2006-10-23 | 2008-06-02 | 대한민국 | Isolated strains of Akabane virus, Chuzan virus and Aino virus, and the method for preparing the viral vaccine by mixing the same |
| CN105969739B (en) * | 2016-07-05 | 2019-10-01 | 中国农业科学院特产研究所 | One plant of Akabane Disease virus vaccine strain and application thereof for being adapted to Vero cell culture |
| CN114989269B (en) * | 2022-06-30 | 2023-09-19 | 天康制药股份有限公司 | Bovine akabane immunogenicity antigen and vaccine |
| CN115141812B (en) * | 2022-08-05 | 2023-02-28 | 云南省畜牧兽医科学院 | Acremonium carbonum vaccine strain and application thereof |
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