Akabane disease virus strain and use thereof

Abstract

Provided are an Akabane disease virus strain and a use thereof. The Akabane disease virus strain is Akabane disease virus AKAV / JL / 2022, and the accession number thereof is CGMCC No. 45375. The Akabane

Description

AKABANE DISEASE VIRUS STRAIN AND APPLICATION THEREOF Technical Field The present invention relates to the technical field of veterinary biological products, and particularly to an Akabane disease virus strain and an application thereof. Background Akabane disease is a viral infectious disease caused by Akabane disease virus (AKAV). The virus is mainly mechanically transmitted by blood-sucking insects (such as Mosquitoes and Culicoides), and can also be transmitted vertically by a mother. Generally speaking, AKAV mainly causes reproductive disturbance and congenital malformation in ruminants such as cattle and sheep. AKAV also causes premature birth, abortion, stillbirth in diseased pregnant females, as well as arthrogryposis, abnormal body shapes, cerebral defects and other symptoms in aborted fetuses. Most of the adult bulls and rams or non-pregnant cows and ewes infected with the virus are manifested as recessive infection, and the infected animals carry the virus but have no obvious clinical symptoms. Akabane disease affects the development of fetus. When a cow in early stage of pregnancy is infected, the smaller the gestational age is, the more likely it is to cause abortion, and the larger the gestational age is, the more likely it is to cause premature birth; when a cow in middle stage of pregnancy is infected, dystocia will often occur, which is caused by the arthrogryposis, rigidity and deformity of the limbs of a calf, and even bom naturally, the calf is difficult to stand on its own; when a cow in later stage of pregnancy is infected, the calf born is clever and alert, but unable to stand, and is complicated with ataxia and amyotrophy. When a sheep is infected, arthrogryposis and neurological symptoms usually occur at the same time. Akabane disease vims belongs to the genus of Orthobunya vims in the family of Peribunya viridae under the order of Bunya virales. The vims is mostly transmitted by arthropods, and some Bunya viruses will pose a significant threat to human health. However, no human infection with Akabane disease virus has been reported so far. AKAV genome is a single strand of negative stranded RNA, composed of three RNA fragments, i.e., fragments L, M and S. Among the three RNAfragments, fragment M which encodes the glycoprotein for inducing the production of a neutralizing antibody and a hemagglutination inhibiting antibody is most variable in isolates, and fragment S which encodes a nucleocapsid protein and a non-structural protein is highly conservative. The research at present shows that AKAV has only one serotype. Based on the sequence of fragment S, AKAV isolates are divided into five gene groups (I-V), among which, gene group I can be further divided into subgroups la and lb, and la is more common. An AKAV virion is spherical, is 90-100 nm in diameter, and has an envelope and spikes. No feasible and effective treatment for Akabane disease is available, because the calf is born with deformed limbs and astasia, and cannot be treated by any method. Generally, a calf that can suckle will not die, but the growth rate will be relatively slow, and a lot of human and material resources will be consumed, so the economic value is not high. If the calf has relatively serious symptoms and has dysphagia, the calf will eventually die. Therefore, once cattle and sheep are infected, the infected animals are most likely to be culled. On the other hand, adult male and non-pregnant female animals infected with Akabane virus are mostly manifested as recessive infection and have no obvious clinical symptoms, and virus variation may occur, so the prevention and control of Akabane disease are difficult due to the recessive infection and virus variation. Therefore, it is of great significance to develop an effective Akabane disease vaccine for the prevention and control of Akabane disease. Summary In a first aspect, the present invention provides an Akabane disease virus strain, which is an Akabane virus AKAV / JL / 2022, and the preservation number thereof is CGMCC No.45375. The strain is an epidemic Akabane disease virus strain obtained by plaque cloning, purification, isolation and cultivation, which can be passaged stably in cells, has strong pathogenicity and good immunogenicity, can induce an organism to produce a high-titer neutralizing antibody, and provides a basis for the preparation of an effective vaccine and the prevention and control of Akabane disease. The strain has been preserved at China General Microbiological Culture Collection Center (CGMCC) on December 25, 2022. The address of the preservation unit is: Institute of Microbiology, Chinese Academy of Sciences, Building 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the postcode is: 100101, the classification designation is: Akabane virus, and the preservation number is: CGMCC No.45375. The primary Akabane disease virus strain of the present invention is isolated from fetal membrane tissue collected from cattle positive for Akabane disease in Jilin province. A method for isolating the AKAV / JL / 2022 strain of the present invention comprises the following steps: (1) Pathological sample processing: collecting fetal membrane of aborted cattle with the symptoms of Akabane disease, preparing the fetal membrane into a suspension with DMEM, conducting centrifuging after grinding, freezing and thawing, filtering the supernatant and then adding penicillin-streptomycin solution to obtain a virus suspension. (2) Isolation and screening: taking MDBK adherent cells forming a confluent monolayer, discarding the original culture solution, adding the virus suspension obtained above, placing the mixture in an incubator at 37°Cand 5% CO2, discarding the inoculum after adsorption, and adding DMEM containing penicillin and streptomycin for cultivation. Observing the cells daily for cytopathic effect (CPE); if no CPE is observed, collecting the culture solution on the 5th day, and continuing with passage culture after freezing and thawing for 3 times; if any CPE is observed, collecting a virus liquid to obtain a primary vims strain; The criterion for judging CPE is: the degree of CPE is higher than 80%. In the early stage of CPE, typical cell enlargement and roundness can be observed, the refractivity is enhanced, and then some cells begin to fuse; in the late stage of CPE, the cells begin to show large areas of fusion and disruption, and finally the fused cells die and shed; (3) Plaque cloning and purification: using DMEM to dilute the vims liquid of Fl strain by a 10-fold gradient into 101 to 10’5, discarding the culture solution in a 6-well plate where MDBK cells grow into a confluent monolayer, rinsing with a maintenance medium for 2 times, adding 400 ul of virus liquid diluted to the above-mentioned different dilution factors into each well, placing the well plate in the incubator for adsorption, discarding the liquid after 1 hour, adding a first layer of nutrient agar with a thickness of about 3 mm, wherein the nutrient agar is prepared by uniformly mixing 2% low melting point agarose with 2xDMEM at an equal volume, standing at room temperature for 30 min, inverting the well plate for cultivation at 37°C and 5% CO2 for 3 days, adding a second layer of nutrient agar when CPE is observed, standing at room temperature for 30 min, inverting the well plate for cultivation away from light at 37°C and 5% CO2, and observing and recording the growth of plaques daily; After plaques are formed, selecting small and isolated plaques in the cell wells, sucking out the plaques with a 10 pl pipette tip, putting the plaques into 1.0 ml of serum-free DMEM together with agarose, conducting freezing and thawing repeatedly for 3 times to fully release the virus, inoculating the virus into new blank MDBK cells for virus proliferation, repeating this cloning process for three times, selecting plaques randomly for passage culture, and selecting a strain with stable passage and high virus content. It can be seen that the first generation of the primary Akabane disease virus strain of the present invention causes obvious CPE in the cells. The S-gene sequence of the isolated AKAV / JL / 2022 strain is identified by RT-PCR. As identified, the strain is different from strain CH-01 (login number: MW194117.1) and strain GXDH01 (login number: MH174977.1) of gene subtype la published by China in 2020, but has a highest homology of 98.5% with AKAV / S / ISR / -170 / 18 (login number: MW822048.1) published by Israel in 2018, which belongs to the same gene subtype lb. In the process of plaque cloning and purification, a large number of plaque samples are screened for passage according to the present invention, and the degree of CPE of cells infected with different strains is observed during passage. The results show that some strains cannot carry out longterm passage, and the passage stability thereof is poor; while the cells infected with other strains have a low degree of CPE, and the virulence thereof is low. After a lot of screening, only two strains with good passage stability and high virulence are found, the virus contents of the two strains are determined respectively, the strain with the highest virus content is selected, and the preserved strain AKAV / JL / 2022 with strong pathogenicity and good immunogenicity is obtained after verification by immunity test. In a second aspect, the present invention provides an application of the above-mentioned Akabane disease virus strain in preparation of drugs or reagents, wherein the drugs or reagents are used for treating, preventing or diagnosing Akabane disease. In a third aspect, the present invention provides a product, wherein the product contains the Akabane disease virus strain; and the product is a drug, a feed or a feed additive. In a fourth aspect, the present invention provides a vaccine, wherein the vaccine contains the Akabane disease virus strain or a culture thereof. As a preferred embodiment of the present invention, the vaccine is an inactivated vaccine. As a preferred embodiment of the present invention, the vaccine is a veterinary vaccine. As a preferred embodiment of the present invention, the titer of the Akabane disease virus strain in the vaccine or the culture thereof is higher than 10° TCID50 / mL. As a preferred embodiment of the present invention, after the vaccine is inoculated, the titer level of a neutralizing antibody against Akabane virus is not less than 1:128. As a preferred embodiment of the present invention, the vaccine also contains an adjuvant. Preferably, the adjuvant is an ISA201VG adjuvant or an oil adjuvant. In a fifth aspect, the present invention provides a method for preparing the vaccine in any one of the above-mentioned embodiments, comprising: Inoculating the Akabane disease virus strain into cells for cultivation to prepare a virus liquid culture; then inactivating the virus liquid culture, mixing the culture with the adjuvant, and emulsifying the mixture to prepare the vaccine. Preferably, MDBK cells are used for virus cultivation. Preferably, a virus liquid is harvested when the degree of CPE reaches 80%-90%. Preferably, P-propiolactone is used for inactivation, and 0.25%o P-propiolactone is more preferred. Preferably, the steps for inactivation include: Mixing the virus liquid culture with P-propiolactone, conducting inactivation at 80-100 r / min and 0-8°C, and then placing the mixture in a water bath at 35-40°C for hydrolysis. Preferably, inactivation is conducted for more than 24 hours; and / or hydrolysis is conducted for more than 24 hours. Preferably, the inactivated virus liquid culture is mixed with the adjuvant at a weight ratio of 1:0.5-1.5, and the mixture is emulsified at SO-33 °C to prepare the vaccine. Preferably, the time for emulsification is more than 30 min. In a sixth aspect, the present invention provides an application of the vaccine or the method for preparing the vaccine in any one of the above-mentioned embodiments in preparation of drugs or reagents used for diagnosing, preventing or treating Akabane disease. Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an Akabane disease virus strain which has good passage stability, strong pathogenicity and good immunogenicity, and can induce an organism to produce a high-titer neutralizing antibody. When prepared into a vaccine, the strain can be used for specifically preventing Akabane disease, which has an important medical value for the prevention, control and treatment of Akabane disease in pastoral areas, and has a far-reaching and great practical significance. Description of Drawings Fig. 1 shows optical microscope photographs of MDBK cells infected with a primary AKAV / JL / 2022 strain provided by the present invention; A shows a negative control; and B shows MDBK cells infected with a primary AKAV / JL / 2022 strain. Fig. 2 is a growth curve of a Fl0 generation of AKAV / JL / 2022 strain provided by the present invention. Fig. 3 is an electron microscope photograph of an AKAV / JL / 2022 strain provided by the present invention. Fig. 4 shows information of existing Akabane virus strains. Fig. 5 shows homology analysis results of S-gene sequence of an AKAV / JL / 2022 strain. Fig. 6 shows phylogenetic tree analysis results of S-gene sequence of an AKAV / JL / 2022 strain. Detailed Description To make a purpose, a technical solution and advantages of the present invention more clear, the technical solution in the present invention will be clearly and fully described below. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention. The methods used in the following embodiments are conventional methods unless otherwise specified. For specific steps, see: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). The way to obtain various biological materials described in the embodiments is only to provide a way to obtain experimental materials to achieve the purpose specifically disclosed, and shall not be understood as a limitation to the sources of biological materials of the present invention. In fact, the sources of biological materials used are extensive, and any biological material that can be obtained without violating laws and ethics can be for alternative use according to instructions in the embodiments. Embodiment 1: Strain isolation and identification 1.1 Cells and pathological sample MDBK cells were obtained commercially, and pathological sample was collected from fetal membrane of aborted cattle positive for AKAV in a cattle farm of Jilin province in 2022. 1.2 Pathological sample processing Before the cells were inoculated, the fetal membrane of aborted cattle tested positive for AKAV by RT-PCR was prepared into a suspension with DMEM at a volume ratio of 1:5, ground by a grinder, frozen and thawed at -80°C once, and centrifuged at 4°C and 12000 r / min for 30 min; the supernatant was filtered with a 0.22 pm filter to obtain a virus suspension; and the virus suspension was added with penicillin-streptomycin solution (streptomycin 100 pg / ml, penicillin 100 U / ml) and stored in a -4°C refrigerator for later use. In the process of virus isolation, the suspension of the fetal membrane was placed at -80°C quick-freezing, which accelerated the release of the virus from the fetal membrane, reduced the loss of live virus during freezing, and increased the probability of successful virus isolation. 1.3 Virus isolation and screening MDBK adherent cells (25 cm2) forming a confluent monolayer were taken, the original culture solution was discarded, the cells were washed once with PBS, 1 ml of the virus suspension obtained above was added, the mixture was placed in an incubator at 37°C and 5% CO2, the inoculum was discarded after adsorption for 1 hour, and 10 ml of DMEM containing penicillin-streptomycin solution (streptomycin 100 pg / ml, penicillin 100 U / ml) was added for cultivation. The cells were observed daily for cytopathic effect (CPE); if no CPE was observed, the culture solution was collected on the 5th day, and blind passage was continued after freezing and thawing for 3 times. It could be seen that the first generation of the isolated primary AKAV / JL / 2022 strain caused obvious CPE in the cells. As shown in Fig. 1, in the early stage of CPE, typical cell enlargement and roundness could be observed, the refractivity was enhanced, and then some cells began to fuse; in the late stage of CPE, the cells began to show large areas of fusion and disruption, and finally the fused cells died and shed. When the degree of CPE was higher than 80%, freezing and thawing were conducted for 3 times to harvest a virus liquid, i.e. Fl generation of virus, and the Fl generation of virus was passaged on the MDBK adherent cells for habituated culture. 1.4 Virus plaque purification (1) DMEM was used to dilute the virus liquid of Fl generation of Akabane virus by a 10-fold gradient into 10’1 to 10'5, the culture solution in a 6-well plate where MDBK cells grew into a confluent monolayer was discarded, the cells were rinsed with a maintenance medium for 2 times, 400 ul of virus liquid diluted to the above-mentioned different dilution factors was added into each well, the well plate was placed in the incubator for adsorption, the liquid was discarded after 1 hour, a first layer of nutrient agar with a thickness of about 3 mm was added, wherein the nutrient agar was prepared by uniformly mixing 2% low melting point agarose with 2xDMEM at an equal volume, the well plate was stood at room temperature for 30 min and inverted for cultivation in the CO2 incubator at 37°C and 5% CO2 for 3 days, a second layer of nutrient agar was added when CPE was observed, the well plate was stood at room temperature for 30 min and inverted for cultivation away from light at 37°C and 5% CO2, and the growth of plaques was observed and recorded daily; (2) After plaques were fonned, small and isolated plaques in the cell wells were selected, sucked out with a 10 pl pipette tip, put into 1.0 ml of serum-free DMEM together with agarose, frozen and thawed repeatedly for 3 times to fully release the virus; and the virus was inoculated into new blank MDBK cells for virus proliferation. This cloning process was repeated for three times; a large number of plaques from the fourth generation of clones were randomly selected for passage culture; and the degree of CPE of cells infected with different strains was observed during passage. The results showed that some strains couldn't carry out long-term passage, while the cells infected with other strains had a low degree of CPE. After screening, only two strains that could carry out long-term passage through MDBK cells were found, the virus contents of die two strains were determined respectively after continuous passage to the 20th generation, and the strain with the highest virus content was selected, i.e. the purified Akabane virus AKAV / JL / 2022 was obtained. 1.5 Determination of virus titer A 96-well cell culture plate where MDBK cells grew into a good confluent monolayer was taken, cell culture solution was discarded, and the cells were washed twice with PBS. Serum-free DMEM was used to respectively dilute the virus liquid after continuous passage by a 10-fold gradient, virus liquid with a dilution of 10'2-10'6 was inoculated respectively into the 96-well cell culture plate, 4 wells were inoculated for each dilution, and a positive control and a negative control were set up at the same time. The culture plate was placed in an incubator at 37°C and 5% CO2 for cultivation for 3-5 days, and CPE was observed and recorded daily. The virus titer was calculated by a Reed-Muench method. Experimental results showed that the virus titers of the 3rd, 4th, 5th, 7th, 10th, 12th, 15 th, 17th and 20th generations of two plaque strains after continuous passage were respectively 104 5 TCID50 / mL, 105 5 TCID50 / mL, 106 0 TCID50 / mL, 105 5 TCID50 / mL, 1060 TCID50 / mL, 105 5 TCID50 / mL, 1060 TCID50 / mL, 1060 TCID50 / mL, 10575 TCID50 / mL and 1045 TCID50 / mL, 10425 TCID50 / mL, IO4-75 TCID50 / mL, 105 TCID50 / mL, IO4-0 TCID50 / mL, 105 TCID50 / mL, 104 5 TCID50 / mL, 10475 TCID50 / mL, 10455 TCID50 / mL. The results showed that one of the Akabane vims plaque strains AKAV / JL / 2022 could be passaged stably in MDBK cells, could be used to obtain a vims liquid with a high titer, and could be used as a candidate strain for an Akabane disease vaccine. 1.6 Determination of vims growth curve In order to study the growth characteristics of the vims, AKAV / JL / 2022 vims was inoculated into MDBK cells to plot a growth curve. The vims liquid of isolated F10 generation was taken to inoculate the MDBK cells at a virus load of 1% and placed in an incubator at 37°Cand 5% CO2, the inoculum was discarded after adsorption for 1 hour, the maintenance medium was added to continue cultivation, samples were taken at 8-hour intervals until 48 hour after inoculation, the vims liquid was collected at different time points to determine TCID50, and the growth curve of the vims on MDBK cells was plotted according to the determination results, as shown in Fig. 2. 1.7 Observation by electron microscope The vims liquid of Akabane vims AKAV / JL / 2022 was taken and centrifuged at 10000 r / min for 30 minutes, the supernatant was taken and centrifuged at 40000 r / min for five hours, and then the supernatant was discarded; the precipitate was dissolved by 0.5 ml of deionized water, negatively stained with 2% sodium phosphotungstate for 30 minutes, dried naturally at room temperature, and observed under a transmission electron microscope. As shown in Fig. 3, it could be seen that the isolate was approximately round, with a morphological size of 100 nm, an envelope and spikes, which was consistent with the features of Akabane virus. 1.8 PCR identification and sequence analysis In this experiment, a specific detection primer mentioned in an article themed with Genetic analysis of Akabane virus isolates from cattle in Korea published by the National Veterinary Research and Quarantine Service of Anyang, Gyeonggi-do, South Korea in Veterinary Microbiology in 2010 was used. The primer was designed according to the gene sequence encoding the S-protein of Akabane virus, and the target gene fragment size was 794 bp. PCR identification of the isolated AKAV / JL / 2022 virus strain was conducted using the above-mentioned specific detection primer, and the electrophoresis results of the PCR identification of this strain showed that a target band with a gene fragment size of about 794 bp was obviously obtained in a PCR product. The product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, the results were spliced to be compared with the gene sequences of 13 Akabane virus strains from Japan, South Korea, Israel and other countries published in GenBank database (as shown in Fig. 4) for homology, and a phylogenetic tree was drawn, as shown in Fig. 5 and Fig. 6. The results showed that the isolate AKAV / JL / 2022 has a highest homology of 98.5% with AKAV / S / ISR / -170 / 18 (login number: MW822048.1) published by Israel in 2018, has a homology of 97.4% with AKAV / S / ISR / -01 (login number: AY378155.1) published by Israel in 2001, has a homology of 94.7% and 95.3% respectively with CH-01 (login number: MW194117.1) and GXDH01 (login number: MH 174977.1) of gene subtype la published by China in 2020, has a homology of 96.3% and 96.4% respectively with another two strains of subtype la, i.e. a Taiwan strain NT-14 (login number: AF529883.1) and a Japanese strain Iriki (login number: AB297855.1), has a homology of 94.4% and 94.9% respectively with Japanese strains Okayama2004 (AB289320) and Obe-1 (AF034942.1) of gene type II, has a homology of 95.3% and 95.2% respectively with South Korean strains 93FMX (FJ498797) and AK7 (FJ498795.1) of gene type II, has a homology of 92.2% with an Australian strain B8935 (MH734940.1) of gene type III, and has a lowest homology of 83.9% with a Kenyan strain MP496 (AB297885.1) of gene type IV. The phylogenetic tree also showed that AKAV / JL / 2022 was in the same branch with AKAV / S / ISR / -170 / 18 (login number: MW822048.1) and AKAV / S / ISR / -01 (login number: AY378155.I) published by Israel, indicating that the isolate belongs to gene subtype lb. 1.9 Virus challenge experiment in suckling mice Virus liquid (106 TCID50 / ml) of the 5th, 10th and 15th generations of AKAV / JL / 2022 isolate was centrifuged at 10000 rpm for 15 minutes and diluted with serum-free DMEM by a 1 O-fold gradient, virus liquid with a dilution of 1 to 10'7 was inoculated respectively into suckling mice of 1-3 days old, 4 mice were inoculated for each dilution, 10 ul of virus liquid was intracranially inoculated for each mouse, and blank DMEM was set up as the negative control. The death of the suckling mice was observed every day for 14 days. The results showed that the suckling mice began to have neurological symptoms, convulsions and stiff limbs from the third day onwards, and died in succession. The median lethal doses were calculated by the Reed-Muench method after 14 days according to the death situation as shown in Table 1, and the median lethal doses of F5, F10 and Fl5 generations were respectively 106 LD50 / ml, 106 LD50 / ml and 105 83 LD50 / ml. Table 1 Death situation of suckling mice infected with AKAV / JL / 2022 strain Generation Vims content F5 (VERO) F10 (VERO) Fl5 (VERO) Blank 0 / 4 0 / 4 0 / 4 6.0 TCIDso / ml 4 / 4 4 / 4 4 / 4 5.0 TCIDso / ml 4 / 4 4 / 4 4 / 4 4.0 TCIDso / ml 4 / 4 4 / 4 4 / 4 3.0 TCIDso / ml 4 / 4 3 / 4 4 / 4 2.0 TCIDso / ml 0 / 4 1 / 4 1 / 4 1.0 TCIDso / ml 2 / 4 3 / 4 0 / 4 0.10 TCIDso / ml 2 / 4 0 / 4 1 / 4 0.01TCID50 / ml 0 / 4 0 / 4 0 / 4 The brain tissue of dead suckling mice inoculated with virus of different dilutions in different generations was tested by PCR in an aseptic condition. The results showed that the mice were all tested positive by PCR, 5 indicating that the AKAV / JL / 2022 isolate had a high virulence. After passage through MDBK cells for 15 generations, the change in virulence was small. The AKAV / JL / 2022 isolate could be used as a test strain for an Akabane disease vaccine and could be used for the preparation of other diagnostic reagents. 10 Embodiment 2: Preparation of virus inactivated vaccine 2.1 Preparation of virus seed of Akabane virus MDBK adherent cells growing into a good confluent monolayer was taken, the original culture solution was discarded, and F9 virus liquid of the AKAV / JL / 2022 strain was added at a ratio of 0.5%-2% (v / v) and 15 cultivated at 37°C; when the degree of CPE was higher than or equal to 80%, the virus liquid was harvested, and freezing and thawing were conducted for 3 times to obtain a basic virus seed; the basic virus seed was diluted with DM EM culture solution by a 10-fold gradient, five dilutions (i.e. 102-106) were used to inoculate the virus liquid respectively into the 96-well cell culture plate where MDBK cells grew into a monolayer, 4 wells were inoculated for each dilution, 100 ul of DMEM was added after 100 ul of diluent was added into each well, and a negative control and a positive control were set up at the same time. After cultivation in an incubator at 37°C and 5% CO2 for 5 days, the cells were observed for CPE under a microscope, and TCID50 was calculated by the Reed-Muench method. As calculated, the virus content of the virus seed was not less than 106 TCID50 / ml. 2.2 Purity test of virus liquid for vaccine preparation The test was carried out according to the appendix of the current Chinese Veterinary Pharmacopoeia. The results showed that the basic virus seed was free from bacteria, mycoplasma and exogenous virus contamination. 2.3 Inactivation of virus liquid for vaccine preparation 0.25%o P-propiolactone was used for inactivation for 4 hours on a shaker at a rotational speed of 80-100 r / min, and inactivation was conducted again at room temperature for 24 hours to hydrolyze residual P-propiolactone and obtain an inactivated virus liquid. The inactivated virus liquid was diluted with DMEM culture solution and inoculated into well-grown MDBK adherent cells with the culture solution at a ratio of 10% (V / V), the cells were cultivated in an incubator at 37°C and 5% CO2 for 5 days and observed daily, the culture was frozen and thawed at -80°C for 3 times, blind passage was conducted for 3 generations according to the above method, and each generation of culture was observed for CPE. The results showed that no CPE was caused and the inactivation was complete. 2.4 Vaccine preparation (1) Preparation of aqueous phase: the inactivated virus liquid was heated to 31±1°C for later use. (2) Preparation of oil phase: an oil adjuvant was used and heated to 31±1°C for later use. (3) Emulsification: the oil phase and the aqueous phase were mixed at a mass ratio of adjuvant: aqueous phase = 1:1. The specific operation was: feeding the adjuvant into an emulsification tank, feeding the aqueous phase slowly into the emulsification tank at a positive pressure under a stirring condition, stirring at 30-33°C for 30 min until the aqueous phase was fully mixed with the adjuvant, and emulsifying the mixture into a two-phase oil emulsion vaccine. Then the vaccine was packaged quantitatively, capped, sealed, labeled, and stored at 2-8°C. 2.5 Vaccine product testing (1) Appearance: slightly viscous milky white emulsion. (2) Dosage form: W / O / W. (3) Stability: 10 ml of vaccine was absorbed, added into a centrifuge tube and centrifuged at 3000 r / min for 15 minutes, and the aqueous phase separated at the bottom of the tube was not more than 0.5 ml. (4) Viscidity: as tested according to the appendix of the current Chinese Veterinary Pharmacopoeia, the viscidity met the specification. (5) Load inspection: as tested according to the appendix of the current Chinese Veterinary Pharmacopoeia, the load met the specification. (6) Sterility testing: as tested according to the appendix of the current Chinese Veterinary Pharmacopoeia, the product was sterile. (7) Safety inspection: 2 ml of vaccine was injected subcutaneously and respectively into two guinea pigs which were 350-450 g in weight; 0.5 ml of vaccine was injected subcutaneously and respectively into two guinea pigs which were 18-22 g in weight. After 7 days of daily observation, the guinea pigs have no death or obvious local or systemic adverse reactions caused by vaccine injection 2.6 Neutralizing antibody titer detection 8 healthy female guinea pigs which were 350-450 g in weight (the titer of the neutralizing antibody against Akabane virus is not greater than 1:4) were selected and randomly divided into 2 groups. The 4 guinea pigs in the first group were intramuscularly and respectively injected with 1.0 ml of vaccine, and served as an immune group; the 4 guinea pigs in the second group were respectively injected with 1.0 ml of DMEM, and served as an control group; after 21 days, the guinea pigs were immunized once again in the same way; heart blood was collected 21 days after the second immunization, serum was isolated, the titer level of the neutralizing antibody against Akabane virus was determined, and the results were shown in Table 2. It could be seen from the results that the titer level of the neutralizing antibody against Akabane virus in the serum of the 4 guinea pigs in the immune group was not less than 1:128, while the titer of the neutralizing antibody in the control group was not greater than 1:2. Table 2 Results of guinea pig serum neutralizing antibody titer detection Group No. Titer of neutralizing antibody 1 1:128 2 1:256 Immune group 3 1:128 4 1:360 DI <1:2 D2 <1:2 Control group D3 <1:2 D4 <1:2 The above results indicate that after being immunized with the inactivated vaccine prepared by the Akabane disease virus strain provided by the present invention, the guinea pigs can be induced to produce a high-titer neutralizing antibody against Akabane disease (>128). Therefore, the 5 inactivated vaccine provided by the invention can effectively protect the guinea pigs from being infected with Akabane disease virus for a long time. Finally, it should be noted that the above embodiments are only used for describing the technical solution of the present invention rather than limitation. Although the present invention is described in detail by referring 10 to the above embodiments, those ordinary skilled in the art should understand that the technical solution recorded in each of the above embodiments can be still amended, or some technical features therein can be replaced equivalently. However, these amendments or replacements do not enable the essence of the corresponding technical solution to depart 15 from the spirit and the scope of the technical solution of various embodiments of the present invention.

Claims

1. An Akabane disease virus strain, wherein the Akabane disease virus strain is Akabane virus AKAV / JL / 2022, and the preservation number thereof is CGMCC No.45375.

2. An application of the Akabane disease virus strain according to claim 1 in preparation of drugs or reagents, wherein the drugs or reagents are used for treating, preventing or diagnosing Akabane disease.

3. A product, wherein the product contains the Akabane disease virus strain according to claim 1; and the product is a drug, a feed or a feed additive.

4. A vaccine, wherein the vaccine contains the Akabane disease virus strain according to claim 1 or a culture thereof.

5. The vaccine according to claim 4, wherein the vaccine is an inactivated vaccine.

6. The vaccine according to claim 4 or 5, wherein the titer of the Akabane disease virus strain or the culture thereof is higher than 1060 TCID50 / mL.

7. The vaccine according to any one of claims 4-6, wherein after the vaccine is inoculated, the titer level of a neutralizing antibody against Akabane virus is not less than 1:128.

8. The vaccine according to any one of claims 4-7, wherein the vaccine also contains an adjuvant.

9. A method for preparing the vaccine according to claim 8, comprising:inoculating the Akabane disease virus strain of claim 1 into cells for cultivation to prepare a virus liquid culture; then inactivating the virus liquid culture, mixing the culture with the adjuvant, and emulsifying themixture to prepare the vaccine.

10. An application of the vaccine according to any one of claims 4-8 or the method for preparing the vaccine according to claim 9 in preparation of drugs or reagents used for diagnosing, preventing or treating Akabane 5 disease.

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