Recombinant vesicular stomatitis virus carrying schmallenberg virus gene as well as preparation and application of recombinant vesicular stomatitis virus

Abstract

The invention discloses a recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene as well as preparation and application thereof, and belongs to the technical field of recombinant viruses. The invention aims to provide a recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene. The invention provides a recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene, which is characterized in that a vesicular stomatitis virus is used as a starting virus, and a protein with an amino acid sequence as shown in SEQ ID NO.2 is used for replacing a protein with an amino acid sequence as shown in SEQ ID NO.4 to obtain the recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene. The recombinant virus can replace the Schmallenberg virus to establish a virus neutralization test and is used for detecting a neutralizing antibody of the Schmallenberg virus, so that a reliable tool is provided for serological detection of cattle and sheep and potency evaluation of a candidate vaccine of the Schmallenberg virus.

Description

Technical Field

[0001] The invention belongs to the technical field of recombinant virus modification, and specifically relates to a recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene and a preparation and application thereof. Background Art

[0002] Schmallenberg disease is a teratogenic infectious disease of ruminants caused by Schmallenberg virus (SBV). Adult animals including cattle, sheep, goats, etc. are mainly asymptomatic or subclinical after being infected with SBV. Some animals will have symptoms such as fever, physical decline, loss of appetite, diarrhea, and decreased milk production, which can have a serious impact on their production performance. The greatest harm of SBV comes from the miscarriage, stillbirth, mummification, and congenital malformation of offspring caused by its infection of pregnant female livestock, mainly manifested as joint bending and hydrocephalus syndrome. Schmallenberg disease is a vector-borne disease, and Culicoides is its main transmission vector. After SBV first appeared in Germany and the Netherlands in 2011, it quickly spread to more than a dozen European countries and showed a cyclical epidemic trend of outbreaks every two to three years, causing serious harm to the entire European ruminant breeding industry. There are no reports of SBV epidemics in my country. Since the activities of insect vectors are random and spread quickly, once SBV is introduced into my country, it can be expected to spread rapidly among ruminants such as cattle and sheep and cause serious losses. Therefore, my country urgently needs to develop scientific and reliable detection methods and preventive preparations to prevent the introduction of SBV. However, since SBV is currently only prevalent in some European countries, many countries, including my country, lack resources for prevalent strains, which greatly limits the prevention and control research of the virus. Summary of the invention

[0003] The purpose of the present invention is to provide a recombinant vesicular stomatitis virus carrying Schmallenberg virus gene.

[0004] The invention provides a recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene. The vesicular stomatitis virus is used as a starting virus, and a protein having an amino acid sequence as shown in SEQ ID NO.2 is used to replace a protein having amino acids as shown in SEQ ID NO.4 to obtain the recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene.

[0005] The present invention provides a recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene. The vesicular stomatitis virus is used as a starting virus, and the nucleotides of a gene sequence such as SEQ ID NO.1 are replaced by nucleotides of a gene such as SEQ ID NO.3 to obtain the recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene.

[0006] The present invention provides a microbial preparation of the above-mentioned recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene.

[0007] The present invention provides an application of the above-mentioned recombinant vesicular stomatitis virus carrying Schmallenberg virus gene or the above-mentioned microbial preparation in the detection of evaluating the antibody level against Schmallenberg virus.

[0008] The present invention provides a use of the above-mentioned recombinant vesicular stomatitis virus carrying Schmallenberg virus gene or the above-mentioned biological preparation in preparing a kit for evaluating the efficacy of a drug or vaccine for treating or preventing Schmallenberg virus.

[0009] The present invention provides an application of the above-mentioned recombinant vesicular stomatitis virus carrying Schmallenberg virus gene or the above-mentioned biological preparation in preparing a kit for screening drugs or vaccines for treating or preventing Schmallenberg virus infection.

[0010] The present invention provides a kit, which contains the above-mentioned recombinant vesicular stomatitis virus carrying Schmallenberg virus gene or the above-mentioned microbial preparation, Schmallenberg virus positive serum, Schmallenberg virus antibody or medicine or vaccine for treating or preventing Schmallenberg virus infection.

[0011] The present invention provides a method for preparing a recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene, wherein the specific steps of the method are as follows:

[0012] Step 1: Remove the nucleotides in the gene sequence of plasmid pVSV-eGFP as shown in SEQ ID NO.3, and insert the nucleotides in the gene sequence as shown in SEQ ID NO.1 by homologous recombination;

[0013] Step 2: Virus rescue: pVSVΔG-eGFP-SBV M plasmid and auxiliary plasmids pVSV N, pVSV P, pVSV L and pVSV G were co-transfected into BSR cells at a ratio of 2:2:2:1:1;

[0014] Step 3: Collect the cell culture fluid obtained in step 2 and passage it in Vero cells to obtain recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene.

[0015] It is further defined that the transfected cells obtained in step 2 are cultured with DMEM containing 10% FBS, and 6 hours after transfection, new DMEM containing 10% FBS is replaced to continue culturing the cells.

[0016] It is further defined that in step 3, when more than 90% of the cells show green fluorescence, the cell culture fluid is collected and continuously passaged in Vero cells. When obvious CPE is observed and reaches more than 90%, the recombinant virus harvested is a recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene.

[0017] Beneficial effect: The present invention utilizes the reverse genetic technology of vesicular stomatitis virus to replace the protein shown in SEQ ID NO.4 of vesicular stomatitis virus with the protein shown in SEQ ID NO.2 of Schmallenberg virus for the first time, and rescues the vesicular stomatitis virus strain of recombinant Schmallenberg virus. Relying on the expression of Schmallenberg virus envelope glycoprotein, the recombinant virus obtains the cell tropism of Schmallenberg virus and can infect a variety of Schmallenberg virus susceptible cells. In view of this, the recombinant virus can replace Schmallenberg virus to establish virus neutralization test, and is used for the detection of Schmallenberg virus neutralizing antibodies, thereby providing a reliable tool for serological detection of cattle and sheep and the efficacy evaluation of Schmallenberg virus candidate vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Construct the resulting map for the vector;

[0019] Figure 2 This is the genome structure of pVSVΔG-eGFP-SBV M;

[0020] Figure 3 This is a diagram showing the cytopathic effect of rVSVΔG-eGFP-SBV M during passage in Vero cells;

[0021] Figure 4 This is the result of RT-PCR identification of recombinant rVSVΔG-eGFP-SBV M;

[0022] Figure 5 The figure is the result of SDS-PAGE analysis of purified virus particles stained with Coomassie blue;

[0023] Figure 6 This is a diagram showing the expression results of the protein shown in SEQ ID NO.2 of SBV in infected cells detected by Western blot;

[0024] Figure 7 This is a laser confocal observation of the expression and localization of the protein shown in SEQ ID NO.2 of SBV in infected cells;

[0025] Figure 8 The figure shows the morphological comparison and size analysis results of rVSVΔG-eGFP-SBV M and rVSV-eGFP virus particles;

[0026] Fig. 9 This is the growth kinetic curve result of rVSVΔG-eGFP-SBV M and rVSV-eGFP in Vero cells;

[0027] Fig.10 This is a graph showing the tropism of rVSVΔG-eGFP-SBV M and rVSV-eGFP to different host cells;

[0028] Fig.11 . The specific reaction result of rVSVΔG-eGFP-SBV M and SBV positive bovine serum;

[0029] Fig.12 This is a diagram showing the results of the rVSVΔG-eGFP-SBV M neutralization test validation. DETAILED DESCRIPTION

[0030] pVSV N, pVSV P, pVSV L and pVSV G are described in the article Immunogenicity of a recombinantVSV-Vectored SARS-CoV vaccine induced robust immunity in rhesus monkeys after single-dose immunization.

[0031] pVSV-eGFP is recorded in "Construction and Immunogenicity Study of Recombinant Vesicular Stomatitis Virus with Nipah Virus Envelope Glycoprotein" and is an alias for the pCI-Rz-VSV-eGFP-FL vector.

[0032] Example 1. Construction of recombinant VSV full-length cDNA expressing SBV envelope glycoprotein

[0033] The full-length cDNA recombinant plasmid pVSV-eGFP expressing eGFP was digested with endonuclease NheI to remove the SEQ ID NO.3 gene. The digested plasmid was ligated with T4 DNA ligase and named pVSVΔG-eGFP after sequencing. The plasmid did not contain the SEQ ID NO.3 gene of VSV. It was then digested with endonuclease Nhe I to obtain a linearized cloning vector ( Figure 1 ).

[0034] According to the genome sequence of the SBV reference strain, the sequence shown in SEQ ID NO.1 encoding SBV was synthesized, and the sequence was optimized for mammalian cell-preferred codons (SEQ ID NO.1). The optimized SBV partial gene was cloned into the pCAGGS vector and named pCAGGS-SBV M.

[0035] SEQ ID NO.1:

[0036]

[0037] Amino acid sequence of SEQ ID NO.1 gene sequence (SEQ ID NO.2):

[0038]

[0039] SEQ FROM NO.3:

[0040]

[0041] Amino acid sequence of SEQ ID NO.3 gene sequence (SEQ ID NO.4):

[0042] MKCLLYLAFLFIGVNCKFTIVFPHNRKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQ SCGYATVTDAEAAIVQVTPHHVLVDEYTGEWVDSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMA DKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPVFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEI GPNGVLRTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDETLFFGDTGLSKNPIEFVEGWFSSWKSSIASFCFIIGLIIGLFLVLRVGIYLCIKLKHTKKRQIYTDIEMNRLGK.

[0043] According to the sequence shown in SBV SEQ ID NO.1, upstream and downstream primers were designed (Table 1), and the target gene was amplified using the cloning plasmid pCAGGS-SBVM as a template. The PCR product was homologously recombined with the vector (i.e., linearized pVSVΔG-eGFP), and the homologous recombination reaction system was shown in Table 2. The recombination reaction was carried out in a PCR instrument at 37°C for 30 minutes, and then the temperature was lowered to 4°C after the reaction. The homologous recombination product was cloned into XL10 competent cells, a single clone was picked and the plasmid was extracted, and the endonuclease Nhe I was used for digestion and identification, and the positive plasmid was sent for sequencing. After sequencing and identification, it was named pVSVΔG-eGFP-SBV M ( Figure 2 ).

[0044] Table 1 Primers required for specific amplification of the sequence shown in SEQ ID NO.1 of SBV

[0045]

[0046]

[0047] Table 2 Primers required for constructing and expressing SBV chimeric recombinant VSV full-length cDNA

[0048]

[0049] Note: Border part - NheI restriction site

[0050] Example 2. Rescue and passage of recombinant viruses

[0051] 1. Using liposome transfection, the pVSVΔG-eGFP-SBV M full-length plasmid obtained in Example 1 and the auxiliary plasmids pVSV N, pVSV P, pVSV L and pVSV G were co-transfected into BSR cells at a ratio of 2:2:2:1:1, i.e. 2 μg, 2 μg, 2 μg, 1 μg, 1 μg per well, respectively, and cultured with DMEM containing 10% FBS. After 6 hours of transfection, the cells were cultured with new DMEM containing 10% FBS. When more than 90% of the cells showed green fluorescence, the cell culture fluid was collected and continued to be passaged in Vero cells. When obvious CPE was observed and reached more than 90%, the recombinant viruses were harvested and named rVSVΔG-eGFP-SBV M, respectively, and stored at -80°C ( Figure 3 ).

[0052] 2. RT-PCR Identification of Recombinant Viruses

[0053] The RNA of the harvested recombinant virus rVSVΔG-eGFP-SBV M was extracted and reverse transcribed, and PCR amplification was performed using the corresponding primers (Table 1). The PCR product was a target fragment of approximately 4212 bp in size ( Figure 4 ). In addition, sequencing confirmed that the sequence of the exogenous gene inserted into the recombinant virus was consistent with the reference sequence, proving that the gene shown in SEQ ID NO.1 of the target gene has been correctly recombined into the VSV genome.

[0054] 3. Expression of SBV envelope glycoprotein Gc in recombinant virus

[0055] SBV envelope glycoprotein Gc is the main target of neutralizing antibodies. In order to verify the expression of this protein in the recombinant virus rVSVΔG-eGFP-SBV M, Gc protein was detected by western blot and IFA respectively.

[0056] The recombinant virus was purified and concentrated by density gradient centrifugation, and the purified virus was analyzed by SDS-PAGE. The parent VSV virus was used as a control. The results showed that the recombinant virus had a Gc protein band of the expected size ( Figure 5). At the same time, western blot analysis showed that the recombinant virus could react specifically with mouse anti-Gc protein serum ( Figure 6 ). Laser confocal microscopy showed that cells infected with the recombinant virus rVSVΔG-eGFP-SBV M were able to specifically bind to mouse anti-Gc protein serum and exhibit red fluorescence; while Vero cells infected with the parent rVSV-eGFP did not express Gc, resulting in no red fluorescence ( Figure 7 ). The above results showed that rVSVΔG-eGFP-SBV M could correctly express SBV envelope glycoprotein Gc.

[0057] 4. Observation of the Morphology of Virus Particles by Electron Microscopy

[0058] The purified recombinant virus rVSVΔG-eGFP-SBV M and the parent rVSV-eGFP were used to prepare electron microscopy samples by tungsten phosphate negative staining, and the morphology of the virus particles was observed by transmission electron microscopy. The results showed that the width of the recombinant virus was not significantly different from that of the wild-type VSV, but the length was significantly larger than that of the wild-type VSV ( Figure 8 ).

[0059] 5. In vitro growth kinetics of recombinant viruses

[0060] In order to compare the growth kinetics of the recombinant virus rVSVΔG-eGFP-SBV M and the parental rVSV-eGFP on Vero cells, the recombinant virus and the parental virus were inoculated into Vero cells at an infection dose of MOI = 0.01. From 0 to 108 h after infection, the supernatant of the infected cells was collected every 12 h and the samples were titrated on Vero cells to calculate the titer TCID 50 The virus growth curve was drawn. The results showed that compared with the parent virus VSV, the titer of the recombinant virus rVSVΔG-eGFP-SBV M in Vero cells was lower and the rising trend was slower, indicating that the replacement of VSV G protein by exogenous protein affected the replication rate of VSV. The titer reached a peak at 84h after infection, with a titer of 10 7 TCID 50 / mL; while the parent rVSV-eGFP reached its peak at 36h after infection, with a titer of up to 10 9 TCID 50 / mL( Fig. 9 ).

[0061] Example 3. Application of recombinant virus: SBV tropism for cells of different species

[0062] The recombinant virus rVSVΔG-eGFP-SBV M and the parent rVSV-eGFP were inoculated into bovine MDBK cells, bovine BT cells, sheep MDOK cells, canine MDCK cells, porcine PK-15 cells, mouse BSR cells, and human 293T cells at an MOI of 0.01, respectively. Green fluorescence observation was performed 72 hours after inoculation. The results showed that the parent VSV was susceptible to all cells, and the recombinant virus was able to infect all cells except MDBK ( Fig.10 ), which is consistent with the characteristic of SBV having a wide range of host tropism. Therefore, the recombinant virus rVSVΔG-eGFP-SBV M can be used as a model virus to replace SBV to study the molecular mechanism of virus invasion of different host cells.

[0063] Example 4. Detection of SBV neutralizing antibodies using a recombinant virus neutralization assay

[0064] In order to verify the ability of the recombinant virus rVSVΔG-eGFP-SBV M to detect SBV neutralizing antibodies, SBV standard positive bovine serum was used as the primary antibody, and the recombinant virus was analyzed by western blot. The results showed that the recombinant virus was able to react specifically with SBV positive serum ( Fig.11 In order to further verify the ability of the recombinant virus to determine the SBV neutralizing antibody titer in serum, the neutralization titer of 2 8 The SBV standard serum was diluted 2× and the diluted serum was mixed with an equal volume of 100 TCID 50 The recombinant virus rVSVΔG-eGFP-SBV M was mixed and incubated at 37°C for 1 hour. 40 μL of the mixture of virus and serum was inoculated into Vero cells in a 96-well cell plate and incubated at 37°C and 5% CO2 for 1 hour. 100 μL of 10% FBS DMEM culture medium was added to each well and placed in an incubator at 37°C and 5% CO2 for 36 hours. The green fluorescence expressed by the cells was observed. The neutralization titer (VNT) was the highest serum dilution that inhibited 50% fluorescence. The results are shown in Fig.12 The results showed that the titer of the standard serum was 2 8.32 , which is consistent with the neutralization titer calibrated by SBV, indicating that the recombinant virus rVSVΔG-eGFP-SBV M can replace SBV for the detection of neutralizing antibodies.

[0065] Example 5. Evaluation of the efficacy of drugs or vaccines for treating or preventing Schmallenberg virus

[0066] Evaluate the efficacy of existing candidate drugs or vaccines in preventing and treating SBV infection in animal models.

[0067] Experimental Materials:

[0068] (1) rVSVΔG-eGFP-SBV M recombinant virus (used to simulate SBV infection);

[0069] (2) Known drug candidates (such as antiviral drugs) or vaccine candidates;

[0070] (3) Animal models (animals with obvious immune effects, such as mice or guinea pigs);

[0071] (4) Control drug / vaccine (a drug or vaccine with known efficacy as a positive control).

[0072] Experimental design:

[0073] 1. Animal grouping:

[0074] (1) Drug group: inoculated with candidate drugs to treat SBV infection.

[0075] (2) Vaccine group: vaccinated with candidate vaccines to prevent SBV infection.

[0076] (3) Control group:

[0077] 1) Negative control group: No drugs or vaccines were given, but the virus was inoculated to evaluate the effect of natural infection.

[0078] 2). Positive control group: inoculated with known effective drugs or vaccines.

[0079] 2. Vaccination plan:

[0080] (1) Drug group: After inoculation with the rVSVΔG-eGFP-SBV M recombinant virus, drug intervention was given at different time points (e.g., 1 hour, 24 hours after inoculation, etc.).

[0081] (2) Vaccine group: A prime-boost strategy was used, with booster vaccination 2-4 weeks after the initial vaccination, followed by vaccination with the rVSVΔG-eGFP-SBV M recombinant virus.

[0082] (3) Control group: inoculated with the virus but not receiving any drug or vaccine intervention.

[0083] 3. Experimental observation:

[0084] (1) Clinical symptom assessment: Observe changes in the animal's body temperature, weight, behavioral activities, etc., and record the severity of the symptoms.

[0085] (2) Viral load detection: Collect blood, lung, brain and other tissues to detect viral RNA levels (such as by RT-PCR) or viral titers (such as by viral titer determination). Effective drugs or vaccines should be able to significantly reduce viral load.

[0086] (3) Immune response assessment: Neutralizing antibody detection: After vaccination, the immune response of the vaccine is tested through serum neutralization experiments to evaluate the neutralizing antibody titer.

[0087] 4. Experimental endpoint:

[0088] (1) Viral load: evaluates the inhibitory effect of drugs or vaccines on viral replication.

[0089] (2) Immune protection: Evaluate the effects of candidate drugs or vaccines on clinical symptoms and immune protection in animals.

[0090] (3) Neutralizing antibody titer: to evaluate the immune effect of the vaccine.

[0091] Example 6. Screening for drugs or vaccines for treating or preventing Schmallenberg virus infection

[0092] In order to screen out the best candidate that can effectively treat or prevent SBV infection from multiple candidate drugs or vaccines.

[0093] Experimental Materials:

[0094] (1) rVSVΔG-eGFP-SBV M recombinant virus

[0095] (2) Multiple drug candidates (such as antiviral drugs)

[0096] (3) Multiple candidate vaccines (such as protein vaccines, viral vector vaccines, etc.)

[0097] (4) Animal models (such as mice or guinea pigs)

[0098] (5) Control group: animals not vaccinated with drugs or vaccines

[0099] Experimental design:

[0100] 1.Drug screening experiment:

[0101] Animal Grouping:

[0102] (1) Using multiple drug candidates, each drug is divided into multiple groups and administered at different time points (e.g., 1 hour, 24 hours after virus inoculation).

[0103] (2) Negative control group: only the virus is inoculated and the natural infection process is observed.

[0104] (3) Positive control group: treated with known effective antiviral drugs.

[0105] Experimental process:

[0106] (1) Virus inoculation: Inoculate rVSVΔG-eGFP-SBV M recombinant virus.

[0107] (2) Drug intervention: After virus inoculation, drug intervention is given at different time points.

[0108] (3) Viral load detection: Detect the viral load in different tissues to evaluate whether the drug can inhibit viral replication.

[0109] (4) Immune response assessment: Evaluate the effect of the drug on the immune system through serum neutralizing antibody testing.

[0110] (5) Clinical symptom observation: monitor changes in body temperature, weight and clinical symptoms, and evaluate the therapeutic effect of the drug.

[0111] 2. Vaccine screening experiment:

[0112] Animal Grouping:

[0113] (1) Vaccine with different vaccine candidates and set up multiple groups.

[0114] (2) Negative control group: not vaccinated, but inoculated with virus.

[0115] (3) Positive control group: inoculated with a vaccine known to be effective.

[0116] Experimental process:

[0117] (1) Vaccination: Use a prime-boost strategy to vaccinate different vaccine candidates.

[0118] (2) Virus inoculation: Inoculate rVSVΔG-eGFP-SBV M recombinant virus for challenge.

[0119] (3) Immune response assessment: Detect immune responses, including neutralizing antibody titer and T cell response, through ELISA, flow cytometry and other methods.

[0120] (4) Viral load detection: Collect tissue samples at different time points and detect viral load.

[0121] (5) Clinical symptom observation: record changes in clinical symptoms and evaluate the preventive effect of the vaccine.

[0122] 3. Data Analysis:

[0123] Statistical analysis: Use appropriate statistical methods (e.g., ANOVA, t-test) to compare different drug or vaccine groups and screen out the best candidates.

Claims

1. A recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene, characterized in that: Vesicular stomatitis virus was used as the starting virus, and the protein with the amino acid sequence shown in SEQ ID NO.2 was used to replace the protein with the amino acid sequence shown in SEQ ID NO.4 to obtain a recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene.

2. A recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene, characterized in that: Using vesicular stomatitis virus as the starting virus, a recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene is obtained by replacing the nucleotides in the gene sequence shown in SEQ ID NO.1 with the nucleotides in the gene shown in SEQ ID NO.

3.

3. A microbial preparation containing the recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene according to claim 1.

4. Use of the recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene according to claim 1 or 2 or the microbial preparation according to claim 3 in the detection of evaluating the level of antibodies against Schmallenberg virus.

5. Use of the recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene according to claim 1 or 2 or the biological preparation according to claim 3 in the preparation of a kit for evaluating the efficacy of a drug or vaccine for treating or preventing Schmallenberg virus.

6. Use of the recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene according to claim 1 or 2 or the biological preparation according to claim 3 in the preparation of a kit for screening drugs or vaccines for treating or preventing Schmallenberg virus infection.

7. A kit, characterized in that: The kit contains the recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene according to claim 1 or 2 or the microbial preparation according to claim 3, Schmallenberg virus positive serum, Schmallenberg virus antibodies, or a drug or vaccine for treating or preventing Schmallenberg virus infection.

8. A method for preparing a recombinant vesicular stomatitis virus carrying a Schmallenberg virus gene, characterized in that: The specific steps of the method described are as follows: Step 1: Remove the nucleotides in the gene sequence of plasmid pVSV-eGFP as shown in SEQ ID NO.3, and insert the nucleotides in the gene sequence as shown in SEQ ID NO.1 by homologous recombination; Step 2: Virus rescue: pVSVΔG-eGFP-SBV M plasmid and auxiliary plasmids pVSV N, pVSV P, pVSV L and pVSVG were co-transfected into BSR cells at a ratio of 2:2:2:1:1; Step 3: Collect the cell culture fluid obtained in step 2 and passage it in Vero cells to obtain recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene.

9. The method according to claim 8, characterized in that The transfected cells obtained in step 2 were cultured with DMEM containing 10% FBS. After 6 hours of transfection, new DMEM containing 10% FBS was replaced to continue culturing the cells.

10. The method according to claim 8, characterized in that In step 3, when more than 90% of the cells show green fluorescence, the cell culture fluid is collected and continuously passaged in Vero cells. When obvious CPE is observed and reaches more than 90%, the recombinant virus harvested is a recombinant vesicular stomatitis virus carrying the Schmallenberg virus gene.

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