O-type foot-and-mouth disease virus strain containing VP1 T193A mutation site, construction method of O-type foot-and-mouth disease virus strain and application of O-type foot-and-mouth disease virus strain in vaccine antigen escape research

Abstract

The invention belongs to the field of veterinary biological products, and particularly relates to an O-type foot-and-mouth disease virus strain containing a VP1 T193A mutation site, a construction method of the O-type foot-and-mouth disease virus strain and application of the O-type foot-and-mouth disease virus strain in vaccine antigen escape research. According to the O-type foot-and-mouth disease virus mutant strain provided by the invention, threonine at the 193rd site of VP1 protein of a wild O-type foot-and-mouth disease virus strain is mutated into alanine. The amino acid sequence of the mutated VP1 protein is as shown in SEQ ID NO: 2. The lesion time, virus titer and growth curve of the mutant strain rVP1-T193A and rWT constructed by the invention are highly similar, but the neutralizing antibody titer of immune serum is reduced by 4.26 times, the corresponding r value is 0.23 and less than 0.3, and immune escape may be caused. Animal immune challenge protection experiments also prove that the VP1 T193A causes the FMDV to escape from vaccine immunity. The mutation significantly reduces the sensitivity of the virus to a neutralizing antibody, and can be used for researching a virus immune escape mechanism and optimizing vaccine design.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary biological products, and particularly relates to an O-type foot-and-mouth disease virus strain containing a VP1 T193A mutation site, a construction method thereof and application thereof in vaccine antigen escape research. BACKGROUND

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease of pigs, cattle, sheep and other major domestic and wild even-toed animals caused by foot-and-mouth disease virus (FMDV). The disease spreads rapidly, is highly contagious, and has a very high incidence. The World Organization for Animal Health (OIE) has listed it as a reportable animal disease. The outbreak and prevalence of FMD seriously endanger the productivity of livestock and the quality of livestock products, affect international trade in livestock and their products, and cause huge economic losses to the livestock breeding industry in the affected areas. Therefore, effectively preventing and controlling FMD is of great strategic significance to the sustainable and healthy development of the global livestock industry.

[0003] At present, the important means of controlling and preventing FMD is immunization with inactivated vaccine, which plays a very important role in the prevention and purification of FMD. However, FMD has many serotypes, and due to the lack of correction function of RNA-dependent RNA polymerase (RDRP) RDRP, the genome of the virus is highly variable, and immune pressure can also promote the development of FMDV towards vaccine escape. In recent years, mutant strains have been able to escape vaccine immunity.

[0004] The VP1 protein of foot-and-mouth disease virus (FMDV) is the main antigen target, and its G-H loop (residues 141-160) and C-terminal linear epitope (residues 200-213) are the core recognition regions of neutralizing antibodies. Studies have shown that amino acid mutations of VP1 (such as mutations of key sites in the G-H loop) can affect antibody binding ability by changing epitope conformation or charge distribution, leading to immune escape. SUMMARY

[0005] The O-type foot-and-mouth disease virus (FMDV) mutant strain constructed by reverse genetics technology in the present application has a threonine (T) at position 193 of the VP1 protein mutated to alanine (A). The mutant strain reveals that the strain has a certain immune escape ability and significantly reduces the sensitivity of the virus to neutralizing antibodies.

[0006] The present application specifically adopts the following technical solutions: The application provides an O type foot-and-mouth disease virus mutant, which is obtained by mutating threonine at the 193th site of VP1 protein of a wild O type foot-and-mouth disease virus strain into alanine. The wild O type foot-and-mouth disease virus strain is FMDV / O / 17002 strain, and the accession number of the wild O type foot-and-mouth disease virus strain is CCTCC NO:V202060. The amino acid sequence of the mutated VP1 protein is shown as SEQ ID NO:2. The nucleotide sequence of the mutated VP1 protein is shown as SEQ ID NO:4.

[0007] The application also provides a construction method of the O type foot-and-mouth disease virus mutant, which is obtained by introducing T193A mutation by using reverse genetics technology. The application introduces VP1 T193A mutation on the infectious clone skeleton of FMDV strain FMDV / O / 17002 to construct the mutant rVP1-T193A. The specific steps include: Step 1: primers are designed to introduce mutation sites into a semi-long plasmid containing VP1 gene of the wild O type foot-and-mouth disease virus strain to obtain a mutant semi-long plasmid. The wild O type foot-and-mouth disease virus strain is FMDV / O / 17002 strain, and the amino acid sequence of the VP1 protein of the wild O type foot-and-mouth disease virus strain is shown as SEQ ID NO:1. The semi-long plasmid is obtained by connecting the nucleotide of L-VP4-VP3-VP2-VP1 of the wild O type foot-and-mouth disease virus strain with the vector pcDNA3.1. The mutation is to change the base ACG of the 193th amino acid of the VP1 protein into GCG.

[0008] Step 2: the mutant semi-long plasmid obtained in step 1 and a full-length plasmid containing the full-length gene of the wild O type foot-and-mouth disease virus strain are respectively double-digested and then connected to obtain a mutant full-length plasmid. The full-length plasmid is obtained by connecting the nucleotide of the full-length gene of the wild O type foot-and-mouth disease virus strain with the vector pcDNA3.1. The double digestion is performed by using Sbf I and Pac I restriction endonuclease.

[0009] Step 3: the full-length mutant plasmid obtained in step 2 is transfected into BHK-21 cells to perform virus rescue to obtain a recombinant virus rVP1-T193A, which is the O type foot-and-mouth disease virus mutant.

[0010] The application also provides application of the mutant in evaluation of protection efficacy of O type foot-and-mouth disease virus vaccine and / or research on antigen escape of O type foot-and-mouth disease virus vaccine.

[0011] In addition, the application also provides application of the mutant in preparation of a broad-spectrum foot-and-mouth disease virus vaccine.

[0012] The application also provides an inactivated vaccine containing the mutant strain, which induces neutralizing antibody titers more than 4 times lower than wild type.

[0013] The application has the following advantages: 1. The neutralizing antibody sensitivity of the mutant strain constructed by the application is reduced: through micro-neutralization test (VNT), it is found that the neutralization titer (NT50) of polyclonal antibodies of rVP1-T193A is 4.26 times lower than that of wild type (rWT) (P<0.001), and the antigen ratio (r1) is 0.23, indicating that it significantly escapes the immune response induced by the existing vaccine. p <0.001), and the antigen ratio (r1) is 0.23, indicating that it significantly escapes the immune response induced by the existing vaccine.

[0014] 2. The structural mechanism of the mutant strain constructed by the application: molecular dynamics simulation shows that the T193A mutation destroys the hydrogen bond network of VP1 and VP3 Q96, leading to the change of C-terminal epitope conformation, hindering the binding of antibodies ( Figure 2 ).

[0015] 3. The application shows that rVP1-T193A can break through the vaccine immunity of rWT through an immune pig challenge model, and the protection efficiency is 20% (1 / 5).

[0016] 4. The application of the mutant strain constructed by the application in vaccine antigen escape research: the mutant strain can be used to evaluate the gap of vaccine protection efficacy, and guide the design of broad-spectrum vaccine.

[0017] 5. The application of the mutant strain constructed by the application in vaccine optimization: by introducing the T193A mutation in reverse, conserved epitopes or multivalent vaccines can be screened and designed, and the cross-protection ability can be improved. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a schematic diagram of the FMDV VP1 mutation site of the application.

[0019] Figure 2 It is a schematic diagram of the hydrogen bond before and after the mutation of VP1 193.

[0020] Figure 3 It is an electrophoresis map of the enzyme digestion product of the recombinant plasmid. M is a DNA marker of DL 15000, 1 is the enzyme digestion band of the original full-length plasmid pSK-HB2017P1, and 2 and 3 are the enzyme digestion bands of the 1st and 2nd mutant plasmids of pSK-HB2017P1-VP1-T193A.

[0021] Figure 4 It is the cell morphology 24h after transfection of the original full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-T193A to BHK-21 cells.

[0022] Figure 5Indirect immunofluorescence results for rWT and rVP1-T193A mutant.

[0023] Figure 6 Plaque phenotype for rWT and rVP1-T193A mutant.

[0024] Figure 7 Multi-step growth curve for rWT and rVP1-T193A mutant.

[0025] Figure 8 Neutralizing antibody titers for rWT and rVP1-T193A mutant against rWT vaccine immune serum.

[0026] Figure 9 Hoof lesion pictures for rWT and rVP1-T193A challenge after immunization with rWT inactivated vaccine.

[0027] Preserved biological material: FMDV / O / 17002; Preserved time: September 17, 2020; Preserved unit name: China Center for Type Culture Collection; Preserved number: CCTCC NO: V202060; Preserved unit address: Wuhan, China, Wuhan University; Classification name: Foot-and-mouth disease virus O / 17002 strain (FMDV / O / 17002 strain). DETAILED DESCRIPTION

[0028] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0029] CutSmart, Sbf I, Pac I are purchased from New England Biolabs (Beijing) Co., Ltd. 3-day-old Kunming mice are purchased from the Lanzhou Institute of Animal Health, Chinese Academy of Agricultural Sciences.

[0030] Example 1 1. Construction of O-type foot-and-mouth disease virus strain rVP1-T193A containing VP1 T193A mutation site The FMDV VP1 mutation site is as Figure 1 , 2The amino acid sequence of the original VP1 and the amino acid sequence of the mutant VP1 are shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0031] 1.1, Recombinant plasmid construction The VP1 gene fragment containing the T193A mutation was amplified by PCR using the semi-long plasmid pSK-HB2017P1 (pSK-HB207P1 is obtained by linking the nucleotide sequence of L-VP4-VP3-VP2-VP1 of FMDV / O / 17002 (CCTCC NO: V202060) with the vector pcDNA3.1) as the template, and the PCR amplification system was as follows: 2xM5-Mutase Mix (Beijing Joinery Biotech Co., Ltd.) 10 μL, forward mutation primer (10 μM) 0.4 μL, reverse mutation primer (10 μM) 0.4 μL, semi-long plasmid pSK-HB2017P1 1-10 ng, ddH2O supplemented to 20 μL; the PCR program was as follows: 95℃ for 2 min, (94℃ for 25 s, 60℃ for 25 s, 68℃ for 3 min, 25 cycles), 68℃ for 5 min. The primer sequences used were as follows: forward mutation primer: 5'-CCCTATTG G CTGTCCACCCGAGTGAGGC-3' (containing an ACT→GCT mutation); reverse mutation primer: 5'-GTGGACAG C CAATAGGGGCCGAGGACAGT-3'.

[0032] 8 μL of the above PCR product was taken, 1 μL of 10xM5-Remase Buffer and 1 μL of M5 Remase enzyme (Beijing Joinery Biotech Co., Ltd.) were added, and the mixture was incubated at 37℃ for 1 h to remove the original plasmid. The PCR product after removal of the original plasmid was transformed into E. coli DH5α, plated, and a single colony was picked and the plasmid was extracted to obtain the mutant plasmid pSK-HB2017P1-VP1-T193A.

[0033] Using Sbf Ⅰ and Pac Ⅰ, the recombinant mutant plasmid pSK-HB2017P1-VP1-T193A and the full-length plasmid pOHB2017 (pSK-HB2017 is obtained by linking the full-length nucleotide sequence of FMDV / O / 17002 with the vector pcDNA3.1) were double-digested, and the double-digestion system was as follows: CutSmart 5 μL, plasmid 1 μg, Sbf Ⅰ 1 μL, PacI1μL, ddH2O was supplemented to 50μL, and the reaction conditions were 37℃ for 20 min. After double enzyme digestion, the enzyme digestion products were subjected to agarose gel electrophoresis, the gel was cut, and the 6000bp target band of pSK-HB2017P1-VP1-T193A after enzyme digestion and the 8000bp target band of full-length plasmid pOHB2017 after enzyme digestion were recovered, and the recombinant plasmid pOHB2017-VP1-T193A was obtained by ligation.

[0034] The recombinant plasmid pOHB2017-VP1-T193A was subjected to double enzyme digestion using Sbf I and Pac I, and the identified correct plasmid was sent to Genechem Biotech Co., Ltd. for sequencing identification.

[0035] The results showed that the enzyme digestion bands of the recombinant plasmid pOHB2017-VP1-T193A were consistent with those of the original full-length plasmid pSK-HB2017P1, and two bands of 8000bp and 6000bp were cut out, which were consistent with the expected size (see Figure 3 ), and the sequencing results of the recombinant plasmid pOHB2017-VP1-T193A also showed that the VP1 T193A mutation was successfully introduced into the 8000bp target band of the full-length plasmid pOHB2017.

[0036] Among them, the nucleotide sequence of the original VP1 and the nucleotide sequence of the mutant VP1 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0037] 1.2, Rescue of Recombinant Virus The conventional monolayer BHK-21 was transferred to a T25 cell bottle, and when the cell density grew to 70%-80%, the full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-T193A were transfected into BHK-21 cells using transfection reagent jetPRIME (purchased from Polyplus Transfection® company) respectively (the specific operation method is referred to the instruction manual). 4h after transfection, the transfection solution was replaced with 2% FBS DMEM, and the cells were continuously cultured in a 37℃, 5% CO2 incubator, and the cytopathic effect was observed. The cells were harvested 72h after transfection, freeze-thawed twice, continuously passaged on BHK-21, and stored in a -80 refrigerator for standby.

[0038] The results show that the original full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-T193A both appear typical cytopathogenic effect (CPE) after 24 hours of transfection of BHK-21 cells, the cells of the transfection reagent control (MOCK) are in a fibrous distribution, and the cells are basically full, while the diseased cells are large and round, in a grape-like distribution, and a large number of cells die (see Figure 4 The genetically engineered viruses rescued after transfection of BHK-21 cells by the original full-length plasmid pOHB2017 and the recombinant plasmid pOHB2017-VP1-T193A are named rWT and rVP1-T193A, respectively.

[0039] 1.3, PCR identification of recombinant viruses The virus supernatant of the fourth generation after continuous passage of rWT and rVP1-T193A is used to extract the viral RNA of the supernatant using an Omega Viral RNA Kit (purchased from Omega Bio-Tek Company), and RT-PCR amplification (RT-PCR kit purchased from Takara, RR055A) is performed using primers VP1-3253-F: 5'-CGCTCGGCAACAGACCAC-3'; VP1-3907-R: 5'-GTTCAAGGACTGTTTCACAGGTG-3' to obtain the PCR amplification product of VP1, and the amplification product is sent to Jieke Biotechnology Co., Ltd. for sequencing identification to verify the correctness of the recombinant virus. The reaction system of the RT-PCR is as follows: PrimeScript 1 Step Enzyme Mix 2 μL, 2×1 Step Buffer 25 μL, VP1-3253-F (20 μM) 1 μL, VP1-3907-R (20 μM) 1 μL, template RNA 1 μg, and RNase Free dH2O is added to 50 μL.

[0040] The reaction program of the RT-PCR is as follows: 50°C for 30 min; 94°C for 2 min; 94°C for 30 sec, 60°C for 30 sec, 72°C for 90 sec, 30 cycles.

[0041] The sequencing results show that the rVP1-T193A recombinant virus contains the VP1 T193A mutation, indicating that the recombinant FMDV containing the VP1 T193A substitution is successfully constructed, and the sequencing sequences of VP1 of rWT and rVP1-T193A are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. It should be noted that the sequencing sequence is a partial sequence of the PCR amplification product of the above-mentioned VP1.

[0042] 1.4, indirect immunofluorescence identification of recombinant viruses When the six-well plate BHK-21 monolayer cells grow to 70%-80% density, the parent virus rWT and the recombinant virus rVP-T193A are inoculated respectively. The virus-inoculated cells are detected for specific protein expression by indirect immunofluorescence. The specific steps are as follows: S1: After 12 hours of virus inoculation, the culture solution is discarded, and the cells are washed with PBS for 3 times. Pre-cooled anhydrous ethanol is added, and the cells are fixed at -20°C for 30 minutes; S2: The cells are washed with PBS for 3 times, and 5% BSA is added for room temperature blocking for 1 hour; S3: The cells are washed with PBS for 3 times, and 1:500 diluted anti-FMDV non-structural protein 3D polyclonal antibody (FMDV-3D polyclonal antibody, FMDV 3D protein is expressed from E. coli and purified, and the serum obtained after immunization of 8-week-old New Zealand rabbits twice is the rabbit anti-FMDV 3D polyclonal antibody) is added for 37°C incubation for 1 hour; S4: The cells are washed with PBS for 3 times, and 1:1000 diluted FITC-labeled goat anti-rabbit IgG secondary antibody is added for 37°C incubation for 1 hour; S5: The cells are washed with PBS for 3 times, and 1:1000 diluted DAPI is added for room temperature incubation for 10 minutes. The cells are washed with PBS for 3 times to remove the excess DAPI, and then are placed under a fluorescence microscope for photographing.

[0043] The results show that the BHK-21 cells inoculated with the parent virus rWT and the recombinant virus rVP-T193A are specifically combined with the FMDV-3D polyclonal antibody, and green fluorescence can be observed, while the control cells have no fluorescence (see Figure 5 ), which indicates that the recombinant FMDV is successfully constructed, and the mutation of VP1 T193A does not affect the rescue of the infectious FMDV.

[0044] 2. Plaque phenotype and multi-step growth curve of the recombinant virus The 4th generation parent virus rWT and the recombinant virus rVP1-T193A are diluted by 10 times respectively, and then the viruses of different dilutions are inoculated into the 6-well plate BHK-21 or PK-15 cells covered with monolayer cells at 200 μL per well. The cells are incubated at 37°C in a 5% CO2 incubator for 1 hour, and are shaken every 10 minutes to avoid cell drying. After the incubation, the virus solution is discarded, and 2 mL of astragalus gum mixed solution (MEM and 1.2% astragalus gum mixed at 1:1, and 1% FBS is added) is added. After 48 hours of culture in a 37°C incubator, the culture solution is discarded, the cells are gently washed with PBS for 3 times, pre-cooled anhydrous ethanol is added, and the cells are fixed at -20°C for 30 minutes. The anhydrous ethanol is discarded, the cells are gently washed with PBS for 3 times, 1% crystal violet is used for overnight staining, the cells are washed with PBS for 5 times, and the plaques of the virus are observed, and the plaque forming units (PFU / mL) of the virus are calculated.

[0045] The 4th generation parental virus rWT and recombinant virus rVP1-T193A were used to infect the monolayer BHK-21 cells or PK-15 cells in a 6-well plate at an MOI of 0.1, and after adsorption for 1 h, the virus liquid was removed, washed with PBS for 3 times, DMEM was added, and the culture was placed in a 37°C, 5% CO2 incubator, and the supernatant was collected at 4 h, 8 h, 12 h, 16 h, 20 h and 24 h after inoculation, and the virus titer was determined (TCID 50 ) in 96-well monolayer BHK-21 cells (or PK-15 cells), and the multi-step growth curve of the virus was drawn.

[0046] The results show that the plaque phenotype and multi-step growth curve of the parental virus rWT and the recombinant virus rVP1-T193A are similar (see Figure 6 and Figure 7 ). It is shown that the VP1 T193A mutation does not significantly affect the replication ability of the recombinant FMDV.

[0047] 3, Neutralizing antibody titer detection The laboratory-preserved rWT vaccine-immunized pig serum (rWT was cultured using suspended BHK-21 cells, inactivated and emulsified after the determination of 146s, 8-week-old healthy pigs were immunized, and the immune serum was obtained 28 days after immunization) was used to detect the neutralizing antibody titer of the 4th generation parental virus rWT and the recombinant virus rVP1-T193A. The specific steps are as follows: (1) The immune pig serum was inactivated at 56°C for 30 min, and then 2-fold dilution was performed, and the virus was diluted to 2000 TCID 50 / mL, the diluted virus was mixed with different dilutions of serum in equal volume, incubated at 37°C for 1 h, then added to 96-well plate monolayer BHK-21 cells, 100 μL per well, and placed in a 7°C, 5% CO2 incubator for 72 h.

[0048] (2) After 72 h, the cytopathic effect of each well was observed and recorded, and the neutralizing antibody titers of the parental virus rWT and the recombinant virus rVP1-T193A were calculated using the Reed-Muench method.

[0049] The results show that the neutralizing antibody titer of the parental virus rWT to the serum is 1:23442.3, and the neutralizing antibody titer of the recombinant virus rVP1-T193A to the serum is 1:5495.4, and the corresponding r1 value is 0.23, which is less than 0.3, indicating that the VP1 T19A mutation leads to the mismatch of FMDV and vaccine, and may cause immune escape (see Figure 8 ).

[0050] 4, Animal immunization and challenge protection experiment Select 8 weeks of age of common pathogen antibody negative pigs (purchased from Gansu Province Linxia Hui Autonomous Prefecture Kangle County some pig farm) 9, using the parent strain rWT inactivated and emulsified vaccine to immunize pigs, 42 days after immunization, O type FMDV specific antibody detection of pigs. After determining the antibody positive, using the parent virus rWT and recombinant virus rVP1-T193A to immunized pigs (8000 LD50 / head), observe the clinical symptoms of pigs to score, record the incidence.

[0051] The results show that: the vaccine can 100% protection of pigs against the infection of parent virus rWT (4 / 4), and the protection rate of recombinant virus rVP1-T193A is only 20% (1 / 5). It is proved that VP1 T193A mutation leads to FMDV can escape from the vaccine immunity (see Figure 9 ).

[0052] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, rather than limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An O type foot-and-mouth disease virus mutant, characterized in that, The threonine at position 193 of the VP1 protein of a wild type O foot-and-mouth disease virus strain is mutated to alanine.

2. The O type foot-and-mouth disease virus mutant of claim 1, wherein, The wild type O foot-and-mouth disease virus strain is FMDV / O / 17002 strain, and the accession number of the wild type O foot-and-mouth disease virus strain is CCTCC NO:V202060.

3. The O type foot-and-mouth disease virus mutant of claim 1, wherein, The nucleotide sequence of the mutated VP1 protein is shown in SEQ ID NO:

4.

4. The method for constructing a mutant strain of type O foot-and-mouth disease virus as described in claim 1, characterized in that, The mutation site is introduced by reverse genetics technology.

5. The construction method according to claim 4, characterized in that, The method comprises the following steps: Step 1: design primers to introduce the mutation site into a semi-long plasmid containing the VP1 gene of the wild type O foot-and-mouth disease virus strain, to obtain a mutant semi-long plasmid; Step 2: link the mutant semi-long plasmid obtained in step 1 and the full-length plasmid containing the full-length gene of the wild type O foot-and-mouth disease virus strain after double enzyme digestion, to obtain a mutant full-length plasmid; Step 3: transfect the full-length mutant plasmid obtained in step 2 into BHK-21 cells, perform virus rescue, and obtain a mutant O foot-and-mouth disease virus strain.

6. The construction method of claim 5, wherein, In step 1, the wild type O foot-and-mouth disease virus strain is FMDV / O / 17002 strain, and the accession number of the wild type O foot-and-mouth disease virus strain is CCTCC NO:V202060; the amino acid sequence of the VP1 protein of the wild type O foot-and-mouth disease virus strain is shown in SEQ ID NO:

1.

7. The construction method of claim 5, wherein, In step 1, the mutation is to change the base ACG encoding the 193rd amino acid of the VP1 protein to GCG.

8. Use of the mutant strain of any one of claims 1-3 in evaluation of the protection efficacy of foot-and-mouth disease virus vaccine and / or foot-and-mouth disease virus vaccine antigen escape research.

9. Use of the mutant strain of any one of claims 1-3 in preparation of a broad-spectrum foot-and-mouth disease virus vaccine.

10. An inactivated vaccine comprising the mutant strain of any one of claims 1-3.

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