WO2022027749A1 - 耐热表型稳定遗传、携带负标记的重组口蹄疫病毒无毒株及o/a型口蹄疫二价灭活疫苗 - Google Patents

耐热表型稳定遗传、携带负标记的重组口蹄疫病毒无毒株及o/a型口蹄疫二价灭活疫苗 Download PDF

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WO2022027749A1
WO2022027749A1 PCT/CN2020/111715 CN2020111715W WO2022027749A1 WO 2022027749 A1 WO2022027749 A1 WO 2022027749A1 CN 2020111715 W CN2020111715 W CN 2020111715W WO 2022027749 A1 WO2022027749 A1 WO 2022027749A1
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fmdv
type
mouth disease
foot
virus
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French (fr)
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于力
杨德成
王海伟
周国辉
孙超
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中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心）
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    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32161—Methods of inactivation or attenuation
    • C12N2770/32162—Methods of inactivation or attenuation by genetic engineering

Definitions

  • the present invention relates to viral full-length cDNA infectious cloning plasmids and recombinant virus strains rescued by the full-length cDNA infectious cloning plasmids, in particular to FMDV full-length cDNA infectious cloning plasmids and a heat-resistant table rescued by reverse genetic technology
  • a recombinant foot-and-mouth disease virus avirulent strain with stable genetic type and 3B epitope negative marker the present invention further relates to the recombinant foot-and-mouth disease virus avirulent strain in the preparation of O/A type foot-and-mouth disease bivalent inactivated vaccine or in the preparation of differential diagnosis of foot-and-mouth disease vaccine inoculated animals
  • the use in a reagent for naturally infecting animals belongs to the field of construction and application of a recombinant foot-and-mouth disease virus avirulent strain stably carrying a marker on the capsid.
  • Foot-and-Mouth Disease is caused by Foot-and-Mouth Disease Virus (FMDV). Infectious Diseases (Grubman and Baxt. 2004. Clinical Micro. Rev. 17:465-493).
  • FMDV Foot-and-Mouth Disease Virus
  • the disease is prevalent in many countries in Asia, Africa and the Middle East.
  • the financial burden of epidemic prevention, losses caused by disease outbreaks, and restrictions on international trade have a huge impact on the economy and society of endemic countries.
  • the introduction of the disease into epidemic-free countries often leads to catastrophic consequences.
  • Considering the highly contagious nature of FMD the serious harm it causes to endemic countries, and the huge threat to disease-free countries, it is known as a political and economic disease internationally, and it has always been highly valued by governments around the world.
  • FMDV is a member of MicroRNA virus family and foot-and-mouth disease virus genus. There are seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3) and dozens of subtypes. There is no immune crossover between the serotype strains. Protection, there is only partial cross-protection between subtype strains.
  • the viral genome is a single-stranded positive-stranded RNA with a total length of about 8.5kb, consisting of a 5' non-coding region (5'UTR), an open reading frame (ORF) and a 3'UTR.
  • the ORF encodes a total of 4 structural proteins ( VP4, VP2, VP3 and VP1) and eight nonstructural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C and 3D).
  • Immunization with inactivated vaccines is an effective means of preventing and controlling foot-and-mouth disease in endemic countries. Ring immunization is often used in epidemic-free countries to reduce losses caused by excessive animal slaughter.
  • the seed virus for vaccine production needs to be replaced in time, and for virulent strains, this replacement is affected by It is limited and time-consuming; third, the 146S capsid of foot-and-mouth disease virus is easily depolymerized, which affects the immune protection efficacy of the vaccine and the shelf life of the vaccine. It has been reported that the method of improving heat resistance through artificial mutation can improve the stability of the capsid of foot-and-mouth disease virus. (Abhay Kotecha et al., nature structural &molecular biology, 2015), but its thermostable phenotype is unstable (Katherine A.
  • Non-structural proteins such as 3A and 3B of foot-and-mouth disease virus are difficult to remove, resulting in residues in vaccines. After animals undergo multiple immunizations, specific antibodies will be induced, making it difficult for differential diagnosis techniques based on non-structural protein antibody detection to distinguish vaccines Vaccinated animals and naturally infected animals, which brings difficulties to the implementation of foot-and-mouth disease immunization prevention and control programs and the quarantine in live animal trade.
  • FMDV attenuation research has encountered an insurmountable obstacle, namely: the attenuated phenotypes produced are limited by animal species, and the failure to find three Effective methods and approaches for attenuating the main cloven-hoofed animals, pigs, cattle and sheep.
  • Existing studies on genetic engineering of FMD virus attenuated mainly focus on the non-structural proteins L, 3A and 3D of the virus, but none of them have been able to solve this problem.
  • One of the objectives of the present invention is to provide a universal platform for FMDV cDNA infectious cloning plasmids and a recombinant FMD that does not replicate in susceptible animals and has 3A and 3B protein epitope negative markers constructed and rescued by using the reverse genetic manipulation platform avirulent strains of the virus;
  • the second purpose of the present invention is to provide O-type FMDV full-length cDNA infectious cloning plasmid and the heat-resistant phenotype rescued by reverse genetic technology from the cDNA infectious cloning plasmid with stable inheritance, no replication in susceptible animals, and 3A and 3B protein epitope negatively labeled O-type recombinant FMDV avirulent strain;
  • the third object of the present invention is to provide a full-length cDNA infectious cloning plasmid of type A FMDV and the heat-resistant phenotype rescued by reverse genetic technology from the cDNA infectious cloning plasmid, which is stable and does not replicate in susceptible animals.
  • the fourth object of the present invention is to provide a bivalent inactivated vaccine for preventing and treating O/A type foot-and-mouth disease.
  • the present invention first provides an infectious cloning plasmid of FMDV full-length cDNA, wherein domain 4 of IRES of FMDV genome is replaced with domain 4 of IRES of bovine rhinovirus, and the coding of amino acids 84-143 of FMDV virus non-structural protein 3A is deleted.
  • the coding regions of 3B1 and 3B2 proteins of FMDV were replaced with the coding regions of 3B3 proteins of FMDV, and Pst I restriction sites were introduced on both sides of the coding region of FMDV structural protein P1.
  • the present invention has carried out codon optimization on the coding sequence of the 3B3 protein used to replace 3B1 and 3B2, and the optimized nucleotide sequence is SEQ ID NO.1.
  • the nucleotide sequence of the FMDV full-length cDNA infectious cloning plasmid is shown in SEQ ID NO.2.
  • the present invention further utilizes the FMDV full-length cDNA infectious cloning plasmid to rescue a recombinant foot-and-mouth disease virus avirulent strain by reverse genetic method.
  • the recombinant FMDV avirulent strain carries a heat-resistant capsid, does not replicate in susceptible animals, and has 3A and Characterization of 3B epitope negative tagging.
  • the method for using the FMDV full-length cDNA infectious cloning plasmid to rescue the avirulent strain of the recombinant foot-and-mouth disease virus by reverse genetics includes: linearizing the FMDV full-length cDNA infectious cloning plasmid and transcribing in vitro to obtain the genome of the recombinant virus RNA transfected cells to rescue recombinant FMDV; wherein, the cells can be BHK-21 cells.
  • the present invention provides an O-type FMDV full-length cDNA infectious cloning plasmid, wherein the structural protein P1 coding region of the O-type FMDV full-length cDNA infectious cloning plasmid is replaced with the O-type FMDV structural protein P1 coding region; Among them, tyrosine (Y) at position 79 of the structural protein VP2 of O-type FMDV was mutated to histidine (H) and serine (S) at position 93 was mutated to tyrosine (Y).
  • nucleotide sequence of the O-type FMDV infectious full-length cDNA clone plasmid is shown in SEQ ID NO.3.
  • the present invention further utilizes the O-type FMDV full-length cDNA infectious cloning plasmid to rescue the O-type recombinant FMDV avirulent strain by reverse genetic method.
  • the O-type recombinant FMDV avirulent strain has a genetically stable heat-resistant phenotype and is susceptible to It does not replicate in animals and has the characteristics of negative labeling of 3A and 3B epitopes.
  • the method for using the O-type FMDV full-length cDNA infectious cloning plasmid to rescue the recombinant O-type FMDV avirulent strain by reverse genetics includes: linearizing the O-type FMDV full-length cDNA infectious cloning plasmid and transcribing in vitro The genomic RNA of the recombinant virus is obtained, and the recombinant O-type FMDV virus is rescued by transfecting cells; wherein, the cells can be BHK-21 cells.
  • the present invention provides a type A FMDV full-length cDNA infectious cloning plasmid, wherein the structural protein P1 coding region of the FMDV full-length cDNA infectious cloning plasmid is replaced with the structural protein P1 coding region of type A FMDV;
  • the alanine (A) at position 3 of the structural protein VP1 of type A FMDV was mutated to threonine (T) and the asparagine (N) at position 17 was mutated to histidine (H).
  • nucleotide sequence of the type A FMDV full-length cDNA infectious cloning plasmid is shown in SEQ ID NO.4.
  • the present invention further utilizes the A-type FMDV full-length cDNA infectious cloning plasmid to rescue the A-type recombinant FMDV avirulent strain, and the A-type recombinant FMDV avirulent strain has a genetically stable heat-resistant phenotype, does not replicate in susceptible animals, and Features negative tagging of 3A and 3B epitopes.
  • the method for using the type A FMDV full-length cDNA infectious cloning plasmid to rescue the recombinant type A FMDV avirulent strain by reverse genetics includes: linearizing the type A FMDV full-length cDNA infectious cloning plasmid and transcribing in vitro The genomic RNA of the recombinant virus is obtained, and the recombinant type A FMDV is rescued by transfecting cells; wherein, the cells can be BHK-21 cells.
  • the present invention also provides a method for constructing a recombinant O-type foot-and-mouth disease virus mutant with stable inheritance of capsid heat-resistant phenotype, comprising: changing the amino acid at position 79 of the structural protein VP2 of O-type FMDV from tyrosine (Y) Mutation to histidine (H) and the amino acid at position 93 from serine (S) to tyrosine (Y); the capsid heat-resistant phenotype of the recombinant O-type FMDV mutant strain constructed by this method can be stable inheritance.
  • the present invention further provides a method for constructing a recombinant type A FMDV mutant strain with stable inheritance of capsid heat-resistant phenotype, comprising: mutating the amino acid at position 3 of the structural protein VP1 of type A FMDV from alanine (A) It is threonine (T) and the amino acid at position 17 is mutated from asparagine (N) to histidine (H); Can be genetically stable.
  • the present invention further provides the purposes of described recombinant FMDV strain in the following aspects, including:
  • the bivalent inactivated vaccine of the present invention may further comprise a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient may be a functional molecule as a vehicle, adjuvant, carrier or diluent.
  • Pharmaceutically acceptable excipients may be transfection enhancers, including surfactants, such as immunostimulatory complexes (ISCOMS), incomplete Freunds adjuvant, LPS analogs (including monophosphoryl lipid A) ), muramyl peptides, quinone analogs, squalene, squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations or nanoparticles Wait;
  • ISCOMS immunostimulatory complexes
  • LPS analogs including monophosphoryl lipid A
  • the vaccines of the invention are administered intranasally, intrathecally, and intraarticularly, or a combination thereof.
  • the vaccine can be prepared in an acceptable formulation for administration according to normal veterinary practice. The veterinarian can readily determine the dosage regimen and route of administration most suitable for a particular animal.
  • the bivalent inactivated vaccine of the present invention can be administered using conventional syringes, needle-free injection devices, and the like.
  • the inventor's previous research found that the IRES chimeric virus FMDV (R4) produced by replacing the FMDV IRES domain 4 (IRES-Fd4) with the bovine rhinovirus (BRBV) IRES domain 4 (IRES-Bd4), with 10 6 TCID 50 /head dose of neck muscle inoculation of natural host pigs did not show any clinical symptoms of foot-and-mouth disease, did not produce viremia, did not excrete the virus in the oral and nasal cavity, and the virus did not induce antibodies in pigs, indicating that the IRES chimeric virus FMDV ( R4) Loss of replication ability in susceptible animals (CN108085302A, WO/2018/090994).
  • the present invention adopts the most sensitive intradermal inoculation route of the heel and heel, and uses a higher inoculation dose of FMDV (R4) (10 7 TCID 50 /head or 10 8 TCID 50 / head) and inoculated the wild-type virus O/YS/CHA/05 at a dose of 10 5 TCID 50 /head by the same route as a virulent control. Does not replicate, cannot establish infection, and therefore completely loses pathogenicity in pigs.
  • the present invention conducts a comparative study on the molecular markers on the 3B protein;
  • the sequence is shown in SEQ ID NO.1) to replace the 3B1 and 3B2 proteins to remove the main antigenic region of the 3B protein to realize the molecular negative labeling of foot-and-mouth disease virus and then use the antibody detection ELISA method established by monoclonal antibody 2H1 to vaccinate animals and natural infection
  • Differential diagnosis of animals is a feasible method and strategy.
  • the present invention uses the full-length cDNA infectious cloning plasmids pYS and pQSA of O-type and A-type FMDV to construct and rescue the heat-resistant mutants rO/YS-S2093Y and rA of the 93rd amino acid site-directed mutation of VP2 protein respectively.
  • the present invention conducts multiple rounds of heat stress screening on mutant viruses rO/YS-S2093Y and rA/QSA-Q2093C, and finally obtains O-type FMDV heat-resistant mutants rO/YS ⁇ and A type FMDV thermostable mutant rA/QSA ⁇ .
  • the heat-resistant mutant strains rO/YS ⁇ and rA/QSA ⁇ obtained by screening under heat-resistant stress were successively propagated in BHK-21 cells for 10 generations without stress respectively for the stability test of heat-resistant phenotype (treated at 56 °C for 1 h).
  • the heat resistance of the tenth generation (P10) virus of the heat-resistant mutant strain did not change from the primary (P0) virus, indicating that its heat-resistant phenotype is genetically stable.
  • the sequence determination of the structural protein coding region of the heat-resistant mutant showed that the P10 virus of the O-type FMDV heat-resistant mutant rO/YS ⁇ was the same as the P0 virus, only containing two amino acid mutations of Y2079H and S2093Y; the heat-resistant mutant rA/YS ⁇
  • the P10 generation virus of QSA ⁇ like the P0 virus, only contains two amino acid mutations A1003T (the 3rd amino acid A of VP1 protein is mutated to T) and N1017H (17th amino acid N of VP1 protein is mutated to H), while the mutant virus has two amino acid mutations.
  • Q2093C has a reverse mutation.
  • pYS and pQSA with O and A FMDV infectious cDNAs, respectively, and performed single and combinatorial mutations at the mutation sites of the heat-tolerant mutants to determine the molecular determinants of O and A FMDV heat tolerance. It was found that Y2079H mutation alone could not increase the heat resistance of O-type FMDV. Since S2093Y mutation alone could not be stably inherited, heat resistance analysis was not performed; however, the combined mutation of Y2079H and S2093Y significantly enhanced the heat resistance of O-type FMDV.
  • the present invention finds that although the Y2079H mutation does not produce a thermostable phenotype, it can synergize with the S2093Y thermostable mutation to stably inherit, so the capsid thermostable phenotype of O-type FMDV is determined synergistically; the combined mutations of A1003T and N1017H synergistically determine The capsid of type A FMDV has a thermostable phenotype and is stably inherited.
  • the present invention further constructs a universal plasmid pIRES with 3A and 3B epitope negative markers and capsid heat-resistant phenotype based on the FMDV infectious cloning plasmid with the IRES domain 4 replaced.
  • Bd4 3AB m -Pst I can create a recombinant FMDV avirulent strain with molecular markers and stable capsid heat-resistant phenotype for any popular strain.
  • the present invention performs the following genetic engineering operations on the basis of the infectious cDNA clone plasmid pFMDV (R4) of FMDV (R4): deletion of non-structural
  • R4 of FMDV (R4) deletion of non-structural
  • a codon-optimized 3B3 coding sequence was used to replace 3B1 and 3B2, resulting in the deletion of 3B1/3B2 and the duplication of 3B3, thereby introducing a 3A&3B bimolecular marker;
  • the enzyme cleavage site Pst I is introduced on the side to replace the coding gene of the structural protein P1 of any other FMDV strain, and at the same time, a mutation that determines the stability of the viral capsid is introduced in the structural protein coding region, thereby obtaining 3A&3B marked capsid stability
  • the O-type Southeast Asian genotype Mya-98 strain O/M98/CHA/2010 and the A-type Sea-97 genotype G2 subtype strain A were selected according to the current prevalence of foot-and-mouth disease in Asia. /JLYS/CHA/2014, replaced the P1 genes of pIRES Bd4 3AB m -Pst I with their heat-resistant modified P1 genes, respectively, and the constructed recombinant plasmids were named pIRES Bd4 3AB m -O/M98 and pIRES Bd4 3AB m -A/J14.
  • the genomic RNA of the recombinant virus was obtained by in vitro transcription, and transfected into BHK-21 cells to rescue the recombinant virus, which were named rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A respectively. /J14.
  • the three recombinant viruses were continuously passed on for 10 generations on BHK-21 cells, and it was confirmed by nucleotide sequence determination and analysis that the recombinant viruses had a high degree of genetic stability.
  • the introduced 3A & 3B molecular markers, the substituted IRES domain 4, and the thermostable phenotype modification site were all stably inherited.
  • the present invention compares the in vitro growth characteristics of the constructed recombinant labeled virus with wild-type virus O/YS/CHA/05 and chimeric virus FMDV (R4), and the result finds that the recombinant virus rIRES Bd4 3AB m-
  • the replication kinetics of PanAsia, rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 were similar in BHK-21 cells, but the time of peak replication of these recombinantly labeled viruses was about 4 hours later than that of wild-type virus.
  • the specific monoclonal antibody 3A10 and the specific monoclonal antibody 2H1 that recognize the labeled molecule 3A previously prepared by the inventors were used to label the cells infected with the 3A&3B virus. Indirect immunofluorescence detection was performed.
  • the present invention further uses a heat inactivation test to determine the thermal stability phenotype of the recombinant labeled virus prepared by the present invention, and the measurement results show that the heat resistant modified recombinant labeled virus has significant capsid heat resistance stability characteristics.
  • the present invention uses rIRES Bd4 3AB m -O/PanAsia at a pathogenic dose of 10,000 times higher than that of the wild-type virus WT (10 5 TCID 50 /head).
  • the virus dose (10 9 TCID 50 /head) was intradermally inoculated into the hoof of pigs for pathogenicity evaluation; the results showed that the recombinant marker virus rIRES Bd4 3AB m -O/PanAsia did not replicate in pigs and could not establish infection, so it proved that The recombinant marker virus carrying the thermostable phenotype has no pathogenicity to pigs.
  • the present invention was applied in pigs, cattle, The virulence of these two recombinantly labeled viruses was evaluated in three susceptible animals in sheep by high-dose and sensitive approaches; the results showed that the virus rIRES Bd4 3AB with a thermostable phenotype and 3A&3B markers with IRES domain 4 replaced m -O/M98 and rIRES Bd4 3AB m -A/J14 lost the ability to replicate in pigs, cattle, and sheep, and thus completely lost their pathogenicity, so they were avirulent strains of FMDV.
  • the present invention uses recombinant marker viruses rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 through BEI After inactivation, it was mixed with an equal volume of ISA 201VG adjuvant to prepare an O/A bivalent inactivated vaccine, and a commercial O/A bivalent inactivated vaccine (Zhongnong Wit) was used as a vaccine control to inoculate 10 pigs respectively, and another 6 The first pig was inoculated with PBS as a negative control; the results of the immune protection efficacy test showed that all animals inoculated with the bivalent inactivated vaccine of the present invention could detect O-type and A-type FMDV-specific neutralizing antibodies 7 days after inoculation (dpv).
  • control pigs inoculated with PBS were divided into 2 groups of 3 pigs, 1-3 after inoculation of 10 8.5 TCID 50 /head dose of FMDV O/M98/CHA/2010 or FMDV A/JLYS/CHA/2014 All patients had typical clinical symptoms of FMD.
  • the bivalent inactivated vaccine prepared by the avirulent FMDV strain of the present invention is the same as the commercial bivalent inactivated vaccine, and the vaccinated animals have no clinical manifestations of FMD and obtain immune protection after being challenged with O-type or A-type FMDV.
  • the FMDV avirulent strains rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 carrying heat-resistant phenotypes and 3A&3B markers created by the present invention have excellent immunogenicity, and inoculated animals can induce high levels of The neutralizing antibody can effectively resist the attack of the parental virulent strain, and can be used as a seed virus for the production of foot-and-mouth disease inactivated vaccine.
  • the present invention constructs and rescues a safe, stable and negatively labeled infectious cDNA clone plasmid pIRES Bd4 3AB m -Pst I of the foot-and-mouth disease inactivated vaccine seed virus, which carries the molecular determinants of the virus capsid heat-resistant phenotype, Molecular determinants of the phenotype of virus in vivo loss of replication ability and negative labeling elements for the deletion of viral non-structural protein 3A & 3B epitopes, and the introduction of Pst I restriction sites on both sides of the viral structural protein P1 coding region for rapid cloning and replacement of other arbitrary Structural protein coding region of a foot-and-mouth disease virus strain.
  • the recombinant viruses rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 constructed by replacing the coding region of the capsid protein of the two dominant current strains with this universal plasmid have the following characteristics: in BHK-21 cells
  • the stability of viral capsids was significantly enhanced after heat-resistant modification; pigs, cattle and sheep were inoculated with high dose (10 9 TCID 50 /head) virus through sensitive route without infection; the seed virus thus prepared It has the following excellent features: it not only solves the hidden safety hazard of plant dispersal in the production of foot-and-mouth disease inactivated vaccine, but also greatly reduces the production cost because the vaccine production process does not require high-standard biosafety protection; Sexual modification greatly increases the stability of the 146S capsid, which is conducive to the maintenance of the immunogenicity of the inactivated vaccine, the preservation of the prepared vaccine, the improvement of the quality of the vaccine, and the reduction of the cost
  • the present invention uses these two recombinant viruses as seed viruses to prepare O/A bivalent inactivated vaccines, immunized animals can induce high levels of neutralizing antibodies, and can be challenged with homologous strains to obtain immune protection; 3A and 3B monoclonal antibodies are used
  • the established blocking ELISA method is used to detect the vaccinated animals, which can realize the differential diagnosis between the vaccinated animals and the naturally infected animals.
  • the method for preparing a safe, stable and negatively labeled FMDV avirulent strain provided by the invention can rapidly create the seed virus of an inactivated vaccine against any FMDV epidemic strain within 2 weeks, and is used to prepare a safe, stable and differentially diagnosed FMDV.
  • Inactivated vaccines can provide safe, effective and practical technical means for the prevention, control and purification of global foot-and-mouth disease.
  • Figure 1 Immunofluorescence assay to detect the binding ability of FMDV non-structural protein 3B mutant strain to mAb 2H1.
  • FIG. 3 Schematic diagram of the construction of FMDV avirulent strains labeled with 3A & 3B.
  • Figure 5 3B antibody response of O/A foot-and-mouth disease bivalent inactivated vaccine prepared from 3A&3B-labeled, heat-resistant recombinant FMDV avirulent strains, and commercial inactivated bivalent vaccine vaccinated animals, and the differential diagnosis results of monoclonal antibody blocking ELISA.
  • A/QSA/CHA/2009 which has up to 97.6% nucleotide sequence homology with the VP1 gene of the reference strain A/HuBWH/CHA/2009 (GenBank accession number: JF792355), its full-length cDNA infectious clone is pQSA.
  • the FMDV strain O/M98/CHA/2010 has up to 99.2% homology to the VP1 nucleotide sequence of the reference strain O/BY/CHA/2010 (GenBank accession number: JN998085).
  • A/JLYS/CHA/2014 (Liang et al., Archives Virology, 2016), which has up to 99.6 homology to the VP1 nucleotide sequence of the reference strain A/GDMM/CHA/2013 (GenBank accession number: KF450794) %.
  • Monoclonal antibodies 10B10 (Yang et al., Archives Virology, 2017; CN107177558A (application number: 201710349810.1)
  • 3A10 Wang et al., Research in Veterinary Science, 2019; CN109295006A (patent application number: 201811126938.2)) and 2H1 ( CN109295995A (application number: 201811126187.4) was prepared by the inventor's laboratory.
  • IRES chimeric virus FMDV (R4) produced by replacing the FMDV IRES domain 4 (IRES-Fd4) with the bovine rhinovirus (BRBV) IRES domain 4 (IRES-Bd4) was administered at a dose of 10 6 TCID 50 per head
  • the natural host pigs inoculated with neck muscles did not show any clinical symptoms of foot-and-mouth disease, did not produce viremia, did not excrete toxins in the oral and nasal cavity, and the virus did not induce antibodies in pigs, indicating that the virus lost its ability to replicate in susceptible animals.
  • Chinese invention patent (CN108085302A) and international PCT patent (application publication number: WO/2018/090994).
  • this experiment compared the molecular markers on 3B protein.
  • the inventor's laboratory obtained a monoclonal antibody 2H1 against FMDV non-structural protein 3B2 in the preliminary research, and the epitope recognized by the research confirmed that the epitope motif is 34 KPLKVK 39 , K 34 , K 37 and V 38 are the key amino acids of the epitope (CN109295005A, invention patent application number: 201811126187.4).
  • the key amino acids K 37 and V 38 of the 2H1 epitope were simultaneously replaced with alanine (A) to construct the eukaryotic expression plasmid pCI with inactivation of the 3B1 epitope -3B1 m 23.
  • A alanine
  • the analysis of the antigenicity of 3B protein by DNAstar software showed that the antigenic determinants were mainly concentrated in the 3B1 and 3B2 parts while the 3B3 part contained less antigenic determinants.
  • 3B1 and 3B2 are important for FMDV replication, deletion will seriously affect FMDV replication, so another 3B3 protein was selected to replace 3B1 and 3B2 proteins; in order to maintain the genetic stability of the virus, to prevent the two 3B3 coding gene sequences from being identical Source recombination, and the 3B3 protein coding sequence added to replace 3B1 and 3B2 was genetically optimized (the optimized gene sequence is shown in SEQ ID NO. 1).
  • the optimized 3B3 coding gene was introduced into the recombinant virus produced by the O-type FMDV infectious clone pYS, and its replication ability was similar to that of its parental strain; then the modified 3B coding sequence was used to construct a eukaryotic expression vector, named pCI-3B33.
  • the eukaryotic expression plasmids pCI-3B1 m 23 and pCI-3B33 expressing the mutant 3B protein and the control plasmid pCI-3B123 (WT) expressing the wild-type 3B protein were transfected into BHK-21 cells, respectively.
  • the indirect immunofluorescence detection results are shown in Figure 1.
  • the wild-type 3B123 protein can bind to the 2H1 antibody and generate a strong specific fluorescent signal
  • the mutant 3B1 m 23 protein can weakly bind to the 2H1 antibody to generate a weak fluorescent signal
  • 3B33 The protein did not bind to the 2H1 antibody at all and did not show any fluorescent signal.
  • the heat pressure screening of the virus is to heat at a specific temperature for 30 minutes to inactivate 99.99% of the virus titer in a unit volume, and repeat the heat inactivation test under the condition of gradually increasing the temperature until the temperature before and after heating. Virus titers were approximately the same. This experiment performed multiple rounds of thermal screening at temperatures of 51°C, 53°C and 56°C until the emergence of highly heat-adapted virus mutants.
  • the proportion of the 146S intact viral capsid remaining was determined, which was a key indicator for judging the thermal stability of the virus 146S antigen.
  • the samples were taken and cooled in an ice bath immediately.
  • the content of viral 146S was determined by sucrose density gradient centrifugation, and the percentage of intact viral 146S capsids remaining after heat inactivation was calculated.
  • this experiment performed multiple rounds of heat stress screening on mutant viruses rO/YS-S2093Y and rA/QSA-Q2093C, and finally obtained O-type FMDV heat-resistant mutants rO/YS ⁇ and A type FMDV thermotolerant mutant rA/QSA ⁇ ( Figures 2A and 2B).
  • the heat-resistant mutant strains rO/YS ⁇ and rA/QSA ⁇ obtained by the screening of heat-resistant stress were successively passaged in BHK-21 cells for 10 generations without pressure for thermal stability test (1h at 56 °C), and the two heat-resistant mutants were tested for thermal stability.
  • the heat tolerance of the tenth generation (P10) virus of the strain did not change from the primary (P0) virus (Fig. 2), indicating that its heat resistant phenotype is genetically stable.
  • the sequence determination of the structural protein coding region of the heat-resistant mutant showed that the P10 virus of the O-type FMDV heat-resistant mutant rO/YS ⁇ was the same as the P0 virus, only containing two amino acid mutations of Y2079H and S2093Y; the heat-resistant mutant rA/YS ⁇
  • the P10 generation virus of QSA ⁇ like the P0 virus, only contains two amino acid mutations A1003T (the 3rd amino acid A of VP1 protein is mutated to T) and N1017H (17th amino acid N of VP1 protein is mutated to H), while the mutant virus has two amino acid mutations.
  • Q2093C has a reverse mutation.
  • pYS and pQSA with O and A FMDV infectious cDNAs, respectively, and performed single and combinatorial mutations at the mutation sites of the heat-tolerant mutants to determine the molecular determinants of O and A FMDV heat tolerance.
  • the results are shown in Figures 2C and 2D, Y2079H mutation alone could not increase the heat resistance of type O FMDV ( Figure 2C), and the heat resistance analysis was not performed because the S2093Y mutation alone could not be stably inherited; however, the combined mutation of Y2079H and S2093Y significantly enhanced the heat resistance.
  • O-type FMDV thermal properties Fig. 2C).
  • thermostable phenotype can be stably inherited.
  • Example 4 Construction and rescue of FMDV avirulent strains carrying thermostable phenotype and negative labelling of non-structural protein 3A&3B epitope deletion, verification of thermostable stability and detection of in vitro growth characteristics
  • the construction of the full-length cDNA infectious clone of the 3A&3B double-molecular-labeled FMDV avirulent strain is as follows: First, using pYS as a template, using 5458(EcoR I)-F and d3AB-1-R primers for PCR amplification, the obtained The PCR product was recovered and purified by gel and used as a template to amplify with primers 5458(EcoR I)-F, d3AB-2-R and d3AB-3-R, and the obtained PCR product was called fragment A; - 2H1-F and 8221(EcoR V)-R amplification, the obtained PCR product is called fragment B; finally, using the purified A and B fragments as templates, primers 5458(EcoR I)-F and 8221(EcoR V) -R was subjected to fusion PCR amplification to produce a fragment of approximately 2.4 Kb in size with deletion of amino acids 84-143 of
  • the genomic cDNAs of FMDV strains O/M98/CHA/2010 and A/JLYS/CHA/2014 were constructed and used as templates, respectively, using P1-F and O/M98P1-R or A/J14P1-R as primers to amplify the
  • the P1 genes of the two strains were mutated for heat resistance.
  • the products were amplified by PCR, recovered and purified by gel, and then cloned into pIRES Bd4 3AB m - digested by Pst I with In-fusion recombinase (Clontech).
  • the recombinants were named pIRES Bd4 3AB m -O/M98 and pIRES Bd4 3AB m -A/J14 after the sequence analysis confirmed that they were correct.
  • the primers used in the present invention are shown in Table 2.
  • the recombinant plasmids pIRES Bd4 3AB m -O/M98 and pIRES Bd4 3AB m -A/J14 were linearized by restriction endonuclease EcoR V, and then transcribed in vitro according to the instructions of RiboMAX TM Large Scale RNA Production Systems-T7 system. It is: 6 ⁇ L of 25mmol/L rNTP, 4 ⁇ L of 5 ⁇ buffer, 2 ⁇ L of T7 RNA polymerase mixture, 8 ⁇ L (2 ⁇ g) of linearized recombinant plasmid, and the total volume is 20 ⁇ L.
  • the reaction mixture After fully mixing the reaction mixture, it was incubated at 37°C for 2.5 h, digested with RNase-Free DNase for 15 min, the DNA template was removed, and the in vitro transcription product was purified by phenol-chloroform extraction.
  • the BHK-21 cells in the 6-well plate grew to 80% to 90% monolayer, wash the cells twice with PBS, and add 1.5 mL of DMEM cell culture medium containing 2% fetal bovine serum.
  • the RNA obtained by in vitro transcription was transfected into BHK-21 cells according to the instructions of QIAGEN's Effectene Transfection Reagent transfection kit for virus rescue. The transfected cells were cultured at 37°C under 5% CO 2 to observe cytopathic changes.
  • the virus was harvested about 3 days later, and then inoculated into BHK-21 cells after repeated freezing and thawing 3 times until the virus could produce stable CPE.
  • the rescued recombinant viruses were identified as rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14, and the strains with correct sequences were verified by full-length genome sequence analysis, which were used for subsequent experiments.
  • Wild-type virus O/YS/CHA/05, FMDV(R4) and recombinant virus rIRES Bd4 3AB m -PanAsia, rIRES Bd4 3AB m -O/M98, and rIRES Bd4 3AB m -A/J14 were inoculated at a dose of 0.05MOI respectively.
  • BHK-21 cells with good logarithmic growth phase were adsorbed at 37°C for 1 h and then washed with PBS to remove unadsorbed viruses, and added DMEM containing 2% fetal bovine serum to maintain the culture at 2h, 4h, 6h, and 8h after inoculation, respectively.
  • the culture supernatant was harvested, and the TCID 50 titers of the viruses harvested at different time points were determined. The determination was repeated 3 times at each time point, and the average value was calculated. Taking the time of virus infection of cells as the abscissa and the logarithm of the TCID 50 titer of the virus at different time points as the ordinate, the growth curve of virus replication was drawn.
  • Recombinant FMDV strains were inoculated into BHK-21 cells at a dose of 0.05MOI, adsorbed at 37°C for 1 h, washed twice with PBS, and maintained in DMEM containing 2% fetal bovine serum. After the cells showed obvious cytopathic changes, freeze-thaw was repeated 3 times, and the supernatant was harvested by centrifugation, and passaged to the 10th passage in BHK-21 cells. Viral RNA was extracted, amplified by RT-PCR and sequenced.
  • BHK-21 cells inoculated with virus were cultured for 12 h, harvested and lysed, and transferred to nitrocellulose membrane after SDS-PAGE electrophoresis.
  • the membrane was blocked with 5% skim milk, incubated with MAb 10B10, 3A10 or 2H1 (1:1000 dilution) as the primary antibody, washed with PBST after 1 h at 37°C, and added with HRP-labeled anti-mouse IgG-antibody (anti-mouse IgG-antibody).
  • IgG-HRP, 1:5000 dilution was treated at 37 °C for 1 h, and ECL luminescent solution was used for color identification.
  • ⁇ -actin antibody (1:1000 dilution) was selected as the internal reference as the primary antibody, and anti-mouse IgG-HRP (1:5000 dilution) was also used as the enzyme-labeled antibody.
  • BHK-21 cells grown in 96-well plates were inoculated with virus, rinsed twice with PBS after 6 h, fixed with absolute ethanol at -20 °C for 15 min, added with 1:100 diluted monoclonal antibody 10B10, 3A10 or 2H1, and incubated at 37 °C Incubate for 60 min in a wet box, wash 5 times with PBS, add FITC-labeled goat anti-mouse IgG (1:100) and incubate in a humid box at 37°C for 45 min, cover with 70% glycerol, and observe under a fluorescence microscope.
  • the present invention is based on the infectious cDNA clone plasmid pFMDV (R4) of FMDV (R4) (Fig. 3A, 3B), as follows
  • the full-length infectious cDNA cloning plasmid of the 3A&3B-labeled avirulent FMDV was obtained and named pIRES Bd4 3AB m -Pst I, and the recombinant virus rescued by this plasmid was named rIRES Bd4 3AB m -PanAsia.
  • the O-type Southeast Asian genotype Mya-98 strain O/M98/CHA/2010 and the A-type Sea-97 genotype G2 subtype strain A were selected according to the current prevalence of foot-and-mouth disease in Asia. /JLYS/CHA/2014, replaced the P1 genes of pIRES Bd4 3AB m -Pst I with their heat-resistant modified P1 genes, respectively, and the constructed recombinant plasmids were named pIRES Bd4 3AB m -O/M98 and pIRES Bd4 3AB m -A/J14.
  • the genomic RNA of the recombinant virus was obtained by in vitro transcription, transfected into BHK-21 cells to rescue the recombinant virus, and named rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/ J14 ( Figure 3A). Furthermore, the three recombinant viruses were continuously passed on for 10 generations on BHK-21 cells, and it was confirmed by nucleotide sequence determination and analysis that the recombinant viruses had a high degree of genetic stability. Importantly, the introduced 3A & 3B molecular markers, the substituted IRES domain 4 and the thermostable modification site can all be inherited stably.
  • the specific monoclonal antibody 3A10 (Chinese Invention Patent Publication No. CN109295006A (Patent Application No.: 201811126938.2)) prepared by the inventors to recognize the marker molecule 3A and a specific monoclonal antibody that recognizes 3B
  • the specific monoclonal antibody 2H1 (Chinese invention patent CN109295005A (patent application number: 201811126187.4)) was used for indirect immunofluorescence detection of 3A&3B labeled virus-infected cells.
  • the epitope recognized by 3A10 is known to be the 5-aa peptide sequence 126 ERTLP 130 located on the FMDV 3A protein (Wang et al., Research in Veterinary Science, 2019; Chinese Patent Application Publication No. CN109293748A (Patent Application No.: 201811126944.8)), Therefore, mAb 3A10 loses reactivity with 126 ERTLP 130 -deleted 3A protein; and the epitope recognized by 2H1 is the 6-aa peptide sequence 34 KPLKVK 39 located on FMDV 3B2 protein (patent application publication number CN109293747A (patent application number: 201811126936.3) ), so mAb 2H1 lost reactivity with the 34 KPLKVK 39 -deleted 3B2 protein.
  • the immunofluorescence detection results are shown in Figure 4B.
  • the 3A&3B labeled viruses like the wild-type strain O/YS/CHA/05, can be detected by the FMDV-shared monoclonal antibody 10B10 (Yang et al. , Archives Virology, 2017; Chinese Invention Patent Application Publication No. CN107177558A (Patent Application No.
  • the thermal stability of the labeled virus was determined using a heat inactivation assay.
  • the cell culture supernatants of wild-type virus and labeled virus with a titer of about 10 7 TCID 50 /ml were heat-inactivated at 42°C for 4 hours, and the intact 146S FMDV particles in the samples after heat treatment were quantitatively determined by FMDV146S sucrose density gradient centrifugation. proportion.
  • 9 pigs were randomly selected and divided into 3 groups, with 3 pigs in each group: in the first group, the wild-type strain FMDV O/YS/CHA/05 was intradermally inoculated on the heels of pigs at a dose of 10 5 TCID 50 per head; In three groups, pig trotters were intradermally inoculated with wild-type strain O/M98/CHA/2010 or A/JLYS/CHA/2014 at a dose of 10 7 TCID 50 /head.
  • the remaining 25 pigs were randomly divided into 5 groups of 5 pigs, and each group of pigs was inoculated with 10 7 TCID 50 /head or 10 8 TCID 50 /head dose of FMDV(R4), 10 9 TCID 50 /head dose in each group of pigs
  • the body temperature of the inoculated pigs was measured, clinical manifestations were observed, and nasal swabs, oral swabs and blood were collected.
  • Virus rIRES Bd4 3AB m-O/PanAsia with IRES domain 4 replaced, thermostable mutation, 3B-tagged virus rIRES Bd4 3AB m -O/PanAsia is not pathogenic to pigs
  • thermostable mutation, 3B-tagged virus rIRES Bd4 3AB m -O/PanAsia is not pathogenic to pigs
  • pigs 15#, 16#) were intradermally inoculated into the hoof with a dose 10,000 times higher than the wild-type virus titer (10 9 TCID 50 /head).
  • rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 virus rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 have no pathogenicity to pigs, cattle and sheep.
  • Pigs (21#, 22# and 23#; or 31#, 32# and 33#) were first inoculated with wild-type virus O/M98/CHA/2010 or A/JLYS/CHA/2014 at a dose of 10 7 TCID 50 /head ) as a control, after inoculation 48h all appear body temperature rise and typical foot-and-mouth disease symptoms, in 3-4 days after inoculation, the content of viral RNA in pig blood and mouth and nasal swabs is significantly higher than that of healthy pigs (2.6 log 10 viral RNA). CN/ml), indicating that severe viremia had occurred at this time, producing a high load of oral and nasal excretion, and induction of higher titers of neutralizing antibodies 10 days after vaccination.
  • the marker viruses rIRES Bd4 3AB m -O/M98 or rIRES Bd4 3AB m -A/J14, with IRES domain 4 replaced, were inoculated by the most sensitive intradermal route of the heel at a very high dose of 10 9 TCID 50 per head.
  • Pigs 24#, 25#, 26#, 27#, 28#; or 34#, 35#, 36#, 37#, 38#
  • Blood and oral and nasal swab samples collected every day after vaccination were all negative for viral RNA (copy number was lower than 2.6 log 10 viral RNA CN/ml), and no virus neutralizing antibody was produced during the observation period ( ⁇ 1:8).
  • viruses rIRES Bd4 3AB m -O/M98 or rIRES Bd4 3AB m -A/J14 carrying a thermostable phenotype and 3B marker with IRES domain 4 replaced using 100 times the wild-type pathogenic virus titer (10 9 TCID 50 /head) inoculated cattle (04#, 05#, 06#, 07 and 08#; or 14#, 15#, 16#, 17# and 18#) via the most sensitive intradermal route of the tongue.
  • wild-type pathogenic virus titer 10 9 TCID 50 /head
  • inoculated cattle 04#, 05#, 06#, 07 and 08#; or 14#, 15#, 16#, 17# and 18#
  • there were no clinical manifestations of FMD no elevated body temperature, no viremia, no oral and nasal detoxification (Table 3 and Table 4), and no virus-neutralizing antibody production ( ⁇ 1:8).
  • viruses rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 which carry a thermostable phenotype and 3B marker with IRES domain 4 replaced, do not replicate in cattle and cannot establish infection, therefore Cattle completely lose their pathogenicity.
  • viruses rIRES Bd4 3AB m -O/M98 or rIRES Bd4 3AB m -A/J14 carrying a thermostable phenotype and a 3B marker with IRES domain 4 replaced by the most sensitive hoof-crown intradermal route showed a 100-fold increase in wild-type Sheep (05 # , 06#, 07#, 08# and 09#; or 16#, 17#, 18#, 19# and 20 #) , during the 10-day observation period, there were no clinical manifestations of FMD and elevated body temperature, no viremia, no oral and nasal detoxification (Table 3 and Table 4), and no virus-neutralizing antibody production ( ⁇ 1:8).
  • viruses rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 which carry a thermostable phenotype and 3B marker with IRES domain 4 replaced, do not replicate in sheep and cannot establish infection, therefore Complete loss of pathogenicity to sheep.
  • BHK-21 cells were cultured in suspension in an Erlenmeyer flask, and when they grew to 3.5 ⁇ 10 6 cells/ml, the recombinant virus rIRES Bd4 3AB m -O/M98 or rIRES Bd4 3AB m -A/J14 was inoculated at 0.01 MOI at 37°C
  • the virus was harvested after 16 h of suspension culture, and the virus titer was determined by TCID 50 analysis after repeated freezing and thawing three times.
  • the virus was diluted to 10 9.25 TCID 50 /ml, BEI (final concentration 2 mmol/L) was added, inactivated at 30° C.
  • the virus strain was prepared into a 3A/3B-labeled, heat-resistant and stable capsid foot-and-mouth disease virus O/A bivalent inactivated vaccine, which was stored at 4°C until use.
  • 20 heads were randomly selected and divided into 2 groups, 10 heads in each group: one group was inoculated with (2ml/head) O/A bivalent inactivated vaccine (rIRES Bd4 3AB m -O/M98 strain + rIRES Bd4 3AB strain in the neck muscle behind the ear) m -A/J14 strain), another group was inoculated (2ml/head) with the commercial O/A bivalent inactivated vaccine (Re-O/MYA98/JSCZ/2013 strain + Re-A/WH strain) /09 strain, Zhongnong Wit), and the remaining 6 pigs were inoculated with PBS as challenge control.
  • the two vaccine experimental groups were randomly divided into 2 groups, 5 immunized pigs were selected in each group, and 3 PBS-inoculated control pigs were added to each group, so that the neck muscles of 8 pigs in each group were inoculated with 2ml 10 8.5 TCID 50 /ml dose of O/M98/CHA/2010 strain or A/JLYS/CHA/2014 strain.
  • body temperature was measured, clinical manifestations were observed, and nasal swabs, buccal swabs and blood were collected.
  • the collected nasal swabs, oral swabs and serum samples were used to extract total RNA by TRIZOL method.
  • the cDNA obtained by reverse transcription with Oligo (dT 15 ) primer was used as the template, and the FMDV-specific primer (3DF: 5'GGA TGC CGT CTG was used as the template).
  • GTT GTT 3'; 3DR: 5'CGT AGG AGA TCA TGG TGT AAG AGT 3') was detected by fluorescence quantitative PCR.
  • the specific operation of fluorescence quantitative PCR is as follows Green qPCR Super Mix-UDG with ROX (Invitrogen) kit instructions were performed, and the content of viral genomic RNA in the sample was calculated by the standard curve function. Serum and mouth and nose from healthy pigs
  • PCR-amplified FMDV RNA copy number of swab samples 2.6 The log10 was taken as the background value, and the viral RNA copy number/ml (viral RNA CN/ml) was higher than this value to be judged as FMDV RNA positive.
  • the micro-cell neutralization test was carried out by the method of diluting serum of fixed virus. First, the TCID 50 of FMDV was measured with BHK-21 cells; then, the serum was inactivated at 56 ° C for 30 min, and doubling dilution was done with PBS; the virus of 100 TCID 50 was mixed with equal volumes of serum diluted with different dilutions, and the temperature was 37 ° C.
  • modified viruses rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 were inactivated by BEI. It was mixed with equal volume of ISA 201VG adjuvant to prepare O/A type foot-and-mouth disease bivalent inactivated vaccine, and commercialized O/A bivalent inactivated vaccine (Zhongnong Wit) was used as the vaccine control, respectively inoculated 10 pigs, and the other 6 pigs.
  • Pigs were inoculated with PBS as a negative control, and the results are shown in Tables 5 and 6.
  • O-type and A-type FMDV-specific neutralizing antibodies could be detected at 7 days after vaccination (dpv), and the level of neutralizing antibodies reached a peak at 21 days after vaccination (1:256).
  • the O/A type foot-and-mouth disease bivalent inactivated vaccine inoculated pigs (201#-210#) of the present invention and the commercial bivalent inactivated vaccine inoculated pigs (101#-110#) induce a similar level of FMDV neutralizing antibodies, but No FMDV neutralizing antibodies ( ⁇ 8) were detected in animals inoculated with PBS (301#-306#). Moreover, none of the 26 pigs had side effects after vaccination.
  • the O/A foot-and-mouth disease bivalent inactivated vaccine prepared with the modified avirulent strain of FMDV in this experiment like the commercial bivalent inactivated vaccine, the vaccinated animals have no clinical FMD after challenge with O or A FMDV. performance and immune protection (Table 5, Table 6).
  • the FMDV avirulent strains rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A/J14 carrying heat-resistant phenotype and 3B marker created by the present invention have excellent immunogenicity, and inoculated animals can induce high
  • the level of neutralizing antibodies can effectively resist the challenge of parental virulent strains, and can be used as a seed virus for the production of foot-and-mouth disease inactivated vaccines.
  • the invention uses the established FMDV 3B monoclonal antibody blocking ELISA antibody detection method, and completely eliminates the 3B epitope (the short peptide located in 3B2) recognized by the 2H1 monoclonal antibody through the deletion of the main antigen regions 3B1 and 3B2 of the 3B protein of the FMDV avirulent strain.
  • the blocking ELISA method established in the present invention cannot detect the animals immunized with the virus inactivated vaccine, thereby eliminating the interference of the residual 3B protein in the vaccine to the differential diagnosis.
  • this experiment used this 3B deletion negative marker FMDV O/A bivalent inactivated vaccine to inoculate pigs, cattle and sheep with 2 times the dose (4ml/head) respectively. , 4 consecutive immunizations at 28-day intervals, the serum was collected 30 days after the last immunization, and the production and level of non-structural protein 3B antibodies were evaluated by a blocking ELISA method based on the 3B protein monoclonal antibody 2H1.
  • any FMDV strains with 3B deletion markers constructed by the platform established by the present invention include the modified viruses rIRES Bd4 3AB m -PanAsia, rIRES Bd4 3AB m -O/M98 and rIRES Bd4 3AB m -A obtained by the present invention /J14, using it as a seed virus to produce an inactivated foot-and-mouth disease vaccine to immunize animals can achieve differential diagnosis between vaccinated animals and naturally infected animals.

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Abstract

提供了FMDV病毒cDNA感染性克隆质粒、耐热表型稳定遗传、携带负标记的重组口蹄疫病毒无毒株及O/A型口蹄疫二价灭活疫苗。该质粒携带有病毒衣壳耐热表型的分子决定因素、在体内失去复制能力的分子因素、3A和3B蛋白表位缺失的负标记因素及在P1编码区两侧引入Pst I酶切位点，可针对任何流行株替换其衣壳蛋白编码区快速构建并拯救热稳定而且被标记的口蹄疫病毒无毒株，用作口蹄疫灭活疫苗种子病毒。该二价灭活疫苗接种动物后，能诱导高水平中和抗体并产生免疫保护，进行抗体检测可实现疫苗接种动物与自然感染动物的鉴别诊断。

Description

耐热表型稳定遗传、携带负标记的重组口蹄疫病毒无毒株及O/A型口蹄疫二价灭活疫苗 技术领域
本发明涉及病毒全长cDNA感染性克隆质粒以及用该全长cDNA感染性克隆质粒拯救获得的重组病毒株，尤其涉及FMDV全长cDNA感染性克隆质粒以及采用反向遗传技术拯救获得的耐热表型稳定遗传、携带3B表位负标记的重组口蹄疫病毒无毒株，本发明进一步涉及该重组口蹄疫病毒无毒株在制备O/A型口蹄疫二价灭活疫苗或者在制备鉴别诊断口蹄疫疫苗接种动物与自然感染动物的试剂中的用途，属于衣壳稳定携带标记重组口蹄疫病毒无毒株的构建及应用领域。
背景技术
口蹄疫(Foot-and-Mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)引起的，主要危害猪、牛、羊等偶蹄动物的一种急性、热性、高度接触性传染病(Grubman and Baxt.2004.Clinical Micro.Rev.17:465-493)。该病在亚洲、非洲和中东的许多国家流行，防疫的财政负担、疾病爆发带来的损失以及国际贸易的限制对流行国家经济社会的影响均是巨大的，无疫国家传入该病常导致灾难性后果。考虑到FMD的高度传染性、对流行国家造成的严重危害以及对无疫国家的巨大威胁，它在国际上被称为政治经济病，历来受到各国政府的高度重视，OIE曾将其列在一类动物传染病的首位。
FMDV属于微RNA病毒科、口蹄疫病毒属的成员，共有七个血清型(A、O、C、Asia1、SAT1、SAT2和SAT3)和数十个亚型，各血清型毒株之间无免疫交叉保护、各亚型毒株之间仅存在部分交叉保护。该病毒基因组为单股正链RNA，全长约为8.5kb，由5'非编码区(5'UTR)、开放阅读框(ORF)和3'UTR组成，其ORF共编码4个结构蛋白(VP4、VP2、VP3和VP1)和8个非结构蛋白(L、2A、2B、2C、3A、3B、3C和3D)。
灭活疫苗的免疫接种是流行国家口蹄疫防控的有效手段，无疫国家突然爆发时也常采用环形免疫以减少动物宰杀数量过大造成的损失。然而，口蹄疫灭活疫苗在生产和使用过程中存在四个主要问题，亟待解决：一是，使用强毒株作为生产灭活疫苗的种毒，存在工厂泄露而散毒的风险，一经发生将带来严重后果；二是，病毒发生变异或新的流行毒株传入，会突破原来的免疫屏障导致免疫失败，需要及时更换生产疫苗的种毒，而对于强毒株来说这种更换是受限制且耗时的；第三，口蹄疫病毒146S衣壳易解聚，从而影响疫苗的免疫保护效力以及疫苗的保质期，已有报告通过人工突变提升耐热性的方法可以改善口蹄疫病毒衣壳的稳定性(Abhay Kotecha et al.,nature structural&molecular biology,2015)，但其耐热表型是不稳定的(Katherine A.Scott et al.,Journal of Virology,2016)；第四，在疫苗制备过程中，口蹄疫病毒的3A、3B等非结构蛋白难以清除干净导致在疫苗中残留，在动物经历多次免疫接种后会诱导产生特异性抗体，使得以非结构蛋白抗体检测为基准的鉴别诊断技术难以区分疫苗接种动物与自然感染动物，这就给口蹄疫免疫防控计划的实施以及活畜贸易中的检疫带来了难题。
有关FMDV减毒的研究，在过去60年里世界上有许多国家做出了巨大努力，虽然取得一些重要进展但一直没能取得成功。无论是用传统的传代适应技术还是用现代的遗传工程技术，FMDV减毒研究均遇到了一个无法克服的障碍，即：产生的减毒表型受动物种属的局限、没能找到对三种主要偶蹄动物猪、牛、羊均减毒的有效方法和途径。已有的口蹄疫病毒基因工程减毒研究主要集中在病毒的非结构蛋白L、3A和3D上，但是均没能解决这一难题。
发明内容
本发明的目的之一是提供一个FMDV cDNA感染性克隆质粒通用平台以及采用该反向遗传操作平台构建并拯救得到的在易感动物体内不复制以及具有3A和3B蛋白表位负标记的重组口蹄疫病毒无毒株；
本发明的目的之二是提供O型FMDV全长cDNA感染性克隆质粒以及由该cDNA感染性克隆质粒采用反向遗传技术拯救得到的耐热表型稳定遗传、在易感动物体内不复制以及具有3A和3B蛋白表位负标记的O型重组FMDV无毒株；
本发明的目的之三是提供A型FMDV全长cDNA感染性克隆质粒以及由该cDNA感染性克隆质粒采用反向遗传技术拯救得到的耐热表型稳定遗传、在易感动物体内不复制以及具有3A和3B蛋白表位负标记的A型重组FMDV无毒株；
本发明的目的之四是提供防治O/A型口蹄疫的二价灭活疫苗。
本发明的上述目的是通过以下技术方案来实现的：
本发明首先提供了FMDV全长cDNA感染性克隆质粒，其中，将FMDV基因组IRES的结构域4用牛鼻病毒的IRES的结构域4替换，缺失FMDV病毒非结构蛋白3A的84-143位氨基酸的编码区，用FMDV的3B3蛋白的编码区取代FMDV的3B1和3B2蛋白的编码区以及在FMDV结构蛋白P1编码区两侧引入Pst I酶切位点。
为了保持病毒的遗传稳定性，防止两个3B3编码序列发生同源重组，本发明对用来替换3B1和3B2的3B3蛋白的编码序列进行了密码子优化，其优化后的核苷酸序列为SEQ ID NO.1所示。
作为本发明一种优选的具体实施方式，所述FMDV全长cDNA感染性克隆质粒的核苷酸序列为SEQ ID NO.2所示。
本发明进一步利用FMDV全长cDNA感染性克隆质粒采用反向遗传方法拯救得到重组口蹄疫病毒无毒株，该重组FMDV无毒株携带有耐热衣壳、在易感动物体内不复制以及具有3A和3B表位负标记的特性。
所述的利用FMDV全长cDNA感染性克隆质粒采用反向遗传方法拯救得到重组口蹄疫病毒无毒株的方法包括：将该FMDV全长cDNA感染性克隆质粒线性化后经体外转录获得重组病毒的基因组RNA，转染细胞拯救出重组FMDV；其中，所述的细胞可以是BHK-21细胞。
进一步的，本发明提供了O型FMDV全长cDNA感染性克隆质粒，其中，将所述的FMDV全长cDNA感染性克隆质粒的结构蛋白P1编码区用O型FMDV的结构蛋白P1编码区替换；其中，将O型FMDV的结构蛋白VP2的第79位的酪氨酸(Y)突变为组氨酸(H)以及将第93位的丝氨酸(S)突变为酪氨酸(Y)。
作为一种优选的具体实施方式，所述的O型FMDV感染性全长cDNA克隆质粒的核苷酸序列为SEQ ID NO.3所示。
本发明进一步利用该O型FMDV全长cDNA感染性克隆质粒采用反向遗传方法拯救得到O型重组FMDV无毒株，该O型重组FMDV无毒株具有遗传稳定的耐热表型、在易感动物体内不复制以及具有3A和3B表位负标记的特性。
所述的利用O型FMDV全长cDNA感染性克隆质粒采用反向遗传方法拯救得到重组O型FMDV无毒株的方法包括：将该O型FMDV全长cDNA感染性克隆质粒线性化后经体外转录获得重组病毒的基因组RNA，转染细胞拯救出重组O型FMDV病毒；其中，所述的细胞可以是BHK-21细胞。
进一步的，本发明提供了A型FMDV全长cDNA感染性克隆质粒，其中，将所述的FMDV全长cDNA感染性克隆质粒的结构蛋白P1编码区用A型FMDV的结构蛋白P1编码区替换；其中，将A型FMDV的结构蛋白VP1的第3位的丙氨酸(A)突变为苏氨酸(T)以及将第17位的天冬酰胺(N)突变为组氨酸(H)。
作为一种优选的具体实施方式，所述的A型FMDV全长cDNA感染性克隆质粒的核苷酸序列为SEQ ID NO.4所示。
本发明进一步利用该A型FMDV全长cDNA感染性克隆质粒拯救得到A型重组FMDV无毒株，该A型重组FMDV无毒株具有遗传稳定的耐热表型、在易感动物体内不复制以及具有3A和3B表位负标记的特性。
所述的利用A型FMDV全长cDNA感染性克隆质粒采用反向遗传方法拯救得到重组A型FMDV无毒株的方法包括：将该A型FMDV全长cDNA感染性克隆质粒线性化后经体外转录获得重组病毒的基因组RNA，转染细胞拯救出重组A型FMDV；其中，所述的细胞可以是BHK-21细胞。
本发明还提供了一种构建衣壳耐热表型稳定遗传的重组O型口蹄疫病毒突变株的方法，包括，将O型FMDV的结构蛋白VP2的第79位的氨基酸由酪氨酸(Y)突变为组氨酸(H)以及将第93位的氨基酸由丝氨酸(S)突变为酪氨酸(Y)；由该方法构建得到的重组O型FMDV突变株的衣壳耐热表型能够进行稳定遗传。
本发明进一步提供了一种构建衣壳耐热表型稳定遗传的重组A型FMDV突变株的方法，包括，将A型FMDV的结构蛋白VP1的第3位的氨基酸由丙氨酸(A)突变为苏氨酸(T)以及将第17位的氨基酸由天冬酰胺(N)突变为组氨酸(H)；由该方法构建得到的重组A型FMDV突变株的衣壳耐热表型也能够稳定遗传。
本发明更进一步提供了所述的重组FMDV毒株在以下各方面的用途，包括：
应用所述重组FMDV无毒株制备防治口蹄疫的药物或者用于制备鉴别诊断疫苗接种动物与 自然感染动物的试剂中的用途；
应用所述重组O型FMDV无毒株制备防治O型口蹄疫的药物或者用于制备鉴别诊断疫苗接种动物与自然感染动物的试剂中的用途；
应用所述的A型重组FMDV无毒株制备防治A型口蹄疫的药物或者用于制备鉴别诊断疫苗接种动物与自然感染动物的试剂中的用途；
同时应用所述重组O型FMDV无毒株和所述A型重组FMDV无毒株制备得到防治O型口蹄疫和A型口蹄疫的二价灭活疫苗；
其中，本发明所述二价灭活疫苗还可包含药学上可接受的赋形剂，药学上可接受的赋形剂可为作为媒介物、佐剂、载体或稀释剂的功能分子。药学上可接受的赋形剂可为转染促进剂，包括表面活性剂，如免疫刺激复合物(ISCOMS)、弗氏(Freunds)不完全佐剂、LPS类似物(包括单磷酰脂质A)、胞壁酰肽、醌类似物、角鲨烯(squalene)、角鲨烯(squalene)、透明质酸、脂质、脂质体、钙离子、病毒蛋白、聚阴离子、聚阳离子或纳米颗粒等；
可通过不同途径包括口服、胃肠外、舌下、经皮肤、经直肠、经粘膜、局部、通过吸入、通过颊部施用、胸膜内、静脉内、动脉内、腹膜内、皮下、肌内、鼻内、鞘内以及关节内或其组合施用本发明的疫苗。对于兽医学使用，可按照正常的兽医学操作将疫苗制备成可接受的制剂施用。兽医可容易地确定最适合于特定动物的给药方案和施用途径。可利用传统注射器、无针注射装置等施用本发明二价灭活疫苗。
本发明概述
本发明人的前期研究发现，将FMDV IRES结构域4(IRES-Fd4)替换为牛鼻病毒(BRBV)IRES结构域4(IRES-Bd4)产生的IRES嵌合病毒FMDV(R4)，以10 6TCID 50/头剂量颈部肌肉接种自然宿主猪，不表现任何口蹄疫临床症状、不产生病毒血症、口腔鼻腔不排毒，而且该病毒在猪体内也不诱生抗体，这表明IRES嵌合病毒FMDV(R4)在易感动物体内失去复制能力(CN108085302A,WO/2018/090994)。为进一步评价FMDV(R4)对猪的致病力，本发明采用最敏感的蹄踵皮内接种途径、使用更高的FMDV(R4)接种剂量(10 7TCID 50/头或10 8TCID 50/头)并以相同途径接种10 5TCID 50/头剂量的野生型病毒O/YS/CHA/05作为强毒对照，超高剂量、最敏感途径的接种试验结果表明，FMDV(R4)在猪体内不复制、不能够建立感染、因此对猪完全失去致病力。
为了构建可用于鉴别诊断的分子标记病毒，本发明对在3B蛋白上的分子标记方式进行了比较研究；比较结果表明，用一个经过基因优化的3B3蛋白(该优化后的编码基因的核苷酸序列为SEQ ID NO.1所示)替换3B1和3B2蛋白去除3B蛋白的主要抗原性区域以实现对口蹄疫病毒的分子负标记再用单抗2H1建立的抗体检测ELISA方法进行疫苗接种动物与自然感染动物的鉴别诊断，是可行的方法和策略。
Abhay Kotecha等人发现S2093Y(VP2蛋白的第93位氨基酸S突变为Y)突变能显著增加FMDV衣壳的耐热稳定性(Abhay Kotecha et al.,nature structural&molecular biology,2015)。随后Katherine A.Scott等人发现，S2093Y突变作为SAT2型FMDV耐热表型的分子决定因素不能稳定遗传(Katherine A.Scott et al.,Journal of Virology,2016)。有鉴于此，本发明用O型和A型FMDV的全长cDNA感染性克隆质粒pYS和pQSA分别构建并拯救了VP2蛋白的第93位氨基酸定点突变的耐热突变株rO/YS-S2093Y和rA/QSA-Q2093C，发现这些突变株的耐热表型同样也是不稳定的，在体外传代过程中病毒的耐热性能逐渐减弱，传至第5代时耐热特性全部消失；对不同代次的传代病毒进行序列测定发现，突变株rO/YS-S2093Y在传至第3代时S2093Y突变为S2093H，突变株rA/QSA-Q2093C在传至第5代时Q2093C发生了回复突变，因此导致突变株的耐热表型消失。本发明在O型和A型FMDV进行的突变研究支持Katherine A等人在SAT2型FMDV的研究结果，进一步表明Abhay Kotecha等人发现的VP2蛋白的第93位氨基酸作为耐热表型分子决定因素不能够稳定遗传。
为了获得遗传稳定的FMDV耐热突变株，本发明对突变病毒rO/YS-S2093Y和rA/QSA-Q2093C进行多轮热压力筛选，最终获得了O型FMDV耐热突变株rO/YSδ和A型FMDV耐热突变株rA/QSAδ。将经耐热压力筛选获得的耐热突变株rO/YSδ和rA/QSAδ分别在BHK-21细胞中无压力连续传10代进行耐热表型稳定性检测(56℃作用1h)表明，两个耐热突变株的第10代(P10)病毒与原代(P0)病毒的耐热性没有发生变化，表明其耐热表型具有遗传稳定性。对耐热突变株的结构蛋白编码区进行序列测定显示：O型FMDV耐热突变株rO/YSδ的P10代病毒和P0病毒一样，只含有Y2079H和S2093Y 2个氨基酸突变；耐热突变株rA/QSAδ的P10代 病毒和P0病毒一样，仅含有A1003T(VP1蛋白的第3位氨基酸A突变为T)和N1017H(VP1蛋白的第17位氨基酸N突变为H)2个氨基酸突变，而突变病毒的Q2093C发生了回复突变。分别用O型和A型FMDV感染性cDNA克隆pYS和pQSA，对耐热突变株的突变位点进行单一突变和组合突变以确定决定O型和A型FMDV耐热的分子决定因素。结果发现，Y2079H单独突变不能增加O型FMDV的耐热特性，由于S2093Y单独突变不能稳定遗传因此未进行耐热性分析；然而，Y2079H和S2093Y组合突变显著增强O型FMDV的耐热特性。A1003T或N1017H单独突变不能增加A型FMDV的耐热特性，而二者的组合突变却显著增强A型FMDV的耐热特性。由此，本发明发现，Y2079H突变虽然不产生耐热表型但可协同S2093Y耐热突变稳定遗传，因此协同决定O型FMDV的衣壳耐热稳定性表型；A1003T和N1017H的组合突变协同决定A型FMDV的衣壳耐热稳定性表型并可稳定遗传。
基于本发明人上述的研究结果，本发明进一步以IRES结构域4被替换的FMDV感染性克隆质粒为基础，构建了带有3A和3B表位负标记以及衣壳耐热表型的通用质粒pIRES Bd43AB m-Pst I，可针对任何流行毒株创制携带分子标记而且衣壳耐热表型稳定的重组FMDV无毒株。
为了构建带有可鉴别诊断标记的FMDV无毒株作为口蹄疫灭活疫苗种子病毒，本发明以FMDV(R4)的感染性cDNA克隆质粒pFMDV(R4)为基础进行如下的基因工程操作：缺失非结构蛋白3A的84-143位氨基酸，用一个编码序列密码子优化的3B3取代3B1和3B2、导致3B1/3B2的缺失和3B3的重复，由此引入3A&3B双分子标记；同时，在结构蛋白编码区两侧引入酶切位点Pst I，用于替换任何其他FMDV毒株的结构蛋白P1的编码基因，同时在结构蛋白编码区引入决定病毒衣壳稳定性的突变，由此获得3A&3B标记的衣壳稳定的FMDV无毒株的全长cDNA感染性克隆质粒，命名为pIRES Bd43AB m-Pst I，由该质粒拯救的重组病毒命名为rIRES Bd43AB m-PanAsia。在此感染性cDNA克隆平台上，根据当前亚洲地区口蹄疫的流行情况，选择O型东南亚基因型Mya-98毒株O/M98/CHA/2010和A型Sea-97基因型G2亚型毒株A/JLYS/CHA/2014，用它们经耐热性修饰的P1基因分别替换pIRES Bd43AB m-Pst I的P1基因，构建的重组质粒分别命名为pIRES Bd43AB m-O/M98和pIRES Bd43AB m-A/J14。这些重组质粒经DNA序列测定验证正确后，经体外转录获得重组病毒的基因组RNA，转染BHK-21细胞拯救出重组病毒，分别命名为rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14。进而在BHK-21细胞上将这3个重组病毒连续传10代，经核苷酸序列测定与分析证实，重组病毒具有高度的遗传稳定性。重要的是，所引入的3A&3B分子标记、置换的IRES结构域4以及耐热表型修饰位点均可以稳定遗传。
本发明将构建的重组标记病毒与野生型病毒O/YS/CHA/05和嵌合病毒FMDV(R4)的体外生长特性进行比较，结果发现，含有3A&3B双分子标记的重组病毒rIRES Bd43AB m-PanAsia、rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14在BHK-21细胞上的复制动力学相似，可是这些重组标记病毒复制达到高峰的时间较野生型病毒推迟约4小时。为了对重组标记病毒的3A&3B双分子标记进行功能性验证，采用本发明人以前制备的识别标记分子3A的特异性单克隆抗体3A10和识别3B的特异性单克隆抗体2H1对3A&3B标记病毒感染的细胞进行间接免疫荧光检测。免疫荧光检测结果显示，在病毒感染的BHK-21细胞中，3A&3B标记病毒与野生型毒株O/YS/CHA/05一样均能够被FMDV共享型单抗10B10(Yang et al.,Archives Virology,2017；CN107177558A)所识别；然而，识别3B蛋白的单抗2H1和识别3A蛋白的单抗3A10仅能识别野生型病毒O/YS/CHA/05，而不识别3A&3B负标记的重组病毒rIRES Bd43AB m-PanAsia、rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14，这从功能上表明本发明重组标记病毒的3A&3B分子负标记已被成功地引入；同时，用这些单抗对病毒感染细胞进行的Western blot分析也证实了免疫荧光检测结果，表明3A&3B表位负标记的FMDV失去了对单抗3A10和2H1的反应性。
本发明进一步用热灭活试验测定本发明制备的重组标记病毒的热稳定性表型，测定结果表明，经耐热修饰的重组标记病毒具有显著的衣壳耐热稳定性特征。
为了分析携带耐热表型的重组标记病毒对易感动物的毒力，本发明用高于野生型病毒WT致病剂量(10 5TCID 50/头)10000倍的rIRES Bd43AB m-O/PanAsia病毒剂量(10 9TCID 50/头)于蹄部皮内接种猪进行致病力评价；结果表明，重组标记病毒rIRES Bd43AB m-O/PanAsia在猪体内没有复制，不能够建立感染，因此证明该携带耐热表型的重组标记病毒对猪无致病力。
为检测IRES结构域4被替换的携带耐热表型和3A&3B分子负标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14的毒力，本发明分别在猪、牛、羊三种易感动物上采用高剂量和敏感途径对这两株重组标记病毒的致病力进行评价；结果表明，IRES结构域4被替换的携带耐热表型和3A&3B标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14在猪、 牛、羊体内均丧失复制能力、因此完全失去致病力，因而是FMDV无毒株。这些结果也表明，用本发明构建的通用感染性克隆质粒平台pIRES Bd43AB m-Pst I可针对一个国家或地区的FMDV优势流行毒株快速创制携带耐热表型和3A&3B负标记的无毒株，用作灭活疫苗生产的种子病毒。
为了评价携带耐热表型、3A&3B标记的FMDV无毒株制备的灭活疫苗的免疫保护效力，本发明将重组标记病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14经BEI灭活后与ISA 201VG佐剂等体积混合配制成O/A二价灭活疫苗，并用商业化O/A二价灭活疫苗(中农威特)作为疫苗对照，分别接种10头猪，另外6头猪接种PBS作为阴性对照；免疫保护效力试验结果显示，所有接种本发明二价灭活疫苗的动物在接种后7天(dpv)可检出O型和A型FMDV特异性中和抗体，接种后21天中和抗体水平达到高峰(1：256)。本发明的二价灭活疫苗与商业化二价灭活疫苗诱导猪产生FMDV中和抗体的水平相近、但接种PBS的动物未检出FMDV中和抗体，而且，26头猪在接种后均没有出现副反应。在免疫接种后28天，将免疫的动物分成两组，分别用O型和A型同源野生型FMDV毒株进行攻击，观察免疫保护的效果。接种PBS的6头对照猪分成2组每组3头，在接种10 8.5TCID 50/头剂量的O型FMDV O/M98/CHA/2010或A型FMDV A/JLYS/CHA/2014后1-3天，均出现典型的FMD临床症状。然而，本发明的FMDV无毒株制备的二价灭活疫苗同商品化二价灭活疫苗一样，接种动物在用O型或A型FMDV攻击后均无FMD临床表现、获得免疫保护。因此，本发明创制的携带耐热表型和3A&3B标记的FMDV无毒株rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14具有优良的免疫原性，接种动物可诱导产生高水平的中和抗体、能有效抵抗亲本强毒株的攻击，可作为生产口蹄疫灭活疫苗的种子病毒。
综上，本发明构建并拯救安全、稳定、负标记的口蹄疫灭活疫苗种子病毒的感染性cDNA克隆质粒pIRES Bd43AB m-Pst I，其携带有病毒衣壳耐热表型的分子决定因素、病毒在体内失去复制能力表型的分子决定因素以及病毒非结构蛋白3A&3B表位缺失的负标记元素，而且在病毒结构蛋白P1编码区两侧引入Pst I酶切位点用于快速克隆置换其他任一口蹄疫病毒株的结构蛋白编码区。用此通用质粒针对当前两个优势流行株的衣壳蛋白编码区进行替换构建的重组病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14，具有如下特征：在BHK-21细胞上复制正常；经耐热修饰后病毒衣壳的稳定性显著增强；用高剂量(10 9TCID 50/头)病毒经敏感途径接种猪、牛和羊均不发生感染；由此制备的种子病毒具有如下的优良特征：不但解决了口蹄疫灭活疫苗生产中工厂散毒的安全性隐患，由于疫苗生产过程无需高标准的生物安全防护、也大大降低了生产成本；对病毒衣壳进行的耐热性修饰，大大增加了146S衣壳的稳定性，从而有利于灭活疫苗免疫原性的维持以及配制的成品疫苗的保存、提升疫苗的质量、降低疫苗的成本；用配套的3B单抗阻断ELISA方法检测免疫接种动物的血清，可实现疫苗接种动物与自然感染动物的鉴别诊断，可满足口蹄疫免疫防控计划中清群和净化的需求。
本发明用这两个重组病毒作为种子病毒制备O/A二价灭活疫苗，免疫动物可诱导高水平的中和抗体、用同源性毒株攻击均获得免疫保护；用3A和3B单抗建立的阻断ELISA方法对该疫苗接种动物进行检测，可实现对疫苗接种动物与自然感染动物的鉴别诊断。本发明提供的安全、稳定、携带负标记的FMDV无毒株制备方法可针对任何FMDV流行毒株在2周内快速创制灭活疫苗的种子病毒，用于制备安全、稳定、可鉴别诊断的FMDV灭活疫苗，可为全球口蹄疫的预防、控制和净化提供安全有效而且实用的技术手段。
附图说明
图1免疫荧光试验检测FMDV非结构蛋白3B突变株与单抗2H1的结合能力。
图2FMDV热稳定突变株的筛选及其分子决定因素的确定。
图3 3A&3B标记的FMDV无毒株的构建示意图。
图4重组FMDV毒株3A&3B标记以及耐热表型的验证及其体外生长特性。
图5由3A&3B标记、耐热的重组FMDV无毒株制备的O/A型口蹄疫二价灭活疫苗、商品化灭活二价疫苗接种动物的3B抗体应答以及单抗阻断ELISA鉴别诊断结果。
具体实施方式
以下结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
试验材料
细胞、毒株和抗体：BHK-21细胞在含5％CO 2条件下于37℃培养，培养液为含有10％FBS的DMEM。O型FMDV O/YS/CHA/05毒株(GenBank登录号：HM008917)，其感染性cDNA克隆pYS见中国发明专利(CN101838658A(专利申请号201010160669.9))。FMDV(R4)感染性cDNA克隆pFMDV(R4)见中国发明专利(CN108085302A(申请号201711093774.3))和国际PCT专利(国际公布号：WO/2018/090994A1)。A/QSA/CHA/2009，其与参考毒株A/HuBWH/CHA/2009(GenBank登录号：JF792355)的VP1基因的核苷酸序列同源性高达97.6％，它的全长cDNA感染性克隆为pQSA。FMDV毒株O/M98/CHA/2010，与参考毒株O/BY/CHA/2010(GenBank登录号：JN998085)的VP1核苷酸序列同源性高达99.2％。A/JLYS/CHA/2014(Liang et al.,Archives Virology,2016)，其与参考毒株A/GDMM/CHA/2013(GenBank登录号：KF450794)的VP1核苷酸序列的同源性高达99.6％。单克隆抗体10B10(Yang et al.,Archives Virology,2017；CN107177558A(申请号：201710349810.1))、3A10(Wang et al.,Research in Veterinary Science,2019；CN109295006A(专利申请号：201811126938.2))和2H1(CN109295995A(申请号：201811126187.4))由本发明人实验室制备。
实施例1 FMDV(R4)病毒株对猪致病力的评价
前期研究发现，将FMDV IRES结构域4(IRES-Fd4)替换为牛鼻病毒(BRBV)IRES结构域4(IRES-Bd4)产生的IRES嵌合病毒FMDV(R4)，以10 6TCID 50/头剂量颈部肌肉接种自然宿主猪，不表现任何口蹄疫临床症状、不产生病毒血症、口腔鼻腔不排毒、而且该病毒在猪体内也不诱生抗体，表明病毒在易感动物体内失去复制能力，详见中国发明专利(CN108085302A)和国际PCT专利(申请公开号：WO/2018/090994)。为进一步评价FMDV(R4)对猪的致病力，采用最敏感的蹄踵皮内接种途径、使用更高的FMDV(R4)接种剂量(10 7TCID 50/头或10 8TCID 50/头)，并以相同途径接种10 5TCID 50/头剂量的野生型病毒O/YS/CHA/05作为强毒对照，结果如表1所示。接种10 5TCID 50野生型病毒的猪(11#、12#和13#)在接种后2天出现典型的口蹄疫症状，体温均高于40℃，食欲下降、精神沉郁，四蹄及鼻部出现水疱；用Real-time PCR方法检测病毒RNA，在病毒接种后1天血液及口、鼻拭子中病毒RNA的拷贝数即显著高于健康猪的背景值(2.6 log 10viral RNA CN/ml)，表明接种猪此时已形成病毒血症并经口腔和鼻腔排毒，接种后3-4天病毒血症以及口腔和鼻腔排毒达到高峰；接种后10天，接种猪均产生高水平的中和抗体(>1:128)。然而，经同样的蹄踵皮内途径，用高于野生型病毒滴度100倍(10 7TCID 50/头)的FMDV(R4)剂量接种猪(01#、02#、03#、04#、5#)、甚至用高于野生型病毒滴度1000倍(10 8TCID 50/头)的FMDV(R4)剂量接种猪(06#、07#、08#、09#、10#)，在10天观察期内也无体温升高(均低于40℃)以及FMD临床表现，用Real-time PCR在每天采集的血液及口鼻拭子样品中没有检出病毒RNA，血清中也无病毒中和抗体产生(<1:8)。这种超高剂量、最敏感途径的接种试验进一步表明，FMDV(R4)在猪体内不复制、不能够建立感染、因此对猪完全失去致病力。
表1 野生型病毒O/YS/CHA/05和IRES嵌合病毒FMDV(R4)接种猪的临床表现
Figure PCTCN2020111715-appb-000001
Figure PCTCN2020111715-appb-000002
实施例2 3B蛋白上的分子标记方式的比较
为了构建可用于鉴别诊断的分子标记病毒，本试验对在3B蛋白上的分子标记方式进行了比较研究。本发明人实验室的前期研究获得了一株针对FMDV非结构蛋白3B2的单克隆抗体2H1,对其识别的抗原表位进行研究确定，该表位基序为 34KPLKVK 39，K 34、K 37和V 38为该表位的关键氨基酸(CN109295005A，发明专利申请号：201811126187.4)。为了灭活单抗2H1识别的表位从而引入抗原分子标记，用丙氨酸(A)同时取代2H1表位的关键氨基酸K 37和V 38，构建此3B1表位失活的真核表达质粒pCI-3B1 m23。另外，用DNAstar软件对3B蛋白的抗原性进行分析显示，其抗原决定簇主要集中在3B1和3B2部分而3B3部分包含的抗原决定簇较少。由于3B1和3B2对于FMDV复制是重要的，缺失后将会严重影响FMDV的复制，因此选用另外一个3B3蛋白替换3B1和3B2蛋白；为了保持病毒的遗传稳定性，防止两个3B3编码基因序列发生同源重组，对此被加入替换3B1和3B2的3B3蛋白编码序列进行了基因优化(优化后的基因序列为SEQ ID NO.1所示)。将优化后的3B3编码基因导入O型FMDV感染性克隆pYS产生的重组病毒，其复制能力与其亲本毒株相近；进而用该修饰3B的编码序列构建真核表达载体，称作pCI-3B33。将表达3B蛋白突变体的真核表达质粒pCI-3B1 m23和pCI-3B33以及表达野生型3B蛋白的对照质粒pCI-3B123(WT)分别转染BHK-21细胞，48h后用2H1抗体进行间接免疫荧光检测。间接免疫荧光检测结果如图1所示，野生型3B123蛋白能够与2H1抗体结合并产生强的特异性荧光信号，突变的3B1 m23蛋白可与2H1抗体微弱地结合产生微弱的荧光信号，而3B33蛋白与2H1抗体完全不结合，不显示任何荧光信号。这些试验结果表明，用一个经过基因优化的3B3蛋白替换3B1和3B2蛋白去除3B蛋白的主要抗原性区域以实现对口蹄疫病毒的分子负标记、用单抗2H1建立的抗体检测ELISA进行疫苗接种动物与自然感染动物的鉴别诊断，是可行的方法和策略。
实施例3 遗传稳定FMDV耐热突变株的筛选及耐热表型分子决定因素的确定
1试验方法
1.1口蹄疫病毒的热压力筛选方法
病毒的热压力筛选，就是在特定的温度下加热30分钟，使单位体积中的病毒滴度99.99％失活，并且在逐渐升高温度的情况下进行重复的热失活试验，直到加热前后的病毒滴度大致相同为止。本试验在51℃、53℃和56℃的温度下进行多轮热筛选，直到高度热适应性病毒突变株的出现。
1.2病毒衣壳稳定性检测方法
将病毒通过较高温度处理后测定146S完整病毒衣壳存留的比例，这是判断病毒146S抗原热稳定性的关键指标。将分装好的热压力适应病毒与亲本株病毒对照一同稀释到10 7TCID 50/ml，在42℃条件下进行热灭活处理，分别在15、30、45、60、120、180、240min取样并立即放入冰浴中冷却，采用蔗糖密度梯度离心法测定病毒146S含量、并计算经这些时间热灭活后完整病毒146S衣壳存留的比例。
2.试验结果
Abhay Kotecha等人发现S2093Y突变能显著增加FMDV衣壳的耐热稳定性(Abhay Kotecha et al.,nature structural&molecular biology,2015)。随后Katherine A.Scott等人发现，S2093Y突变作为SAT2型FMDV耐热表型的分子决定因素不能稳定遗传(Katherine A.Scott et al.,Journal of Virology,2016)。有鉴于此，本试验用O型和A型FMDV的全长cDNA感染性克隆质粒pYS和pQSA分别构建并拯救了VP2蛋白的第93位氨基酸定点突变的耐热突变株rO/YS-S2093Y和rA/QSA-Q2093C，发现他们的耐热表型同样也是不稳定的，在体外传代过程中病毒的耐热性能逐渐减弱，传至第5代时耐热特性全部消失；对不同代次的传代病毒进行序列测定发现，突变株rO/YS-S2093Y在传至第3代时S2093Y突变为S2093H，突变株rA/QSA-Q2093C在传至第5代时Q2093C发生了回复突变，因此导致突变株的耐热表型消失。本发明在O型和A型FMDV进 行的突变研究支持Katherine A等人在SAT2型FMDV的研究结果，进一步表明Abhay Kotecha等人发现的VP2蛋白的第93位氨基酸作为耐热表型分子决定因素不能够稳定遗传。
为了获得遗传稳定的FMDV耐热突变株，本试验对突变病毒rO/YS-S2093Y和rA/QSA-Q2093C进行多轮热压力筛选，最终获得了O型FMDV耐热突变株rO/YSδ和A型FMDV耐热突变株rA/QSAδ(图2A和2B)。将经耐热压力筛选获得的耐热突变株rO/YSδ和rA/QSAδ分别在BHK-21细胞中无压力连续传10代进行热稳定性检测(56℃作用1h)表明，两个耐热突变株的第10代(P10)病毒与原代(P0)病毒的耐热性没有发生变化(图2)，表明其耐热表型具有遗传稳定性。对耐热突变株的结构蛋白编码区进行序列测定显示：O型FMDV耐热突变株rO/YSδ的P10代病毒和P0病毒一样，只含有Y2079H和S2093Y 2个氨基酸突变；耐热突变株rA/QSAδ的P10代病毒和P0病毒一样，仅含有A1003T(VP1蛋白的第3位氨基酸A突变为T)和N1017H(VP1蛋白的第17位氨基酸N突变为H)2个氨基酸突变，而突变病毒的Q2093C发生了回复突变。分别用O型和A型FMDV感染性cDNA克隆pYS和pQSA，对耐热突变株的突变位点进行单一突变和组合突变以确定决定O型和A型FMDV耐热的分子决定因素。结果如图2C和2D所示，Y2079H单独突变不能增加O型FMDV的耐热特性(图2C)，由于S2093Y单独突变不能稳定遗传因此未进行耐热性分析；然而，Y2079H和S2093Y组合突变显著增强O型FMDV耐热特性(图2C)。A1003T和N1017H单独突变不能增加O型FMDV的耐热特性，而组合突变却显著增强A型FMDV的耐热特性。以上试验结果表明，Y2079H突变虽然不产生耐热表型，但可协同S2093Y耐热突变稳定遗传，因此协同决定O型FMDV的衣壳耐热稳定性表型；A1003T和N1017H突变协同决定A型FMDV的衣壳耐热稳定性表型并可稳定遗传。
实施例4 携带耐热稳定表型以及非结构蛋白3A&3B表位缺失负标记的FMDV无毒株的构建和拯救以及耐热稳定性的验证及其体外生长特性检测
1试验方法
1.1 3A&3B标记的FMDV无毒株的全长cDNA感染性克隆的构建
3A&3B双分子标记FMDV无毒株的全长cDNA感染性克隆的构建，过程如下：首先，以pYS为模板，使用5458(EcoR I)-F和d3AB-1-R引物进行PCR扩增，获得的PCR产物经胶回收纯化作为模板用引物5458(EcoR I)-F、d3AB-2-R和d3AB-3-R扩增，获得的PCR产物称作片段A；之后以pYS为模板，用引物d3B-2H1-F和8221(EcoR V)-R扩增，获得的PCR产物称作片段B；最后以纯化的A、B片段作为模板，用引物5458(EcoR I)-F和8221(EcoR V)-R进行融合PCR扩增，产生一个缺失3A蛋白84-143位氨基酸、而且用一个经密码子修饰的3B3取代3B1和3B2的大小约为2.4Kb的片段；将该2.4Kb片段经EcoR I和EcoR V双酶切，克隆至经同样酶切的pFMDV(R4)载体中，经序列分析确定为正确的克隆被命名为pIRES Bd43AB m；然后，以pIRES Bd43AB m为模板，用引物L(Pst I)-F和L(Pst I)-R，2A(Pst I)-F和2A(Pst I)-R进行PCR扩增，在结构蛋白P1编码区两侧引入PstI酶切位点，经序列分析确定为正确的克隆被命名为pIRES Bd43AB m-Pst I。
构建FMDV毒株O/M98/CHA/2010和A/JLYS/CHA/2014的基因组cDNA，分别作为模板，以P1-F与O/M98P1-R或A/J14P1-R为引物，分别扩增这2个毒株的P1基因并对其进行耐热性突变，产物经PCR扩增、胶回收纯化后，用In-fusion重组酶(Clontech)分别克隆至经Pst I酶切的pIRES Bd43AB m-Pst I载体中，重组子经序列分析确定正确后，分别命名为pIRES Bd43AB m-O/M98和pIRES Bd43AB m-A/J14。本发明所使用的引物见表2。
表2 用于构建3A&3B标记FMDV无毒株的引物及其序列
Figure PCTCN2020111715-appb-000003
Figure PCTCN2020111715-appb-000004
1.2病毒的拯救
重组质粒pIRES Bd43AB m-O/M98和pIRES Bd43AB m-A/J14经限制性内切酶EcoRⅤ酶切线性化后，按照RiboMAX TM Large Scale RNA Production Systems-T7系统说明书进行体外转录，反应体系为：25mmol/L rNTP 6μL，5×缓冲液4μL，T7RNA聚合酶混合液2μL，线性化的重组质粒8μL(2μg)，总体积为20μL。将反应物充分混匀后，于37℃温育2.5h，用RNase-Free DNase消化15min，除去DNA模板，按酚氯仿抽提方法纯化体外转录产物。当6孔板中的BHK-21细胞生长至80％～90％单层时，用PBS洗2遍细胞，加1.5mL含2％胎牛血清的DMEM细胞培养液。将体外转录获得的RNA按QIAGEN公司的Effectene Transfection Reagent转染试剂盒说明书转染BHK-21细胞，进行病毒拯救。转染的细胞在5％CO 2条件下于37℃培养，观察细胞病变，大约3d左右收获病毒，反复冻融3次后接种BHK-21细胞传代，直到病毒能产生稳定的CPE。获得拯救的重组病毒，经全长基因组序列测定验证序列正确的毒株，分别命名为rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14，用于后续试验。
1.3病毒生长曲线的绘制
野生型病毒O/YS/CHA/05、FMDV(R4)与重组病毒rIRES Bd43AB m-PanAsia,rIRES Bd43AB m-O/M98、rIRES Bd43AB m-A/J14，按0.05MOI剂量分别接种处于对数生长期状态良好的BHK-21细胞，于37℃吸附1h后用PBS洗去未吸附的病毒，加入含2％胎牛血清的DMEM维持培养，分别在接种后2h、4h、6h、8h、10h、12h、16h、20h收获培养上清，测定不同时间点收获病毒的TCID 50滴度，每个时间点重复测定3次、计算平均值。以病毒感染细胞的时间为横坐标、病毒在不同时间点TCID 50滴度的对数值为纵坐标，绘制病毒复制的生长曲线。
1.4重组FMDV病毒株体外传代及遗传稳定性检测
重组FMDV病毒株按0.05MOI剂量分别接种于BHK-21细胞，于37℃吸附1h后用PBS洗涤2次，加入含2％胎牛血清的DMEM维持培养。待细胞出现明显的细胞病变后，反复冻融3次，离心收获上清，在BHK-21细胞传代至第10代。提取病毒RNA，进行RT-PCR扩增及序列测定。
1.5 Western blot检测
接种病毒的BHK-21细胞培养12h后，收获细胞并进行裂解处理，SDS-PAGE电泳后转印至硝酸纤维素膜上。用5％脱脂乳封闭膜，分别以MAb 10B10、3A10或2H1(1：1000稀释)作为一抗进行温育，37℃作用1h后用PBST洗涤，加入HRP标记的抗鼠IgG-抗体(抗鼠IgG-HRP，1：5000稀释)于37℃作用1h，用ECL发光液进行显色鉴定。另外，内参选择β-actin抗体(1：1000稀释)作为一抗，同样以抗鼠IgG-HRP(1：5000稀释)作为酶标抗体。
1.6间接免疫荧光
生长在96孔板中的BHK-21细胞接种病毒，6h后用PBS漂洗2次，用-20℃无水乙醇固定15min，加入1:100稀释的单克隆抗体10B10、3A10或2H1，在37℃湿盒中温育60min，用PBS 洗5次，加FITC标记的山羊抗鼠IgG(1:100)于37℃湿盒中温育45min，用70％甘油封片，在荧光显微镜下观察。
2.试验结果
2.1携带耐热稳定表型以及非结构蛋白3B负标记的FMDV无毒株的构建和拯救
为了构建带有可鉴别诊断标记的口蹄疫病毒无毒株作为口蹄疫灭活疫苗种子病毒，本发明以FMDV(R4)的感染性cDNA克隆质粒pFMDV(R4)(图3A、3B)为基础，进行如下的基因工程操作：
缺失非结构蛋白3A的84-143位氨基酸，而且由一个编码序列密码子优化的3B3取代3B1和3B2、导致3B1/3B2的缺失和3B3的重复，由此引入分子标记(图3C、3D)；同时，在结构蛋白编码区两侧引入酶切位点Pst I，可用于替换任何其他FMDV毒株的结构蛋白P1的编码基因，同时在结构蛋白编码区引入决定病毒衣壳稳定性的突变，由此获得3A&3B标记的FMDV无毒株的全长感染性cDNA克隆质粒，命名为pIRES Bd43AB m-Pst I，由该质粒拯救的重组病毒命名为rIRES Bd43AB m-PanAsia。
在此感染性cDNA克隆平台上，根据当前亚洲地区口蹄疫的流行情况，选择O型东南亚基因型Mya-98毒株O/M98/CHA/2010和A型Sea-97基因型G2亚型毒株A/JLYS/CHA/2014，用它们经耐热性修饰的P1基因分别替换pIRES Bd43AB m-Pst I的P1基因，构建的重组质粒分别命名为pIRES Bd43AB m-O/M98和pIRES Bd43AB m-A/J14。这些重组质粒经DNA测序验证正确后，经体外转录获得重组病毒的基因组RNA，转染BHK-21细胞拯救出重组病毒，分别命名为rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14(图3A)。进而，在BHK-21细胞上将这3个重组病毒连续传10代，经核苷酸序列测定与分析证实，重组病毒具有高度的遗传稳定性。重要的是，引入的3A&3B分子标记、置换的IRES结构域4以及耐热修饰位点均可以稳定遗传。
2.2重组FMDV的3A&3B标记以及耐热稳定性的验证及其体外生长特性
与野生型病毒O/YS/CHA/05和嵌合病毒FMDV(R4)的体外生长特性进行比较，含有3A&3B双分子标记的重组病毒rIRES Bd43AB m-PanAsia、rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14在BHK-21细胞上的复制动力学相似，可是这些标记病毒复制达到高峰的时间较野生型病毒推迟约4小时(图4A)。
为了对重组病毒的3A&3B双分子标记进行功能性验证，用本发明人制备的识别标记分子3A的特异性单克隆抗体3A10(中国发明专利公开号CN109295006A(专利申请号：201811126938.2))和识别3B的特异性单克隆抗体2H1(中国发明专利CN109295005A(专利申请号：201811126187.4))，对3A&3B标记病毒感染的细胞进行间接免疫荧光检测。已知3A10识别的表位是位于FMDV 3A蛋白上的5-aa肽序列 126ERTLP 130(Wang et al.，Research in Veterinary Science,2019；中国专利申请公开号CN109293748A(专利申请号：201811126944.8))，因此单抗3A10与 126ERTLP 130缺失的3A蛋白失去反应性；而2H1识别的表位是位于FMDV 3B2蛋白上的6-aa肽序列 34KPLKVK 39(专利申请公开号CN109293747A(专利申请号：201811126936.3))，因此单抗2H1与 34KPLKVK 39缺失的3B2蛋白失去反应性。免疫荧光检测结果如图4B所示，在病毒感染的BHK-21细胞中，3A&3B标记病毒与野生型毒株O/YS/CHA/05一样均能够被FMDV共享型单抗10B10(Yang et al.,Archives Virology,2017；中国发明专利申请公开号CN107177558A(专利申请号201710349810.1))所识别；然而，识别3B蛋白的单抗2H1和识别3A蛋白的单抗3A10仅能识别野生型病毒O/YS/CHA/05，而不识别3A&3B负标记的病毒rIRES Bd43AB m-PanAsia、rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14(图4B)，这从功能上表明重组病毒的3A&3B分子标记已被成功地引入。同时，用这些单抗对病毒感染细胞进行的Western blot分析(图4C)，也证实了免疫荧光检测结果，表明3A&3B负标记的FMDV失去了对单抗3A10和2H1的反应性。
用热灭活试验测定标记病毒的热稳定性。将滴度约为10 7TCID 50/ml的野生型病毒和标记病毒的细胞培养上清液，于42℃热灭活4h，用FMDV146S蔗糖密度梯度离心方法定量测定热处理后样品中完整146S FMDV颗粒的比例。结果如图4D、4E和4F所示，标记病毒rIRES Bd43AB m-A/J14、rIRES Bd43AB m-O/M98和rIRES Bd43AB m-PanAsia在42℃处理4h后保留的完整146S颗粒百分比分别为68.9％、85.7％和82.2％，而它们的亲本野生型病毒保留的146S颗粒百分比分别为3.6％、5.3％和5.6％，结果表明经耐热修饰的重组标记病毒具有显著的衣壳耐热稳定性特征。
实施例5 重组病毒株对易感宿主的毒力评价
1.试验方法
1.1猪体毒力评价试验
健康仔猪34头，20-30公斤，血清FMDV抗体阴性。随机选取9头分成3组，每组3头：第一组，猪蹄踵皮内接种野生型毒株FMDV O/YS/CHA/05，剂量为10 5TCID 50/头；第二组和第三组，猪蹄踵皮内接种野生型毒株O/M98/CHA/2010或A/JLYS/CHA/2014，剂量为10 7TCID 50/头。剩余25头猪，随机分成5组每组5头，每组猪于蹄踵皮内接种10 7TCID 50/头或10 8TCID 50/头剂量的FMDV(R4)、10 9TCID 50/头剂量的重组病毒rIRES Bd43AB m-PanAsia或rIRES Bd43AB m-O/M98或rIRES Bd43AB m-A/J14。在接种后10天内，每天测量接种猪的体温、观察临床表现并采集鼻腔拭子、口腔拭子及血液。
1.2牛体毒力评价试验
健康荷斯坦奶牛16头，200-250公斤，血清FMDV抗体阴性。随机选取6头分成两组每组3头，每组牛舌面皮内分别接种剂量为10 7TCID 50/头的野生型毒株O/M98/CHA/2010或A/JLYS/CHA/2014。剩余10头牛分成两组每组5头，每组牛舌面皮内接种剂量为10 9TCID 50/头的重组病毒rIRES Bd43AB m-O/M98或rIRES Bd43AB m-A/J14。在接种后10天内，每天测量牛的体温、观察临床表现并采集鼻腔拭子、口腔拭子及血液。
1.3羊体毒力评价试验
健康绵羊16只，30-40公斤，血清FMDV抗体阴性。随机选取6只分成两组每组3只，每组羊蹄冠部皮内注射10 7TCID 50/头剂量的野生型毒株O/M98/CHA/2010或A/JLYS/CHA/2014。剩余10只羊分成两组每组5只，蹄冠部皮内注射10 9TCID 50/头剂量的重组病毒rIRES Bd43AB m-O/M98或rIRES Bd43AB m-A/J14。在接种后10天内，每天测量羊的体温、观察临床表现并采集鼻腔拭子、口腔拭子及血液。
2试验结果
2.1 IRES结构域4被替换、耐热突变、3B标记的病毒rIRES Bd43AB m-O/PanAsia对猪无致病力为了分析IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/PanAsia对易感动物的毒力，本试验用高于野生型病毒10000倍滴度(10 9TCID 50/头)的剂量于蹄部皮内接种猪(15#、16#、17#、18#和19#)进行致病力评价，结果如表2所示。在观察期内，接种猪均无体温升高(温度低于40℃)，也无任何FMD临床表现；检测血液及口、鼻拭子中的病毒RNA，在接种后1-10天测得的病毒RNA均呈阴性；另外，在观察期内接种猪均无FMDV中和抗体产生(<1:8)。这表明，标记病毒rIRES Bd43AB m-O/PanAsia在猪体内没有复制，不能够建立感染，因此对猪无致病力。
2.2 IRES结构域4被替换、耐热突变、3B标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14对猪、牛、羊均无致病力
为检测IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14的毒力，本试验分别在猪、牛、羊三种易感动物上对这两株重组病毒的致病力进行评价，结果如表3和表4所示。首先用野生型病毒O/M98/CHA/2010或A/JLYS/CHA/2014以10 7TCID 50/头的剂量接种猪(21#、22#和23#；或31#、32#和33#)作为对照，在接种后48h均出现体温升高以及典型的口蹄疫症状，在接种后3-4天猪血液及口、鼻拭子中病毒RNA的含量显著高于健康猪(2.6 log 10viral RNA CN/ml)，表明此时已发生严重的病毒血症、产生高负载的口腔和鼻腔排毒，在接种后10天诱导产生较高滴度的中和抗体。然而，IRES结构域4被替换的标记病毒rIRES Bd43AB m-O/M98或rIRES Bd43AB m-A/J14，以10 9TCID 50/头的极高剂量经最敏感的蹄踵皮内途径接种猪(24#、25#、26#、27#、28#；或34#、35#、36#、37#、38#)，在观察期内均无FMD的临床症状和体温升高，在接种后1-10天每天采集的血液及口、鼻拭子样品检测病毒RNA均为阴性(拷贝数低于2.6 log 10viral RNA CN/ml)，在观察期内均无病毒中和抗体产生(<1:8)。这些结果表明，IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14在猪体内不复制、不能够建立感染，因此对猪完全失去致病力。
为了检测IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98和 rIRES Bd43AB m-A/J14对牛的致病力，首先用野生型病毒O/Mya98/CHA/2010或A/JLYS/CHA/2014以10 7TCID 50/头的剂量经舌部皮内接种牛(01#、02#和03#；或11#、12#和13#)作为阳性对照，接种后3天均出现发热并形成病毒血症，4-6天出现典型的口蹄疫临床症状(表3和表4)。然而，IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98或rIRES Bd43AB m-A/J14使用100倍野生型致病病毒滴度(10 9TCID 50/头)的剂量经最敏感的舌部皮内途径接种牛(04#、05#、06#、07和08#；或14#、15#、16#、17#和18#)，在10天观察期内均无FMD临床表现和体温升高、无病毒血症、无口腔鼻腔排毒(表3和表4)，也无病毒中和抗体产生(<1:8)。这表明，IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14在牛体内不复制、不能够建立感染，因此对牛完全失去致病力。
为了检测IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14对羊的致病力，首先用野生型病毒O/Mya98/CHA/2010或A/JLYS/CHA/2014，以10 7TCID 50/头的剂量、经蹄冠部皮内途径接种绵羊(01#、02#和03#；或10#、11#和12#)作为阳性对照，接种羊在2-4dpi均出现体温升高、蹄部或齿痕部水泡、病毒血症、以及口腔鼻腔排毒(表3和表4)，但发病羊跛行不明显。然而，IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98或rIRES Bd43AB m-A/J14经最敏感的蹄冠部皮内途径以100倍野生型致病病毒滴度(10 9TCID 50/头)的剂量接种绵羊(05#、06#、07#、08#和09#；或16#、17#、18#、19#和20#)，在10天观察期内均无FMD临床表现和体温升高、无病毒血症、无口腔鼻腔排毒(表3和表4)，也无病毒中和抗体产生(<1:8)。这表明，IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14，在羊体内不复制、不能够建立感染，因此对羊完全失去致病力。
上述结果表明，IRES结构域4被替换的携带耐热表型和3B标记的病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14在猪、牛、羊体内均丧失复制能力、因此完全失去致病力，因而称作FMDV无毒株。这些结果也表明，用本发明的通用感染性克隆质粒平台pIRES Bd43AB m-Pst I，可针对一个国家或地区的FMDV优势流行毒株快速创制携带无毒和耐热表型以及3B负标记的无毒株，用作灭活疫苗生产的种子病毒。
表3 野毒株O/M98/CHA/2010及修饰病毒rIRES Bd43AB m-O/M98接种猪牛羊的临床表现
Figure PCTCN2020111715-appb-000005
Figure PCTCN2020111715-appb-000006
表4 野毒株A/JLYS/CHA/2014及修饰病毒rIRES Bd43AB m-A/J14接种猪牛羊的临床表现
Figure PCTCN2020111715-appb-000007
实施例6 标记耐热无毒株O/A型口蹄疫二价灭活疫苗的制备及免疫保护效力试验
1试验方法
1.1标记、耐热、无毒株O/A型口蹄疫二价灭活疫苗的制备
在三角摇瓶中悬浮培养BHK-21细胞，当生长至3.5×10 6cells/ml时，按0.01MOI接种重组病毒rIRES Bd43AB m-O/M98或rIRES Bd43AB m-A/J14，37℃悬浮培养16h收获病毒，反复冻融三次后经TCID 50分析测定病毒的滴度。将病毒稀释至10 9.25TCID 50/ml后加入BEI(终浓度2mmol/L)，于30℃灭活28h，期间每2h摇晃，之后加入2％的硫代硫酸钠溶液终止灭活。病毒灭活经检验合格后，将rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14等体积混合，加入相同体积的Montanide ISA 201VG佐剂(法国SEPPIC)混匀，由此用无毒株制备成3A/3B标记的、衣壳耐热稳定的口蹄疫病毒O/A二价灭活疫苗，置于4℃保存待用。
1.2猪的免疫接种及攻毒试验
健康架子猪26头，30-40公斤，血清FMDV抗体阴性。随机选取20头分为2组，每组10头：一组于耳后颈部肌肉接种(2ml/头)O/A二价灭活疫苗(rIRES Bd43AB m-O/M98株+rIRES Bd43AB m-A/J14株)，另一组耳后颈部肌肉接种(2ml/头)商品化的O/A二价灭活疫苗(Re-O/MYA98/JSCZ/2013株+Re-A/WH/09株，中农威特)，剩余6头猪接种PBS作为攻毒对照。在接种后28天，两个疫苗实验组各自随机分成2组，每组选取5头免疫猪、各加上3头PBS 接种对照猪，这样每组8头猪颈部肌肉接种2ml 10 8.5TCID 50/ml剂量的O/M98/CHA/2010毒株或A/JLYS/CHA/2014毒株。在攻毒后10天内，每天测量体温、观察临床表现并采集鼻腔拭子、口腔拭子及血液。
1.3疫苗重复免疫接种试验
FMDV血清抗体阴性的健康猪、牛、羊各20个，随机分成2组每组10个。一组颈部肌肉接种2倍使用剂量(4ml/头)的标记无毒株O/A二价灭活疫苗，另一组颈部肌肉接种2倍使用剂量(4ml/头)的商品化的O/A二价灭活疫苗。间隔28天接种一次、连续接种4次，最后一次接种后30天采集接种动物的血清，置-20℃冰箱备用。
1.4临床症状观察
每天仔细观察动物临床发病情况并作记录，按照Elizabeth Rieder等描述的方法(Elizabeth Rieder et al,J Virol,2005)进行临床打分：蹄部发病每蹄记1分，鼻部、舌部或唇部发病记1分，最大分值为5分。
1.5口、鼻排毒及病毒血症的检测
采集的鼻腔拭子、口腔拭子及血清样品，用TRIZOL法提取总RNA，使用Oligo(dT 15)引物反转录获得的cDNA作为模板，以FMDV特异性引物(3DF：5'GGA TGC CGT CTG GTT GTT 3'；3DR：5'CGT AGG AGA TCA TGG TGT AAG AGT 3')进行荧光定量PCR检测。荧光定量PCR的具体操作按照
Figure PCTCN2020111715-appb-000008
Green qPCR Super Mix-UDG with ROX(Invitrogen)试剂盒说明书进行，通过标准曲线函数来计算样品中病毒基因组RNA的含量。以健康猪的血清和口、鼻
拭子样品进行PCR扩增的FMDV RNA拷贝数2.6
Figure PCTCN2020111715-appb-000009
log10作为背景值，病毒RNA拷贝数/ml(viral RNA CN/ml)高于此数值判定为FMDV RNA阳性。
1.6微量细胞中和试验
采用固定病毒稀释血清的方法进行微量细胞中和试验。先用BHK-21细胞测定FMDV的TCID 50；然后，将血清于56℃灭活30min，用PBS做倍比稀释；用100TCID 50的病毒分别与等体积不同稀释度稀释的血清混合，37℃温箱中温育1h；将上述温育的血清-病毒混合液分别接种BHK-21细胞，每孔100μL，每个稀释度设8孔重复，在37℃于5％CO 2培养箱中培养，每日观察细胞病变(CPE)，72h后做最终判定。另外，设病毒、阳性血清和正常细胞对照，根据CPE变化按Reed-Muench方法(Reed and Muench.,1938)计算病毒的中和滴度，即能保护50％BHK-21细胞不出现CPE的血清稀释浓度。
1.7口蹄疫病毒3B单抗阻断ELISA(CN109295005A，专利申请号：201811126187.4)
将单抗3A10用碳酸钠缓冲液(pH＝9.6)稀释后包被ELISA板，100μL/孔，4℃过夜；用PBST洗涤5次，加入原核表达的口蹄疫病毒非结构蛋白3AB抗原，每孔50μL，室温孵育1h；PBST洗涤5次，加入10倍稀释的待检血清，50μL/孔，同时设有阴性和阳性血清对照，37℃孵育1h,PBST洗涤5次；加入工作浓度的HRP标记的检测单抗2H1，50μL/孔，37℃孵育1h；PBST洗涤5次后，每孔加入50μL的TMB底物溶液避光显色15min，加等量浓硫酸终止反应，用酶标仪测定450nm波长下的光吸收值(OD 450nm值)。结果判定：计算抑制百分率(Percentage inhibition,PI)并判定阴阳性，PI＝(阴性对照OD值-样品OD值)/(阴性对照OD值-阳性对照OD值)，PI≥50％为阳性，PI<50％为阴性。
2试验结果
2.1 FMDV的标记耐热无毒株O/A型二价灭活疫苗能诱导有效的免疫保护
为了评价携带耐热表型、3A&3B标记的FMDV无毒株制备的灭活疫苗的免疫保护效力，将修饰病毒rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14经BEI灭活后与ISA 201VG佐剂等体积混合配制成O/A型口蹄疫二价灭活疫苗，并用商业化O/A二价灭活疫苗(中农威特)作为疫苗对照，分别接种10头猪，另外6头猪接种PBS作为阴性对照，结果如表5和表6所示。所有接种灭活疫苗的动物，在接种后7天(dpv)可检出O型和A型FMDV特异性中和抗体，接种后21天中和抗体水平达到高峰(1：256)。本发明的O/A型口蹄疫二价灭活疫苗接种猪(201#-210#)与商业化二价灭活疫苗接种猪(101#-110#)诱导产生FMDV中和抗体的水平相近，但接种PBS的动物(301#-306#)未检出FMDV中和抗体(<8)。而且，26头猪在接种后均没有出现副反 应。
在免疫接种后28天，将免疫的动物分成两组，分别用O型和A型同源野生型FMDV毒株进行攻击，观察免疫保护的效果。接种PBS的6头对照猪分成2组每组3头，在接种10 8.5TCID 50/头剂量的O型FMDV O/M98/CHA/2010或A型FMDV A/JLYS/CHA/2014后1-3天，均出现典型的FMD临床症状(表3和表4)。然而，本试验用修饰的FMDV无毒株制备的O/A型口蹄疫二价灭活疫苗，同商品化二价灭活疫苗一样，接种动物在用O型或A型FMDV攻击后均无FMD临床表现、获得免疫保护(表5，表6)。因此，本发明创制的携带耐热表型和3B标记的FMDV无毒株rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14，具有优良的免疫原性，接种动物可诱导产生高水平的中和抗体、可有效抵抗亲本强毒株的攻击，可作为口蹄疫灭活疫苗生产的种子病毒。
表5 O/A二价灭活疫苗接种猪诱导的O型FMDV中和抗体及免疫保护
Figure PCTCN2020111715-appb-000010
表6 O/A二价灭活疫苗接种猪诱导的A型FMDV中和抗体及免疫保护
Figure PCTCN2020111715-appb-000011
2.2单抗阻断ELISA检测FMDV标记灭活疫苗接种动物的3B抗体应答
FMD灭活疫苗在制备过程中会残留少量的非结构蛋白，动物经多次重复免疫接种后会诱导产生一定水平的非结构蛋白特异性抗体，给疫苗接种动物与自然感染动物的鉴别诊断造成干扰从而不能进行有效的鉴别。本发明使用所建立的FMDV 3B单抗阻断ELISA抗体检测方法，通过FMDV无毒株3B蛋白主要抗原区3B1和3B2的缺失，彻底消除了2H1单抗识别的3B表位(位于3B2的短肽 34KPLKVK 39)，这样，用本发明建立的阻断ELISA方法不能检出该病毒灭活疫苗免疫接种的动物，从而排除了疫苗中残留的3B蛋白对鉴别诊断的干扰。为了验证这种标记疫苗的鉴别诊断设计，本试验用这种3B缺失负标记的FMDV O/A型二价灭活疫苗分别以2倍剂量(4ml/头)接种猪、牛、羊三种动物，以28天间隔连续免疫4次，在最后一次免疫后30天采集血清，用基于3B蛋白单抗2H1建立的阻断ELISA方法评估非结构蛋白3B抗体的产生及其水平，同时选择商业化O/A二价灭活疫苗(没有3B缺失的负标记)接种动物的血清作为常规疫苗接种 动物的检测对照、选择病毒感染动物的血清作为阳性检测对照。结果如图5所示，经3B单抗阻断ELISA检测，所有感染野生型病毒的动物均产生高滴度的针对3B蛋白2H1表位的抗体(阻断率大于75％)；接种商业化O/A二价灭活疫苗的动物，有一部分的猪(2/10)、牛(4/10)或羊(4/10)产生了针对3B蛋白2H1表位的抗体(阻断率大于50％)；而所有接种本发明制备的3B缺失负标记疫苗的动物，均不产生针对3B蛋白2H1表位的抗体(阻断率小于25％)。这些结果表明，用本发明建立的平台构建的任何3B缺失标记的FMDV毒株，包括本发明获得的修饰病毒rIRES Bd43AB m-PanAsia、rIRES Bd43AB m-O/M98和rIRES Bd43AB m-A/J14，用其作为种子病毒生产口蹄疫灭活疫苗免疫接种动物，均可实现疫苗接种动物与自然感染动物的鉴别诊断。

Claims (18)

  1. FMDV全长cDNA感染性克隆质粒，其特征在于，FMDV基因组IRES的结构域4用牛鼻病毒IRES的结构域4进行替换，缺失FMDV非结构蛋白3A的84-143位氨基酸的编码区，用FMDV的3B3蛋白的编码区取代FMDV的3B1和3B2蛋白的编码区以及在FMDV结构蛋白P1编码区两侧引入Pst I酶切位点。
  2. 按照权利要求1所述的FMDV全长cDNA感染性克隆质粒，其特征在于，所述的病毒3B3蛋白的编码区是密码子优化后的序列，其核苷酸序列为SEQ ID NO.1所示。
  3. 按照权利要求1或2所述的FMDV全长cDNA感染性克隆质粒，其特征在于，其核苷酸序列为SEQ ID NO.2所示。
  4. 由权利要求1-3任何一项所述的FMDV全长cDNA感染性克隆质粒采用反向遗传方法拯救获得的携带负标记的重组FMDV无毒株。
  5. O型FMDV全长cDNA感染性克隆质粒，其特征在于，将权利要求1的FMDV全长cDNA感染性克隆质粒的结构蛋白P1编码区用O型FMDV的结构蛋白P1编码区替换；其中，将O型FMDV的结构蛋白VP2的第79位的酪氨酸突变为组氨酸以及将第93位的丝氨酸突变为酪氨酸。
  6. 按照权利要求5所述的O型FMDV感染性全长cDNA克隆质粒，其特征在于，其核苷酸序列为SEQ ID NO.3所示。
  7. 由权利要求5或6所述的O型FMDV感染性全长cDNA克隆质粒采用反向遗传方法拯救获得的具有耐热表型和3B表位缺失负标记的重组O型口蹄疫病毒无毒株。
  8. A型FMDV全长cDNA感染性克隆质粒，其特征在于，将权利要求1的FMDV全长cDNA感染性克隆质粒的结构蛋白P1编码区用A型FMDV的结构蛋白P1编码区替换；其中，将A型FMDV的结构蛋白VP1的第3位的丙氨酸突变为苏氨酸以及将第17位的天冬酰胺突变为组氨酸。
  9. 按照权利要求8所述的A型FMDV感染性全长cDNA克隆质粒，其特征在于，其核苷酸序列为SEQ ID NO.4所示。
  10. 由权利要求8或9所述的A型FMDV全长cDNA感染性克隆质粒采用反向遗传方法拯救获得的具有耐热表型和3B表位缺失负标记的重组A型口蹄疫病毒无毒株。
  11. 防治O型口蹄疫和A型口蹄疫的二价灭活疫苗，其特征在于，包括防治上有效量的权利要求7所述的重组O型口蹄疫病毒无毒株和权利要求10所述的重组A型口蹄疫病毒无毒株以及免疫佐剂。
  12. 权利要求4所述的重组口蹄疫病毒无毒株在制备防治口蹄疫的药物中的用途或者用于制备鉴别诊断口蹄疫疫苗接种动物与自然感染动物的试剂中的用途。
  13. 权利要求7所述的重组O型口蹄疫病毒无毒株在制备防治O型口蹄疫的药物中的用途或者用于制备鉴别诊断口蹄疫疫苗接种动物与自然感染动物的试剂中的用途。
  14. 权利要求10所述的A型重组口蹄疫病毒无毒株在制备防治A型口蹄疫的药物中的用途或者用于制备鉴别诊断口蹄疫疫苗接种动物与自然感染动物的试剂中的用途。
  15. 衣壳耐热表型稳定遗传的重组O型口蹄疫病毒株，其特征在于，该O型FMDV的结构蛋白VP2的第79位的氨基酸由酪氨酸突变为组氨酸以及第93位的氨基酸由丝氨酸突变为酪氨酸。
  16. 衣壳耐热表型稳定遗传的重组A型口蹄疫病毒株，其特征在于，该A型FMDV的结构蛋白VP1的第3位的氨基酸由丙氨酸突变为苏氨酸以及将第17位的氨基酸由天冬酰胺突变为组氨酸。
  17. 一种获得衣壳耐热表型稳定遗传的重组O型口蹄疫病毒突变株的方法，其特征在于，包括：将O型FMDV的结构蛋白VP2的第79位的氨基酸由酪氨酸突变为组氨酸以及将第93位的氨基酸由丝氨酸突变为酪氨酸。
  18. 一种构建衣壳耐热表型稳定遗传的重组A型口蹄疫病毒突变株的方法，其特征在于，包括：将A型FMDV的结构蛋白VP1的第3位的氨基酸由丙氨酸突变为苏氨酸以及将第17位的氨基酸由天冬酰胺突变为组氨酸。
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