WO2018090994A1 - 口蹄疫病毒温度敏感减毒株及其构建方法和用途 - Google Patents

口蹄疫病毒温度敏感减毒株及其构建方法和用途 Download PDF

Info

Publication number
WO2018090994A1
WO2018090994A1 PCT/CN2017/111936 CN2017111936W WO2018090994A1 WO 2018090994 A1 WO2018090994 A1 WO 2018090994A1 CN 2017111936 W CN2017111936 W CN 2017111936W WO 2018090994 A1 WO2018090994 A1 WO 2018090994A1
Authority
WO
WIPO (PCT)
Prior art keywords
foot
mouth disease
ires
temperature
disease virus
Prior art date
Application number
PCT/CN2017/111936
Other languages
English (en)
French (fr)
Inventor
于力
杨德成
王海伟
周国辉
Original Assignee
中国农业科学院哈尔滨兽医研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国农业科学院哈尔滨兽医研究所 filed Critical 中国农业科学院哈尔滨兽医研究所
Priority to RU2018144892A priority Critical patent/RU2723353C1/ru
Priority to EP17872552.9A priority patent/EP3543332A4/en
Priority to US16/461,004 priority patent/US10918710B2/en
Publication of WO2018090994A1 publication Critical patent/WO2018090994A1/zh
Priority to ZA2019/00154A priority patent/ZA201900154B/en

Links

Images

Classifications

    • 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
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • 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
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • 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 site-directed mutants, chimeras of the foot-and-mouth disease virus genome IRES, and the use thereof in the construction of a temperature-sensitive attenuated strain of foot-and-mouth disease virus, and the present invention further relates to the reduction of temperature-sensitive attenuated strain of foot-and-mouth disease virus as a prevention and control of FMD
  • the use of a toxic vaccine strain or a safe poison for the production of a foot-and-mouth disease inactivated vaccine belongs to the field of prevention and treatment of foot-and-mouth disease.
  • Foot and Mouth Disease is an acute, heat, and highly contagious disease caused by Foot and Mouth Disease Virus (FMDV), which mainly harms cloven-hoofed animals such as pigs, cattle, and sheep (Grubman). And Baxt.2004.Clinical Micro.Rev.17:465-493), once the outbreak of the disease has a serious impact on international trade and the social economy, it is internationally known as a political and economic disease and has always been highly valued by governments. .
  • FMDV Foot and Mouth Disease Virus
  • FMDV belongs to the microRNA virus family and the foot-and-mouth disease virus genus. There are seven serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3), and there is no immune cross protection between the serotype strains.
  • the viral genome is a single-stranded positive-stranded RNA of approximately 8.5 kb in length and consists of a 5' non-coding region (5' UTR), an open reading frame (ORF) and a 3' UTR.
  • the 5' end of the genome of FMDV lacks a cap structure, and the initiation of protein translation depends on the RNA cis-acting element in the 5'UTR, the internal ribosome entry site (IRES), which recruits eukaryotic translations. Initiation factor and synthesis of ribosomal initiation viral proteins.
  • the FMDV IRES is approximately 450 nt in length and includes four domains which are essential for the initiation of FMDV protein translation.
  • the invention provides a method for constructing a temperature-sensitive attenuated strain of Foot-and-Mouth Disease Virus, comprising: constructing a full-length cDNA infectious cloning plasmid of foot-and-mouth disease virus, and in vitro transcripting the foot-and-mouth disease virus genomic RNA, which will be foot-and-mouth disease
  • the viral genomic RNA is transfected into the cell and rescued by the virus; wherein the cytosine (C) at position 351 of the K-region loop of the IRES domain 4 of the foot-and-mouth disease virus genomic RNA ( 351 CUUUAA 356 ) is mutated to Guanine (G) or adenine (A), the base sequence of the K-loop after mutation is 351 GUUUAA 356 or 351 AUUUAA 356 .
  • the present invention provides a temperature-sensitive attenuated strain of foot-and-mouth disease virus obtained by the above construction method.
  • the present invention provides a temperature-sensitive attenuated strain of foot-and-mouth disease virus obtained by the above-mentioned construction method, which is named as rC351G; the temperature-sensitive attenuated strain rC351G of foot-and-mouth disease virus provided by the invention has high genetic stability and is temperature sensitive.
  • the phenotype which is attenuated for all susceptible cloven-hoofed animals (body temperature 38.5-40 ° C), meets the safety requirements of live attenuated vaccines.
  • the invention inoculates the foot-and-mouth disease virus temperature-sensitive attenuated strain rC351G into pigs, and the vaccinated animals do not show any clinical symptoms, but can induce high levels of O-type FMDV neutralizing antibodies and can completely resist the O-type different genotypes which are currently prevalent in China. Attack of FMDV heterologous strain; the temperature-sensitive attenuated strain rC351G of foot-and-mouth disease virus constructed by the invention has good safety, and between the inoculated pig and the healthy pig No horizontal transmission occurs, and the continuous passage of virulence in pigs does not return.
  • the temperature-sensitive attenuated strain rC351G of the foot-and-mouth disease virus constructed by the invention can be prepared into a live attenuated vaccine for controlling foot-and-mouth disease according to the conventional preparation method of live attenuated vaccine, or the foot-and-mouth disease virus temperature-sensitive attenuated strain rC351G is inactivated.
  • the vaccine is safe for the prevention of foot-and-mouth disease; the full-length cDNA infectious clone of the foot-and-mouth disease virus temperature-sensitive attenuated strain rC351G can be rescued and prepared as a live attenuated vaccine for the prevention and treatment of foot-and-mouth disease, or as a safe species for foot-and-mouth disease inactivated vaccine.
  • the invention provides a patent-approved institution for the full-length cDNA infectious cloning plasmid of the foot-and-mouth disease virus for protecting the foot-and-mouth disease virus temperature-sensitive attenuated strain rC351G, and the microbial preservation number is: CGMCC No. 13148; the classification name is: Escherichia coli ( E.coli); preservation time is: October 26, 2016; the depositary is: China General Microbial Culture Collection Management Center; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.
  • the present invention provides an IRES mutant of a temperature-sensitive attenuated strain of foot-and-mouth disease virus, the nucleotide sequence of which is shown in SEQ ID No. 1.
  • the FRESV reverse genetic technique can be used to construct the foot-and-mouth disease.
  • the virus temperature-sensitive attenuated strain, further, the temperature-sensitive attenuated strain of the foot-and-mouth disease virus can be prepared into a live attenuated vaccine according to the conventional preparation method of the live attenuated vaccine for controlling foot-and-mouth disease, or the temperature of the foot-and-mouth disease virus to be constructed is sensitive.
  • the full-length cDNA infectious clone of the attenuated strain was rescued as a safe poison for inactivated vaccine or used to construct a foot-and-mouth disease RNA vaccine.
  • the invention provides a method for constructing another temperature-sensitive attenuated strain of foot-and-mouth disease virus, comprising: constructing a full-length cDNA infectious cloning plasmid of foot-and-mouth disease virus, and in vitro transcripting the foot-and-mouth disease virus genomic RNA, transfecting the foot-and-mouth disease virus genomic RNA into the cell and performing the method. Rescue of the virus; wherein the IRES domain 4 of the foot-and-mouth disease virus genomic RNA obtained by in vitro transcription is replaced with the IRES domain 4 of Bovine rhinitis B virus (BRBV) genomic RNA.
  • BRBV Bovine rhinitis B virus
  • the invention adopts the above construction method to obtain another temperature-sensitive attenuated strain of foot-and-mouth disease virus, which is named as FMDV (R4); the temperature-sensitive attenuated strain FMDV (R4) of foot-and-mouth disease virus provided by the invention has high genetic stability and has The temperature-sensitive phenotype, whose temperature-sensitive properties indicate that it is attenuated for all susceptible cloven-hoofed animals (body temperature 38.5-40 °C), meets the safety requirements of attenuated strains.
  • the invention inoculates pigs with foot-and-mouth disease virus temperature-sensitive attenuated strain FMDV (R4), and the vaccinated animals do not show any clinical symptoms, and no horizontal transmission occurs between the inoculated pigs and the healthy pigs.
  • the vaccinated pigs did not produce antibodies against FMDV, indicating that the FMDV (R4), a temperature-sensitive attenuated strain of foot-and-mouth disease virus constructed by the present invention, is more effective as a foot-and-mouth disease inactivated vaccine.
  • the full-length cDNA infectious clone of FMDV (R4), a temperature-sensitive attenuated strain of foot-and-mouth disease virus can also be rescued as a poison of foot-and-mouth disease inactivated vaccine.
  • the invention provides a patent-approved institution for the full-length cDNA infectious cloning plasmid of the foot-and-mouth disease virus for protecting the foot-and-mouth disease virus temperature-sensitive attenuated strain FMDV (R4) for preservation;
  • the microbiological preservation number is CGMCC NO.13149; E. coli; preservation time is: October 26, 2016;
  • the depositary is: China General Microbial Culture Collection Management Center; deposit address: Microbiology of Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing graduate School.
  • the present invention also provides a chimeric IRES (internal ribosome entry site) obtained by replacing the IRES domain 4 of foot-and-mouth disease virus genomic RNA with IRES domain 4 of bovine rhinovirus genomic RNA, the full-length nucleotide sequence of which is SEQ ID No.2; using FMDV reverse genetics, the chimeric IRES can be used to construct a temperature-sensitive attenuated strain of foot-and-mouth disease virus. Further, the temperature-sensitive attenuated strain of foot-and-mouth disease virus can be constructed according to a live attenuated vaccine.
  • a chimeric IRES internal ribosome entry site
  • the conventional preparation method is prepared as a live attenuated vaccine for controlling foot-and-mouth disease, or a full-length cDNA infectious clone of a temperature-sensitive attenuated strain of foot-and-mouth disease virus is rescued as a safe poison of a foot-and-mouth disease inactivated vaccine.
  • the invention further provides a method for constructing a temperature-sensitive attenuated strain of foot-and-mouth disease virus, comprising: constructing a full-length cDNA infectious cloning plasmid of foot-and-mouth disease virus, and in vitro transcribed the foot-and-mouth disease virus genomic RNA, and transfecting the foot-and-mouth disease virus genomic RNA into the cell.
  • the rescue of the virus is carried out; wherein the K region of the IRES domain 4 of the foot-and-mouth disease virus genomic RNA obtained by in vitro transcription is replaced with the K region of the IRES domain 4 of the BRBV genomic RNA.
  • a temperature-sensitive attenuated strain of foot-and-mouth disease virus obtained by the above construction method which is named rdK
  • the temperature-sensitive attenuated strain rdK of foot-and-mouth disease virus provided by the invention has a temperature-sensitive phenotype and has high genetic stability and is temperature sensitive The characteristics indicate that it is attenuated for all susceptible cloven-hoofed animals (body temperature 38.5-40 ° C), which meets the safety requirements of attenuated strains.
  • the temperature-sensitive attenuated strain rdK is inoculated into pigs, and the vaccinated animals do not exhibit any clinical symptoms, and no horizontal transmission occurs between the inoculated pigs and the healthy pigs.
  • the vaccinated pig does not produce antibodies against FMDV, which indicates that the temperature-sensitive attenuated strain rdK of the foot-and-mouth disease virus constructed by the present invention has better safety as a foot-and-mouth disease inactivated vaccine, and therefore, the foot-and-mouth disease virus can be The full-length cDNA infectious clone of the temperature-sensitive attenuated strain rdK was rescued as a safe poison for the foot-and-mouth disease inactivated vaccine.
  • the invention provides a patent-approved institution for the full-length cDNA infectious cloning plasmid of the foot-and-mouth disease virus for protecting the foot-and-mouth disease virus temperature-sensitive attenuated strain rdK, and the microbial preservation number is CGMCC NO.13150; the classification name is: Escherichia coli ( E.coli); preservation time: October 26, 2016; deposit form The position is: China General Microbial Culture Collection Management Center; deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing.
  • the present invention provides that the K region of the IRES domain 4 of the foot-and-mouth disease virus genomic RNA is replaced with the K region of the IRES domain 4 of the BRBV genomic RNA to obtain a chimeric IRES, the full-length nucleotide sequence of which is SEQ ID No.
  • the chimeric IRES can be applied to the construction of a temperature-sensitive attenuated strain of foot-and-mouth disease virus using conventional FMDV reverse genetics techniques in the art.
  • the O-type FMDV reverse genetics operating system was used to replace the domain 4 of FMDV IRES with the corresponding region of BRBV IRES, and the IRES chimeric mutant FMDV (R4) was successfully constructed and rescued.
  • the virulence of the IRES chimeric virus FMDV (R4) and its wild-type virus FMDV (WT) was compared using a 3-day-old suckling mouse model. The results showed that the IRES domain 4 chimeric virus FMDV (R4) pair
  • the virulence of suckling mice decreased significantly, and its virulence decreased by about 100-fold compared with the parental virus FMDV (WT), demonstrating that subdomain 4 of IRES is a determinant of FMDV virulence.
  • the present invention uses the O-type FMDV reverse genetics operating system for the J, K or N subdomains in domain 4 of FMDV IRES
  • the corresponding regions of BRBV IRES were replaced one by one, and the J, K or N subdomain chimeric FMDV mutants of three IRESs were successfully constructed and rescued, respectively named rdJ, rdK or rdN.
  • the replication kinetics of the three chimeric viruses at different temperatures were examined and analyzed, and a one-step growth curve was drawn.
  • the proliferation characteristics of BHK-21 cells inoculated with chimeric virus rdK were similar to those of parental FMDV (WT) at 33 °C and 37 °C, but their replication ability was significantly decreased at 41 °C, compared with the parental
  • the toxic FMDV (WT) decreased by about 100-fold; on IBRS-2 cells, the replication ability of the chimeric virus rdK decreased significantly at 37 °C, which was about 100-fold lower than that of the parental virus, and the replication of rdK at 41 °C.
  • the ability is almost lost.
  • the replication characteristics of the chimeric virus rdK (instead of rdJ and rdN) are consistent with the replication characteristics of the chimeric virus FMDV (R4), indicating that the K region of the IRES domain 4 determines the temperature sensitive properties of the chimeric virus FMDV (R4).
  • the virulence test results of the suckling mice showed that the virulence of rdK decreased by about 16 times compared with the virulence of FMDV (WT). Taken together, the above results indicate that the K region of IRES domain 4 determines the temperature-sensitive attenuated phenotype of the IRES chimeric virus FMDV (R4).
  • the K regions of the IRES domain 4 of FMDV and BRBV are each composed of a stem-loop structure.
  • the present invention uses the reverse genetic manipulation technique to use the stem and the loop of the K region of the FMDV IRES with the stem and loop of the K region of the BRBV IRES, respectively.
  • the two chimeric viruses rescued were named rK (Stem) and rK (Loop), respectively, and the replication kinetics of the two chimeric viruses at different temperatures of different cells were detected.
  • the chimeric virus rK (Stem) has similar proliferation characteristics to the parental FMDV (WT) in BHK-21 or IBRS-2 cells at 33 ° C, 37 ° C and 41 ° C;
  • the replication ability of the recombinant virus rK (Loop) on both cells gradually decreased with increasing temperature, and its replication characteristics were extremely similar to those of rdK.
  • the above results indicate that the loop structure of the K region of IRES domain 4 determines the temperature sensitivity of the chimeric virus FMDV (R4).
  • the virulence of rK (Loop) was determined to be unstable in the mice, and a T351C reversion mutation occurred at the IRES 351 position, which was in response to the IRES 351 after multiple passages of the virus in vitro.
  • this reversion mutation causes the loop structure of the kK region of the rK (Loop) to be close to the loop structure of the K region of the parental virulent strain, so that the pathogenicity of the mutant rK (Loop) to the mouse is restored to the level of the parental virus. .
  • the ring of the IRES K region of FMDV (- 351 CUUUAA 356 -) is similar to the ring of the IRES K region of BRBV (-UUUAC-), the main difference being that the IRES of FMDV is more than 351 at the beginning of the ring structure of its K region.
  • the present invention first mutated the IRES 356 position A of FMDV to C of the IRES 356 position of BRBV, and the constructed and rescued mutant virus was named rA356C.
  • the replication kinetics of the mutant virus at different temperatures showed that the replication ability of rA356C on both BHK-21 and IBRS-2 cells was similar to that of the parental FMDV (WT) at 33 °C, 37 °C, and 41 °C. , indicating that base 356A of FMDV IRES is independent of the temperature-sensitive phenotype of FMDV.
  • the present invention made the following four mutations for the base C of the 351 site:
  • the final test results showed that the mutation schemes of (1) and (4) failed to rescue the virus due to the destruction of the octanucleotide loop structure, while the (2) and (3) mutation schemes were due to eight
  • the nucleotide loop structure can save the virus, and the rescued recombinant viruses are named rC351A and rC351G.
  • the replication kinetics of the two IRES point mutant viruses at different temperatures were examined.
  • the results showed that the replication characteristics of the 351-base C-point mutant virus of the two IRESs were similar to those of the IRES chimeric virus rK (Loop), indicating that this site is the molecular determinant of the IRES chimeric FMDV temperature-sensitive phenotype.
  • the present invention also found that the virulence of the FMDV temperature-sensitive mutant strain of the IRES C351 site mutation was significantly decreased in the suckling mouse, and the virulence of rC351G was decreased by about 10,000 times and the virulence of rC351A was decreased by 1000 times compared with the wild type virus.
  • the above test results finally indicate that the 351 base C on the ring of the K region of the IRES domain 4 determines the temperature-sensitive attenuated phenotype of FMDV.
  • the present invention combines IRES chimeric or site-directed mutagenesis of recombinant viruses FMDV (R4), rdK, rK (Loop), rC351G, rC351A and the parental virus FMDV (WT) in BHK, respectively.
  • the -21 cells were continuously passaged for 20 passages, and the IRES sequence of the 20th generation virus was measured.
  • the present invention further constructs a full-length cDNA infectious clone of type A and Asia type foot-and-mouth disease virus and rescues the virus of CRY replacement by IRES.
  • the mutant strains were named A-rC351G and Asia1-rC351G, respectively.
  • the temperature sensitivity of the above IRES mutant virus was determined. The results showed that the replication ability of B--21 and IBRS-2 cells decreased with temperature, whether it was A-rC351G or Asia1-rC351G, indicating that the temperature decreased with increasing temperature.
  • the C351G mutation of IRES also enables the temperature-sensitive phenotype of FMDV strains of other serotypes.
  • the virulence test results of A-rC351G and Asia1-rC351G on suckling mice showed that the C351G mutation of IRES can also reduce the virulence of type A and Asia1 FMDV to suckling mice by at least 10,000 times, indicating that O is popular in China.
  • the temperature-sensitive attenuated phenotypes of the three serotypes of FMDV strains, A and Asia1, were determined by IRES C351. Since the C351 site of IRES is conserved across all FMDV strains, it was determined that IRES C351 is a molecular determinant of the temperature-sensitive attenuated phenotype of all serotype FMDV strains.
  • the present invention determines the temperature-sensitive attenuated phenotype of all seven serotype FMDV strains by structural and functional studies and sequence alignment analysis to finally determine the nucleotide C351 on the loop of the K region of FMDV IRES domain 4.
  • the FMDV attenuated mutant strain with C351G substitution in IRES showed high genetic stability on serial cells, and the strains all had temperature-sensitive phenotypes, so all susceptible cloven-hoofed animals (body temperature 38.5-40 °C) Both are attenuated and meet the safety requirements of live attenuated vaccines.
  • the temperature-sensitive attenuating mutant strain of foot-and-mouth disease virus provided by the invention has the safety and effectiveness of inoculation of susceptible animals and is effective after being challenged, and has the advantages of safety advantages, and can be used as a safe vaccine for live attenuated vaccine or inactivated vaccine.
  • Toxin for preventing foot-and-mouth disease; the full-length cDNA infectious clone of the IRES mutant or chimeric IRES sequence provided by the present invention and the temperature-sensitive attenuated strain of foot-and-mouth disease virus can prepare a live attenuated vaccine against foot-and-mouth disease and is used for preparing foot-and-mouth disease inactivated The vaccine is produced by poisoning or used to prepare a foot-and-mouth disease RNA vaccine.
  • Figure 1 shows the results of virulence determination of IRES subdomain 4 chimeric virus FMDV (R4) in suckling mice
  • Figure 2 is a one-step growth curve of IRES chimeric virus FMDV (R4) and its parental virus at different temperatures;
  • Figure 3 is a one-step growth curve of a subdomain chimeric FMDV mutant of IRES domain 4;
  • Figure 4 is a one-step growth curve of the IRES chimeric FMDV mutant strains rK (Stem) and rK (Loop);
  • Figure 5 is a one-step growth curve of the FRESV mutant strains rC351G and rC351A by site-directed mutagenesis
  • Figure 7 shows the results of virulence determination of FMDV temperature-sensitive mutants in suckling mice
  • IRES C351G mutant-mediated translation initiation efficiency analysis detection of IRES-mediated protein translation efficiency using a replication system (A); viral infection of BHK-21 cells (B) and IBRS-2 cells (C) after VP2 The amount of protein expressed and the titer of the virus produced.
  • Figure 10 shows the results of virulence evaluation of attenuated mutant strains and wild type parental virus FMDV (WT) vaccinated pigs.
  • BHK-21 cells and IBRS-2 cells were cultured at 37 ° C under 5% CO 2 , and the culture was DMEM containing 10% FBS.
  • the pOK-12 vector was kindly provided by Messing (1991); the O-type FMDV O/YS/CHA/05 strain (GenBank accession number: HM008917) and the infectious cDNA clone pYS of the virus were available in the literature (Chinese Invention Patent Publication No.: CN101838658A) Acquired by the method disclosed in (ZL201010160669.9)); the infectious cDNA clone of Asia1 type FMDV Asia1/YS/CHA/05 strain (GenBank accession number: GU931682) can be obtained by the literature (Chinese Invention Patent Publication No. CN101724636A (ZL200810171258.2) Obtained in the manner disclosed in )); A-type FMDV A/VN/03/2009 strain (GenBank accession number: GQ406249) was preserved by the inventor
  • a full-length infectious cDNA clone of the chimeric FMDV IRES replacing the BRBV IRES domain 4 subdomain N was constructed using the fusion PCR method.
  • the specific method is as follows: First, using plasmid pYS as a template, 4N-1:U, 4N-1:L as primers to amplify fragment A; using synthetic BRBV IRES gene as template, 4N-2:U, 4N-2: L is a primer, and fragment B is amplified; and fragment C is amplified by using pYS plasmid as a template and 4N-3:U, 4N-3:L as a primer.
  • PCR-amplified fragments A, B, and C were used as templates, and fusion PCR was performed using 4N-1:U and 4N-3:L as primers to amplify a 5'-end FMDV gene fragment containing partial BRBV IRES replacement.
  • the fragment size is approximately 1.9Kb.
  • the fragment was recovered by gel, digested with Bgl II and Nhe I, and cloned into pYS vector, and the correct clone was designated as p(dN).
  • full-length FMDV infectious cDNA clone plasmids containing the BRBV IRES domain 4 subdomains J and K were constructed using the primers in Table 1, and designated p(dJ) and p(dK).
  • PCR reaction procedure 94 ° C for 4 min; 94 ° C for 30 s, 68 ° C for 9 min, 18 cycles; 72 ° C for 10 min.
  • the PCR product is purified.
  • the methylated template of the PCR product was degraded by DpnI (at 37 ° C for 1 h), and the treated PCR product was transformed into DH5 ⁇ competent cells, and the correct recombinant plasmids were identified as pK (loop) and pK (stem). ), pC351G, pC351A, pC351T, p ⁇ C351, pIn-351C, pA-rC351G, pAsia1-rC351G.
  • Recombinant plasmids p(dN), p(dJ) and p(dK), pK(loop), pK(stem), pC351G, pC351A, pC351T, p ⁇ C351, pIn-351C, pA-rC351G, pAsia1-rC351G were restricted after endonuclease EcoRV digestion of linearized, in accordance with the RiboMAX TM Large Scale RNA Production systems- T7 transcription system extracellular specification, the reaction system is: 25mmol / L rNTP 6 ⁇ L, 5 ⁇ buffer, 4 L, the T7 RNA polymerase mixture [mu] L, EcoRV linearized recombinant plasmid 8 ⁇ L (2 ⁇ g) in a total volume of 20 ⁇ L.
  • the reaction mixture was well mixed, incubated at 37 ° C for 2.5 h, digested with RNase-Free DNase for 15 min, the DNA template was removed, and the transcript was purified by phenol chloroform extraction.
  • BHK-21 cells in a 6-well plate were grown to 60% to 90% monolayer, the cells were washed twice with PBS, and 1.5 mL of DMEM cell culture medium containing 2% fetal calf serum was added.
  • RNA obtained by extracellular 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 conditions, and the cytopathic changes were observed.
  • the virus was harvested about 3 days later, and the BHK-21 cells were inoculated by repeated freezing and thawing for 3 times until the virus could produce stable CPE.
  • the recombinant virus was verified by full-length genome sequencing to confirm the exact mutant strain for subsequent experiments.
  • the rescued viruses were named rdN, rdJ, rdK, rK (loop), rK (stem), rC351G, rC351A, rIn-351C, A-rC351G, and Asia1-rC351G, respectively.
  • Wild-type FMDV, recombinant IRES chimeric virus and IRES site-directed mutant virus were inoculated with BHK-21 and IBRS-2 cells in a logarithmic growth phase at a dose of 0.05 MOI, respectively, at 33 ° C, 37 ° C, 41 ° C. After adsorption for 1 h at three different temperatures, the unadsorbed virus solution was washed away with PBS, and cultured in DMEM containing 2% fetal bovine serum, and 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, and 28 h after inoculation, respectively.
  • the virus was harvested at 32h and 40h, and the TCID 50 titer of the virus was measured at different time points. The average value was calculated after repeating the measurement three times at each time point. The time of infection of the virus was taken as the abscissa, and the logarithm of the TCID 50 titer of the virus at different time points was plotted on the ordinate, and a one-step growth curve of virus replication at different temperatures was plotted.
  • Wild-type FMDV, chimeric virus rK (loop) and point mutation viruses rC351A and rC351G were inoculated into BHK-21 cells for 1 hour, washed twice with PBS, and maintained in DMEM containing 2% fetal bovine serum. After the cells have obvious cytopathic effects, the virus is harvested. After repeated freezing and thawing for 3 times, it was passaged down and passed continuously for 20 generations. Viral RNA was extracted every 5 passages for RT-PCR amplification and sequencing.
  • the 96-well plates of BHK-21 cells and IBRS-2 cells transfected with the chimeric viral replicon RNA were separately cultured in an incubator at 33 ° C, 37 ° C, and 41 ° C. After 12 h, the cells were collected and lysed, and according to Renilla- Glo TM Luciferase Assay System kit, detection of Rluc activity on GloMax luminometer, the reaction system is added 50 ⁇ L of reaction solution per 10 ⁇ L of cell lysate, detection parameters: 2s pre-read delay, 10s detection time.
  • BHK-21 cells and IBRS-2 cells inoculated with 100TCID 50 wild-type FMDV and its recombinant virus were cultured for 12 hours, and the cells were harvested, subjected to lysis treatment, subjected to SDS-PAGE electrophoresis, and transferred to a nitrocellulose membrane.
  • MAb 4B2 diluted 1:1000 was used as the primary antibody, and treated at 37 ° C for 1 h.
  • HRP-labeled rabbit anti-mouse IgG secondary antibody (1:5000 dilution) was added at 37. After °C for 1 h, the DAB solution was added for color development after washing.
  • the internal reference selected ⁇ -Actin antibody diluted 1:1000 as a primary antibody
  • HRP-labeled goat anti-mouse IgG (1:10000 dilution
  • the virus was serially diluted with a sterilized PBS in a 10-fold gradient, and 3 day old BALB/c suckling mice were selected and randomly divided into groups of 5 each. Each strain was inoculated with 3 dilutions of virus in turn, 200 ⁇ L of virus solution was injected into each suckling mouse, and an equal amount of PBS was injected into the negative control group. After 7 days of continuous observation, the survival time of the suckling mice was plotted with the death time of the suckling mice as the abscissa and the survival rate of the suckling mice as the ordinate.
  • Each group of pigs was measured for body temperature, observed clinical symptoms, and nasal and swab swabs and blood were collected daily after inoculation.
  • the third group of pigs were bled 3 days, 7 days, 14 days, and 21 days after inoculation, respectively.
  • FMDV serum antibody-negative healthy shelf pigs were 5 pigs, 3 neck muscles were injected with 10 6 TCID 50 doses of rC351G attenuated strain, and 2 head neck muscles were injected with 1 ml PBS solution as a control.
  • the immunized group and the control group were challenged 21 days after inoculation, and a 1000 ID 50 dose of O/Mya-98/CHA/2010 virus was injected into the neck of each pig.
  • the body temperature was measured daily, clinical symptoms were observed, and nasal swabs, buccal swabs, and blood were collected.
  • Fresh nasal, buccal and blood samples were extracted by total RNA using TRIZOL method, and cDNA obtained by reverse transcription of Oligo (dT 15 ) primer was used as a template for foot-and-mouth disease virus-specific primers (3DF: 5'GGA TGC CGT CTG GTT GTT 3'; 3DR: 5'CGT AGG AGA TCA TGG TGT AAG AGT 3') for quantitative PCR detection.
  • the specific operation of real-time PCR The Green qPCR Super Mix-UDG with ROX (Invitrogen) kit instructions were used to calculate the amount of viral genomic RNA in the sample by a standard curve function.
  • the background value of PCR-amplified FMDV RNA in healthy pig blood and snout swab samples was 2.6 Log10, viral RNA copy number/ml (viral RNA CN/ml) above this value was judged to be FMDV RNA positive.
  • Pig whole blood was collected every day, and the separated serum fraction was used to detect FMDV antibody, and the operation was carried out according to the instructions of the O-type FMDV antibody liquid phase blocking ELISA test kit produced by the Foot and Mouth Disease Reference Laboratory of Lanzhou Veterinary Research Institute.
  • Two wells of 1:2 and 1:4 were set as negative controls for each serum dilution plate, four wells of 1:16, 1:32, 1:64, and 1:128 were used as positive controls, and the virus antigen was set to 4-well control.
  • the serum to be tested was serially diluted from 1:8 to 1:1024. Each step of the reaction was carried out according to the instructions. After the reaction was terminated, the OD 450 nm value was measured with a microplate reader.
  • Pig whole blood was collected daily and the separated serum was used to detect antibodies to the FMDV non-structural protein 3ABC.
  • Serum was diluted in 96-well ELISA plates and two wells were placed in the negative control. 6 ⁇ L of serum was diluted into 120 ⁇ L of serum dilution, the dilution was 1:21, and each replicate was set to 1 repetition. A total of 46 serums were diluted in each plate, and the diluted serum was added dropwise to the ELISA coated plate for a certain period of time.
  • OD 450 nm value the light absorption value at a wavelength of 450 nm was measured by a microplate reader.
  • Antibody titer (OD 450 nm sample - OD 450 nm negative) / (OD 450 nm positive - OD 450 nm negative), if this value > 0.2 was positive.
  • the TCID 50 of FMDV O/YS/CHA/05 virus was determined by BHK-21 cells, and then the micro-cell neutralization test was carried out by using fixed virus dilution serum: the serum was inactivated at 56 ° C for 30 min, and diluted with PBS.
  • the virus of 100TCID 50 was mixed with serum of different dilutions of equal volume, and incubated for 1 hour in a 37 ° C incubator; the above serum-virus mixture was inoculated into BHK-21 cells, 100 ⁇ L per well, and 8 wells per titer.
  • the cells were cultured in a 5% CO 2 incubator at 37 ° C, and the cells were observed daily, and finally judged after 72 hours.
  • virus neutralization titer according to the cytopathic effect (CPE) according to the Reed-Muench method (Reed and Muench., 1938), which can protect 50% of BHK-21 cells.
  • CPE cytopathic effect
  • FMDV serum antibody negative pigs were screened using three serotypes (type O, type A and AsiaI type) FMDV antibody indirect ELISA assay. The specific steps are as follows: inactivated and purified FMDV whole virus as antigen, coated with 96-well ELISA plate, 5% skim milk was blocked at 4 ° C overnight; PBST was washed 3 times, and the serum to be tested was added, 100 ⁇ l per well, 37 ° C After incubation for 1 h, after washing 3 times with PBST, 100 ⁇ l of HRP-labeled goat anti-porcine IgG (1:5000) was added as a secondary antibody (Sigma) per well, incubated at 37 ° C for 1 h; washed 3 times with PBST, and added TMB substrate.
  • inactivated and purified FMDV whole virus as antigen coated with 96-well ELISA plate, 5% skim milk was blocked at 4 ° C overnight
  • PBST was washed 3 times, and the serum to
  • the chromogenic solution was 50 ⁇ l/well, and reacted at 37 ° C for 15 min in the dark; the reaction was stopped by adding 50 ⁇ l/well of 2 M H 2 SO 4 , and finally the OD 450 nm value was measured by a microplate reader.
  • IRES chimeric virus FMDV (R4) is a virulent strain
  • the O-type FMDV reverse genetics operating system was used to replace the domain 4 of FMDV IRES with the corresponding region of BRBV IRES, and the IRES chimeric mutant FMDV (R4) was successfully constructed and rescued.
  • the suckling mouse is a well-recognized animal model for evaluating the virulence of FMDV. Therefore, the virulence of the IRES chimeric virus FMDV (R4) and its wild-type virus FMDV (WT) was compared using a 3-day-old suckling mouse model. As shown in Figure 1.
  • FMDV For FMDV (WT), 1TCID 50 toxic dose caused death of all suckling mice, 0.1TCID 50 toxic dose caused part (50%) of suckling mice to die, while 0.01TCID 50 toxic dose did not cause suckling death; for IRES chimerism
  • the virus FMDV (R4) strain 100TCID 50 toxic dose caused all suckling mice to die, 10TCID 50 toxic dose caused part (80%) of the suckling mice to die, and 1TCID 50 toxic dose did not cause the death of suckling mice.
  • the present invention In order to find the cause of the weakened virulence of FMDV (R4), the present invention first analyzed the secondary structure stability of the wild type virus and the chimeric virus FMDV (R4) IRES by M-fold software, and the results showed that the wild type virus IRES ⁇ G value is -196.60kcal/mol, and the ⁇ G value of the chimeric virus FMDV(R4)IRES is -185.40kcal/mol, indicating that the chimeric IRES is less stable than the wild-type IRES and has a temperature-sensitive structural basis;
  • the domain 4 of the chimeric IRES of FMDV (R4) is derived from bovine rhinovirus, which is mainly infected with the upper respiratory tract of cattle and has the property of replicating in a temperature lower than body temperature (33 ° C). Based on the above analysis, FMDV (R4) is a temperature-sensitive attenuated mutant, and the attenuated phenotype of FMDV (R4) is the result of its temperature-sensitive properties.
  • FMDV (R4) replication ability was slightly higher than FMDV (WT) at 33 °C; FMDV (R4) replication ability was slightly lower than FMDV (WT) at 37 °C; At 41 °C, the replication ability of the chimeric virus FMDV (R4) was significantly reduced, which was about 100-fold lower than that of the parental virus FMDV (WT).
  • the chimeric virus FMDV (R4) On IBRS-2 cells, the chimeric virus FMDV (R4) has similar proliferation characteristics to the parental FMDV (WT) at 33 °C; at 37 °C, the replication ability of the chimeric virus FMDV (R4) is Significantly decreased, at least 100 times lower than the parental FMDV (WT); at 41 °C, the chimeric virus FMDV (R4) replication capacity was completely lost, and its parental toxicity FMDV (WT) still achieved a higher replication Degree (10 6.25 TCID 50 /ml).
  • the K region of IRES domain 4 determines the temperature-sensitive attenuated phenotype of FMDV (R4)
  • the present invention uses the O-type FMDV reverse genetics operating system for the J, K and N subdomains in domain 4 of FMDV IRES
  • the corresponding regions of BRBV IRES were replaced one by one, and the J, K and N subdomain chimeric FMDV mutants of three IRESs were successfully constructed and rescued, named rdJ, rdK and rdN.
  • the replication kinetics of the three chimeric viruses at different temperatures were examined and analyzed, and a one-step growth curve was drawn. The results are shown in Figure 3.
  • the chimeric viruses rdJ, rdN and the parental FMDV (WT) have similar proliferation characteristics either on BHK-21 cells or on IBRS-2 cells, or at different temperatures of 33 ° C, 37 ° C and 41 ° C.
  • BHK-21 cells inoculated with chimeric virus rdK had similar replication ability to parental FMDV (WT) at 33 °C and 37 °C, but their replication ability decreased significantly at 41 °C, compared with parental FMDV ( WT) decreased by about 100-fold; on IBRS-2 cells, even at 37 °C, the replication ability of the chimeric virus rdK decreased significantly, about 100-fold lower than that of the parental toxicity, and the replication ability of rdK was almost lost at 41 °C.
  • the replication characteristics of the chimeric virus rdK (instead of rdJ and rdN) are consistent with the replication characteristics of the chimeric virus FMDV (R4), indicating that the K region of the IRES domain 4 determines the temperature sensitive properties of the chimeric virus FMDV (R4).
  • the virulence test results of the suckling mice showed that the virulence of rdK decreased by about 16 times compared with the virulence of FMDV (WT) (Fig. 7). Taken together, the above results indicate that the K region of IRES domain 4 determines the temperature-sensitive attenuated phenotype of the IRES chimeric virus FMDV (R4).
  • the K region of the IRES domain 4 of FMDV and BRBV is composed of a stem-loop structure. composition.
  • the present invention uses the reverse genetic manipulation technique to use the stem and the loop of the K region of the FMDV IRES with the stem and loop of the K region of the BRBV IRES, respectively.
  • the two chimeric viruses rescued were named rK (Stem) and rK (Loop), respectively, and the replication kinetics of the two chimeric viruses at different temperatures were detected.
  • the one-step growth curve is shown in Fig. 4. Shown.
  • the chimeric virus rK (Stem) has similar proliferation characteristics to the parental FMDV (WT) in BHK-21 or IBRS-2 cells at 33 ° C, 37 ° C and 41 ° C;
  • the replication ability of the recombinant virus rK (Loop) on both cells gradually decreased with increasing temperature, and its replication characteristics were extremely similar to those of rdK.
  • the above results indicate that the loop structure of the K region of IRES domain 4 determines the temperature sensitivity of the chimeric virus FMDV (R4).
  • the virulence of rK (Loop) was determined to be unstable in the mice, and a T351C reversion mutation occurred at the IRES 351 position, which was in response to the IRES 351 after multiple passages of the virus in vitro.
  • the ring of the IRES K region of FMDV (- 351 CUUUAA 356 -) is similar to the ring of the IRES K region of BRBV (-UUUAC-), the main difference being that the IRES of FMDV is more than 351 at the beginning of the ring structure of its K region.
  • One base C, 356 is base A and BRBV IRES is base C here.
  • the present invention first mutated the IRES 356 position A of FMDV to C of IRES 356 of BRBV, and named the rescued mutant virus rA356C. The replication kinetics of the mutant virus at different temperatures (Fig.
  • the present invention made the following four mutations for the base C of the 351 site:
  • the replication ability of rC351G and rC351A is 100 times lower than that of the parental toxicity;
  • the replication ability of rC351G and rC351A decreased gradually with the increase of temperature compared with the parental toxicity, and the replication level at 41 °C decreased significantly by 10,000 times compared with the parental toxicity.
  • the above results indicated that the replication characteristics of the 351-base C-point mutant virus of the two IRESs were similar to those of the IRES chimeric virus rK (Loop), indicating that this site is a molecular determinant of the temperature-sensitive phenotype of the IRES chimeric FMDV.
  • the present invention combines IRES chimeric or point-mutated recombinant viruses FMDV (R4), rdK, rK (Loop), rC351G, rC351A and the parental virus FMDV (WT) in BHK, respectively.
  • the -21 cells were continuously passaged for 20 passages, and the IRES sequence of the 20th generation virus was measured.
  • the IRES sequences of FMDV (R4), rdK, rC351G and rC351A were not produced by any mutation, and the recombinant virus still maintained the original temperature-sensitive properties (Fig.
  • IRES C351 is a determinant of temperature-sensitive attenuated phenotype shared by FMDV strains
  • rC351G and IRES C351G were selected as the research objects in the subsequent studies. On The results of the study were all produced using the O-type FMDV strain. In order to verify that the C351G mutation of IRES also determines the temperature-sensitive attenuated phenotype of other serotypes of FMDV strain, a full-length cDNA infectious clone of type A and Asia1 foot-and-mouth disease virus was used to construct and rescue the virus mutant of IRES with C351G replacement.
  • A-rC351G and Asia1-rC351G Named A-rC351G and Asia1-rC351G.
  • the temperature sensitivity of the above IRES mutant virus was measured, and the results are shown in Figures 8A and 8B: whether A-rC351G or Asia1-rC351G, the replication ability in BHK-21 and IBRS-2 cells increased with temperature. The decrease, which indicates that the C351G mutation of IRES can also obtain a temperature-sensitive phenotype of FMDV strains of other serotypes.
  • IRES C351 is the molecular determinant of the temperature-sensitive attenuated phenotype of all serotypes of FMDV strains. factor.
  • the viral genomic RNA is translated as a large polyprotein as an mRNA template, followed by processing of the viral polyprotein and replication of the viral RNA.
  • two viral replicons FMDV (WT)-luc and rC351G-luc were constructed, which contain the normal IRES of the virus and C351G mutant. The two replicons were transfected into BHK-21 and IBRS-2 cells at 33 ° C, 37 ° C and 41 ° C, respectively, and the IRES and its mutant-mediated translation levels were assessed.
  • luciferase signal is produced by transfected replicon RNA rather than newly synthesized RNA
  • a portion of the transfected cells are added with an inhibitor (2 mM GnHCl) that is effective in inhibiting FMDV RNA replication.
  • an inhibitor (2 mM GnHCl) that is effective in inhibiting FMDV RNA replication.
  • Fig. 9A in the BHK-21 cells, the IRES in which the C351G mutation occurred, although the translation efficiency at 33 ° C and 37 ° C was similar to that of the wild type IRES, was markedly decreased at 41 ° C; in the IBRS-2 cells, The IRES-mediated translation initiation efficiency of the C351G mutation decreased with increasing temperature, and the temperature condition at 41 °C decreased most significantly.
  • the translation ability of the IRES mutant C351G at this temperature was almost lost.
  • the change in translation initiation efficiency of the IRES mutant C351G is consistent with the growth dynamics of the mutant strain rC351G at different temperatures in BHK-21 and IBRS-2 cells, and both exhibit temperature sensitivity characteristics, and this temperature sensitivity The characteristics are more intense on the target animal pig cells of the virus.
  • the above results indicate that the IRES mutated by C351G changes its translation initiation efficiency in a temperature-sensitive manner, thereby affecting the replication ability and virulence of the virus in a temperature-sensitive manner.
  • this experiment analyzed the expression kinetics of the VP2 structural protein of the IRES mutant virus rC351G and its parental virulence FMDV (WT). Results As shown in Figures 9B and 9C, the expression level of VP2 protein in BHK-21 and IBRS-2 cells of mutant virus rC351G decreased with increasing temperature, especially in porcine source cell IBRS-2, viral VP2 protein. Expression at 41 ° C was almost lost.
  • the C351G mutation of IRES mediates this temperature-sensitive effect of the translational level of the viral VP2 protein, which is completely consistent with the change in the replication titer of the infectious virion in the porcine source cell IBRS-2 at different temperatures.
  • the translational initiation ability of the IRES C351G mutant is regulated by temperature.
  • Neonatal rat toxicity test results show that, compared to the virulence of FMDV (WT), and decreased virulence rC351G about 104-fold (FIG. 7).
  • the virulence test results of the rC351G strain on the animal pig are shown in FIG.
  • Three pigs (15#, 46#, 18#) were inoculated with 10 5 TCID 50 doses of wild-type virus FMDV (WT), and the body temperature reached 41 °C 48 h after inoculation.
  • Typical symptoms of foot-and-mouth disease appeared, which was characterized by decreased appetite.
  • Blisters were found in the four hoofs and the nose.
  • the viremia was formed in the vaccinated pigs one day after the inoculation.
  • the viral RNA content in the blood and nose and mouth swabs was significantly higher than that in the healthy pigs.
  • rC351G vaccinated pigs did not produce viremia, antibodies against FMDV non-structural protein 3ABC were produced 21 days after inoculation, whereas cohabiting pigs tested negative for 3ABC antibodies 21 days after inoculation, indicating that the attenuated strain rC351G was Inoculated pigs showed low levels of local replication but did not produce viremia and did not detoxify or spread horizontally.
  • the above test results showed that the virulence of rC351G virus in suckling mice was significantly decreased, and the virulence of the pigs was lost.
  • the strain was serially passaged in piglets.
  • the results showed that the attenuated strain rC351G was continuously transmitted in pigs for 3 generations, and 3 pigs were inoculated in each generation. All the pigs were normal in body temperature during the 5-day observation period and had no clinical symptoms. Serum antibodies collected from the third-generation vaccinated pigs at 3, 7, 14, and 21 days after inoculation were tested.
  • the two control pigs (65#, 73#) increased their body temperature to 41 °C 2 days after the challenge, the appetite decreased, and the spirit was depressed.
  • Blisters appeared in the four hooves of the two pigs 3 days after the challenge;
  • the control pigs developed viremia, and the detection of FMDV RNA in blood and snout swabs was positive.
  • the viremia reached a peak of 8.7 log 10 viral RNA CN/ml 3 days after challenge.
  • the virus can be isolated from both blood and nose and mouth swabs.
  • the three pigs (60#, 66#, 68#) inoculated with the attenuated strain rC351G did not have any clinical symptoms and elevated body temperature within 7 days after the challenge, and the FMDV RNA in the blood and nose and mouth swabs of the immunized pigs.
  • the detection was also negative, and the virus isolation was also negative; the FMDV antibody test results after challenge showed that the rC351G immunization group increased the titer of O-type FMDV neutralizing antibody from 1:128 to 1:512 at 7 days after challenge.
  • the LPBE antibody increased from 1:180 to 1:720, while the PBS control group rapidly increased the FMDV neutralizing antibody level from less than 1:8 to 1:128 on the 7th day after challenge, and the LPBE antibody also rapidly increased from 1:8 to 1. :180.
  • the results of the challenge test showed that the attenuated strain rC351G virus-immunized pigs could provide complete anti-infective protection against the current attack of different genotypes of O-type FMDV.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

提供了口蹄疫病毒温度敏感减毒株及其构建方法和用途。所述减毒株是通过以下方式构建的：将FMDV IRES结构域4的K区环上的一个胞嘧啶突变为鸟嘌呤或腺嘌呤以获得温度敏感减毒株，或者以牛鼻病毒基因组的IRES结构域4的K区置换口蹄疫病毒基因组的IRES结构域4的K区得到嵌合IRES序列，从而获得温度敏感减毒株。

Description

口蹄疫病毒温度敏感减毒株及其构建方法和用途 技术领域
本发明涉及口蹄疫病毒基因组IRES的定点突变体、嵌合体及其在构建口蹄疫病毒温度敏感减毒株中的用途，本发明进一步涉及所构建的口蹄疫病毒温度敏感减毒株在作为FMD防控的减毒疫苗株或用作生产口蹄疫灭活疫苗的安全种毒的用途，属于口蹄疫的防治领域。
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)，该病一旦爆发会对国际贸易和社会经济造成严重影响，因此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。
FMDV属于微RNA病毒科、口蹄疫病毒属成员，共有七个血清型(A、O、C、Asia1、SAT1、SAT2和SAT3)，各血清型毒株之间无免疫交叉保护。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。FMDV的基因组5'末端缺乏帽子结构，其蛋白翻译的起始依赖于5'UTR中的RNA顺式作用元件—内部核糖体进入位点(Internal ribosome entry site，IRES)，它通过招募真核翻译起始因子及核糖体起始病毒蛋白的合成。FMDV IRES长度约为450nt，包括4个结构域，它是FMDV蛋白翻译起始的必需元件。
免疫接种是控制FMD流行的重要手段。然而，国内外使用的商业化口蹄疫灭活疫苗存在以下缺点：1)使用强毒株作为疫苗生产种毒，存在灭活不彻底散毒或工厂生产中散毒的风险；2)灭活的FMDV抗原免疫原性差，免疫保护期短，免疫动物在再次感染FMDV时易于形成持续性感染，持续感染动物可能成为再次流行的传染源，这会严重影响口蹄疫免疫清除计划的实施效果；3)口蹄疫病毒抗原易发生变异，疫苗生产种毒的更新赶不上病毒变异速度，这会对灭活疫苗的免疫效果产生较大的影响。人类和动物疫苗的研究历史表明，只有弱毒疫苗才能诱导快速、坚强、持久的免疫应答，可完全克服灭活疫苗存在的缺点和不足，通过使用弱毒疫苗在世界上已成功消灭了天花和牛瘟、控制了小儿麻痹症等病毒性疾病。虽然口蹄疫在世界许多国家和 地区流行并且时而在无疫国家爆发，至今被广泛使用的疫苗仍然是免疫期短且成本高的灭活疫苗，国内外尚未成功研制出安全有效的FMDV减毒活疫苗。
在过去的100年中，研究人员一直试图开发一种减毒活疫苗用于口蹄疫的防控，但由于该病毒毒力致弱表型不稳定、致弱毒株不能诱导有效的保护性免疫应答、减毒株在不同动物种属间的致弱程度不同、存在毒力返强的危险等原因，FMDV弱毒疫苗的研发至今尚未取得成功。近年来，随着分子病毒学技术的发展以及对口蹄疫病毒研究的不断深入，尤其是口蹄疫病毒感染性cDNA克隆的使用，可通过结构功能研究确定病毒毒力的决定因素，随后在口蹄疫病毒基因组中引入特定的改变、在体内外评估其减毒表型，这使得开发具有良好免疫原性、且在所有宿主动物中均成功减毒的口蹄疫病毒减毒活疫苗成为可能。开发一种实用性FMDV减毒活疫苗需要满足以下条件：1)对所有种属的易感动物均是减毒的，不引起临床症状；2)免疫后诱导坚强的免疫应答，对免疫动物能够提供抗感染保护；3)免疫接种动物不排毒，减毒疫苗株在接种动物与健康动物个体之间不传播，不能返祖。这些要求的指标极具挑战性，但它们是一种口蹄疫减毒活疫苗研究取得成功并且获得应用的前提条件。
发明内容
本发明提供了一种口蹄疫病毒(Foot-and-Mouth Disease Virus)温度敏感减毒株的构建方法，包括：构建口蹄疫病毒全长cDNA感染性克隆质粒，经体外转录得到口蹄疫病毒基因组RNA，将口蹄疫病毒基因组RNA转染细胞并进行病毒的拯救；其中，所述体外转录得到的口蹄疫病毒基因组RNA的IRES结构域4的K区环(351CUUUAA356)上的351位的胞嘧啶(C)突变为鸟嘌呤(G)或腺嘌呤(A)，突变后K区环的碱基序列为351GUUUAA356或351AUUUAA356。
本发明提供了采用上述构建方法得到的口蹄疫病毒温度敏感减毒株。具体的，本发明提供了用所述构建方法获得口蹄疫病毒温度敏感减毒株，其命名为rC351G；本发明所提供的口蹄疫病毒温度敏感减毒株rC351G具有高度的遗传稳定性且均具有温度敏感表型，其对所有易感的偶蹄动物(体温38.5-40℃)均是减毒的，符合减毒活疫苗的安全性需求。本发明将口蹄疫病毒温度敏感减毒株rC351G接种猪，接种动物不表现任何临床症状，但是能够诱导产生高水平的O型FMDV中和抗体并能够完全抵抗当前在中国优势流行的O型不同基因型FMDV异源毒株的攻击；本发明构建的口蹄疫病毒温度敏感减毒株rC351G具有良好的安全性，其在接种猪与健康猪之间 不发生水平传播，并且在猪体内连续传代毒力不返强。因此，本发明所构建的口蹄疫病毒温度敏感减毒株rC351G可按照减毒活疫苗的常规制备方法制备成减毒活疫苗用于防治口蹄疫，或者是将口蹄疫病毒温度敏感减毒株rC351G作为灭活疫苗的安全种毒应用于预防口蹄疫；可将口蹄疫病毒温度敏感减毒株rC351G的全长cDNA感染性克隆经拯救后制备成减毒活疫苗用于防治口蹄疫、或者作为口蹄疫灭活疫苗的安全种毒、或者用于构建口蹄疫RNA疫苗。
本发明将用于拯救口蹄疫病毒温度敏感减毒株rC351G的口蹄疫病毒全长cDNA感染性克隆质粒提交专利认可的机构进行保藏，其微生物保藏编号是：CGMCC NO.13148；分类命名是：大肠杆菌(E.coli)；保藏时间是：2016年10月26日；保藏单位是：中国普通微生物菌种保藏管理中心；保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
本发明提供了口蹄疫病毒温度敏感减毒株的IRES突变体，其核苷酸序列为SEQ ID No.1所示；采用FMDV反向遗传技术手段，能够将所述的IRES突变体用于构建口蹄疫病毒温度敏感减毒株，进一步的，可以将构建的口蹄疫病毒温度敏感减毒株按照减毒活疫苗的常规制备方法制备成减毒活疫苗用于防治口蹄疫、或者是将构建的口蹄疫病毒温度敏感减毒株的全长cDNA感染性克隆经拯救后作为灭活疫苗的安全种毒、或者用于构建口蹄疫RNA疫苗。
本发明提供了另一种口蹄疫病毒温度敏感减毒株的构建方法，包括：构建口蹄疫病毒全长cDNA感染性克隆质粒，经体外转录得到口蹄疫病毒基因组RNA，将口蹄疫病毒基因组RNA转染细胞并进行病毒的拯救；其中，所述经体外转录得到口蹄疫病毒基因组RNA的IRES结构域4用牛鼻病毒(Bovine rhinitis B virus，BRBV)基因组RNA的IRES结构域4进行了替换。
本发明采用上述构建方法得到另一株口蹄疫病毒温度敏感减毒株，其命名为FMDV(R4)；本发明所提供的口蹄疫病毒温度敏感减毒株FMDV(R4)具有高度的遗传稳定性且具有温度敏感表型，其温度敏感特性表明其对所有易感的偶蹄动物(体温38.5-40℃)均是减毒的，符合减毒株的安全性需求。本发明将口蹄疫病毒温度敏感减毒株FMDV(R4)接种猪，接种动物不表现任何临床症状，接种猪与健康猪之间不发生水平传播。更重要的是，接种猪也不产生针对FMDV的抗体，这表明本发明所构建的口蹄疫病毒温度敏感减毒株FMDV(R4)作为口蹄疫灭活疫苗种毒具有更加良好的 安全性，也可将口蹄疫病毒温度敏感减毒株FMDV(R4)的全长cDNA感染性克隆经拯救后作为口蹄疫灭活疫苗的种毒。
本发明将用于拯救口蹄疫病毒温度敏感减毒株FMDV(R4)的口蹄疫病毒全长cDNA感染性克隆质粒提交专利认可的机构进行保藏；其微生物保藏编号是：CGMCC NO.13149；分类命名是：大肠杆菌(E.coli)；保藏时间是：2016年10月26日；保藏单位是：中国普通微生物菌种保藏管理中心；保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
本发明还提供了以牛鼻病毒基因组RNA的IRES结构域4置换口蹄疫病毒基因组RNA的IRES结构域4所得到的嵌合IRES(内部核糖体进入位点)，其核苷酸全长序列为SEQ ID No.2所示；采用FMDV反向遗传技术手段，该嵌合IRES能够用于构建口蹄疫病毒温度敏感减毒株，进一步的，可以将所构建的口蹄疫病毒温度敏感减毒株按照减毒活疫苗的常规制备方法制备成减毒活疫苗用于防治口蹄疫，或者是将构建的口蹄疫病毒温度敏感减毒株的全长cDNA感染性克隆经拯救后作为口蹄疫灭活疫苗的安全种毒。
本发明更进一步的提供了一种口蹄疫病毒温度敏感减毒株的构建方法，包括：构建口蹄疫病毒全长cDNA感染性克隆质粒，经体外转录得到口蹄疫病毒基因组RNA，将口蹄疫病毒基因组RNA转染细胞并进行病毒的拯救；其中，经体外转录得到口蹄疫病毒基因组RNA的IRES结构域4的K区用BRBV基因组RNA的IRES结构域4的K区进行了替换。
采用上述构建方法得到的一株口蹄疫病毒温度敏感减毒株，其命名为rdK；本发明所提供的口蹄疫病毒温度敏感减毒株rdK具有温度敏感表型且具有高度的遗传稳定性，其温度敏感特性表明它对所有易感的偶蹄动物(体温38.5-40℃)均是减毒的，符合减毒株的安全性需求。本发明将温度敏感减毒株rdK接种猪，接种动物不表现任何临床症状，接种猪与健康猪之间不发生水平传播。更重要的是，接种猪也不产生针对FMDV的抗体，这表明本发明所构建的口蹄疫病毒温度敏感减毒株rdK作为口蹄疫灭活疫苗种毒具有更加良好的安全性，因此，可以将口蹄疫病毒温度敏感减毒株rdK的全长cDNA感染性克隆经拯救后作为口蹄疫灭活疫苗的安全种毒。
本发明将用于拯救口蹄疫病毒温度敏感减毒株rdK的口蹄疫病毒全长cDNA感染性克隆质粒提交专利认可的机构进行保藏，其微生物保藏编号是：CGMCC NO.13150；分类命名是：大肠杆菌(E.coli)；保藏时间是：2016年10月26日；保藏单 位是：中国普通微生物菌种保藏管理中心；保藏地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
本发明提供了将口蹄疫病毒基因组RNA的IRES结构域4的K区用BRBV基因组RNA的IRES结构域4的K区进行替换得到嵌合IRES，其核苷酸全长序列为SEQ ID No.3所示；采用本领域的常规FMDV反向遗传技术手段，该嵌合IRES能够应用于构建口蹄疫病毒温度敏感减毒株。
本发明技术方案的详细描述
本发明用O型FMDV反向遗传操作系统，对FMDV IRES的结构域4用BRBV IRES的对应区域进行置换，成功构建并拯救了IRES嵌合的突变株FMDV(R4)。用3日龄乳鼠为模型，对IRES嵌合病毒FMDV(R4)及其野生型病毒FMDV(WT)的毒力进行比较测定，测定结果表明，IRES结构域4嵌合病毒FMDV(R4)对乳鼠的毒力显著下降，与亲本病毒FMDV(WT)相比其毒力下降约100倍，证明IRES的亚结构域4是FMDV毒力的决定因素。
为了确定FMDV(R4)的复制是否具有温度敏感特性，绘制了FMDV(R4)在仓鼠源BHK-21和猪源IBRS-2两种细胞上不同温度下的一步生长曲线。试验结果证实了IRES嵌合减毒株FMDV(R4)是一个温度敏感突变株，而且这种温度敏感特性在易感宿主动物猪源细胞IBRS-2上表现的尤为明显。
为进一步剖析IRES嵌合病毒FMDV(R4)温度敏感减毒表型的分子决定因素，本发明用O型FMDV反向遗传操作系统对FMDV IRES的结构域4中的J、K或N亚结构域用BRBV IRES的对应区域进行逐一置换，成功构建并拯救了三株IRES的J、K或N亚结构域嵌合的FMDV突变株，分别命名为rdJ、rdK或rdN。对这三株嵌合病毒在不同温度下的复制动力学进行检测分析，并绘制一步生长曲线。结果发现，无论在BHK-21细胞上还是在IBRS-2细胞上，或在33℃、37℃和41℃的不同温度条件下，嵌合病毒rdJ、rdN与亲本毒FMDV(WT)均具有相似的增殖特性；而嵌合病毒rdK接种的BHK-21细胞在33℃和37℃条件下与亲本毒FMDV(WT)具有相似的复制能力，但在41℃条件下其复制能力明显下降、比亲本毒FMDV(WT)下降约100倍；在IBRS-2细胞上，即使37℃条件下嵌合病毒rdK的复制能力就显著下降，与亲本毒相比下降约100倍，在41℃时rdK的复制能力几乎丧失。嵌合病毒rdK(而不是rdJ和rdN) 的复制特性与嵌合病毒FMDV(R4)的复制特征一致，表明IRES结构域4的K区决定嵌合病毒FMDV(R4)的温度敏感特性。同时，乳鼠毒力实验结果表明，与FMDV(WT)的毒力相比，rdK的毒力下降约106倍。综合以上结果表明，IRES结构域4的K区决定IRES嵌合病毒FMDV(R4)的温度敏感减毒表型。
FMDV和BRBV的IRES结构域4的K区，均由一个茎-环(stem-loop)结构组成。为了确定K区茎-环结构中与FMDV温度敏感减毒表型相关的区域，本发明用反向遗传操作技术将FMDV IRES之K区的茎、环分别用BRBV IRES之K区的茎、环进行替换，拯救的两株嵌合病毒分别命名为rK(Stem)和rK(Loop)，进而对这两株嵌合病毒在不同细胞不同温度下的复制动力学进行检测。嵌合病毒rK(Stem)无论在BHK-21或在IBRS-2细胞上还是在33℃、37℃和41℃不同温度条件下，均与亲本毒FMDV(WT)具有相似的增殖特性；而嵌合病毒rK(Loop)在两种细胞上的复制能力则随温度的升高而逐渐下降，其复制特性与rdK极其相近。上述结果表明，IRES结构域4之K区的环结构决定嵌合病毒FMDV(R4)的温度敏感性。对rK(Loop)的毒力进行测定发现，该病毒在乳鼠体内不稳定，在IRES 351位出现T351C回复性突变，这与该病毒在体外细胞上多次传代后IRES 351位所发生的回复突变相一致；这种回复性突变导致rK(Loop)K区的环结构接近亲本强毒株K区的环结构，使突变株rK(Loop)对乳鼠的致病力恢复到亲本病毒的水平。
FMDV的IRES K区的环(-351CUUUAA356-)与BRBV的IRES K区的环(-UUUAC-)结构相近，其主要差别是FMDV的IRES在其K区环结构的起始处351位多一个碱基C、在356位为碱基A而BRBV IRES此处为碱基C。为了精细确定决定口蹄疫病毒温度敏感表型的分子因素，本发明首先将FMDV的IRES 356位A突变为BRBV的IRES 356位的C，构建并拯救的突变病毒命名为rA356C。该突变病毒在不同温度下的复制动力学显示，在33℃、37℃、41℃条件下，rA356C在BHK-21和IBRS-2两种细胞上的复制能力均与亲本毒FMDV(WT)相近，表明FMDV IRES的碱基356A与FMDV的温度敏感表型无关。
为确定IRES的C351碱基与FMDV温度敏感表型的相关性，本发明对351位点的碱基C做了如下4种突变：
(1)缺失碱基C351；(2)碱基C351突变为碱基A；(3)碱基C351突变为 碱基G；(4)碱基C351突变为碱基U。
最终试验结果发现，第(1)种和第(4)种的突变方案由于八核苷酸环结构被破坏而没能拯救出病毒，而第(2)和第(3)种突变方案由于八核苷酸环结构不变均能拯救出病毒，所拯救的重组病毒分别命名为rC351A、rC351G。对这两株IRES点突变病毒在不同温度下的复制动力学进行检测。检测结果表明，两个IRES的351位碱基C点突变病毒的复制特性与IRES嵌合病毒rK(Loop)相近，表明该位点是IRES嵌合FMDV温度敏感表型的分子决定因素。本发明同时也发现，IRES C351位点突变的FMDV温度敏感突变株对乳鼠的毒力显著下降，与野生型病毒相比rC351G的毒力下降约10000倍、rC351A的毒力下降1000倍。以上试验结果最终表明，IRES结构域4的K区的环上的351位碱基C决定FMDV的温度敏感减毒表型。
为检测FMDV温度敏感减毒株的遗传稳定性，本发明将IRES嵌合或定点突变的重组病毒FMDV(R4)、rdK、rK(Loop)、rC351G、rC351A以及亲本病毒FMDV(WT)分别在BHK-21细胞上连续传20代，并对第20代病毒的IRES序列进行测定。测定结果表明，IRES嵌合或定点突变的FMDV温度敏感减毒株FMDV(R4)、rdK、rC351G和rC351A具有高度的遗传稳定性；而IRES的K区的环嵌合的FMDV温度敏感减毒株rK(Loop)是不稳定的，传至第20代时部分发生回复突变、传至第25代时病毒群完全发生回复突变。
为了验证IRES的C351G突变也决定其他血清型FMDV毒株的温度敏感性减毒表型，本发明进一步用A型和Asia1型口蹄疫病毒全长cDNA感染性克隆构建并拯救了IRES发生C351G替换的病毒突变株，分别命名为A-rC351G和Asia1-rC351G。对上述IRES突变病毒的温度敏感性进行测定，测定结果表明，无论是A-rC351G还是Asia1-rC351G，在BHK-21和IBRS-2细胞中的复制能力均随温度的升高而下降，这表明IRES的C351G突变也能够使其他血清型的FMDV毒株获得温度敏感性表型。另外，A-rC351G和Asia1-rC351G对乳鼠的致病力检测结果显示，IRES的C351G突变同样能够使A型和Asia1型FMDV对乳鼠的毒力下降至少10000倍，说明在中国流行的O、A和Asia1三个血清型FMDV毒株的温度敏感减毒表型均是由IRES C351决定的。由于IRES的C351位点在所有FMDV毒株均是保守的，由此确定IRES C351是所有血清型FMDV毒株温度敏感减毒表型的分子决定因素。
本发明通过结构与功能研究和序列比对分析，最终确定FMDV IRES结构域4的K区的环上的核苷酸C351决定所有七个血清型FMDV毒株的温度敏感减毒表型。IRES发生C351G替换的FMDV减毒突变株在细胞上连续传代显示出高度的遗传稳定性，并且该类毒株均具有温度敏感表型，因此对所有易感的偶蹄动物(体温38.5-40℃)均是减毒的，符合减毒活疫苗的安全性需求。
本发明所提供的口蹄疫病毒温度敏感减毒突变株，接种易感动物并攻毒后展现的安全性和有效性，具有显著的安全性优势，可作为减毒活疫苗或灭活疫苗的安全种毒用于预防口蹄疫；本发明所提供的IRES突变体或嵌合IRES序列及其口蹄疫病毒温度敏感减毒株的全长cDNA感染性克隆，可制备口蹄疫减毒活疫苗、用于制备口蹄疫灭活疫苗生产用种毒、或用于制备口蹄疫RNA疫苗。
附图说明
图1 IRES亚结构域4嵌合病毒FMDV(R4)对乳鼠的毒力测定结果；
图2 IRES嵌合病毒FMDV(R4)及其亲本病毒在不同温度下的一步生长曲线图；
图3 IRES结构域4的亚结构域嵌合FMDV突变株的一步生长曲线图；
图4 IRES嵌合FMDV突变株rK(Stem)和rK(Loop)的一步生长曲线图；
图5 IRES定点诱变的FMDV突变株rC351G和rC351A的一步生长曲线图；
图6 IRES嵌合及定点突变FMDV毒株在BHK-21细胞上的遗传稳定性(A)及温度敏感性(B)检测结果；
图7 FMDV温度敏感突变株对乳鼠的毒力测定结果；
图8 A型和Asia1型FMDV的IRES C351G突变株的温度敏感性(A和B)及其对乳鼠的致病力(C和D)测定结果；
图9 IRES C351G突变体介导的翻译起始效率分析：利用复制子系统检测IRES介导的蛋白翻译效率(A)；病毒感染BHK-21细胞(B)和IBRS-2细胞(C)后VP2蛋白的表达量及产生病毒的滴度。
图10减毒突变株及其野生型亲本病毒FMDV(WT)接种猪的毒力评价测定结果。
具体实施方式
下面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。但是应理解所述实施例仅是范例性的，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技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。
1、试验材料与试验方法
1.1细胞、载体和毒株
BHK-21细胞和IBRS-2细胞在含5％CO2条件下于37℃培养，培养液为含有10％FBS的DMEM。pOK-12载体由Messing(1991)惠赠；O型FMDV O/YS/CHA/05毒株(GenBank登录号：HM008917)及该病毒的感染性cDNA克隆pYS可通过文献(中国发明专利公开号：CN101838658A(ZL201010160669.9))中披露的方式获取；Asia1型FMDV Asia1/YS/CHA/05毒株(GenBank登录号：GU931682)的感染性cDNA克隆可通过文献(中国发明专利公开号CN101724636A(ZL200810171258.2))中所披露的方式获取；A型FMDV A/VN/03/2009毒株(GenBank登录号：GQ406249)由本发明人实验室保存。
1.2引物设计及合成
根据牛鼻病毒IRES基因序列(GenBank登录号：EU236594)和O型FMDVO/YS/CHA/05株的基因组序列，设计用于扩增两种病毒IRES不同结构域的引物(表1)以及定点突变引物(表2)，所有引物均由上海英俊生物技术有限公司合成。
表1构建IRES嵌合病毒的引物及其序列
Figure PCTCN2017111936-appb-000001
Figure PCTCN2017111936-appb-000002
表2定点突变的引物及其序列
Figure PCTCN2017111936-appb-000003
1.3嵌合病毒全长cDNA感染性克隆质粒的构建
利用融合PCR方法，构建置换BRBV IRES domain 4亚结构域N的嵌合FMDV IRES的全长感染性cDNA克隆。具体方法如下：首先，以质粒pYS为模板，以4N-1:U、4N-1:L为引物，扩增出片段A；以人工合成的BRBV IRES基因为模板，以4N-2:U、4N-2:L为引物，扩增出片段B；以pYS质粒为模板，以4N-3:U、4N-3:L为引物，扩增出片段C。纯化PCR扩增的片段A、B、C用作模板，以4N-1:U和4N-3:L为引物进行融合PCR，扩增出含部分BRBV IRES置换的FMDV 5’端基因片段，该片段大小约为1.9Kb。胶回收该片段，经Bgl II和Nhe I双酶切，克隆至pYS载体中，序列测定正确的克隆命名为p(dN)。同样，利用表1中引物分别构建含有BRBV IRES domain4亚结构域J和K的全长FMDV感染性cDNA克隆质粒，命名为p(dJ)和p(dK)。
1.4 A型FMDV全长cDNA感染性克隆的构建
根据口蹄疫病毒A/QSA/CHA/09株的全基因组测序结果，利用人工合成基因的方法合成基因组全长，并在全基因组cDNA的5＇末端引入T7启动子序列和SpeI酶切位点(5'act agt TAA TAC GAC TCA CTA TAGGG 3')，在全基因组cDNA的3＇末端引入EcoRV酶切位点(gat atc)，用于全基因组cDNA的线性化。全基因组经限制性内切酶SpeI和EcoRV酶切后，克隆于低拷贝载体pOK12中，构建完成的感染性cDNA克隆命名为pQSA。
1.5定点突变
按照Quik
Figure PCTCN2017111936-appb-000004
Site-Directed Mutagenesis Kit说明书，通过PCR的方法在感染性cDNA克隆上利用表2引物分别引入突变位点。PCR反应程序：94℃4min；94℃30s，68℃9min，18个循环；72℃10min。反应完成后，纯化PCR产物。用DpnI降解PCR产物中甲基化的模板(37℃作用1h)，将处理后的PCR产物转化DH5α感受态细胞，挑菌，经测序鉴定正确重组质粒分别命名为pK(loop)、pK(stem)、pC351G、pC351A、pC351T、p△C351、pIn-351C、pA-rC351G、pAsia1-rC351G。
1.6病毒的拯救
重组质粒p(dN)、p(dJ)和p(dK)、pK(loop)、pK(stem)、pC351G、pC351A、pC351T、p△C351、pIn-351C、pA-rC351G、pAsia1-rC351G经限制性内切酶EcoRV酶切线性 化后，按照RiboMAXTM Large Scale RNA Production Systems-T7系统说明书胞外转录，反应体系为：25mmol/L rNTP 6μL，5×缓冲液4μL，T7RNA聚合酶混合液2μL，EcoRⅤ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转录产物。当6孔板中的BHK-21细胞生长至60％～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。将细胞外转录获得的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％CO2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次后传代接种BHK-21细胞，直到病毒能产生稳定的CPE。重组病毒经全长基因组测序验证准确的突变株用于后续试验。获得拯救的病毒分别命名为rdN、rdJ、rdK、rK(loop)、rK(stem)、rC351G、rC351A、rIn-351C、A-rC351G、Asia1-rC351G。
1.7一步生长曲线
野生型FMDV、重组的IRES嵌合病毒以及IRES定点突变病毒，按0.05MOI剂量分别接种处于对数生长期状态良好的BHK-21、IBRS-2细胞，分别置于33℃、37℃、41℃三种不同的温度下吸附1h，再用PBS洗去未吸附的病毒液，加入含2％胎牛血清的DMEM维持培养，并分别在接种后4h、8h、12h、16h、20h、24h、28h、32h、40h收获病毒，测定不同时间点收获病毒的TCID50滴度，每个时间点重复测定3次后计算平均值。以病毒的感染细胞的时间为横坐标，以病毒在不同时间点的TCID50滴度的对数值为纵坐标，绘制不同温度下病毒复制的一步生长曲线。
1.8病毒传代及遗传稳定性检测
取野生型FMDV、嵌合病毒rK(loop)和点突变病毒rC351A、rC351G分别接种于BHK-21细胞中感作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，收获病毒。反复冻融3次后，向下传代，连续传20代。每隔5代提取病毒RNA，进行RT-PCR扩增及序列测定。
1.9荧光素酶活性检测
将转染嵌合病毒复制子RNA的BHK-21细胞和IBRS-2细胞的96孔板，分别培 养在33℃、37℃、41℃温箱中，12h后收集细胞并裂解，并按照Renilla-GloTM Luciferase Assay System海肾荧光素酶检测试剂盒说明书，在GloMax发光仪上检测Rluc活性，反应体系为每10μL细胞裂解液加入50μL反应液，检测参数：2s预读延迟，10s检测时间。
1.10 Western blot
将接种100TCID50野生型FMDV及其重组病毒的BHK-21细胞和IBRS-2细胞，培养12h后收获取细胞，经裂解处理后，进行SDS-PAGE电泳，并转印至硝酸纤维素膜上。用5％脱脂乳封闭后，以MAb 4B2(1：1000稀释)作为一抗于，37℃作用1h，用PBST洗涤后，加入HRP标记山兔抗鼠IgG二抗(1：5000稀释)于37℃作用1h，洗涤后加入DAB溶液显色。另外，内参选择β-Actin抗体(1：1000稀释)作为一抗，以HRP标记山羊抗小鼠IgG(1：10000稀释)作为二抗。
1.11乳鼠毒力试验
用灭菌PBS以10倍梯度系列稀释病毒，选用3日龄BALB/c乳鼠并随机分组，每组5只。每个毒株依次接种3个稀释度的病毒，每只乳鼠注射200μL病毒液，阴性对照组注射等量PBS。连续观察7天后，以乳鼠死亡时间为横坐标，以乳鼠存活率为纵坐标，绘制乳鼠的存活曲线。
1.12本动物猪毒力评价试验、安全性试验、免疫接种及攻毒试验
毒力评价试验
20-30公斤的FMDV血清抗体阴性的健康架子猪20头，随机分成4组，每组5头。一组取3头猪颈部肌肉注射105TCID50/头剂量的野生型毒株FMDV(WT)，另三组每组取3头猪颈部肌肉注射106TCID50/头剂量的IRES突变株rC351G，FMDV(R4)或rdK，24h后每组分别放入另2头猪作为同居动物。在接种后7天内，每天测量猪只体温、观察临床症状并采集鼻腔拭子、口腔拭子及血液。
安全性试验
30-40公斤的FMDV血清抗体阴性的健康架子猪9头随机分成3组，每组3头。第一组耳后肌肉注射106TCID50剂量的rC351G减毒株。在接种后5天进行剖杀，取 扁桃体组织、血浆、口鼻分泌物混合进行匀浆处理，取2ml注射第二组猪。同样，第二组猪在接种后5天进行剖杀，取相同组织样品混合进行匀浆处理，取2ml注射第三组猪。每组猪在接种后每天测量体温、观察临床症状以及采集口鼻拭子和血液，第三组猪分别在接种后3天、7天、14天、21天采血。
免疫接种及攻毒试验
为了对减毒株rC351G的免疫保护效果进行评价，用该毒株接种3头猪、以接种PBS的2头猪作为攻毒对照，在接种后21天使用当前在中国呈优势流行的O型FMDV毒株O/Mya-98/CHA/2010进行攻毒，以评价其免疫保护效果，具体方法如下：
20-30公斤的FMDV血清抗体阴性的健康架子猪5头，3头颈部肌肉注射106TCID50剂量的rC351G减毒株，2头颈部肌肉注射1ml PBS溶液作为对照。免疫组和对照组在接种后21天攻毒，每头猪颈部注射1000ID50剂量的O/Mya-98/CHA/2010病毒。在攻毒后7天内，每天测量体温、观察临床症状并采集鼻腔拭子、口腔拭子及血液。
1.121临床症状观察
每天仔细观察猪只临床发病情况并作记录，按照Pacheco andMason等描述的方法(Pacheco and Mason et al,J.Vet.Sci.,2010)进行临床打分：蹄部发病每只记3分，鼻部、舌部以及唇部发病各记3分，最大分值为20分。
1.122病毒血症及排毒情况检测
新鲜的鼻腔、口腔拭子及血液样品经TRIZOL法提取总RNA，使用Oligo(dT15)引物反转录获得的cDNA作为模板，以口蹄疫病毒特异性引物(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 PCTCN2017111936-appb-000005
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。以健康猪血液和口鼻拭子样品PCR扩增FMDV RNA的背景值为2.6
Figure PCTCN2017111936-appb-000006
log10，病毒RNA拷贝数/ml(viral RNA CN/ml)高于此数值判为FMDV RNA阳性。
1.123 FMDV特异性抗体检测
每天采集猪只全血，分离的血清部分用于检测FMDV抗体，按照兰州兽医研究所口蹄疫参考实验室生产的O型FMDV抗体液相阻断ELISA检测试剂盒的说明书进行操作。每一块血清稀释板设置1：2和1：4两孔作为阴性对照，1：16、1：32、1：64、1：128四孔作为阳性对照，另外病毒抗原设置4孔对照。被检血清做从1：8到1：1024依次做2倍倍比稀释。每步反应按说明书操作，反应终止后，用酶标仪测定OD450nm值。
1.124 FMDV非结构蛋白3ABC抗体检测
每天采集猪只全血，分离的血清用于检测FMDV非结构蛋白3ABC的抗体。按照兰州兽医研究所口蹄疫参考实验室3ABC-I-ELISA的说明书进行操作。血清在96孔ELISA板中被稀释，阴阳性对照各设两孔。将6μL血清稀释到120μL血清稀释液中，稀释度为1：21，每份设置1个重复，每块板子共稀释46份血清，将已稀释血清滴加到ELISA包被板上，作用一定时间后依次加入各种酶标试剂和底物，最后用酶标仪测定450nm波长下的光吸收值(OD450nm值)。抗体效价＝(OD450nm样品－OD450nm阴性)/(OD450nm阳性－OD450nm阴性)，若此值＞0.2判为阳性。
1.125微量细胞中和试验
首先用BHK-21细胞测定FMDV O/YS/CHA/05病毒的TCID50，然后采用固定病毒稀释血清的方法进行微量细胞中和试验：将血清于56℃灭活30min，用PBS做倍比稀释；用100TCID50的病毒分别与等体积不同稀释度的血清混合，37℃温箱中温育1h；将上述血清-病毒混合液分别接种BHK-21细胞，每孔100μL，每滴度设8孔重复，在37℃于5％CO2培养箱中培养，每日观察细胞，72h后做最终判定。另外，设病毒、阳性血清和正常细胞对照，根据细胞病变效应(CPE)情况按Reed-Muench方法(Reed and Muench.,1938)计算病毒中和滴度，即能保护50％BHK-21细胞不出现CPE的血清稀释浓度。
1.126间接ELISA
使用三种血清型(O型、A型和AsiaI型)FMDV抗体间接ELISA检测方法筛选FMDV血清抗体阴性猪。具体步骤如下：灭活并提纯的FMDV全病毒作为抗原， 包被96孔ELISA酶标板，加5％脱脂乳4℃过夜封闭；PBST洗涤3次后加入待检血清，每孔100μl，37℃温育1h，用PBST洗涤3次后，每孔加入100μl HRP标记的羊抗猪IgG(1：5000)作为二抗(Sigma)，37℃温育1h；用PBST洗涤3次，加入TMB底物显色溶液50μl/孔，37℃避光反应15min；加入50μl/孔2M H2SO4终止反应，最后用酶标仪测定OD450nm值。
2.试验结果
2.1 IRES嵌合病毒FMDV(R4)是毒力致弱毒株
本发明用O型FMDV反向遗传操作系统，对FMDV IRES的结构域4用BRBV IRES的对应区域进行置换，成功构建并拯救了IRES嵌合的突变株FMDV(R4)。乳鼠是公认的评价FMDV毒力的动物模型，因此用3日龄乳鼠为模型，对IRES嵌合病毒FMDV(R4)及其野生型病毒FMDV(WT)的毒力进行比较测定，测定结果如图1所示。对于FMDV(WT)，1TCID50接毒剂量引起全部乳鼠死亡，0.1TCID50接毒剂量引起部分(50％)乳鼠死亡，而0.01TCID50接毒剂量不引起乳鼠死亡；对于IRES嵌合病毒FMDV(R4)株，100TCID50接毒剂量引起全部乳鼠死亡，10TCID50接毒剂量引起部分(80％)乳鼠死亡，而1TCID50接毒剂量不引起乳鼠死亡。上述结果表明，IRES结构域4嵌合病毒FMDV(R4)对乳鼠的毒力显著下降，与亲本病毒FMDV(WT)相比其毒力下降约100倍，证明IRES的亚结构域4是FMDV毒力的决定因素。
2.2 FMDV(R4)是温度敏感突变株
为了寻找FMDV(R4)毒力减弱的原因，本发明首先用M-fold软件对野生型病毒与嵌合病毒FMDV(R4)IRES的二级结构稳定性进行分析，结果显示，野生型病毒IRES的△G值为-196.60kcal/mol，嵌合病毒FMDV(R4)IRES的△G值为-185.40kcal/mol，表明嵌合IRES比野生型IRES的稳定性差，具备温度敏感性的结构基础；另外，FMDV(R4)的嵌合IRES之结构域4来自牛鼻病毒，而牛鼻病毒主要感染牛上呼吸道，具有在低于体温的环境(33℃)下复制的特性。综合上述分析认为，FMDV(R4)是一个温度敏感减毒突变株，FMDV(R4)的减毒表型是其获得温度敏感特性的结果。
为了探究FMDV(R4)的复制是否具有温度敏感特性，绘制了FMDV(R4)在仓鼠源 BHK-21和猪源IBRS-2两种细胞上不同温度下的一步生长曲线，结果如图2所示。在BHK-21细胞上，在33℃条件下，FMDV(R4)复制能力略微高于FMDV(WT)；在37℃的条件下，FMDV(R4)复制能力略微低于FMDV(WT)；然而在41℃条件下，嵌合病毒FMDV(R4)的复制能力明显下降，较之亲本病毒FMDV(WT)下降约100倍。在IBRS-2细胞上，在33℃的条件下嵌合病毒FMDV(R4)与亲本毒FMDV(WT)具有相似的增殖特性；在37℃条件下，嵌合病毒FMDV(R4)的复制能力就明显下降，比亲本毒FMDV(WT)下降至少100倍；在41℃的条件下，嵌合病毒FMDV(R4)复制能力完全丧失，而其亲本毒FMDV(WT)仍能达到较高的复制滴度(106.25TCID50/ml)。以上试验结果证实了IRES嵌合减毒株FMDV(R4)是一个温度敏感突变株，而且这种温度敏感性在易感宿主动物猪源细胞IBRS-2上表现的尤为明显。
2.3 IRES结构域4的K区决定FMDV(R4)的温度敏感性减毒表型
为了进一步剖析IRES嵌合病毒FMDV(R4)温度敏感减毒表型的分子决定因素，本发明用O型FMDV反向遗传操作系统对FMDV IRES的结构域4中的J、K和N亚结构域用BRBV IRES的对应区域进行逐一置换，成功构建并拯救了三株IRES的J、K和N亚结构域嵌合的FMDV突变株，分别命名为rdJ、rdK和rdN。对这三株嵌合病毒在不同温度下的复制动力学进行检测分析，并绘制一步生长曲线，结果如图3所示。无论在BHK-21细胞上还是在IBRS-2细胞上，或在33℃、37℃和41℃的不同温度条件下，嵌合病毒rdJ、rdN与亲本毒FMDV(WT)均具有相似的增殖特性；而嵌合病毒rdK接种的BHK-21细胞在33℃和37℃条件下与亲本毒FMDV(WT)具有相似的复制能力，但在41℃条件下其复制能力明显下降、比亲本毒FMDV(WT)下降约100倍；在IBRS-2细胞上，即使37℃条件下嵌合病毒rdK的复制能力就显著下降，与亲本毒相比下降约100倍，在41℃时rdK的复制能力几乎丧失。嵌合病毒rdK(而不是rdJ和rdN)的复制特性与嵌合病毒FMDV(R4)的复制特征一致，表明IRES结构域4的K区决定嵌合病毒FMDV(R4)的温度敏感特性。同时，乳鼠毒力实验结果表明，与FMDV(WT)的毒力相比，rdK的毒力下降约106倍(图7)。综合以上结果表明IRES结构域4的K区决定IRES嵌合病毒FMDV(R4)的温度敏感减毒表型。
2.4 IRES结构域4的K区的环结构决定FMDV(R4)的温度敏感减毒表型
FMDV和BRBV的IRES结构域4的K区，均由一个茎-环(stem-loop)结构 组成。为了确定K区茎-环结构中与FMDV温度敏感减毒表型相关的区域，本发明采用反向遗传操作技术将FMDV IRES的K区的茎、环分别用BRBV IRES之K区的茎、环进行替换，拯救的两株嵌合病毒分别命名为rK(Stem)和rK(Loop)，进而对这两株嵌合病毒在不同细胞不同温度下的复制动力学进行检测，一步生长曲线如图4所示。嵌合病毒rK(Stem)无论在BHK-21或在IBRS-2细胞上还是在33℃、37℃和41℃不同温度条件下，均与亲本毒FMDV(WT)具有相似的增殖特性；而嵌合病毒rK(Loop)在两种细胞上的复制能力则随温度的升高而逐渐下降，其复制特性与rdK极其相近。上述结果表明，IRES结构域4的K区的环结构决定嵌合病毒FMDV(R4)的温度敏感性。对rK(Loop)的毒力进行测定发现，该病毒在乳鼠体内不稳定，在IRES 351位出现T351C回复性突变，这与该病毒在体外细胞上多次传代后IRES 351位所发生的回复突变相一致(图6A)；这种回复性突变导致rK(Loop)K区的环结构接近亲本强毒株K区的环结构，使突变株rK(Loop)对乳鼠的致病力恢复到亲本病毒的水平(图7)。
2.5 IRES结构域4的K区环上的351位碱基C决定FMDV的温度敏感减毒表型
FMDV的IRES K区的环(-351CUUUAA356-)与BRBV的IRES K区的环(-UUUAC-)结构相近，其主要差别是FMDV的IRES在其K区环结构的起始处351位多一个碱基C、356位为碱基A而BRBV IRES此处为碱基C。为了准确确定决定口蹄疫病毒温度敏感表型的分子因素，本发明首先将FMDV的IRES 356位A突变为BRBV的IRES 356位的C，将构建并拯救的突变病毒命名为rA356C。该突变病毒在不同温度下的复制动力学(图5)显示，在33℃、37℃、41℃条件下，rA356C在BHK-21和IBRS-2两种细胞上的复制能力均与亲本毒FMDV(WT)相近，表明FMDV IRES的碱基356A与FMDV的温度敏感表型无关。
为确定IRES的C351碱基与FMDV温度敏感表型的相关性，本发明对351位点的碱基C做了如下4种突变：
(1)缺失碱基C351；(2)碱基C351突变为碱基A；(3)碱基C351突变为碱基G；(4)碱基C351突变为碱基U。
最终试验结果发现，第(1)种和第(4)种的突变方案由于八核苷酸环结构被破坏(图5A)而没能拯救出病毒，而第(2)和第(3)种突变方案由于八核苷酸环结构不变(图5A)均拯救出病毒，拯救的重组病毒分别命名为rC351A、rC351G。对 这两株IRES点突变病毒在不同温度下的复制动力学进行检测，结果如图5所示。在BHK-21细胞上33℃和37℃的条件下，rC351G和rC351A与其亲本毒具有相近的增殖特性，当温度升高到41℃时rC351G和rC351A比亲本毒的复制能力下降100倍；在猪源IBRS-2细胞上，rC351G和rC351A与亲本毒相比其复制能力随温度的升高逐渐下降，在41℃条件下的复制水平与亲本毒相比显著下降达10000倍。上述结果表明，两个IRES的351位碱基C点突变病毒的复制特性与IRES嵌合病毒rK(Loop)相近，表明该位点是IRES嵌合FMDV温度敏感表型的分子决定因素。同时也发现，IRES C351位点突变的FMDV温度敏感突变株对乳鼠的毒力显著下降，与野生型病毒相比，rC351G的毒力下降约10000倍、rC351A的毒力下降约1000倍。综合以上试验结果最终表明，IRES结构域4的K区的环上的351位碱基C，决定FMDV的温度敏感性减毒表型。
2.6温度敏感减毒株体外连续传代的遗传稳定性
为检测FMDV温度敏感减毒株的遗传稳定性，本发明将IRES嵌合或点突变的重组病毒FMDV(R4)、rdK、rK(Loop)、rC351G、rC351A以及亲本病毒FMDV(WT)分别在BHK-21细胞上连续传20代，并对第20代病毒的IRES序列进行测定。在BHK-21细胞上传20代，FMDV(R4)、rdK、rC351G和rC351A的IRES序列均无任何突变产生，而且重组病毒仍保持原有的温度敏感特性(图6B)及乳鼠致病力减毒表型(图7)。然而，rK(Loop)虽然在传至第15代时尚无任何突变产生，第20代时已有部分病毒在IRES 351位出现T351C突变，将病毒再传5代则T351C突变病毒成为优势克隆(图6A)；同时，rK(Loop)第25代病毒的温度敏感特性丧失(图6B)，对乳鼠的致病力回复到与野生型病毒相近的水平(图7)。上述试验结果表明，IRES嵌合或定点突变的FMDV温度敏感减毒株FMDV(R4)、rdK、rC351G和rC351A具有高度的遗传稳定性；而IRES的K区的环嵌合的FMDV温度敏感减毒株rK(Loop)是不稳定的，传至第20代时部分发生回复突变、传至第25代时病毒群完全发生回复突变。
2.7 IRES C351是FMDV毒株共享的温度敏感性减毒表型的决定位点
虽然rC351G和rC351A均具有高度的遗传稳定性，但rC351G的减毒效果比rC351A更为显著，因此后续研究均选择rC351G以及IRES C351G作为研究对象。上 述研究结果，均是用O型FMDV毒株产生的。为了验证IRES的C351G突变也决定其他血清型FMDV毒株的温度敏感性减毒表型，用A型和Asia1型口蹄疫病毒全长cDNA感染性克隆构建并拯救了IRES发生C351G替换的病毒突变株，分别命名为A-rC351G和Asia1-rC351G。对上述IRES突变病毒的温度敏感性进行测定，结果如图8A和8B所示：无论是A-rC351G还是Asia1-rC351G，在BHK-21和IBRS-2细胞中的复制能力均随温度的升高而下降，这表明IRES的C351G突变也能够使其他血清型的FMDV毒株获得温度敏感性表型。另外，A-rC351G和Asia1-rC351G对乳鼠的致病力检测结果显示，IRES的C351G突变同样能够使A型和Asia1型FMDV对乳鼠的毒力下降至少10000倍(图8C和8D)，说明在中国流行的O、A和Asia1三个血清型FMDV毒株的温度敏感减毒表型均是由IRES C351决定的。由于IRES的C351位点以及围绕该位点的功能性茎-环结构在所有FMDV毒株均是保守的，由此推断，IRES C351是所有血清型FMDV毒株温度敏感减毒表型的分子决定因素。
2.8 IRES C351G突变体介导的翻译起始能力受温度调控
口蹄疫病毒感染细胞后，病毒基因组RNA作为mRNA模板翻译一个大的多聚蛋白，随后进行病毒多聚蛋白的加工以及病毒RNA的复制。为了确定病毒生命周期中的哪个步骤导致FMDV突变株rC351G的毒力减弱，构建了两个病毒复制子FMDV(WT)-luc和rC351G-luc，这两个复制子分别含有病毒的正常IRES及其C351G突变体。将这两个复制子在33℃、37℃和41℃不同温度下分别转染BHK-21和IBRS-2细胞，评估IRES及其突变体介导的翻译水平。为了区分荧光素酶信号是转染的复制子RNA而不是新合成的RNA产生的，其中一部分转染细胞加入能够有效抑制FMDV RNA复制的抑制剂(2mM GnHCl)。结果如图9A所示，在BHK-21细胞中，发生C351G突变的IRES尽管在33℃和37℃的翻译效率与野生型IRES相似，但是在41℃时明显下降；在IBRS-2细胞中，发生C351G突变的IRES介导的翻译起始效率随温度的升高而降低，在41℃温度条件下降最明显，IRES突变体C351G在此温度下的翻译能力几乎丧失。IRES突变体C351G这种翻译起始效率的改变，与突变株病毒rC351G在BHK-21和IBRS-2细胞中不同温度下的生长动态规律相一致，均呈现温度敏感性特征，而且这种温度敏感特性在病毒的靶动物猪细胞上表现的更为强烈。以上结果表明，C351G发生突变的IRES以温度敏感性方式改变其翻译起始效率，从而以温度敏感性方式影响病毒的复制能力和毒力。
为了进一步证实C351G突变介导IRES翻译起始的温度敏感效应，本试验分析了IRES突变病毒rC351G与其亲本毒FMDV(WT)之VP2结构蛋白的表达动力学。结果如图9B和9C所示，突变病毒rC351G在BHK-21和IBRS-2细胞中VP2蛋白的表达量均随温度的升高而降低，尤其是在猪源细胞IBRS-2中，病毒VP2蛋白在41℃条件下的表达几乎丧失。IRES的C351G突变介导病毒VP2蛋白翻译水平的这种温度敏感效应，与不同温度下IRES突变株rC351G在猪源细胞IBRS-2中感染性病毒粒子复制滴度的变化，是完全一致的，表明IRES C351G突变体的翻译起始能力受温度的调控。
2.9突变株rC351G对乳鼠和本动物猪的毒力试验结果
乳鼠毒力试验结果表明，与FMDV(WT)的毒力相比，rC351G的毒力下降约104倍(图7)。
rC351G毒株对本动物猪的的毒力试验结果如图10所示。分别接种105TCID50剂量野生型病毒FMDV(WT)的3头猪(15#、46#、18#)，在接种后48h体温均达到41℃，出现典型的口蹄疫症状，具体表现为食欲下降、精神沉郁，四蹄及鼻部均出现水疱；接种猪在接种后1天即形成病毒血症，血液及口鼻拭子中病毒RNA的含量显著高于健康猪2.6log10viral RNA CN/ml的水平，接种后4天病毒血症达到高峰为8.7log10RNA copies/ml。2头同居猪(10#、35#)的感染和发病较WT接种猪滞后，在接种后4天(同居后3天)体温达到41℃，四蹄均出现水疱；同居猪在接种后3天血液及口鼻拭子中的病毒RNA为阳性，接种后5天达到高峰7.1log10viral RNA CN/ml的水平。然而，用相当于上述野生型病毒接种10倍剂量的减毒突变株rC351G(106TCID50/头)接种3头猪(36#、49#、59#)，2头猪(57#、28#)用作同居对照，在接种后7天内均无任何临床症状，也无体温升高现象；接种猪以及同居猪的血液及口鼻拭子中病毒RNA检测均为阴性。虽然rC351G接种猪不产生病毒血症，但在接种后21天均可产生针对FMDV非结构蛋白3ABC的抗体，而同居猪在接种后21天检测3ABC抗体均为阴性，这表明减毒株rC351G在接种猪体内呈低水平局部复制但不产生病毒血症不排毒也不发生水平传播。以上试验结果表明，rC351G病毒对乳鼠的毒力显著下降，对本动物猪的毒力丧失。
2.10减毒株rC351G的稳定性和安全性评价试验结果
1)体外细胞传代
为检测减毒株rC351G的遗传稳定性，将其在BHK-21细胞上连续传20代，并对 第20代病毒的全基因组序列进行测定。结果表明，rC351G株具有高度的遗传稳定性，在体外连续传20代其IRES C351G不发生回复突变，而且其减毒表型也不发生改变。
2)同居动物不感染
同居试验结果如图1所示，健康猪(57#、28#)与rC351G接种猪(36#、49#、59#)同居后不表现任何临床症状，7天内采集的血液样品及口鼻拭子检测病毒RNA均为阴性，在21天检测血清中针对FMDV非结构蛋白3ABC的抗体也为阴性，这表明同居猪未发生FMDV rC351G的感染。以上同居试验结果表明，由于减毒突变株rC351G接种猪后不产生病毒血症、不排毒、因而对易感动物失去毒力也不能够水平传播，具有优良的安全性。
3)本动物连续传代毒力不返强
为进一步评价减毒株rC351G的安全性，对该毒株在仔猪体内进行连续传代。结果表明，减毒株rC351G在猪体连续传3代，每代接种3头猪，所有猪在5天观察期内体温均正常、且无任何临床症状。检测第三代接种猪在接种后3、7、14、和21天采集的血清抗体，结果显示：FMDV结构蛋白抗体为阴性(LPBE值小于1:8)，FMDV非结构蛋白3ABC的抗体也为阴性(OD值<0.2)，在接种后3、7、14、21天血液及口鼻拭子中检测病毒RNA也为阴性。以上试验结果表明，减毒株rC351G在猪体内传代毒力不返强，这在前面证明同居不传播的基础上进一步表明人工接种传代也不发病，在体内具有良好的安全性。
2.11减毒株rC351G免疫猪的免疫保护效力试验结果
rC351G免疫猪的免疫保护效力试验结果如表3所示。
表3 rC351G免疫猪对FMDV O/Mya-98/CHA/2010株攻击的保护效果
Figure PCTCN2017111936-appb-000007
根据试验结果可见，2头对照猪(65#、73#)在攻毒后2天体温升高至41℃， 食欲下降、精神沉郁，攻毒后3天2头猪四蹄均出现水疱；在攻毒后48h，对照猪即形成病毒血症，血液及口鼻拭子中FMDV RNA的检测即为阳性，病毒血症在攻毒后3天达到高峰为8.7log10viral RNA CN/ml，此时血液及口鼻拭子中均可分离到病毒。然而，减毒株rC351G接种的3头猪(60#、66#、68#)在攻毒后7天内均无任何临床症状及体温升高现象，且免疫猪血液及口鼻拭子中FMDV RNA的检测也为阴性，病毒分离也为阴性；攻毒后的FMDV抗体检测结果显示，rC351G免疫组在攻毒后7天O型FMDV中和抗体滴度由1:128小幅上升到1:512，LPBE抗体由1:180上升到1:720，而PBS对照组在攻毒后7天FMDV中和抗体水平由小于1:8迅速上升到1:128、LPBE抗体也由1:8迅速上升到1:180。攻毒试验结果表明，减毒株rC351G病毒免疫猪能够对当前流行的O型FMDV不同基因型毒株的攻击提供完全的抗感染保护。
2.12 FMDV(R4)和rdK对本动物猪的毒力试验结果
用减毒突变株FMDV(R4)和rdK(106TCID50/头)分别接种3头猪，24h后每组另外放入2头猪用作同居动物。结果如图10所示，接种后7天所有猪只均无任何临床症状，也无体温升高现象；接种猪以及同居猪的血液及口鼻拭子中病毒RNA检测均为阴性。接种后21天FMDV抗体检测结果显示，所有接种猪和同居猪FMDV中和抗体均为阴性(<1:8)。以上试验结果表明，减毒突变株FMDV(R4)和rdK本动物猪失去感染性，接种猪不产生病毒血症、不排毒、不发生水平传播、也不诱生抗体，因此作为灭活疫苗的种毒具有更好的安全性。

Claims (12)

  1. 一种口蹄疫病毒(Foot and Mouth Disease Virus)温度敏感减毒株的构建方法，包括：构建口蹄疫病毒全长cDNA感染性克隆质粒，经体外转录得到口蹄疫病毒基因组RNA，将口蹄疫病毒基因组RNA转染细胞并进行病毒的拯救；其特征在于：所述体外转录得到的口蹄疫病毒基因组RNA的IRES结构域4的K区环上的351位的胞嘧啶突变为鸟嘌呤或腺嘌呤，突变后K区环的碱基序列为351GUUUAA356或351AUUUAA356。
  2. 按照权利要求1所述构建方法得到的口蹄疫病毒温度敏感减毒株，其特征在于：用于拯救该温度敏感减毒株的全长cDNA感染性克隆质粒的微生物保藏编号是：CGMCC NO.13148。
  3. 权利要求2所述的口蹄疫病毒温度敏感减毒株含有的IRES突变体，其特征在于：其核苷酸序列为SEQ ID No.1所示。
  4. 一种口蹄疫病毒温度敏感减毒株的构建方法，包括：构建口蹄疫病毒全长cDNA感染性克隆质粒，经体外转录得到口蹄疫病毒基因组RNA，将口蹄疫病毒基因组RNA转染细胞并进行病毒的拯救；其特征在于：所述经体外转录得到口蹄疫病毒基因组RNA的IRES结构域4用牛鼻病毒基因组RNA的IRES结构域4进行替换。
  5. 由权利要求4所述构建方法得到的口蹄疫病毒温度敏感减毒株，其特征在于：用于拯救该口蹄疫病毒温度敏感减毒株的全长cDNA感染性克隆质粒的微生物保藏编号是：CGMCC NO.13149。
  6. 以牛鼻病毒基因组的IRES结构域4置换口蹄疫病毒基因组的IRES结构域4所得到的嵌合IRES序列，其特征在于：其核苷酸序列为SEQ ID No.2所示。
  7. 一种口蹄疫病毒温度敏感减毒株的构建方法，包括：构建口蹄疫病毒全长cDNA感染性克隆质粒，经体外转录得到口蹄疫病毒基因组RNA，将口蹄疫病毒基因组RNA转染细胞并进行病毒的拯救；其特征在于：将经体外转录得到口蹄疫病毒基因组RNA的IRES结构域4的K区用牛鼻病毒基因组RNA的IRES结构域4的K区进行了替换。
  8. 按照权利要求7所述构建方法得到的口蹄疫病毒温度敏感减毒株，其特征在于：用于拯救该口蹄疫病毒温度敏感减毒株的全长cDNA感染性克隆质粒的微生物保藏编号是：CGMCC NO.13150。
  9. 将口蹄疫病毒基因组的IRES结构域4的K区用牛鼻病毒基因组的IRES结构域 4的K区进行替换得到的嵌合IRES序列，其特征在于：其核苷酸序列为SEQ ID No.3所示。
  10. 权利要求3所述的IRES突变体在制备口蹄疫减毒活疫苗中的用途、权利要求3所述的IRES突变体在制备口蹄疫RNA疫苗中的用途、或权利要求3所述的IRES突变体在制备口蹄疫灭活疫苗生产用种毒的用途；权利要求6或权利要求9所述的嵌合IRES序列在制备口蹄疫减毒活疫苗中的用途；权利要求6或权利要求9所述的嵌合IRES序列在制备口蹄疫RNA疫苗中的用途、或者权利要求6或权利要求9所述的嵌合IRES序列在制备口蹄疫灭活疫苗生产用种毒的用途。
  11. 权利要求2、5或8任何一项所述的口蹄疫病毒温度敏感减毒株的全长cDNA感染性克隆在构建口蹄疫RNA疫苗中的用途；或者，权利要求2、5或8任何一项所述的口蹄疫病毒温度敏感减毒株的全长cDNA感染性克隆经拯救后制备口蹄疫减毒活疫苗中的用途；或者权利要求2、5或8任何一项所述的口蹄疫病毒温度敏感减毒株的全长cDNA感染性克隆经拯救后制备口蹄疫灭活疫苗生产用种毒的用途。
  12. 权利要求2、5或8任何一项所述的口蹄疫病毒温度敏感减毒株在制备口蹄疫减毒活疫苗中的用途；或者权利要求2、5或8任何一项所述的口蹄疫病毒温度敏感减毒株在制备口蹄疫灭活疫苗生产用种毒的用途。
PCT/CN2017/111936 2016-11-21 2017-11-20 口蹄疫病毒温度敏感减毒株及其构建方法和用途 WO2018090994A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
RU2018144892A RU2723353C1 (ru) 2016-11-21 2017-11-20 Термочувствительные аттенуированные штаммы вируса ящура (fmdv), способ их конструирования и применение
EP17872552.9A EP3543332A4 (en) 2016-11-21 2017-11-20 TEMPERATURE-SENSITIVE WEAKENING TRUNK OF THE FOOT AND CLAUS DISEASE VIRUS AND CONSTRUCTION METHODS THEREFOR AND USES THEREOF
US16/461,004 US10918710B2 (en) 2016-11-21 2017-11-20 Temperature-sensitive attenuated FMDV strains, construction method and application thereof
ZA2019/00154A ZA201900154B (en) 2016-11-21 2019-01-09 Temperature-sensitive attenuating strain of foot-and-mouth disease virus, construction method therefor and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201611047932.7 2016-11-21
CN201611047932 2016-11-21

Publications (1)

Publication Number Publication Date
WO2018090994A1 true WO2018090994A1 (zh) 2018-05-24

Family

ID=62145253

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/111936 WO2018090994A1 (zh) 2016-11-21 2017-11-20 口蹄疫病毒温度敏感减毒株及其构建方法和用途

Country Status (6)

Country Link
US (1) US10918710B2 (zh)
EP (1) EP3543332A4 (zh)
CN (1) CN108085302B (zh)
RU (1) RU2723353C1 (zh)
WO (1) WO2018090994A1 (zh)
ZA (1) ZA201900154B (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112080590A (zh) * 2020-09-29 2020-12-15 广东省农业科学院动物卫生研究所 一种用于检测b型牛鼻病毒的荧光pcr引物、探针及试剂盒

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108085302B (zh) * 2016-11-21 2020-02-14 中国农业科学院哈尔滨兽医研究所 口蹄疫病毒温度敏感减毒株及其构建方法和用途
CN108642068B (zh) * 2018-04-19 2022-02-22 新疆畜牧科学院兽医研究所（新疆畜牧科学院动物临床医学研究中心） 口蹄疫病毒人工致弱株b的全基因组序列和引物及应用
CN109536461B (zh) * 2018-11-23 2020-07-03 中国农业科学院兰州兽医研究所 一种o型口蹄疫病毒突变株及其制备方法和应用
KR102245638B1 (ko) * 2019-10-25 2021-04-29 부산대학교 산학협력단 구제역바이러스 유전자의 특정 변이 부위를 이용한 구제역바이러스 감염숙주 종판별용 바이오마커 조성물
CN111303251B (zh) * 2020-02-25 2021-07-30 中国农业科学院兰州兽医研究所 一种口蹄疫病毒样颗粒体外组装的方法及其应用
WO2022011032A1 (en) * 2020-07-07 2022-01-13 Codagenix Inc. Method of producing modified virus genomes and producing modified viruses
CN111961654B (zh) * 2020-08-05 2022-03-04 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） 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
CN111944770B (zh) * 2020-08-27 2022-04-15 中国农业科学院兰州兽医研究所 一种口蹄疫病毒致弱突变株及其制备方法和应用
CN115896043B (zh) * 2022-07-07 2023-06-20 中国农业科学院兰州兽医研究所 一种o型口蹄疫疫苗候选株及其构建方法和应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101724636A (zh) 2008-10-30 2010-06-09 中国农业科学院哈尔滨兽医研究所 Asial型口蹄疫病毒感染性cDNA及其构建方法和应用
CN101838658A (zh) 2010-04-30 2010-09-22 中国农业科学院哈尔滨兽医研究所 O型口蹄疫病毒变异株及其编码基因和应用
CN103849637A (zh) * 2012-11-29 2014-06-11 于力 O型口蹄疫病毒耐酸突变株、其携带的衣壳蛋白及其编码基因和应用
CN106318955A (zh) * 2015-07-06 2017-01-11 中国农业科学院哈尔滨兽医研究所 表达人肠道病毒71型衣壳蛋白的重组腺病毒、由其制备的疫苗和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003046138A2 (en) * 2001-11-26 2003-06-05 Duke University Genetically stable expression vector
CN108085302B (zh) * 2016-11-21 2020-02-14 中国农业科学院哈尔滨兽医研究所 口蹄疫病毒温度敏感减毒株及其构建方法和用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101724636A (zh) 2008-10-30 2010-06-09 中国农业科学院哈尔滨兽医研究所 Asial型口蹄疫病毒感染性cDNA及其构建方法和应用
CN101838658A (zh) 2010-04-30 2010-09-22 中国农业科学院哈尔滨兽医研究所 O型口蹄疫病毒变异株及其编码基因和应用
CN103849637A (zh) * 2012-11-29 2014-06-11 于力 O型口蹄疫病毒耐酸突变株、其携带的衣壳蛋白及其编码基因和应用
CN106318955A (zh) * 2015-07-06 2017-01-11 中国农业科学院哈尔滨兽医研究所 表达人肠道病毒71型衣壳蛋白的重组腺病毒、由其制备的疫苗和应用

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. EU236594
"GenBank", Database accession no. GQ406249
"GenBank", Database accession no. GU931682
"GenBank", Database accession no. HM008917
GAO R, : "Encephalomiocarditis virus IRES replacement reveals no effect on the replication and virulence of FMDV.", CHINESE JOURNAL OF PREVENTIVE VETERINARY MEDICINE, vol. 37, no. 11, 30 November 2015 (2015-11-30), pages 834 - 837, XP009514547, ISSN: 1008-0589 *
GAO SUN: "IRES Element Determines Cell Tropism of Foot-and-mouth Disease Virus and Its Molecular Mechanism", DISSERTATION, no. 11, 15 November 2014 (2014-11-15), pages 1 - 42, XP009515642, ISSN: 1674-0246 *
GRUBMANBAXT, CLINICAL MICRO. REV., vol. 17, 2004, pages 465 - 493
MATEO R.: "Engineering viable foot-and -moth disease viruses with increased thermostability as a step in the development if improved vaccines", JOURNAL OF VIROLOGY, vol. 82, no. 24, 15 December 2008 (2008-12-15), pages 12232 - 12240, XP055014603 *
PACHECOMASON ET AL., J. VET. SCI., 2010
See also references of EP3543332A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112080590A (zh) * 2020-09-29 2020-12-15 广东省农业科学院动物卫生研究所 一种用于检测b型牛鼻病毒的荧光pcr引物、探针及试剂盒

Also Published As

Publication number Publication date
RU2723353C1 (ru) 2020-06-10
CN108085302B (zh) 2020-02-14
CN108085302A (zh) 2018-05-29
US10918710B2 (en) 2021-02-16
EP3543332A1 (en) 2019-09-25
US20190275139A1 (en) 2019-09-12
ZA201900154B (en) 2020-05-27
EP3543332A4 (en) 2020-06-17

Similar Documents

Publication Publication Date Title
WO2018090994A1 (zh) 口蹄疫病毒温度敏感减毒株及其构建方法和用途
Liu et al. A 5-year review of Senecavirus A in China since its emergence in 2015
JP6419271B2 (ja) ヒトエンテロウイルスに対する抗原及びワクチン
Pratt et al. Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models
Tsai et al. Enterovirus A71 containing codon-deoptimized VP1 and high-fidelity polymerase as next-generation vaccine candidate
US7618802B2 (en) Compositions of coronaviruses with a recombination-resistant genome
Diaz-San Segundo et al. Synonymous deoptimization of foot-and-mouth disease virus causes attenuation in vivo while inducing a strong neutralizing antibody response
Díaz-San Segundo et al. Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease
Borca et al. Role of arginine-56 within the structural protein VP3 of foot-and-mouth disease virus (FMDV) O1 Campos in virus virulence
US11952401B2 (en) Recombinant foot-and-mouth disease virus with reduced immunosuppression activity, and preparation method and use thereof
WO2020258757A1 (zh) 一种突变株3型鸭甲肝病毒ch-p60-117c株及构建方法
CN111394367B (zh) 一种塞内卡病毒重组核酸、重组疫苗株及其制备方法和应用
CN111511397A (zh) 具有密码子对去优化区域的重组病毒及其用于治疗癌症的用途
Almeida et al. Construction and evaluation of an attenuated vaccine for foot-and-mouth disease: difficulty adapting the leader proteinase-deleted strategy to the serotype O1 virus
Hidajat et al. Next generation sequencing of DNA-launched Chikungunya vaccine virus
Li et al. Evaluation of immunogenicity and protective efficacy of a novel Senecavirus A strain-based inactivated vaccine in mice
Chen et al. Construction and evaluation of recombinant pseudorabies virus expressing African swine fever virus antigen genes
WO2022027749A1 (zh) 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
JP2022023198A (ja) プラス鎖ｒｎａウイルスによって引き起こされる感染疾患に対するワクチン
Diaz-San Segundo et al. Use of synonymous deoptimization to derive modified live attenuated strains of foot and mouth disease virus
Dai et al. Research progress in the development of porcine reproductive and respiratory syndrome virus as a viral vector for foreign gene expression and delivery
Yang et al. A temperature-dependent translation defect caused by internal ribosome entry site mutation attenuates foot-and-mouth disease virus: implications for rational vaccine design
Hu et al. Efficacy of Coxsackievirus A2 vaccine candidates correlating to humoral immunity in mice challenged with a mouse-adapted strain
CN116769735A (zh) 一种甲病毒减毒株的构建方法及应用
Nagendrakumar et al. Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease-and capsid-coding regions

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17872552

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2017872552

Country of ref document: EP