CN116218907A - Enterovirus infectious clone with HiBiT novel reporter gene and its construction method and application - Google Patents

Abstract

The invention discloses enterovirus infectious clone with a novel HiBiT reporter gene, a construction method and application thereof, wherein the enterovirus infectious clone comprises enterovirus EVA71, CVA16, CVA10 and CVA7 infectious clone with the novel HiBiT reporter gene, hiBiT is used as the reporter gene for the first time on the basis of the EVA71, CVA16, CVA7 and CVA10 infectious clone, EVA71-HiBiT, CVA16-HiBiT, CVA7-HiBiT and CVA10-HiBiT infectious clone are prepared, the report virus is further saved through the infectious clone, and the biological activity and genetic stability of the report virus are verified in vitro through cytopathy, plaque, WB and flow experiments. The virus clone or report virus related by the invention has wide application value in the field of anti-enterovirus medicines and neutralizing antibody high-flux screening.

Description

Enterovirus infectious clone with HiBiT novel reporter gene and its construction method law and application

technical field

The invention relates to the field of biomedicine, in particular to enterovirus EVA71, CVA16, CVA10 and CVA7 infectious clones with HiBiT novel reporter genes and their construction methods and applications.

Background technique

Enterovirus (Enterovirus) belongs to the single-stranded positive-sense RNA virus family of Picornaviridae. The Enterovirus genus contains 1513 species, which are Enterovirus A-I and Rhinovirus A-C respectively. The common serotypes include Enterovirus A71 (EVA71), Coxsackievirus A10 ( CVA10), Human rhinovirus A1 (HRVA1), Poliovirus (PV), etc. The viral genome is about 7.5kb in length and consists of a 5' non-coding region (5'UTR), a coding region and a 3' non-coding region (3'UTR). After a virus infects a cell, it releases genomic RNA into the cytoplasm for replication and translation. The viral genome contains a reading frame, which is first translated in the cytoplasm to form polyproteins including structural proteins and nonstructural proteins, and then virally encoded proteases 2A and 3C cut the polyproteins to form four mature structural proteins VP1, VP2, and VP3 and VP4 and seven nonstructural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D. Hand, foot and mouth disease is a common infectious disease caused by a variety of enteroviruses, mainly in infants and young children. The common pathogens of hand, foot and mouth disease are EVA71 and CVA16. Most severe cases and deaths are caused by EVA71 infection. In recent years, the cases of hand, foot and mouth disease caused by CVA6 and CVA10 have increased, and the research on antiviral drugs or antibodies to the pathogen of hand, foot and mouth disease is particularly important.

Infectious cloning has been widely used as a powerful tool in virus research. Through reverse genetics technology, scientists can easily construct the viral genome into a plasmid that is easy to amplify and store, and can also perform a variety of directional modifications on the viral genome, such as gene deletion mutations, foreign tag insertion, and reporter genes insert etc. In addition, compared with the high mutation frequency of viruses, the mutation frequency of infectious clones is lower, thereby avoiding mutations during virus passage. In summary, as a tool for virus research, infectious cloning has a wide range of application values in virus pathogenic mechanism, drug and neutralizing antibody screening, etc.

Infectious clones with reporter genes are constructed by inserting exogenous reporter genes on the basis of viral infectious clones. Reporter genes that are widely used generally fall into two categories: fluorescent proteins and luciferases. Among them, fluorescent proteins include green fluorescent protein (GFP) and red fluorescent protein (mCherry), etc.; luciferases include firefly luciferase (Fluc), Renilla luciferase (Rluc), Gaussian luciferase (Gluc) and new fluorescent Sulfase (NanoLuc, Nluc) etc. The currently published enterovirus infectious clones carrying reporter genes include EVA71 and CVA16 infectious clones carrying GFP, and the CVA16 infectious clones carrying GFP lack the genetic stability of eGFP-CVA16 recombinant viruses, and cannot be stably passaged Reporter viruses are not suitable for high-throughput screening of antiviral drugs. In addition, no CVA10 and CVA7 reporter viruses with GFP have been reported, indicating that GFP is not applicable to most enterovirus infectious clones. Although viruses with GFP can be traced by observing the luminescence in vivo and in vitro, it is difficult to quantitatively analyze the virus through fluorescent proteins, which further limits the application of viruses with reporter genes in virology research and drug screening. Studies have reported that the Gluc-carrying EVA71 infectious clone has a Gluc reporter gene that simplifies the virus quantitative analysis steps, but the Gluc-carrying virus has poor stability due to the large Gluc gene fragment. Therefore, finding a stable reporter gene suitable for most enterovirus infectious clones will provide an important tool for the development of drugs and antibodies related to HFMD.

Nluc luciferase protein includes two subunits, the large subunit LgBiT (17.6kDa) and the small subunit SmBiT, 11 amino acid peptides (1.3kDa), the predecessors optimized the small subunit SmBiT to have a strong affinity with LgBiT The active peptide was named HiBiT. As a tagged protein, HiBiT can be used to detect endogenous protein expression, monitor receptor changes, WB experiments and virus research. Since the HiBiT tag has the characteristics of small size, only 11 amino acids, and little effect on the protein studied, it is speculated that it is suitable for the construction of enterovirus infectious clones with reporter genes.

Contents of the invention

In order to overcome the above-mentioned defects in the prior art, the object of the present invention is to provide an enterovirus infectious clone with HiBiT novel reporter gene and its construction method, and successfully rescue EVA71-HiBiT, CVA16 - Four reporter viruses, HiBiT, CVA10-HiBiT and CVA7-HiBiT. In addition, the infectious clones of EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT constructed in the present invention will also provide important tools and reference ideas for basic research and applied research of other similar enteroviruses.

The object of the present invention can be achieved by the following technical methods:

The first aspect of the present invention provides an enterovirus infectious clone with a HiBiT novel reporter gene, including enterovirus EVA71, CVA16, CVA10 and CVA7 infectious clones with a HiBiT novel reporter gene, namely EVA71-HiBiT, CVA16 - HiBiT, CVA10-HiBiT and CVA7-HiBiT infectious clones; wherein:

The nucleic acid sequence of the HiBiT novel reporter gene is shown in SEQ ID NO.1;

The nucleotide sequences of the enteroviruses EVA71, CVA16, CVA10 and CVA7 are respectively shown in SEQ ID NO.2-5.

The second aspect of the present invention provides a method for constructing the enterovirus infectious clone with the HiBiT novel reporter gene, comprising: the EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT infectious clones are respectively replaced by EVA71 , CVA16, CVA10 and CVA7 infectious clones as the basis, constructed using seamless cloning technology; where:

The EVA71 infectious clone introduces the HiBiT sequence through upstream and downstream primers whose sequences are shown in SEQ ID NO.6 and SEQ ID NO.7;

The CVA16 infectious clone introduces the HiBiT sequence through upstream and downstream primers whose sequences are shown in SEQ ID NO.8 and SEQ ID NO.9;

The CVA10 infectious clone introduces a HiBiT sequence through upstream and downstream primers whose sequences are shown in SEQ ID NO.10 and SEQ ID NO.11;

The CVA7 infectious clone introduces the HiBiT sequence through upstream and downstream primers whose sequences are shown in SEQ ID NO.12 and SEQ ID NO.13.

Further, it also includes the steps of transforming the correct fragments recovered from the gel into Trans2-Blue E. coli competent, shaking the bacteria, sequencing verification after plasmid extraction, and storing the correct plasmids at -20°C.

The third aspect of the present invention provides a reporter virus, including reporter virus EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT, respectively with EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT infectious clone Obtained by rescuing the virus, specifically comprising the following steps:

S1. Prepare EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT infectious clones by seamless cloning method;

S2. EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT were transcribed in vitro to obtain mRNA;

S3, EVA71-HiBiT, CVA10-HiBiT and CVA7-HiBiT mRNA transfected RD cells, CVA16-HiBiT mRNA transfected Vero cells;

S4, the cell supernatant was continuously passaged, and the cell pathological changes were observed;

S5. Report virus and parental virus by plaque titration, and verify growth characteristics;

S6, verifying the difference between the growth characteristics of the reporter virus and the parental virus.

Further, in step S1, based on the infectious clones of EVA71, CVA16, CVA10 and CVA7 preserved in the laboratory, using seamless cloning technology, among the infectious clones, EVA71 was passed through the upstream and downstream primers SEQ ID NO.6 and SEQ ID NO .7 Introduce the HiBiT sequence, CVA16 introduces the HiBiT sequence through the upstream and downstream primers SEQ ID NO.8 and SEQ ID NO.9, CVA10 introduces the HiBiT sequence through the upstream and downstream primers SEQ ID NO.10 and SEQ ID NO.11, and CVA7 introduces the HiBiT sequence through the upstream and downstream primers Primers SEQ ID NO.12 and SEQ ID NO.13 were used to introduce HiBiT sequences, and the correct fragments were recovered from the gel and then transformed into Trans2-Blue E. coli competent, shaken, and sequenced for verification after plasmid extraction, and the sequenced correct plasmids were stored at -20°C .

Further, in step S2, templates for in vitro transcription are prepared by PCR primers, EVA71-HiBiT upstream and downstream PCR primers SEQ ID NO.14 and SEQ ID NO.15, CVA16-HiBiT upstream and downstream PCR primers SEQ ID NO.16 and SEQ ID NO .17, CVA10-HiBiT upstream and downstream PCR primers SEQ ID NO.18 and SEQ ID NO.19, CVA7-HiBiT upstream and downstream PCR primers SEQ ID NO.20 and SEQ ID NO.21, using in vitro transcription kit to synthesize mRNA, quality Store at -80°C after controlling the correct mRNA aliquots.

Further, in step S3, the mRNA transcribed in step S2 is transferred into cells in a 6-well plate, and the cell pathological changes are observed day by day.

Further, in step S6, after infecting the cells with the virus supernatant collected in step S4, the biological activity of the reporter virus was verified by using plaques, 2C antibodies to detect the translation level of viral proteins, and dsRNA antibodies to detect the efficiency of virus-infected cells. This virus is compared.

Virus rescue: refers to cloning the full-length cDNA of the virus into a plasmid vector, and rescuing the virulent virus in vitro through the method of reverse genetics.

The exogenous reporter gene in the virus is easily lost after continuous passage, and this genetic instability will negatively affect the application of the reporter virus. The aforementioned four reporter viruses of the present invention, EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT, were continuously passaged in cells to P10, and the fluorescent expression of different generations of virus-infected cells was detected to determine the genetic stability of the reporter virus.

The fourth aspect of the present invention provides the application of the aforementioned enterovirus EVA71, CVA16, CVA10 and CVA7 infectious clones or reporter viruses carrying the HiBiT novel reporter gene in high-throughput screening of anti-enterovirus drugs or neutralizing antibodies.

Compared with the prior art, the beneficial effects of the present invention are as follows:

1. Enteroviruses belong to small RNA viruses, and the length of the viral genome is about 7.5kb. When preparing enterovirus infectious clones with reporter genes, they are often limited by their small capacity to accommodate foreign genes. HiBiT is the gene of NanoLuc The small subunit, containing only 11 amino acids, can be used as an independent luciferase-tagged protein. This invention proposes a method for constructing an enterovirus infectious clone with a new HiBiT reporter gene. The successful construction includes a HiBiT new reporter gene. EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT infectious clones of the gene.

2. In the infectious clone of the present invention, the poly(A)25 tail is contained after the 3'UTR and the T7 RNA polymerase promoter sequence and the hammerhead ribozyme (Hhrib) sequence are included before the 5'UTR, and the T7 RNA polymerase Enzyme promoter, mRNA can be obtained in vitro; hammerhead ribozyme improves the replication ability of viral RNA.

3. The enterovirus infectious clone with the HiBiT novel reporter gene constructed by the present invention is inserted behind the first amino acid (start codon) of the virus VP4 at the HiBiT sequence, and added at the 5' end of the HiBiT sequence The sequence of synonymous mutations from the second to the fourth amino acid of VP4, adding a 2A protease recognition site (AITTL) at the 3' end of the HiBiT sequence.

4. After the mRNA obtained by in vitro transcription in the present invention is transfected into cells, the mRNA is efficiently replicated in the cells and can be correctly assembled into mature virus particles.

5. The enterovirus infectious clone with HiBiT novel reporter gene constructed by the present invention combined with cytopathic experiments, plaque experiments, WB and flow cytometry can produce infectious EVA71-HiBiT, CVA16-HiBiT, CVA10 -HiBiT and CVA7-HiBiT virus particles, and also proved that the EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT viruses rescued by reverse genetics have growth characteristics and other biological characteristics similar to the parent virus, for EVA71, CVA16, CVA10 and CVA7 virus-related mechanism research and application research provide a powerful tool.

6. The present invention combined with the luciferase detection experiment proves that the EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT viruses rescued by the enterovirus infectious clone with the HiBiT novel reporter gene constructed in the present invention can Continuous and stable passage of more than 10 generations in in vitro cell culture without losing the HiBiT exogenous gene, the EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT viruses rescued by the present invention have greatly improved the stability of the virus, stable The reporter virus is an important basis for virus mechanism research and antiviral drug screening.

Description of drawings

Figure 1 is a schematic diagram of the construction of an enterovirus infectious clone with a new HiBiT reporter gene in the embodiment.

Figure 2 is a diagram of cytopathic effects caused by infection of reporter viruses EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT and parental viruses EVA71, CVA16, CVA10, and CVA7 in the examples.

Fig. 3 is the plaque pattern of reporter virus EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT and parent virus EVA71, CVA16, CVA10, CVA7 in the embodiment.

4 is a WB image of cells infected with reporter viruses EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT and parental viruses EVA71, CVA16, CVA10, and CVA7 in the embodiment.

Fig. 5 is the flow cytometric results of cells infected with reporter viruses EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT and parental viruses EVA71, CVA16, CVA10, and CVA7 in the embodiment.

Fig. 6 is the genetic stability result of reporter virus EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT in the cell in the embodiment, wherein: A is the fluorescence value after different generation EVA71-HiBiT virus infection cells; B C is the fluorescence value of cells infected by different generations of CVA16-HiBiT virus; C is the fluorescence value of cells infected by different generations of CVA10-HiBiT virus; D is the fluorescence value of cells infected by different generations of CVA7-HiBiT virus.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

In the following examples, the nucleotide sequence is as follows:

The nucleotide sequence of the HiBiT tag gene is shown in SEQ ID NO.1:

GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC

The nucleotide sequence of the EVA71 virus is shown in SEQ ID NO.2:

TTAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCTACTGGGCGCAAGCACTCTGGCACCTCGGTACCTTTGTGCGCCTGTTTTATACCCCCCCCGTGAAACTTAGAAGCAGCAAAACCACGATCAATAGCAGGCATAACGCTCCAGTTATGTCTTGATCAAGCACTTCTGTTCCCCGGACTGAGTATCAATAGACTGCTCGCGCGGTTGAAGGAGA AAACGTTCGTTATCCGGCTAGCTACTTCGGAAAACCTAGTAACACCATGAAAGTTGCGGAGAGCTTCGTTCAGCACTCCCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGGCGCTCTAATACGGACATGGTGTGAAGAGTCTACTGAGCTAGT TAGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCCAACTGCGGAGCACACGCCCACAAGCCAGCGGGTAGTGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTTTATATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTGGCCATCCGGTGTGCAACAGAGCAAT TATTTACCCTATTTATTGGTTTTGTACCATTAACCTTGAATTCTGTGACCACCCTTTAATTATATCTTGACCCTTAACACAGCTAAACATGGGATCCCAGGTGTCTACACAGCGCTCCGGTTCTCACGAAAACTCAAACTCAGCCACTGAGGGTTCTACCATAAACTACACCACCATTAATTACTACAAAGACTCCTATGCTGCCACAGCAGGCAAACAGAGTCTCAAGCAGGA TCCAGACAAGTTTGCAAATCCTGTTAAAGACATTCCTACTGAAATGGCAGCGCCACTGAAGTCCCCATCCGCTGAGGCATGTGGATACAGTGATCGAGTGGCGCAATTAACTATTGGCAACTCCACCACCACGCAAGAAGCGGCTAATATCATAGTCGGTTATGGTGAGTGCCCTTCCTACTGCTCCAGATTCTGACGCTACAGCAGTGGATAAACCA ACGCGCCCGGATGTTTCAGTGAACAGGTTTTCACATTGGACACTAAATTGTGGGAGAAATCGTCCAAGGGATGGTACTGGAAGTTCCCGGATGTGTTAACTGAAACTGGGGTTTTTGGGCAAAATGCACAATTCCACTACCTCTACCGATCAGGGTTCTGCATCCACGTGCAGTGCAATGCCAGTAAATTCCACCAAGGAGCACTCCTAGTCGCTG TCCTACCAGATGTCATTGGGACAGTGGCAGGCGGTACAGGGACGGAAGATACCCCACCCCCCTTACAAGCAGACTCAACCCGGCGCCGATGGCTTCGAGTTGCAACACCCGTACGTGCTTGATGCTGGCATCCCAATATCACAGTTAACAGTGTGCCCACACCAGTGGATTAATTTGAGGACCAACAACTGTGCTACAATAATAGTGCCATACATTAACG CACTGCCTTTTGATTCTGCCTTGAACCATTGCAACTTTGGGCCTGTTGGTTGTGCCTATTAGCCCACTAGACTACGACCAAGGAGCGACGCCAGTAATCCCTATACTATCACATTGGCCCCAATGTGAGTATACACCACCTCACCCCACCAGAAATCTGGTCGTGAATGTGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGC

The nucleotide sequence of the CVA16 virus is shown in SEQ ID NO.3:

TTAAACAGCCTGTGGGTTGTTCCCACCCACAGGGCCCACTGGGCGCTAGCACACTGGTTCTGCGGGACCTTTGTGCGCCTGTTTTATAACCCTTCCCTAAGCAGTAACTTAGAAGCTTTGTACAATCATGGCCAATAGTGGGCGTGGCGCGCCAGTCACGTCTTGGTCAAGCACTTCTGTATCCCCGGACTGAGTATCAATAGACTGCTCACGCG GTTGAAGGAGAAAACGTTCGTTACCCGGCTAGCTACTTCGAGAAACCTAGTAGCACCATGAAAGTTGCGGAGTGTTTCGCTCAGCACTTCCCCCGTGTAGATCAGGTCGATGAGTCACTGTAAACCCCACGGGCGACCGTGACAGTGGCTGCGTTGGCGGCCTGCCCATGGGGTAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGA GCTAGTTAGCAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCAGAGCGCGCACCCTCAACCCAGGGGGCGGCGCGTCGTAATGGGTAACTCTGCAGCGGAACCGACTTTGGGTGTCCGTGTTTCCTTTTATTCTTTATTGGCTGCTTATGGTGACAATTGAAAAGTTGTTACCATATAGCTATTGGATTGGCCATCCGGTCTAACAGA GCTATTGTTTACCTATTCATTGGCTTCGTCCCCTCTTAATCTCAAGGCCATTCAAACTCTTGATTACATATTGCTCCTCAACTGTAAGAAatgGGATCCCAGGTCTCCACCCAGCGATCCGGGTCACATGAGAACTCAAACTCTGCATCGGAGGGACCATAAATTACACAACTATAAACTACTACAAGGATGCATACGCTGCAAGTGCGGGGCGCCAG GATATGTCCCAAAGACCCGAAGAAATTTACTGACCCTGTCATGGATGTTATACATGAGATGGCCCCACCGCTAAAGTCTCCAAGCGCTGAGGCGTGTGGTTACAGTGATCGTGTGGCTCAGCTTACCATTGGAAACTCCACTATCACGACACAGGAAGCAGCTAACATAGTCATAGCCTATGGAGAGTGGCCTGAGTACTGCCCAGACACAGATGCAACGGCAGTC GACAAACCCACTCGACCTGACGTATCAGTGAATAGATTTTTCACGTTAGACACTAAATCTTGGGCCAAGGATTCAAAGGGTTGGTATTGGAAATTCCCTGATGTTTTGACAGAGGTAGGTGTTTTCGGTCAAAAATGCTCAGTTCCACTACCTGTATCGATCTGGGTTTTGCGTGCACGTTCAGTGCAACGCTAGTAAATTTCACCAAGGTGCCTTACTA GTGGCTGTGCTGCCTGAGTATGTACTCGGTACTATCGCAGGAGGGACCGGGAACGAGAATTCTCATCCTCCCCTACGCCACCACACAGCTGGTCAGGTTGGCGCAGTTCTGACGCACCCTTACGTGCTAGACGCAGGGATCCCTTTAAGCCAATTAACTGTGTGCCCACACCAGTGGATTAATTTAAGAACTAATAATTGTGCAACCATTATAGTTCCATATATGA ACACAGTTCCTTTTGACTCAGCTCTCAAATCACTGCAACTTTGGTCTACTGGTTTCCCAGTGGTGCCATTAGATTTCAACACAGGTGCCACATCTGAAATTCCCATCACAGTCACCATAGCTCCCATGTGTGCGGAGTTTGCGGGTCTGCGCCAGGCGGTAAAGCAAGGTATACCAACAGAGCTTAAACCTGGTACCAATCAGTTTCTTACTACTGATGATGGTGTCT CCGCACCAATTCTGCCAGGTTTCCATCCAACTCCGCCTATACATATACCAGGGGAAGTACACAACCTATTGGAAATATGTAGAGTGGAGACCATCCTGGAAGTCAATAACCTGAAGACCAATGAGACCACCCCCATGCAGCGCTTGTGTTTCCCAGTGTCGGTGCAGAGTAAAACGGGCGAATTATGTGCTGCTTTCAGAGCAGACCCTGGAAGAGATGGCCCGT GGCAGTCTACAATACTAGGTCAACTCTGCAGATACTACACCCAGTGGTCAGGTTCACTGGAGGTGACATTCATGTTTGCAGGTTCATTCATGGCCACAGGAAAAATGCTCATCGCCTACACCCCACCTGGGGGAAATGTGCCTGCGGACAGAATCACAGCAATGCTAGGAACACATGTGATCTGGGACTTCGGATTGCAGTCTTCTGTGACGTTGG TCGTGCCATGGATCAGTAATACACACTACAGGGCACACGCCCGCGCTGGGTACTTTGACTATTACACCACTGGCATCATAACCATATGGTATCAAACTAACTATGTCGTACCCATTGGTGCTCCCACTACAGCATATATCGTGGCTTTGGCAGCAGCTCAGGATAACTTCACTATGAAACTGTGCAAGGATACCGAGGACATTGAGCAAACGGCTAACATACA AGGGGATCCCATTGCAGATATGATTGACCAAACTGTAAACAATCAAGTGAACCGCTCCTTGACTGCGATGCAAGTACTACCTACAGCTGCCAACACTGAGGCAAGTAGTCCAGATTAGGCACTGGTGTTGTACCAGCACTACAAGCTGCGGAGACAGGGGCGTCTAATGCTAGTGACAAGAACCTCATTGAGACTAGATGTGTGTTGAACCATCATTCC ACGCAAGAGACAGCCATTGGGAATTTCTTTAGCCGTGCTGGTCTTGTCAGCATCATCACAATGCCCACCACGGGTACACAGAATACAGATGGTTATGTTAATTGGGACATTGATTTAATGGGATATGCTCAGCTGCGGCGGAAATGCGAGTTATTCACCTACATGCGCTTTGATGCTGAATTCACATTTGTCGTAGCCAAACCCAATGGTGAGCTAGT CCCCCAATTACTCCAGTACATGTATGTCCCGCCAGGGGCTCCGAAACCTACTTCCAGAGATTCGTTTGCTTGGCAGACAGCCACCAACCCATCTGTGTTTGTGAAAATGACGGACCCGCCAGCTCAAGTGTCAGTCCCCTTCATGTCACCAGCTAGTGCATATCAATGGTTTTATGATGGTTATCCACCTTTGGTGAGCACCTCCAAGCAAATGACCTGGATTAT GGTCAATGCCCGAATAATATGATGGGCACTTTTAGCATTAGGACAGTAGGGACTGAGAAGTCACCACACTCCATTACCCTGAGGGTGTATATGAGAATCAAACACGTCAGGGCATGGATCCCAAAGACCTCTGAGAAATCAACCCTATTTGTTTAAGACTAACCCAAATTATAAAGGAAATGATATTAAGTGTACCAGCACTAGTAGAGACAAGATAACAACGCTGG GAGAGTTTGGACAGCAATCGGGCGCCATATGTGGGCAACTATAGGGTGGTGAATCGACACCTTGCCACCACAACGACTGGTCAAACCTTGTGTGGGAGGACAGCCTTAGGGACTTGTTAGTCTCCTCCACCACCGCCCAGGGATGCGACACCATCGCTAGGTGCGATTGTCAAACCGGTGTATACTATTGCAGCTCCAAAAGGAAACACTACCCGGTTA GTTTCACCAAGCCCAGTCTGATATTTGTGGAAGCTAGTGAGTATTACCCAGCTAGATATCAGTCCCATCTCATGCTTGCTGTGGGTCACTCGGAACCTGGTGACTGTGGTGGCATCCTCAGATGCCAGCATGGTGTGGTAGGAATTGTCTCCACTGGTGGAAATGGTCTTGTGGGGTTTGCCGATGTCAGAGACCTTCTATGGCTGGATGAAGA AGCAATGGAGCAGGGAGTGTCTGACTATATCAAAGGCCTCGGTGACGCTTTTGGTATGGGCTTCACTGATGCAGTGTCTAGGGAAGTAGAGGCATTGAAGAACCATCTAATTGGCTCAGAAGGAGCTGTTGAGAAGATCTTGAAGAACTTGGTGAAACTAATCTCGGCTTTGGTCATAGTTGTTAGGAGTGACTATGACATGGTCACCCTCACGG CTACGCTAGCCCTGATTGGGTGTCATGGAAGCCCTGGGCATGGATTAAAGCGAAAACAGCCTCTATTCTTGGCATTCCCATAGTGCAGAAACAGAGCGCTTCATGGCTAAAGAAGTTTAATGATATGGCCAACGCCGCGAAAGGGCTCGAGTGGATTTCTAGTAAAATTAGCAAGTTCATTGACTGGCTTAAAGAAAAGATTATTCCGGCCGCTAAAGA GAAAGTTGAATTCTTAAACAACCTGAAACAGCTTCCCTTGTTGGAGAACCAGATCTCAAAACCTTGAACAGTCTGCTGCCTCGCAAGAAGATCTAGAAGCTATGTTTGGCAATGTGTCATATTTAGCCCACTTCTGCCGCAAGTTCCAGCCACTCTATGCGGTTGAAGCCAAAGGGTTTTATGCCCTAGAGAAAAGGATGAATAACTACATGCAGTTCAAGCAAACA CCGTATTGAACCTGTATGCTTAGTCATTAGAGGCTCCCCAGGAACAGGTAAATCACTTGCTACGGGCATCATTGCTAGAGCTATTGCTGACAAATACCACTCTAGCGTTTACTCACTCCTCCAGATCCAGACCATTTTGATGGATACAAGCAACAAGTAGTCACTGTTATGGATGATCTCTGTCCAGAACCCAGATGGAAAGGACATGTCCCTATTTTGCCAAAT GGTTTCTACAGTGGACTTTTACCACCCATGGCATCATTGGAAGAGAAAGGAGTGTCTTTCACTCCAAGTTTGTCATCGCATCGACCAATGCTAGCAATATTGTAGTTCCACAGTTTCAGATTCTGATGCGATTCGCAGGCGGTTTTATATGGACTGCGATATAGAGGTGACAGACTCTTACAAGACGGACCTTGGCCGACTCGACGCAGGCAGGGCCGCTA AGCTTTGCACAGAAAACAACACTGCTAATTTTAAGAGATGCAGCCCATTAGTGTGTGGCAAGGCCATTCAATTAAGAGACAGGAAGTCTAAAGTGAGATATAGTATTGACACTGTGGTGTCAGAGTTAATCAGAGAGTATAATAGGTCTGCCATTGGGAACACCCATAGAAGCCCTCTTCCAAGGACCCCTAAGTTCAGGCCTATAAGAATTAGCCTTGAAGAAAAGCCGG CCCCAGATGCCATCAGTGACTTACTTGCTAGTGTGGATAGCGAGGAGGTTCGACAATACTGCAGGGAACAAGGGTGGATAATTCCAGAAACATCGACCAATGTGGAACGTCACCTCAACAGAGCAGTGTTGGTGATGCAGTCCATTGCCACCGTGGTTGCGGTTGTGTCTCTCGTCTAGTCATTTATAAATTGTTTGCTGGATTTCAAAGGTGCCTATCT GGTGCACCTAAGCAAGCTCTCAAAAAGCCTGTGCTAAGGACAGCCACAGTCCAAGGACCGAGCTTGGACTTTGCCTTATCCCTTCTAAGGCGCAACATCAGACAGGTGCAAACTGACCAAGGACATTTCACTATGTTAGGAGTGCGAGATCGCCTAGCCATTTTGCCGCGCCACTCGCAACCAGGAAAAACTATCTGGGTGGAGCACAAATTAATCAATGTGTTAGATG CTGTTGAATTGGTGGATGAGCAAGGTGTAAATTTGGAACTTACACTAGTAACCTTGGACACCAACGAAAAATTTAGAGATGTCACCAAGTTTTTCCAGAGACGATCACCGGGGCAAGCGACGCAACCTTGGTCATCAACACTGAGCACATGCCCTCTATGTTCGTCCCAGTGGGTGATGTTGTACAGTATGGGTTTCTAAATCTCCAGCGGTAAGCCCA CACACCGAACCATGATGTACAATTTCCCCACAAAGGCAGGGCAGTGTGGAGGTGTGGTCACTTCAGTCGGTAAGATTATTGGAATTCATATCGGTGGGAATGGACGCCAAGGCTTCTGTGCTGGACTGAAGAGAGGTTACTTTGCCAGTGAACAAGGAGAGATCCAATGGATGAAGTCCAATAAAGAAACTGGGAGACTGAATATTAATGGTCCAACAC GTACCAAATTGGAGCCCAGTGCATTCTACGATGTGTTTGAGGGCAGCAAAGAACCAGCAGTCTTAACCAGTAAGGATCCCAGACTTGAGGTTGATTTTGAGCAAGCTTTGTTTTCCAAATATGTAGGAAATACCCTGCATGAGCCTGACGAGTATGTGACACAGGCTGCTTCTCACTATGCAAACCAGCTAAAGCAATTAGATATAAACACTAATAAGATGAGTATGGAAG AAGCATGCTACGGCACTGAATATCTAGAGGCTATAGACTTGCACCCAGTGCCGGGTACCCCTATAGTGCCCTGGGTGTCAAGAAAAGAGACATACTTGACCCAACCACTAGAGATACTACCAAAATGAAATTCTACATGGATAAATATGGGTTAGACTTGCCCTATTCCACCTATGTGAAAGATGAGCTTAGATCCTTAGATAAGATTAAGAAAGGGAAATCCC GCTTGATTGAAGCCAGTAGTCTAAATGACTCAGTCTACCTTAGGATGACTTTCGGGCATCTCTATGAAACTTTTCATGCCAACCCGGGGACTGTGACTGGGTCTGCAGTAGGGTGTAATCCTGATGTGTTCTGGAGTAAATTACCAATCCTGTTGCCAGGATCGCTCTTTGCATTTGACTATTCAGGATATGATGCAAGTCTCAGCCCAGTATGGTTTAGAGCT TTGGAAGTGGTTCTCCGAGAGATCGGATACTCAGAGGAAGCTGTATCACTAATAGAAGGGATCAACCACCCCATCATGTGTATCGGAACAGGACGTATTGTGTCCTTGGTGGAATGCCTTCAGGTTGTTCCGGCACTTCCAATCTTCAATTCCATGATCAATAACATAATAATCAGAACCCTTTTGATTAAAACTTTTAAGGGGATCGATTTAGATGAGCTGAAT ATGGTAGCTTATGGAGATGATGTGTTAGCTAGCTATCCATTCCCTATTGACTGCTCGGAGCTAGCCAGAACTGGTAAAGAGTATGGATTAACAATGACACCTGCTGACAAGTCACCTTGCTTTAATGAAGTTACCTGGGAAAATGCTACATTCTTAAAGAGAGGCTTCCTGCCAGATCATCATTTCCCATTTCTTTATCCATCCTACCATGCCCATGAGGGAGAT CCACGAGTCCATTCGCTGGACTAAAGACGCACGCAACACTCAGGACCACGTGCGCTCTCTGTGCCTCTTAGCGTGGCATAATGGGAAGGAGGAATATGAAAAATTTGTGAGCACAATTAGATCAGTTCCTATTGGAAAAGCCTTGCTATACCAAATTTTGAGAACTTGAGAAGAAATTGGCTCGAGTTATTTTAATATACAGTTTAAAAGCTGAACCCCACAGAAGTCT GGTCGTGTTAATGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGCAG

The nucleotide sequence of the CVA10 virus is shown in SEQ ID NO.4:

TTAAACAGCCTGTGGGTTGCACCCACTCACAGGGCCCACTGGGCGCTAGCACTCTGGTACCGTGGTACCTTTGTGCGCCTGTTTTATACCCCCTTCCCGTTTGAACATTAGAAGTAACGCACCTCGATCAGTAGCAGGCGCGGCGCACCAGCCGTGTCTTGATCAAGCACTTCTGTTCCCCGGACCGAGTATCAATAGACTGCTCACGCGGTTGAAGGA GAAAACGTTCGTTATCCGGCTAACTACTTCGAGAAACTTAGTAGCACCATGGAAGTTGCAGAGTGTTTCGCTCAGCACTCCCCCCAGTGTAGATCAGGCTGATGGATCACCGCGTTCCTCACGGGTGACCGTGGCGGTGGTCGCGTTGGCGGCCTGCCCATGGGGCAACCCATGGGACGCTCTAATATGGACATGGTGTGAAGAGTCTATTGA GCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACGTGTCCCCAACCCAGGGGATAGCGTGTCGTAACGGGTAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCCTTTATTCTTATCCTGGCTGCTTATGGTGACAATTGAGAGATTGTTACCATATAGCTATTGGATTGGCCATCCGGTGTCTAACAGAGC TATAATAATATCTTTTTGTTGGGTTCGTACCCCCTCAATTTTGAGGTTGTTCTCACATTAAAGTACATCTTGGTGTTAAATATCAGAAGATGGGAGCACAAGTTTCATCTCAGAGGTCAGGATCCCACGAGACTGGGAATGTGGCTACTGGAGGATCGACCATCAACTTCACCAATATAAATTATTACAAGGATTCATATGCAGCTTCTGCTAGTCGTCAGGACTTC ACACAAGATCCAAAGAAGTTCACTCAACCGGTTTTGGATTCCATCAGAGAGCTGTCTGCCCCCTTGAATTCACCATCAGTTGAAGCGTGTGGATATAGTGACCGAGTGGCACAACTAACGGTGGGTAACTCCTCCATTACGACACAAGAGGCTGCCAATATAGTTTTGGCATACGGTGAGTGGCCTGAGTACTGCCCTGATACTGACGCAACAGCGGTGGACAAG CCTACTCGTCCGGATGTGTCAGTTAATAGATTCTACACTTTAGATTCAAAGATGTGGCAAGAGAACTCTACTGGATGGTATTGGAAGTTCCCAGATGTTTTGAACAAGACCGGGGTGTTTGGTCAGAATGCACAGTTCCACTACTTGTATCGTTCAGGGTTCTGTCTTCATGTTCAATGTAACGCTAGCAAATTTCACCAGGGGGCTCTTCTTGTGGCA GTTATCCCGGAGTTCGTTCTAGCAGGCAGAGGTTCAAACACGAAGCCCAATGAAGCCCCTCACCCGGGATTTAATACCACCTTTCCTGGCACTGCTGGCGCTTCCTTCAACGACCCGTACGTGCTTGACTCTGGGGTACCCTTAGCCAATCCTTAATACCCCACATCAGTGGATCAACTTAAGGACCAACAATTGTGCAACAATAATAGTACCTTATATCAATGCTG TCCCCTTTGATTCAGCCATCAATCATAGCAACTTTGGATTAATAGTTGTGCCTGTGAGTCCGCTGAAATACTCTTCAGGGGCCACCACTGCAATCCCAATCACAGTGACCATAGCGCCCCTCAATTCCGAATTTGGTGGGCTGCGCCAAGCTGTCAGCCAGGGACTCCCTACCGAGTTAAGACCAGGCACCAATCAGTTCCTAACAACGGAAGATGATACTGCCGC ACCCATACTCCCAGGTTTCTCCCCCCACCCCAAGCATCCACATTCCAGGAGAAGTACGCTCATTACTAGAACTGTGTAGGGTGGAAACCATACTGGAAGTGAACAACACGACCGATGCAACCGGTCTGAACAGACTCCTAATTCCAGTCTCGCCCAAAACAAGGCAGATGAACTATGTGCTGCATTCATGGTTGATCCTGGTCGTATCGGGCCCTGGCAATCGA CTTTGGTTGGTCAAATATGTAGGTACTACACACAATGGTCAGGCTCGTTAAAGGTCACATTCATGTTTACAGGCTCTTTTATGGCAACAGGTAAGATGCTGATAGCGTACTCACCCACCAGGCAGTGCTCCAACCAGCCAATAGGGAGACCGCAATGCTCGGGACCACGTCATATGGGATTTTGGATTACAATTCATTCAGTTTCACTGGTGATACCATGGATC AGCAACACACACTTCAGAACAGCCAAAACTGGTGGTAACTATGACTATTACACAGCTGGTGTGGTGACATTATGGTATCAAACAATTATGTAGTCCCGCCTGAGACGCCCGGAGAGGCTTACATTATCGCCATGGGGGCTGCTCAAGACAACTTCACCTTGAAGATATGTAAAGACACGGATGAGGTTACCCAAACAAGCTGTCTTACAAGGCGACCCCGTGGAG GACATCATCCACGACGCTTTGAGCAGCACTGTGCGGCGGGCCATAACTAGTGGTCAAGATGTCAACACAGCGGCCGGTACCGCTCCTAGCTCTCACAGGTTGGAGACTGGTCGTGTTCCCGCCCTACAAGCAGCAGAAACTGGAGCCACTTCTAACGCTACAGATGAGAACATGATAGAAACGCGGTGTGTCATGAACAGAAATGGAGTGTTGGAGGCG ACTATAAGTCATTTCTTCTCACGCTCAGGTTTGGTGGGTGTTGTCAATCTAACTGACGGAGGCACCGATACAACGGGATATGCAGTGTGGGACATTGACATCATGGGTTTTGTGCAACTGCGGCGGAAATGTGAGATGTTCACATACATGAGATTCAACGCTGAGTTCACATTCGTCACTACAACAGAAAATGGCGAGGCAAGGCCATTTATGTTACAGT ATATGTATGTACCTCCAAGGTGCCCCTAAGCCAACGGGTAGAGATGCTTTTCAGTGGCAAACAGCGACAAATCCATCCGTTTTCGTTAAGCTCACAGATCCACCTGCTCAGGTATCAGTCCCCTTCATGTCACCTGCTAGTGCCTACCAATGGTTCTATGACGGGTATCCAACATTTGGACAACACCCGGAAACATCTAATACAACATATGGACAGTGCCCTAACAAC ATGATGGGGACCTTTTGCTGTGAGAGTAGTGAGTAGAGTGGCTAGCCAGCTCAAACTACAGACACGAGTGTATATGAAGCTTAAGCATGTGAGAGCATGGATCCCTAGGCCAATAAAGATCCCAGCCTTACCTCCTAAAGAATTTTCCAAATTATGATAGTAGTAAGATCATACAGCGCAAGAGATCGTGCCAGCATAAAACAAGCTAATATGGGAAAGTTTGGGCAA CAGTCTGGGGCAATATATGTGGGTAATTACAGAGTGGTGAATAGACACTTGGCCACACATAACGATTGGGTAAATCTGGTGTGGGAGGATAGTTTCTAGAGACTTGCTGGTCTCATCTACTACTACAGCACAGGGGTGTGATACCATAGCTCGGTGCAACTGTCAGACAGGTGTGTATTACTGTAACTCTCGTAGGAAACACTACCCGGTCAGCTTTTCCAAA CCCAGCCTGATATTTGTTGAAGCTAGCGAATATTACCCAGCTAGGTACCAGTCACACCTCATGCTTGCCGAGGGTCACTCAGAACCTGGTGACTGTGGTGGCATCCTCAGGTGTCAACACGGTGTGGTTGGGTTAGTTTCCACCGGTGGAAATGGCCTCGTGGGGTTTGCTGATGTGAGGGACTTACTCTGGCTTGATGAGGAGGCTATGGA ACAAGGAGTGTCTGACTACATCAAAGGTCTTGGTGATGCTTTTGGCACGGGCTTCACTGACGCAGTGTCCAGGGAAGTGGAAGCATTGAAAAATCACTTGATTGGCTCTGAGGGGGCTGTCGAGAAAATCTTGAAAAACCTGGTGAAGCTAATTTCAGCATTAGTTATAGTCATCAGGAGTGACTACGATATGGTCACTCTTACAGCTACGCTTGCC CTGATCGGGTGCCATGGGAGTCCTTGGGCGTGGATCAAATCAAAGACAGCTTCCATCTTAGGCATATCCATGGCACAAAAGCAAAGCGCCTCATGGCTGAAGAAGTTCAATGATATGGCAAATGCCGCAAAAGGGCTCGAGTGGATCTCCAATAAAGATCAGCAAGTTTATTGATTGGCTTAAGGAGAAGATTCATCCAGCTGCTAAGGAGAAAGTCGAGTTC TTGAATAACTTAAAGCAACTCCCTCTGTTGGAAAATCAAATTTCCAATCTCGAGCAATCTGCCGCCTCACAGGAAGATCTAGAAGCTATATTTGGCAATGTGTCGTACCTAGCTCATTTTTGCCGCAAGTTCCAACCACTCTACGCAACTGAGGCCAAGAGAGTCTATGCCCTGGAGAAAAGAGTGAACAACTACATGCAGTTCAAGAGCAAACACCGTATTGAACCT GTATGCTTGATCATTAGAGGCTCTCCAGGTACGGGAAAATCACTTGCCACAGGTATTATAGCTAGAGCCATTGCTGATAAGTACCATTCAGTGTCTATTCGCTCCCTCCAGACCCAGATCACTTCGACGGGTATAAACAACAGGTGGTCACAGTCATGGACGATCTCTGCCAGAATCCAGACGGGAAAGATATGTCCTTGTTCTGCCAAATGGTCTCCACA GTGGACTTTATACCACCCATGGCGTCACTGGAAGAAAAAGGCGTATCCTTCACCTCTAAGTTTGTCATTGCATCGACCAACGCTAGCAACATCATAGTCCCCCACAGTCTCAGACTCAGATGCAATCCGCAGGAGATTCTATATGGACTGTGATATAGAAGTAACTGACTCTTACAAGACAGATCTCGGTCGATTGGACGCGGGTAGAGCTGCCAAGCTTTGTTCAGA GAATAACACTGCTAACTTCAAAAGATGCAGTCCACTTGTGTGTGGCAAAGCCATCCAATTGAAAGATAGGAAGTCTAAGGTCAGATATAGTGTTGACACTATGGTATCAGAGCTAATCAGAGAGTACAACAATAGATCTGCTGTTGGAAACACCATAGAAGCACTCTTCCAAGGGCCCCCCAAGTTCAGACCTTATAAGAATCAGCCTCGAGGAGAAGCCAACACCAGATGCTAT CAGTGACCTTCTCGCTAGTGTTGATAGCGAAGAGGTCCGACAGTATTGCAGAGAGCAAGGGTGGATAATCCCAGAAACACCAACCAACGTGGAACGACCTCCAATAGAGCAGTTCTGGTAATGCAGTCCATTGCTACCGTAGTTGCGGTTGTGTCCCTTGTGTATGTCATTTATAAACTGTTTGCCGGATTTCAAGGTGCCTACTCTGGAGCACCCAAGCAGGCGCT CAAGAAGCCTGTGCTAAGAACAGCTACTGTCCAAGGACCTAGCTTGGACTTCGCTTTGTCTCTTCTGAGGCGCAACATCAGACAAGCGCAGACCGACCAGGGACACTTCACCATGCTAGGCATACGGGACCGTCTAGCCATCTTGCCACGCCACTCACAACCAGGGAAGACCATCTGGATAGAGCACAAATTGGTCCAACGTACTAGATGCAGTTGAGTTGGTG GATGAGCAAGGTGTTAATTTGGAACTCACGCTGGTGACCTTGGACACTAATGAGAAGTTTAGGGACATTACCAAGTTCATCCCAGAGACAATAGCTGGTGCTAGTGATGCAACTCTAGTTATCAACACTGAGCATATGCCCTCGATGTTTGTGCCAATAGGTGACGTTGTGCAGTATGGGTTTTTTGAATCTCAGTGGCAAACCCACACACAGAACTATGATGTA CAATTTCCCCACGAAAGCAGGACAGTGTGGGGGGGTAGTCACTTCAGTTGGCAAGATCATTGGAATCCACATTGGCGGGAATGGACGCCAGGGCTTCTGCGCTGGTTTAAAGAGGAGCTACTTTGCCAGCGAGCAAGGAGAGATCCAGTGGATGAAGCCCAACAAAGAGACTGGGAGGCTGAACATCAATGGTCCAACCCGAACCAAACTGGAAC CTAGCGTGTTCCACAATGTGTTCGAGGGTAATAAAGAGCCAGCAGTTCTGACCAGTAAAGACCCCAGGCTTGAGGTTGATTTTGAACAAGCCTTGTTCCAAATATGTGGGCAACACTCTGCATGAGCCTGATGAGTATGTGACACAAGCTGCCCTTCATTACGCAAATCAATTAAAACAACTAGACATAAACACCAGCAAGATGAGCATGGAGGAGGCGTGCTATGGT ACAGAAAAATTTAGAAGCTATAGACCCTACACACCAGTGCTGGATATCCTTAGTGCCTTGGGTATTAAAAAGAGGGATATTCTTGATCCGGTCACCAGGGACACCTCCAAGATGAAACTATACATGGACAAGTATGGACTAGATTTACCCTATTCAACCTATGTGAAGGATGAGCTTAGGTCTCTAGATAAATCAAGAAGGGGAAATCTCGCTTAATTGAGGCCAG CAGCTTGAATGATTCTGTCTACCTTAGAATGACTTTTGGTCATCTTTATGAGGTGTTTCACGCCAACCCGGGAACTATAACCGGGTCTGCAGTGGGGTGTAATCCTGATGTGTTCTGGAGCAAGTTGCCAATTCTACTACCGGGTTCGCTCTTTGCGTTTGACTACTCAAGCTATGATGCAAGTCTTAGTCCTGTATGGTTCAGAGCTTTAGAATTGGT TTTACGAGAGATTGGTTACTCAGAGGAGGCTGTGTCACTCATAGAGGGGATTAACCACACTCACCATGTGTATCGGAATAAGACATACTGTGTCCTTGGTGGGATGCCTTCAGGTTGCTCTGGCACTTCCATTTCCATTTCCATGATTAACAACATAATCATTAGAACTCTCTTGATCAAGACGTTCAAAGGGATAGACTTGGATGAACTAAACATGGTG GCCTACGGAGATGATGTACTGGCTAGCTACCCATTTCCCATCGACTGTTTGGAGTTGGCGAGAACTGGCAAAGAGTATGGACTGACTATGACTCCCGCCGATAAGTCACCCCTGTTTTTAATGAAGTCACCTGGGAGAACGCAACCTTTTTGAAGAGGGGTTTCTACCAGACCATCAGTTCCCTTTTCTAATCCACCCTACCATGCCCATGAGGGAAATCCAC GAGTCCATTCGTTGGACCAAGGATGCACGTAACACTCAAAGACCACGTGCGTTCCCTTTGCTTGTTGGCGTGGCACAATGGAAAGGAGGAATATGAAAAATTTGTGAGCACAATCAGATCAGTTCCTATTGGAAAAGCCTTGGCGATACCAAATTTTGAGAACTTGAGGAGAAATTGGCTCGAATTGTTTTAAACTTACAGCTTAAAGCTGAACCCCACCAGAAAC CTGGTCGTGCAAATGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGC

The nucleotide sequence of the CVA7 virus is shown in SEQ ID NO.5:

TTTAAACAGCCTGTGGGTTGTCCCCCACCCACAGGGCCCACTGGGCGTTAGCACTCTGGTATTACGGTACCTTTGTGCGCCTGTTTTATAACCCCTCCCCAACTGTAAATTAGAAGCAACACACGTCGATCAATAGTAGGCGTGACGCACCAGTCACGTCTTGATCAAGCACTTCTGTTCCCCGGACCGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGA AAACGTTCGTTACCCGACCAACTACTTCGAAAAACCTAGTAGCACCATGAAGGTTGCGGAGTGTTTCGCTCGGCACTTCCCCAGTGTAGATCAGGTCGATGAGTCCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGCTGGCGGCCTGCCTATGGGGTAACCCATAGGACGCTCTAATACGGACATGGTGCGAAGAGTCTATTGAGCTAGTT GGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCGCGCGCCCCCATTCCAGGAGGTAGCGCGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATCTTTATTCTGGCTGCTTATGGTGACAATTGACAGATTGTTACCATATAGCTATTGGATTGGCCATCCGGTCACAAGCAGAGCGATT ATCTACCAAATTTATTGGATTTATCCCACTGCACACTCGTGAATATAACACATTACTGCTGATACTGAGTTTGGACAAAAGCAAAATGGGAGCACAGGTGAGCGGCTGGCGGCTGTTCAAAGAAGATTAGCGCCATTACTACCCTTGGCGCTCAAATATCAACACAAAAATCTGGCTCCCATGAGACGGGTAACATCGCTACTGAGGGATCCACCATAAACTTTACT AATATCAATTACTACAAGGACTCCTACGCTGCCTCAGCTAGCAAGCAAGACTTTACTCAAGATCCGGGGAAATTCACCAGCCCGGTCCTAGATGTTTTTATCAGAGATGGCAGCACCACTGCAATCACCGACAGCAGAAGCATGTGGATACAGTGATAGGGTCGCGCAGCACACAGTAGGTAACTCGACAATTACAACGCAAGAAGCGGCTAATGTGATTGTCGCTT ATGGGGAGTGGCCACAATATTGTCCTGACACTGACGCTACAGCGGTGGACAAGCCCACTCGTCCTGACGTCTCAGTCAATAGATTTTATACACTAGACCAAAGATTGGTCATTCAGTTCAAAGGGGTGGTATTGGAAATTCCCAGATATTTCTAGCGGAAACTGGAGTCTTCGGTCAAAATGCACAGTCCACTTTTTGTATAGGTCAGGGTTTTGCAT TCACGTTCAATGCAATGCCAGCAAGTTCATCAGGGAGCACTATTGGTTGCAGTCTTACCAGAATATGTTACTGGTACTGTGTCTGGCAACACTGGACTTGAGAATACTCACCACCATACGCTGCTACTCAGCCTGGTGCTACAGGGTTTGAGTTGACCAACCCGTACATTGACGCTGGGATCCCGTTGAGCCAGTTGTTAGTGTGCCCTCACCAGTG GATTAACCTTCGAACTAACAACTGTGCCACTATAGTGGTACCATACATTAACAGCGTCCCTTTTGATTCGGCCTTAAACCATTGCAATTTCGGTTTAGTAGTGATTCCAGTCTCACCCCCTTGGGTTCCTGCAAGGGGCCACACCAACAATTCCCATAACCATCACAGTGGCCCCTATGAACTCCGAGTTCTCTGGATTGAGACAAGCTGTTACTCAAGGTTTGCCCAT GGAGTTAAAACCCGGCACCAACCAATTTCTCACCACCGACGATGGTGTATCAGCCCTATACTGCCTGGGTTCCACCCAAACACCAGTAATACATATTCCTGGGGAAGTTAGAAACCTCTTAGAGCTGTGTCAAAATTGACACAATAATGGAAGTTAACAATCTTACCACGAATGAAGCCACTCCCATGGAGCGCTTGCGCATACCCGTACAAGTCCAAACACAGTCGG GTGAATTGTGCGCAGCATTCAAAGCTGATCCTGGGCTTGATGGCCCATGGCAATCCACTATGGTTGGGCAGCTTTGTAGATATTACCCCAATGGTCAGGGTCTTTAAAAATAACTTTCATGTTCACCGGGTCATTTATGGCCACTGGGAAAATGCTGATAGCCTACACTCCTCCTGGTGGCTCTCTGCCCGCCAACAGAATGCAGGCAATGCTG GGCACCCACGTCATCTGGGATTTTGGTTTGCAATCATCTGTCACACTAGTTGTACCATGGATTAGCAACACTCATTATCGCTCGCAGGCCACTGGCAGCTTCTTTGATTACTATGCCACTGGCATAGTATCCTATGGTACCAGACAAATTTCGTCGTCCCTATAGGTGCGCCAACTACTGCTTATATAGTGGTTCTGGGTAGCGCTCAAAAGAATTTCACCATGC GGCTATGCAGGGACACTAGTGAACTGACACAAGCCGCAGAGTATCAGGGAGACGAAATAGTCGACCTAATCGAGAGTGCTGTACAGAATACCACTAAAGCCATTACCAGCTCAATCGACACCAAACTGGTGCTAACACTCAAGCTAGCCAACATCGTATAGGCTTGGGGGAGGTTCCCGCTCTTCAAGCTGCTGAGACAGGATCGTCTTCGCTCGTTTCGGA CAAGAACATGATAGAAACAAGATGTGTCGTAAACAAACACAGCACAGAGGAAACCAGCATTACAAACTTCTACTCCAGGGCGGGCCTAGTGGGGGTTGTGAACATGCCAGTACAAGGAACCAGCAACACAAAGGGTTTCGCAAAGTGGGGGATAGATATAATGGGCTTTGTGCAGATGAGGCGCAAACTTGAGCTCATGACATACATGAGATTCTCCG CCGAGTTTACGTTCGTAGCCAGCACTCCTGGGGGAGAGACTACTAACCTATACTGCAATACATGTATGCACCTCCCGGAGCTCCGCTGCCAACCAGGCGGGATTCATACGAATGGCAAACATCCACTAACCCCTCTATTATCAGCAAGATGGCGGACCCCACCCGCTCAGGTATCGGTTCCATTCCTTTTCTCCTGCATCAGCATATCAGTGGTTCTATGATG GCTACCCCACATTTGGGAAACACCCAATAGATCAGGACTTCCAATATGGCATGTGCCCAAAACAACATGATGGGCACATTCTGTGTGCGCATGATCGGTGGGGCAAACCGACCCAATCAGTTACCATACGTATATACATGAGATTAAAGCATATCCGTGCATGGGTGCCCCGGCCACTGAGGAGTCAGAATTACACTATGAGGAATTACCCCGAACTACAACG GGGGCGCAATAAAATGTACATCAAAAAGCAGAGCTACCATAACAACCTTAGGAAAATTTGGCCAACAATCCGGAGCAATTTATGTGGGGAGCTACAGAGTGGTAAACAGGCATTTGGCAACCCCACAATGACTGGAAAAACCTGGTGTGGGAGGACAGTTCCAGAGATTTGTTAGTCTCATCCACTACTGCCCAAGGCTGTGATACCATCGCCCGGTGTGACT GTCAAACGGGGGTGTATTATTGCAATTCCCGTAGAAAACATTCCAGTTTCTCTAAGCCCAGTTTCGTTTTCGTGGAAGCTAGTGAGTACTACCCGGCAAGGTACCAATCTCACCTCATGTTAGCTCAAGGGCACTCAGAACCCGGTGACTGTGGGGGCATCCTCAGATGCCAGCACGGCGTGGTTGGTATAGTCTCCACGGGCGGGAATG GTCTAGTGGGTTTTGCTGATGTGAGAGACCTTTTGTGGCTAGATGAGGAAGCCATGGAGCAAGGGGTGTCTGATTACATCAAGGGACTTGGTGATGCCTTTGGCACAGGATTTACTGACGCGGTTTCCAGAGAAGTTGAGGCCTTGAAGAACCACTTAATCGGGTCTGAGGGTGCTGTGGAGAAGATCCTCAAAAACCTGGTGAAGCTTTATTTCC GCACTTGTCATCGTCATTAGGAGTGATTATGATATGGTCACATTGACAGCAACACTCGCCCTGATTGGATGTCGCGGAAGCCCGTGGGCCTGGATTAAATCAAAAACAGCATCCATCCTGGGCATTCCAATGGCCCAGAAGCAGGGTGCTACTTGGTTGAAGAAGTTCAACGACATGGCAAATGCTGCAAAAGGGCTCGAGTGGATCTCTAACAAATC AGCAAGTTCATCGATTGGCTCAAGGAAAAGATAAATCCCAGCCGCAAAAGAAAAGGTTGAGTTTTTTGAACAATTTGAAGCAACTTCCCTTACTGGAAAACCAAATCTCCAACCTGGAACAATCAGCTGCCTCGCAAGAAGACCTTGAGGCAATGTTTGGTAATGTGTCGTATCTTGCCCATTTCTGCCGCAAATTTCAACCATTATATGCGACAGAAGCTAAAAGGGTAT ATGCTTTAGAAAAGAGGATGAACAACTACATGCAGTTCAAGAGCAAACACCGTATTGAACCTGTGTTTAATCATCAGAGGCTCCCCAGGCACGGGTAAGTCACTTGCCACAGGCATCATTGCTAGGGCCATTGCTGATAAATACCACTCTAGTGTTTACTCACTTCCGCCAGATCCAGATCACTTTGACGGGTACAAACAGCAAGTTGTCACTGTCATGGATGAT CTCTGCCAAAATCCAGATGGGAAAGACATGTCCTCTGTTCTGTCAAATGGTTTCCACTGTAGACTTCATTCCGCCCATGGCCTCCTTGGAAGAGAAAGGAGTGTCATTCACCTCCAAGTTCGTTATAGCATCAACTAATGCCAGCAACATCATAGTTCCAACAGTGTCAGACTCCGACGCCATCCGTAGGCGATTCTTTATGGACTGTGACATCGAAGTGACTGATTCTTATA AGACAGACTTGGGCAGGCTTGATGCTGGAAGGGCTGCCAAATTGTGCTCAGAAAACAACACCGCTAACTTTAAAAAGATGTAGTCCACTAGTATGTGGGAAAGCTATCCAATTGAGAGATAGGAAATCCAAAGTTCGGTATAGTGTGGACACAGTAATTTCAGAGCTTATTAGAGAATACAATAATAGATCAGCTATTGGGAACACCATAGAAGCACTATTTCAGG GACCCCCAAAATTTAGACCTATTAGAATTAGCCTCGAGGAAAAGCCCGCACCTGATGCCATTAGTGACTTACTTGCTAGTGTTGATAGTGAGGAAGTTCGGCAATATTGCAGGGACCAGGGGTGGATAATACCTGAAACCCCTACAAATGTGGAGCGCCATCTAAATAGGCTGTTCTTGTAATGCAGTCAATTGCTACGGTTGTGGCAGTAGTGTCTCTT GTCTATGTTATTCTACAAGCTCTTTGCAGGCTTCCAGGGTGCTTATTCTGGCGCACCCAAGCAGGTGTTGAAGAGACCGGTACTGAGGACCGCAACTGTGCAAGGGCCGAGTCTTGACTTTGCTCTTTCCCTTCTGCGTAGGAACATCAAACAAGCTCAGACTGATCAAGGTCATTTCACTATGTTGGGTATTCGCGATCGACTGGCCATCTTGCCGCGTCATGCAC AACCAGGAAAGACCATTTGGGTTGAACACAAACTGGTCAATGTCTTAGATGCGGTCGAGTTGGTTGATGAACAAGGAGTTAATCTAGAACTCACCTTAGTCACCTTGGATACCAATGAGAAATTTAGAGATATCACTAAGTTCATCCCAGAGGTAATAAGCGGAGCTAGTGATGCAACCTTAGTGATCAATACTGAGCACATGCCATCTATGTTCGTACCTGTG GGTGACGTGGTGCAATACGGGTTCCTAAATCTTAGTGGTAAACCTACCCACAGAACCATGATGTACAACTTCCCCAAAAAGCGGGCCAGTGTGGTGGTGTGGTTACCTCAGTAGGGAAAATTATTGGAATCCACATTGGTGGGAATGGACGCCAGGGGTTCTGTGCAGGTCTGAAAAGAGGCTATTTTGTAAGTGAGCAAGGGGAAATTCAGTG GATGAAGCCCAATAAGGAGACTGGCAGGTTGAACATCAATGGGCCCACCCGCACAAAACTTGAACCCAGTGTGTTCTATGATGTATTTGAGGGTAACAAGGAGCCAGCTGTCCTTACTAGTAAAGATCCCAGACTAGAGGTAGACTTTGAACAAGCCTTGTTTTTCTAAATACGTGGGAAATGTACTGCACGAACCTGATGAGTACGTGACCCAGGCAGCACTAC ATTATGCTAATCAATTGAAGCAGCTGGACATCAATACCAACAAGATGAGCATGGAGGAAGCGTGCTATGGCACTGAAAATTTAGAAGCTATTGACCTCCACACTAGTGCTGGTTACCCATACAGTACGCTGGGTATCAAGAAGAGGACATCCTTGATCCAACTACTAGAGATGTTTCTAAAATGAAGCTTTACCTGGACAAATATGGCCTAGATCTACCTA CTCCACTTATGTGAAAGATGAGCTTAGATCACTAGACAAAATCAAGAAAGGGAAGTCCCGCCTGATTGAAGCCAGTAGTCTAAATGATTCTGTTTACCTTAGAATGACTTTTGGCCATCTGTATGAGGTGTTTCATGCCAACCCAGGGACGGTAACCGGTTCAGCAGTAGGTTGTAATCCAGACGTGTTTTGGAGTAAACTCCCGATTTTGCTTCCAGGGTCGCTCT TTGCATTTGACTATTCAGGATATGACGCTAGTCTTAGTCCTGTGTGGTTTAGAGCTTTAGAAATAGTCCTGCGGGAGGTCGGTTACTCAGAGGAAGCAGTGTCTCTTATAGAGGGAATCAACCACCCACCACGTGTACCGCAACAAGACCTATTGTGTTCTTGGTGGGATGCCCTCAGGCTGTTCAGGCACCTCTATCTTCAATTCAATGATCAATAATATAATT ATTAGAACCCTCTTGATCAAGACGTTTAAGGGGATAGACCTTGATGAGTTGAATATGGTGGCATACGGAGATGATGTATTAGTAGCTACCCTTTTCCCATTGATTGCCTCAGAATTAGCAAAAACAGGTAAAGAGTGGGTTAACTATGACACCAGCTGATAAATCACCTGTTTCAATGAAGTTACCTGGGAAAATGCCACCTTCCTGAAGAGGGGGTTC CTTCCTGATCATCAGTTCCTTTCTCTCATCCACCCCACAATGCCTATGAGAGAAATCCATGAGTCCATACGTTGGACAAAGGATGCACGCAACACCCAAGACCATGTACGCCTCTCTTTGTCTGTTGGCATGGCATAATGGGAAGGACGAGTACGAAAGATTTGTAAGCACAATTAGATCAGTCCCAATTGGAAAAGCGTTAGCTATACCAAATTTTGAGAATTTGA GAAGAAATTGGCTTGAATTATTTTAAACCTACAGCTTTGAGCTGAACCCCACCAGAAATCTGGTCGTGTTAATGACTGGTGGGGGTAAATTTGTTATAACCAGAATAGC

The experimental methods in this example are conventional methods in the art unless otherwise specified. The present invention will be described in detail below through several specific examples, but are not limited to the following examples.

Example 1

A construction and virus rescue method of an enterovirus infectious clone with a HiBiT novel reporter gene, the steps of which are:

1. Preparation of an enterovirus infectious clone with a new HiBiT reporter gene: using infectious clones of EVA71, CVA16, CVA10 and CVA7 as templates, introducing HiBiT sequences during PCR amplification, and using the amplified fragments Agarose gel electrophoresis with a concentration of 0.7% (w/v), according to the DNA Marker bands, cut the amplified products of the correct size and recovered and purified the cDNA fragments with the Tiangen Enhanced Gel Recovery Kit. The purified cDNA products were transformed into Trans2-Blue Escherichia coli competent, and after single-clonal colonies were picked, the correct clones were preliminarily identified by PCR to expand the strains, and the plasmids were extracted for sequencing verification. Correctly sequenced plasmids were stored at -20°C (Figure 1).

The upstream amplification primer of EVA71 has the sequence shown in SEQ ID NO.6: 5'-GCGGCTGTTCAAGAGATTAGCGCCATTACTACCCTTGGTTCGCAAGTGTCTACACA-3'

The downstream amplification primer of EVA71 has a sequence as shown in SEQ ID NO.7:

5'-CTTCTTGAACAGCCGCCAGCCGCTCACCTGGGATCCCATGTTTAGCTGTGTTAAGGGT-3'

The upstream amplification primer of CVA16 has a sequence as shown in SEQ ID NO.8:

5'-GCGGCTGTTCAAAGAAGATTAGCGCCATTACTACCCTTGGGTCACAAGTCTCCACCC-3'

The downstream amplification primer of CVA16 has a sequence as shown in SEQ ID NO.9:

5'-CTTCTTGAACAGCCGCCAGCCGCTCACCTGGGATCCcatTTCTTACAGTTGAGG AGC-3'

The upstream amplification primer of CVA10 has a sequence as shown in SEQ ID NO.10:

5'-CTGGCGGCTGTTCAAAGAAGATTAGCGCCATTACTACCCTTGGAGCACAAGTTTTCATCTCAGAG-3'

The downstream amplification primer of CVA10 has a sequence as shown in SEQ ID NO.11:

5'-TTGAACAGCCGCCAGCCGCTCACCACCTGAGCACCCATCTTCTGATATTTAACA CCAAGATG-3'

The upstream amplification primer of CVA7 has a sequence as shown in SEQ ID NO.12:

5'-CGGCTGTTCAAAGAAGATTAGCGCCATTACTACCCTTGGCGCTCAAATATCAACA CAA-3'

The downstream amplification primer of CVA7 has a sequence as shown in SEQ ID NO.13:

5'-TCTTCTTGAACAGCCGCCAGCCGCTCACCTGTGCTCCCATTTTGCTTTGTCCA AACTCAG-3'

The PCR reaction system is: TransStart FastPfu DNA Polymerase: 1 μL, 5×TransStartFastPfu Buffer: 10 μL, 2.5mM dNTPs: 5 μL, primer (F+R): 2 μL, DNA template: 1 μL, ddH ₂ O: 31 μL;

The amplification conditions are: 95°C, 2min, 1 cycle; 95°C, 20sec; 55°C, 20sec; 72°C, 1min, 35 cycles; 72°C, 5min.

2. In vitro transcription: PCR amplifies the full length of EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT to prepare DNA templates for in vitro transcription. The T7 High Yield RNA Transcription kit was used to transcribe T7 in vitro to obtain mRNA. After the mRNA was purified by LiCl precipitation, the quality of the mRNA was detected by agarose gel electrophoresis. After the band was correct, the mRNA was aliquoted and stored at -80°C; where:

The upstream full-length amplification primer of EVA71-HiBiT has a sequence as shown in SEQ ID NO.14: 5'-GGGGATCAATTCTCTAGAGCGGC-3'

The downstream full-length amplification primer of EVA71-HiBiT has the sequence shown in SEQ ID NO.15: 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTATTCTGGTTATAACAAATTTACCCCA-3'

The upstream full-length amplification primer of CVA16-HiBiT has the sequence shown in SEQ ID NO.16: 5'-GAGGCCCTTTCGTCTTCAAGAATTG-3'

The downstream full-length amplification primer of CVA16-HiBiT has the sequence shown in SEQ ID NO.17: 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTCTGCTATTCTGGTTATAACAAATTTACCCCC-3'

The upstream full-length amplification primer of CVA10-HiBiT has the sequence shown in SEQ ID NO.18: 5'-GACGAGTTCTTCTGAGGGGATCA

The downstream full-length amplification primer of CVA10-HiBiT has the sequence shown in SEQ ID NO.19: 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTATTCTGGTTATAACAAATTTACCCCCAC-3'

The upstream full-length amplification primer of CVA7-HiBiT has the sequence shown in SEQ ID NO.20: 5'-GGGTGAGCAAAAACAGGAAGGC-3'

The downstream full-length amplification primer of CVA7-HiBiT has the sequence shown in SEQ ID NO.21: 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTGCTATTCTGGTTATAACAAATTTACCCCCCACC-3'

T7 in vitro transcription reaction system: Template: 8 μL, A: 1.5 μL, G: 1.5 μL, C: 1.5 μL, U: 1.5 μL, Buffer: 2 μL, T7 Enzyme: 1 μL; RNase-free ddH ₂ O: 3 μL, 37°C , 2h.

3. Virus rescue: Digest well-growing RD and Vero cells, plate 5×10 ⁵ cells per well in a 6-well plate, culture overnight at 37°C, use Lipo3000 transfection reagents EVA71-HiBiT, CVA10-HiBiT and CVA7 -HiBiT mRNA was transfected into RD cells, and CVA16-HiBiT mRNA was transfected into Vero cells. The cells transfected with mRNA continued to be cultured and the cytopathic effect was observed. After more than 80% of the cells appeared cytopathic, repeated at -80°C Freeze and thaw twice, and centrifuge at 4000g to collect the virus supernatant and store it at -80°C.

Example 2

EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT have similar cytopathic ability to the parental virus, and the steps are:

RD and Vero cells in good growth state were digested, and 5×10 ⁵ cells were plated in each well of a 6-well plate. After culturing overnight at 37°C, the cell supernatant was replaced with DMEM medium containing 2% FBS. EVA71-HiBiT, CVA10-HiBiT and CVA7-HiBiT viruses infected RD cells with MOI=1, CVA16-HiBiT virus infected Vero cells with MOI=1, and set up positive control wells infected with EVA71, CVA16, CVA10 and CVA7 parental viruses and untreated cells. Blank group for the infected group. Continue culturing at 37°C. When the cells showed obvious CPE, use an inverted microscope to take pictures and observe (Figure 2).

Example 3

EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT have plaque sizes similar to the parental virus, and the steps are:

1. Virus gradient dilution: Take 300 μL of the virus stock solution and add it to a 1.5mL EP tube, and use DMEM medium containing 2% FBS to dilute the parental virus and the reporter virus in a 10-fold gradient respectively, and dilute from 10 ^-1 to 10 ^-8 for use;

2. Infect cells with gradient diluted virus liquid: Digest RD and Vero cells in good growth state, spread 3×10 ⁵ cells in each well of a 12-well plate, culture overnight at 37°C, discard the cell culture supernatant, and use sterile After washing twice with PBS, 300 μL of gradient diluted virus solution was added, and three replicate wells were made for each gradient. At the same time, an uninfected blank group was set up, infected at 37°C for 2 hours, and shaken every 15 minutes to promote virus infection.

3. Avicel overlay culture and fixed staining: Discard virus infection supernatant, wash twice with sterile PBS, add 1 mL of Avicel from Avicel and 2×4% DMEM 1:1 mixture, cover cells, place in Cultivated in a 37°C incubator, the spotting time of EVA71, CVA10 and CVA7 was 2 days, and the spotting time of CVA16 was 3 days. The spotting time of the reporter virus and the parental virus were consistent. After plaque formation, the cover was discarded by suction, the cells were fixed with 4% paraformaldehyde for 2 h, and then the paraformaldehyde was discarded and stained with crystal violet for 2 h. After staining, rinse the crystal violet with running water, and take pictures for observation after the culture plate is dry (Figure 3).

Example 4

EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT have protein expression levels similar to those of the parent virus, and the steps are:

1. Virus-infected cells: digest RD and Vero cells in good growth state, spread 3×10 ⁵ cells in each well of a 12-well plate, and after culturing overnight at 37°C, discard the cell culture supernatant, and replace the cell supernatant with DMEM medium with 2% FBS. EVA71-HiBiT, CVA10-HiBiT and CVA7-HiBiT viruses infected RD cells with MOI=0.1, CVA16-HiBiT virus infected Vero cells with MOI=0.1, and set up positive control wells infected with EVA71, CVA16, CVA10 and CVA7 parental viruses and untreated cells. Blank group for the infected group. Continue culturing at 37°C for 24.

2. Prepare the protein loading solution: Discard the supernatant, wash with PBS once, add 300 μL of trypsin to each well, and place in a 37°C incubator to digest the cells. After termination of digestion with complete medium, centrifuge at 1000 g for 5 min, discard the supernatant, and collect the cell pellet. Dilute 5×SDS Loading buffer to 1× with ddH ₂ O, and add 100 μL 1×SDS Loading buffer to the cell pellet. Cook the sample at 100°C for 10 minutes to denature the protein. Centrifuge to collect liquid in the tube wall. The prepared protein samples can be run directly on the gel or stored at -20°C for a short time.

3. SDS-PAGE electrophoresis and antibody incubation: prepare 12% SDS-PAGE gel. Prepare the lower layer of glue first, and flatten the lower layer of glue with ddH ₂ O. After 30 minutes, prepare the upper layer of glue and insert it into a 15-hole comb. Dilute 10×Running buffer with ddH ₂ O 10 times to prepare Running buffer working solution, and add the prepared working solution to the electrophoresis tank. Load the sample at 15 μL/well, and add an equal volume of three-color prestained protein marker, and add 1×SDS Loading buffer to the excess wells. 80V constant voltage electrophoresis for 30min, followed by 110V constant voltage electrophoresis until the bromophenol blue reached the bottom of the gel, and the electrophoresis was terminated. Prepare 1L of fast membrane transfer working solution and add it to the electrophoresis tank. Cut 5cm×8cm NC film for later use. Prepare the transfer membrane "sandwich" in the order of "black side, sponge, filter paper, gel, NC membrane, filter paper, sponge, white side". All components were wetted with the membrane transfer working solution to drive out the air bubbles between the layers, and the "sandwich" was placed in the electrophoresis tank, 400mA, and lateral flow electrophoresis for 30min. Prepare 5% milk blocking solution with PBS solution, block at room temperature for 1 hour, and set the shaker speed at 15 r/min. 3% milk primary antibody dilution was prepared in PBST solution containing 0.5% Tween, the 2C antibody prepared in the author's laboratory was diluted 1:1000, and the housekeeping gene β-tubulin antibody was diluted 1:1000. After adding the primary antibody, incubate overnight at 4°C with the shaker speed set at 15r/min. Incubate with secondary antibody. After the primary antibody incubation, wash with PBST 3 times, 5min each time, and set the shaker speed to 70r/min. HRP Goat-anti-Mouse IgG secondary antibody was diluted with PBST solution 1:10000, added secondary antibody, incubated at room temperature for 1 hour, and the shaker speed was set at 15r/min. After incubation with the secondary antibody, wash with PBST 3 times, 5 min each time, and set the shaker speed at 70 r/min. After washing, prepare a high-sensitivity ECL chemiluminescent solution, use a dual-infrared quantitative analyzer to expose and analyze the experimental results, and the membrane can also be temporarily stored in a 4°C refrigerator (Figure 4).

Example 5

EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, and CVA7-HiBiT have a similar level of viral replication to the parental virus, and the steps are:

1. Virus-infected cells: digest RD and Vero cells in good growth state, spread 3×10 ⁵ cells in each well of a 12-well plate, and after culturing overnight at 37°C, discard the cell culture supernatant, and replace the cell supernatant with DMEM medium with 2% FBS. EVA71-HiBiT, CVA10-HiBiT and CVA7-HiBiT viruses infected RD cells with MOI=0.1, CVA16-HiBiT viruses infected Vero cells with MOI=0.1, and set up positive control wells infected with EVA71, CVA16, CVA10 and CVA7 parental viruses and untreated cells. Blank group for the infected group. Continue culturing at 37°C for 18h.

2. Collect cells and analyze by flow staining: After trypsinizing the cells, stop the digestion with complete medium and transfer to EP tubes, centrifuge at 500g for 5min; discard the supernatant, add Fixation/Permeabilization solution to 100μL/tube, 4°C, 20min; dilute 10×BD Perm/Wash ^TM buffer with ddH ₂ O to 1×, wash twice with 250μL 1×BD Perm/Wash ^TM buffer, centrifuge at 1000g for 5min; dilute dsRNA antibody with 1×BD Perm/Wash ^TM buffer, 50 μL/tube, incubate at 4°C in the dark for 30 min; wash twice with 250 μL 1×BD Perm/Wash ^TM buffer, centrifuge at 500 g for 10 min; dilute with Donkey anti-Mouse IgG (H+L) Secondary Antibody, DyLight ^TM 488 1:500 , 50 μL/tube, incubate at 4°C in the dark for 30 min; wash twice with 250 μL 1×BD Perm/Wash ^TM buffer, centrifuge at 500 g for 10 min; resuspend cells in 300 μL BD Perm/Wash ^TM buffer, filter and analyze on the machine (Fig. 5).

Example 6

EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT are genetically stable, and the steps are:

1. Continuous passage of the virus: report the virus twice at -80°C, freeze and thaw twice, centrifuge at 4000g to collect the virus supernatant as the P0 generation, take half of the volume of the P0 generation virus liquid to continue infecting the cells, and collect the virus liquid when the cell pathology exceeds 80%. For the P1 generation virus, the same steps were continued to pass to the P10 generation. The harvested virus fluid was stored at -80°C.

2. Measure luciferase activity after infecting cells with different generations of viruses: 2.5× ¹⁰⁴ Vero and RD cells were inoculated into 96-well plates, cultured overnight at 37°C, discarded the cell culture supernatant, and replaced the cell supernatant with 2 %FBS in DMEM medium. After infecting cells with different generations of reporter virus for 24 hours, add 20 μL of Promega Nano Glo HiBiT LyticDetection substrate to each well, incubate at room temperature for 5 minutes, and detect the fluorescence value. The stability of the virus was reflected by comparing the fluorescence values generated after different generations of virus infected cells ( FIG. 6 ).

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention.

Claims (10)

1. An enterovirus infectious clone with a new HiBiT reporter gene, including enterovirus EVA71, CVA16, CVA10 and CVA7 infectious clones with a new HiBiT reporter gene, namely EVA71-HiBiT, CVA16-HiBiT, CVA10 - HiBiT and CVA7-HiBiT infectious clones; wherein: The nucleic acid sequence of the HiBiT novel reporter gene is shown in SEQ ID NO.1; The nucleotide sequences of the enteroviruses EVA71, CVA16, CVA10 and CVA7 are respectively shown in SEQ ID NO.2-5. 2. The construction method of the enterovirus infectious clone with HiBiT novel reporter gene described in claim 1, is characterized in that, comprises: described EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT infectious clone Based on the infectious clones of EVA71, CVA16, CVA10 and CVA7 respectively, they were constructed using seamless cloning technology; wherein: the infectious clones of EVA71 passed the upstream and downstream sequences shown in SEQ ID NO.6 and SEQ ID NO.7 The primers are introduced into the HiBiT sequence; the CVA16 infectious clone is introduced into the HiBiT sequence through the upstream and downstream primers shown in SEQ ID NO.8 and SEQ ID NO.9; the CVA10 infectious clone is introduced into the HiBiT sequence through the sequences such as SEQ ID NO.10 and The upstream and downstream primers shown in SEQ ID NO.11 introduce the HiBiT sequence; the CVA7 infectious clone introduces the HiBiT sequence through the upstream and downstream primers shown in SEQ ID NO.12 and SEQ ID NO.13. 3. according to the construction method of the enterovirus infectious clone with HiBiT novel reporter gene described in claim 2, it is characterized in that, also comprise that the correct fragment of gel recovery is transferred into Trans2-Blue escherichia coli competent, shaking bacteria, extracting Plasmid post-sequencing verification, the step of storing the correctly sequenced plasmid at -20°C. 4. A reporter virus, comprising reporter virus EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT, respectively with EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA10-HiBiT described in any one of claims 1 to 3 CVA7-HiBiT infectious clones were obtained by rescuing the virus. 5. the construction method of reporter virus described in claim 4 is characterized in that, comprises the following steps: S1. Prepare EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT infectious clones by seamless cloning method; S2. EVA71-HiBiT, CVA16-HiBiT, CVA10-HiBiT and CVA7-HiBiT were transcribed in vitro to obtain mRNA; S3, EVA71-HiBiT, CVA10-HiBiT and CVA7-HiBiT mRNA transfected RD cells, CVA16-HiBiT mRNA transfected Vero cells; S4, the cell supernatant was continuously passaged, and the cell pathological changes were observed; S5. Report virus and parental virus by plaque titration, and verify growth characteristics; S6, verifying the difference between the growth characteristics of the reporter virus and the parental virus. 6. The construction method of the reporter virus according to claim 5, characterized in that, in step S1, based on EVA71, CVA16, CVA10 and CVA7 infectious clones, wherein: said EVA71 infectious clones have a sequence such as SEQ ID NO The upstream and downstream primers shown in .6 and SEQ ID NO.7 introduce the HiBiT sequence; the CVA16 infectious clone introduces the HiBiT sequence through the upstream and downstream primers shown in SEQ ID NO.8 and SEQ ID NO.9; the CVA10 The infectious clone introduces the HiBiT sequence through the upstream and downstream primers shown in SEQ ID NO.10 and SEQ ID NO.11; the CVA7 infectious clone uses the sequence shown in SEQ ID NO.12 and SEQ ID NO.13 The upstream and downstream primers were introduced into the HiBiT sequence, and the correct fragments were recovered from the gel, and then transformed into Trans2-Blue E. coli competent, shaken, and the plasmid was extracted for sequencing verification, and the sequenced correct plasmid was stored at -20°C. 7. according to the construction method of the described reporter virus of claim 5, it is characterized in that, in step S2, prepare the template of in vitro transcription by PCR primer, the sequence of EVA71-HiBiT upstream and downstream PCR primer is as SEQ ID NO.14 and SEQ ID NO. As shown in 15, the sequences of the upstream and downstream PCR primers of CVA16-HiBiT are shown in SEQ ID NO.16 and SEQ ID NO.17, and the sequences of the upstream and downstream PCR primers of CVA10-HiBiT are shown in SEQ ID NO.18 and SEQ ID NO.19. The sequences of the upstream and downstream PCR primers of CVA7-HiBiT are shown in SEQ ID NO.20 and SEQ ID NO.21. The mRNA was synthesized using an in vitro transcription kit, and the mRNA with correct quality control was aliquoted and stored at -80°C. 8. The construction method of the reporter virus according to claim 5, characterized in that, in step S3, the mRNA transcribed in step S2 is transferred into cells in a 6-well plate, and the cell pathological changes are observed day by day. 9. The construction method of the reporter virus according to claim 5, characterized in that, in step S6, after the virus supernatant collected in step S4 infects cells, detect the translation level of viral protein by plaque, 2C antibody, and detect virus by dsRNA antibody The efficiency of infecting cells verifies the biological activity of the reporter virus and compares it with the parental virus. 10. The enterovirus EVA71, CVA16, CVA10 and CVA7 infectious clone described in any one of claims 1 to 3 with the HiBiT novel reporter gene or the reporter virus described in claim 4 in anti-enterovirus drugs or neutralization Application in high-throughput screening of antibodies.

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