CN116024180A - Coxsackie virus A group 6 type Vero cell adaptation strain and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a coxsackievirus A group 6 type Vero cell adaptation strain and application thereof. The genome of the adaptive strain consists of 5 'and 3' non-coding regions and a viral protein coding region, the full-length nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequences of the coded structural proteins and the non-structural proteins are shown as SEQ ID NO. 2. The adaptation strain is obtained by adapting RD cell adaptation strain to Vero cells, and then carrying out serial passage and end dilution purification, wherein the adaptation strain generates multi-site change relative to a parent strain sequence, nucleotide mutation sites are VP2-G1154A, VP2-C1255T, 2C-C4494T and 3D-A6716T, amino acid mutation sites are VP2-A54T and 2C-S310F, and the adaptation strain can be proliferated in human rhabdomyoma cells (RD) and African green monkey kidney cells (Vero) at the same time and can be applied to screening of vaccine strains.

Description

Coxsackie virus A group 6 type Vero cell adaptation strain and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a coxsackievirus A group 6 type Vero cell adaptation strain and application thereof.

Background

Hand-foot-and-mouth disease (Hand, foot and mouse disease, HFMD) is a contagious disease caused by enteroviruses, and the pathogen can be transmitted by contact with the saliva, feces, respiratory secretions or herpes fluid of a patient. Hand-foot-mouth disease usually occurs in children under 5 years old, and occasionally in adults, and its main clinical symptoms are low fever, ulcers in oral mucosa, and herpes at the hands, feet, buttocks and other parts. Generally, hand-foot-mouth disease is self-limiting, most infants self-heal about one week, but few patients can cause serious complications such as meningitis, encephalitis, acute flaccid paralysis, myocarditis, pulmonary edema and the like, and individual severe infants can rapidly develop the disease and then die. To date, hand-foot-and-mouth disease is still one of the problems threatening public health around the world.

Human enteroviruses are currently classified into seven populations, namely human enterovirus A, B, C, D type and human rhinovirus A, B, C type, according to the homology relationship of phylogenetic tree, and each group can be further classified into different serotypes or genotypes. Coxsackie virus type A6 (Coxsackie virus A6, CV-A6) belongs to the genus Enterovirus of the family Picornaviridae, and has a diameter of about 27-30 nm and is a single-stranded positive strand RNA virus. In recent years, CV-A6 has become the primary pathogen causing hand-foot-and-mouth disease. Whereas the marketed EV71 vaccine has no cross-protective efficacy against HFMD caused by enteroviruses of non-EV 71 type.

CV-A6 capsids consist of four structural proteins containing epitopes that bind to cellular receptors. Changes in the amino acids of the structural protein region can result in changes in the conformation of the viral surface proteins, thereby affecting the binding of the virus to cell surface receptors. Structural proteins VP1, VP2, VP3 contain neutralizing epitopes. In addition, the proteins encoded by the non-structural protein coding regions or the nucleotides in the 5 'and 3' non-coding regions are closely related to viral RNA replication, transcription, translation, and viral assembly and release, and changes in the non-structural protein genes can affect the activity of the relevant enzymes, viral RNA replication, viral assembly, and in turn affect the cellular tropism and growth characteristics of the virus.

CV-A6 can proliferate in human rhabdomyoma cells (RD) and produce cytopathic effects (cytopathic effect, CPE), but it is difficult to proliferate in human vaccine cell matrices such as African green monkey kidney cells (Vero), canine kidney cells (MDCK), human embryonic lung fibroblasts (MRC-5), and human embryonic lung diploid cells (2 BS), which makes research on CV-A6 and development of whole virus inactivated vaccines difficult. The research on key nucleotide sites and amino acid sites affecting proliferation of CV-A6 in Vero cells is beneficial to research on mechanisms of CV-A6 infected cells and research on monovalent or multivalent whole virus inactivated vaccines containing CV-A6 for hand-foot-mouth disease.

Disclosure of Invention

Based on the above, one of the purposes of the invention is to provide a coxsackievirus A group 6 Vero cell adapted strain, the genome sequence of which is shown as SEQ ID NO. 1.

The coxsackievirus A group 6 Vero cell adapted strain provided by the invention is an anus swab sample clinically diagnosed as a hand-foot-mouth disease patient in Xiangyang city of Hubei province, is subjected to centrifugation and antibiotic overnight treatment, then RD cells are used as hosts to separate and culture CV-A6-RD cell isolated strains, and then the Vero cells are used as hosts to carry out coxsackievirus CV-A6 adapted passage, thus obtaining the coxsackievirus A group 6 adapted strain, and the nucleotide and amino acid sequences of the coxsackievirus A group 6 adapted strain are determined by sequencing. The sequence is used as a template to synthesize infectious cDNA in vitro, and T7 RNA polymerase is used to carry out in vitro transcription to generate viral RNA, and the viral RNA is transfected into Vero cells to rescue the virus. And thereby further verifying the relationship between the nucleotide and amino acid sequences of the adapted strain and cell tropism.

The adapted strain was designated as human coxsackievirus CV-A6-712, latin name: coxsackie virus A6-712 and was deposited at the China center for type culture Collection at a deposit address of 2022, 8, 3: the preservation number of the Chinese university of Wuhan is CCTCC NO: V202264.

The adaptation strain encodes 11 proteins, namely structural proteins VP4, VP2, VP3 and VP1, non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D, and amino acid sequences of the proteins are shown as SEQ ID NO:2, the encoded amino acid sequences correspond to:

VP4 protein is the 1 st-69 th amino acid sequence in SEQ ID NO. 24;

VP2 protein is the 70 th-325 th amino acid sequence in SEQ ID NO. 24;

VP3 protein is the 326 th to 565 th amino acid sequence in SEQ ID NO. 24;

VP1 protein is 566-870 th amino acid sequence in SEQ ID NO. 24;

the 2A protein is 871-1020 amino acid sequence in SEQ ID NO. 24;

the 2B protein is 1021-1119 amino acid sequence in SEQ ID NO. 24;

the 2C protein is 1120-1448 amino acid sequence in SEQ ID NO. 24;

the 3A protein is 1449-1534 amino acid sequence in SEQ ID NO. 24;

the 3B protein is the 1535 th-1556 th amino acid sequence in SEQ ID NO. 24;

the 3C protein is the 1557 th to 1739 th amino acid sequence in SEQ ID NO. 24;

the 3D protein is 1740-2201 amino acid sequence in SEQ ID NO. 24.

Another object of the present invention is to provide a biomaterial which is any one of the following (1) to (5):

(1) A nucleic acid molecule with a sequence shown as SEQ ID NO. 1 or a complete complementary sequence shown as SEQ ID NO. 1;

(2) An expression cassette comprising the nucleic acid molecule of (1);

(3) A recombinant vector comprising the nucleic acid molecule of (1);

(4) A recombinant microorganism comprising the nucleic acid molecule of (1);

(5) A cell line comprising the nucleic acid molecule of (1).

The third object of the present invention is to provide a method for culturing the coxsackievirus A group 6 Vero cell-adapted strain, wherein the method uses Vero cells as a host.

The invention aims at providing the application of the Coxsackie virus A group 6 Vero cell adaptation strain in preparing the vaccine for preventing and/or treating diseases caused by the Coxsackie virus.

The fifth object of the present invention is to provide the use of the above-mentioned biological material for the preparation and/or prevention of diseases caused by coxsackievirus.

The sixth purpose of the invention is to provide the application of the coxsackievirus A group 6 Vero cell adaptation strain in CV-A6 library construction.

The seventh object of the present invention is to provide a product comprising the coxsackievirus group-a 6 Vero cell-adapted strain described above and/or the biological material described above.

The eighth object of the present invention is to provide the use of key nucleotide and/or amino acid sites affecting CV-A6 proliferation in Vero cells for studying the mechanism of CV-A6 infection of Vero cells, said sites comprising at least the following:

VP2-1115, VP2-1255, 2C-4494 and 3D-6716 sites in the sequence shown in SEQ ID NO. 1; and/or VP2-54, 2C-130 sites in the sequence shown in SEQ ID NO. 2.

According to the invention, through a virus cell culture technology, a CV-A6 Vero cell adaptation strain (CV-A6-712) and a CV-A6 strain (CV-A6-1023) which can only grow on RD cells are obtained, the CV-A6 Vero cell adaptation strain (CV-A6-712) can be proved to be applied to vaccines after further passage, and meanwhile, key nucleotide and amino acid sites influencing CV-A6 strain in Vero cell proliferation are found through sequencing comparison, so that theoretical basis is provided for researching molecular biological characteristics, cell tropism, cell growth characteristics, disease treatment mechanism, immunogenicity and research and development of related vaccines and screening of antiviral drugs of coxsackieviruses.

Drawings

FIG. 1 is a diagram showing the cell states of CV-A6-712 and CV-A6-1023 inoculated in Vero cells in example 3 of the present invention, wherein a and c are respectively the cytopathic states of CV-A6-712 inoculated in Vero cells for 48h and 72h, b and d are respectively the cytopathic states of CV-A6-1023 inoculated in Vero cells for 48h and 72h, and e and f are respectively the immunofluorescence experimental results of CV-A6-712 and CV-A6-1023 inoculated in Vero cells for 72 h;

FIG. 2 shows one-step growth curves for CV-A6-712 and CV-A6-1023;

FIG. 3 shows IFA detection of viral protein expression of CV-A6-712 and CV-A6-1023 upon passage of RD cells to the P3 generation in example 6 of the present invention, where DAPI indicates nuclei, green indicates viral proteins, merge is co-localization of both DAPI and Green;

FIG. 4 shows the IFA assay of CV-A6-712 and CV-A6-1023 of example 6 of the present invention upon passage of Vero cells to the P3 generation, where DAPI indicates nuclei, green indicates viral proteins, and Merge is the co-localization of both DAPI and Green;

FIG. 5 shows the WB detection of CV-A6 antigen in the supernatant of the P3 passages of RD cell culture in example 6 of the present invention;

FIG. 6 shows the WB assay of CV-A6 antigen in the P3-generation supernatant of Vero cell culture in example 6 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.

The following examples are given for illustration of the invention only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.

In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.

The material sources are as follows:

anal swab sample HEV3391 is derived from Xiangyang Lin of Hubei province and is clinically diagnosed as hand-foot-mouth disease patient.

Example 1

The embodiment provides a method for separating and culturing CV-A6-HEV3391 virus strain, which comprises the following steps:

after a sample HEV3391 derived from a anus swab of a patient clinically diagnosed as a hand-foot-and-mouth disease patient in Xiangyang, hubei province was centrifuged at 3000g for 15min, the sample was treated overnight at 4℃in a clean 1.5mL EP tube with the addition of antibiotics (penicillin final concentration 100U/mL, streptomycin final concentration 100. Mu.g/mL). A24-well plate full of single-layer RD cells was used, the culture solution was discarded, and serum-free MEM cell maintenance solution (manufacturer: niday Water No. 05900) was added, and 100. Mu.L of the antibiotic-treated sample solution was inoculated into RD cells and placed in a 5% carbon dioxide incubator at 37℃for culturing. And after cytopathy reaches 80-90%, repeatedly freezing and thawing for three times, centrifuging to obtain virus liquid, namely the first generation (CV-A6-HEV 3391-R1) of RD cell isolate of the virus sample.

Example 2

The present example provides a method for purifying RD cell isolates of CV-A6-HEV3391-R1, which comprises the steps of purifying virus by three consecutive terminal dilution, and then amplifying and culturing CV-A6-HEV3391-R1 virus clone strains, wherein the specific steps are as follows:

the CV-A6-HEV3391-R1 strain virus solution was diluted 10-fold, a 96-well plate full of single-layered RD cells was prepared, 8 wells were repeated for each virus dilution, and CV-A6-3391-R1 strain was inoculated into the 96-well plate at 100. Mu.L/well for 7 days of culture. And (3) microscopic examination of CPE conditions of each hole, picking clone strains from a compound hole corresponding to the last CPE positive dilution, and transferring 200 mu L of virus harvest liquid in the suction hole into a 1.5mL centrifuge tube. Taking 100 mu L of harvest liquid to dilute 10 times, repeating the steps for three times to perform continuous limiting dilution inoculation, finally obtaining purified virus clone strains, and placing the selected clone virus strains in a refrigerator at the temperature of minus 80 ℃ for preservation.

RD cells with good growth status and 100% confluence were taken (T ₂₅ Bottle), the original bottle of culture solution is discarded and MEM cells without serum are added for maintenanceInoculating 10 mu L of virus liquid, placing the virus liquid in a 37 ℃ and 5% carbon dioxide incubator, inoculating one bottle of cells to each clone strain, repeatedly freezing and thawing for three times when cytopathy reaches 80-90%, centrifuging to obtain virus liquid, and sub-packaging and freezing to obtain the P1 generation.

Example 3

This example describes the adaptive passage of CV-A6-HEV3391-R1 clone strains in Vero cells.

100. Mu.L of CV-A6-HEV3391-R1 clone virus solution was inoculated into a 24-well cell culture plate plated with Vero cells, and cultured in a 5% carbon dioxide incubator at 37℃for 5 days. The observation of whether cytopathic effect appears or not is carried out every day to obtain a virus strain (CV-A6-712) suitable for Vero cells and a virus strain (CV-A6-1023) incapable of being suitable for Vero cells, the detection results of Vero cytopathic effect and indirect Immunofluorescence (IFA) of 2 clone purified strains and the growth curves of Vero cells are respectively shown in figures 1 and 2, wherein a and c in figure 1 are respectively cytopathic states of CV-A6-712 inoculated in Vero cells for 48h and 72h, b and d are respectively cytopathic states of CV-A6-1023 inoculated in Vero cells for 48h and 72h, e and f are respectively immunofluorescence experimental results of CV-A6-712 and CV-A6-1023 inoculated in Vero cells for 72h, and 1 is used: 100 rabbit anti-CV-A6 antisera.

Example 4

The whole genome sequences of CV-A6-712 and CV-A6-1023 were determined and analyzed in this example as follows:

(1) Viral RNA extraction and cDNA synthesis: extracting and purifying viral RNA by using a column type viral RNA extraction and purification kit (product number: B518667) of a biological engineering (Shanghai) Co., ltd.), adding a reagent into a PCR tube as shown in a table 1, uniformly mixing, placing into a PCR instrument, reacting at 65 ℃ for 5min, rapidly quenching on ice, adding the following reagent into the reaction solution as shown in a table 2, uniformly mixing, and placing into the PCR instrument at 30 ℃ for 15min;42 ℃ for 60min; cDNA was synthesized by reverse transcription at 70℃for 15 min.

TABLE 1

Reagent(s)	Volume of
		RNase Free H 2 O	5.0μL
Oligo dT(50μM)	1.0μL
		dNTP Mixture(10mM each)	1.0μL
RNA	5.0μL
		Total Volume	10.0μL

TABLE 2

(2) And (3) PCR amplification: CV-A6 full sequence determination primers are shown in Table 3, and PCR amplification was performed using KOD-Plus-Neo (Code No. KOD-401) manufactured by Toyobo Co., ltd., 94℃for 2min;98℃for 10sec;60 ℃, sec;68 ℃ (extension speed 30sec/kb, cycle number 25-45 cycles).

TABLE 3 Table 3

(3) Agarose gel electrophoresis: after the reaction is finished, carrying out 1% agarose gel electrophoresis, identifying PCR amplified bands, after the PCR products are identified correctly, sending the rest amplified products to a biological engineering technical service company for sequencing, and splicing all the sequenced fragments to obtain a whole genome sequence.

Sequencing results showed that the complete genomic sequence of CV-A6-712 is shown below (SEQ ID NO: 1):

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the amino acid sequence is as follows (SEQ ID NO: 2):

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the complete genome sequence of CV-A6-1023 is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4.

The genome of the CV-A6-712 virus strain is 7453 nucleotide residues, the nucleotide positions of the non-coding region 5' -UTR and the non-coding region 3' -UTR in the genome are respectively 1-748 and 7355-7435,3' -UTR are poly A tails, the length of the encoded polyprotein is 2201 amino acid residue, wherein the amino acid sequences and the positions of each structural protein and the non-structural protein of the virus are respectively:

VP4 protein is the 1 st-69 th amino acid sequence;

VP2 protein is the 70 th-325 th amino acid sequence;

VP3 protein is 326-565 th amino acid sequence;

VP1 protein is 566-870 th amino acid sequence;

the 2A protein is 871-1020 amino acid sequence;

the 2B protein is 1021-1119 amino acid sequence;

the 2C protein is 1120-1448 amino acid sequence;

the 3A protein is 1449-1534 amino acid sequence;

the 3B protein is 1535-1556 th amino acid sequence;

the 3C protein is 1557 th to 1739 th amino acid sequence;

the 3D protein is 1740-2201 amino acid sequence.

Example 5

Analysis of key amino acid sites affecting proliferation of CV-A6 virus in Vero cells:

the whole genome sequences of CV-A6-712 and CV-A6-1023 were aligned by MEGA6 software, and the results are shown in Table 4 below. The 1115 nucleotide of CV-A6-712 strain is adenine (A), the 1255 nucleotide is thymine (T), the 4494 nucleotide is thymine (T), the 6716 nucleotide is thymine (T), the 54 th amino acid of VP2 protein is threonine (Thr), and the 130 th amino acid of 2C protein is phenylalanine (Phe). The 1115 nucleotide of CV-A6-1023 strain is guanine (G), the 1255 nucleotide is cytosine (C), the 4494 nucleotide is cytosine (C), the 6716 nucleotide is adenine (A), the 54 th amino acid of VP2 protein is alanine (Ala), and the 130 th amino acid of 2C protein is serine (Ser).

Table 4 CV-A6-712 and CV-A6-1023 nucleotide and amino acid sequence alignments

Example 6

The serial passaging ability of the CV-A6 strain obtained was evaluated:

CV-A6-712 and CV-A6-1023 strains were serially passaged for 3 passages in RD and Vero cells, and culture supernatants were collected and assayed for viral structural proteins IFA in RD cell culture supernatants using rabbit anti-CV-A6 VLP antisera (1:200). The results are shown in FIGS. 3 and 4, where FIG. 3 shows the IFA detection of viral protein expression of CV-A6-712 and CV-A6-1023 when RD cells were passaged to P3, FIG. 4 shows the IFA detection of viral protein expression of CV-A6-712 and CV-A6-1023 when Vero cells were passaged to P3, where DAPI indicates nuclei, green indicates viral proteins, merge indicates co-localization of both DAPI and Green, and shows that CV-A6-712 strain can observe GFP fluorescence in RD and Vero cells, CV-A6 viral protein expression was detected, and CV-A6-1023 strain only observed fluorescence in RD cells, but not in Vero cells.

The level of CV-A6 antigen expression in RD cell culture third generation supernatants was detected by Western Blot using rabbit anti-CV-A6 virion antisera (1:2000) and the results are shown in FIGS. 5 and 6, wherein FIG. 5 shows the detection of CV-A6 antigen in RD cell culture P3 generation supernatants by WB, FIG. 6 shows the detection of CV-A6 antigen in Vero cell culture P3 generation supernatants by WB, the viral structural protein position is indicated on the left of the figure, the protein molecular weight is indicated on the right of the figure, "R-": cell supernatant of clone infected RD cells, "V-": cell uploading of clone strain infected Vero cell, 731 is another virus control which is preserved in the experiment and verified and can not grow on Vero cell, PC is late generation Vero cell adaptation strain preserved in the experiment and verified, and PC is positive control; MOCK was uninfected RD cell supernatant and was a blank control. From the figure it can be seen that the conclusion is consistent with IFA that CV-A6-712 strain was able to be serially passaged on RD and Vero cells, while CV-A6-1023 strain was able to be serially passaged only on RD cells.

It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A coxsackievirus A group 6 type Vero cell adapted strain is characterized in that the genome sequence is shown as SEQ ID NO. 1.

2. The coxsackievirus group a6 Vero cell adapted strain of claim 1, deposited with the chinese collection for typical cultures under accession number: CCTCC NO: V202264.

3. The coxsackievirus a group 6 Vero cell strain of claim 1, wherein the encoded protein has the amino acid sequence shown in SEQ ID No. 2, and each protein has the amino acid sequence of:

VP4 protein is the 1 st-69 th amino acid sequence in SEQ ID NO. 24;

VP2 protein is the 70 th-325 th amino acid sequence in SEQ ID NO. 24;

VP3 protein is the 326 th to 565 th amino acid sequence in SEQ ID NO. 24;

VP1 protein is 566-870 th amino acid sequence in SEQ ID NO. 24;

the 2A protein is 871-1020 amino acid sequence in SEQ ID NO. 24;

the 2B protein is 1021-1119 amino acid sequence in SEQ ID NO. 24;

the 2C protein is 1120-1448 amino acid sequence in SEQ ID NO. 24;

the 3A protein is 1449-1534 amino acid sequence in SEQ ID NO. 24;

the 3B protein is the 1535 th-1556 th amino acid sequence in SEQ ID NO. 24;

the 3C protein is the 1557 th to 1739 th amino acid sequence in SEQ ID NO. 24;

the 3D protein is 1740-2201 amino acid sequence in SEQ ID NO. 24.

4. A biomaterial characterized by being any one of the following (1) to (5):

(1) A nucleic acid molecule with a sequence shown as SEQ ID NO. 1 or a complete complementary sequence shown as SEQ ID NO. 1;

(2) An expression cassette comprising the nucleic acid molecule of (1);

(3) A recombinant vector comprising the nucleic acid molecule of (1);

(4) A recombinant microorganism comprising the nucleic acid molecule of (1);

(5) A cell line comprising the nucleic acid molecule of (1).

5. A method for culturing a coxsackievirus group a6 Vero cell-adapted strain according to any one of claims 1 to 3, wherein the culturing is carried out using Vero cells as a host.

6. Use of a Vero cell-adapted strain of coxsackievirus group 6 as claimed in any one of claims 1-3 in the preparation of a vaccine for the prevention and/or treatment of diseases caused by coxsackievirus.

7. Use of the biomaterial according to claim 3 for the preparation of a vaccine for the prevention and/or treatment of diseases caused by coxsackie virus.

8. Use of a coxsackievirus group A6 Vero cell-adapted strain of any one of claims 1-3 in CV-A6 library construction.

9. A product comprising a coxsackievirus group a type 6 Vero cell adapted strain of claim 1 and/or a biomaterial of claim 3.

10. Use of a key nucleotide site and/or amino acid site affecting proliferation of CV-A6 in Vero cells for studying the mechanism of CV-A6 infection of Vero cells, wherein said site comprises at least the following:

VP2-1115, VP2-1255, 2C-4494 and 3D-6716 sites in the sequence shown in SEQ ID NO. 1; and/or VP2-54, 2C-130 sites in the sequence shown in SEQ ID NO. 2.

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