Coxsackievirus group A type 5 domesticated strain and its isolation method and application

Abstract

The present invention provides a domesticated strain of Coxsackievirus group A5, its isolation method, and application. The domesticated strain of Coxsackievirus group A5 has a cell growth phenotype of RD+ / Vero+ and a deposit number of CCTCCNo: V202234. Through whole-genome sequencing and alignment, relevant nucleotide and amino acid sites that affect the proliferation of the CV-A5 strain in Vero cells were discovered. This domesticated strain is a Vero cell-adapted strain and can provide a research basis for studying CV-A5-related cell tropism, cell growth characteristics, pathogenic mechanisms, gene-directed modification, establishment of animal models in vaccine development, immunogenicity studies, and screening of antiviral drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of virology, and in particular to a high-titer Coxsackievirus group A type 5 (CV-A5) domesticated strain, an isolation method and an application thereof. Background Art

[0002] Hand, foot and mouth disease (HFMD) is a Class C infectious disease caused by multiple enterovirus serotypes. There are at least 20 types of enterovirus that cause HFMD, including Coxsackievirus A (CV-A) types 2, 4, 5, 6, 10, 12 and 16, Coxsackievirus B (CV-B) type 5, echovirus (Enteric cytopatho-genic human orphan virus, Echo) and enterovirus 71 (Enterovirus71, EV-71).

[0003] HFMD is common in children under 5 years old. Current research indicates that EV-71 is the primary pathogen causing severe illness and mortality from HFMD, while other enterovirus serotypes in the CV-A and CV-B groups are also major pathogens causing HFMD outbreaks and sporadic outbreaks.

[0004] The CV-A5 viral capsid is composed of four structural proteins (VP4, VP2, VP3, and VP1). Structural proteins contain epitopes that recognize and bind to the cell receptor KREMEN. Changes in amino acids in the structural protein region can affect the conformation and function of the corresponding structural proteins, thereby affecting the recognition and binding of the virus to cell surface receptors and cell invasion auxiliary factors, and further affecting the virus's ability to infect and its tissue and cell tropism. The structural proteins VP1, VP2, and VP3 contain neutralizing antigenic epitopes that can induce the body to produce antiviral neutralizing antibodies. In addition, the proteins encoded by the non-structural protein coding region and the genomic nucleotide sequence or the nucleotide sequence of the 5'-UTR and 3'-UTR of the non-coding region are closely related to viral RNA replication, transcription, translation, viral assembly, and release. Changes in the nucleotide and amino acid sequences of non-structural proteins can affect the function and activity of related enzymes, viral RNA replication, viral assembly, and subsequently affect the cellular tropism and biological characteristics of the virus.

[0005] CV-A5 can proliferate and produce a cytopathic effect (CPE) in RD cells, but it is difficult to directly isolate, proliferate, and produce CPE in human vaccine cell matrices such as human embryonic lung diploid cells (2BS), Vero cells, human embryonic lung fibroblasts (MRC-5), and canine mitochondrial canine kidney cells (MDCK). This complicates basic research on CV-A5 and the development of monovalent or polyvalent HFMD whole-virus inactivated and live-attenuated vaccines. HFMD outbreaks severely impact the daily lives of infants and young children and pose a potential threat. The co-circulation of multiple enteroviruses, including CV-A5, creates a significant disease and economic burden. Developing and vaccinating target populations with monovalent or polyvalent viral vaccines to establish a population-wide immune barrier is one of the most effective preventive measures to prevent viral transmission. Vaccination is a relatively cost-effective method for controlling infectious diseases. Currently, there are no reports of clinical studies of monovalent or polyvalent CV-A5 vaccines, hindered by the extremely low isolation rate of CV-A5 in human vaccine cell matrices. Studying the nucleotide or amino acid sites of CV-A5 proliferation in Vero cells and other human vaccine cell matrices, and studying the biological traits and gene expression differences of strains or domesticated strains with different cell tropisms will be beneficial to studying the mechanism of CV-A5 infection of Vero cells, and will also be beneficial to the development of monovalent or polyvalent hand, foot and mouth disease whole virus inactivated vaccines targeting CV-A5 and targeted modification to obtain virus strains that can be used in vaccines. Summary of the Invention

[0006] Based on this, it is necessary to provide a high-titer Coxsackievirus group A type 5 (CV-A5) domesticated strain and its isolation method and application.

[0007] The present invention adopts the following technical solutions:

[0008] The present invention provides a high-titer Coxsackievirus group A type 5 (CV-A5) domesticated strain with a cell growth phenotype of RD+ / Vero+ and a storage number of CV-A5-3487V1 / XY / CHN / 2017. The CV-A5 domesticated strain was deposited with the China Center for Type Culture Collection on May 24, 2022, with a storage number of CCTCC No: V202234, and the deposit address is Wuhan University.

[0009] The virus titer of the domesticated CV-A5 strain is 1×10 6.5 CCID 50 / mL.

[0010] The cell growth phenotype of the CV-A5 domesticated strain is RD+ / Vero-, and its storage number is CV-A5-3487R1 / XY / CHN / 2017. The virus mother strain originated from: isolated from sample 3487 of hand, foot and mouth disease patient in Xiangyang City, China (CHN XY) in 2017.

[0011] The genome nucleotide and amino acid sequence lengths of the CV-A5-3487V1 / XY / CHN / 2017 and CV-A5-3487R1 / XY / CHN / 2017 strains are identical. The genome nucleotides and their common characteristics are as follows:

[0012] The strain genome consists of the untranscribed 5'-UTR and 3'-UTR regions, as well as the coding regions for structural proteins 1A, 1B, 1C, and 1D (corresponding to VP4, VP2, VP3, and VP1, respectively), and nonstructural proteins 2A, 2B, 2C, and 3A, 3B, 3C, and 3D. The key nucleotide and amino acid residues that affect the proliferation of the CV-A5 domesticated strain in Vero cells are shown in Table 4.

[0013] The present invention also provides a method for isolating and culturing the above-mentioned Coxsackievirus group A type 5 domesticated strain, comprising the following steps: centrifuging a human enterovirus sample No. 3487 from a hand, foot and mouth disease patient in Xiangyang City, China (CHN XY) in 2017, adding antibiotics to obtain a mother virus liquid; culturing RD cells until the cell confluence is 85-95%, discarding the culture medium, adding a cell maintenance medium, inoculating the mother virus liquid, and placing the cells in a 37°C, 5% carbon dioxide incubator for culture; when the cell pathological rate reaches 80-90%, repeatedly freezing and thawing three times, centrifuging to remove the cell culture medium, obtaining a domesticated virus liquid, and obtaining a first-generation RD cell isolate CV-A5-3487R1 / XY / CHN / 2017; continuing to place CV-A5-3487R1 / XY / CHN / 2017 in an RD cell culture chamber. Subculture was performed to obtain the third-generation virus strain CV-A5-3487R3 / XY / CHN / 2017; CV-A5-3487R1 / XY / CHN / 2017 and CV-A5-3487R3 / XY / CHN / 2017 virus solutions were incubated on Vero cells in a cell culture plate at the same multiplicity of infection and cultured in a 37°C, 5% carbon dioxide incubator. The results showed that CV-A5-3487R1 / XY / CHN / 2017 could not grow in Vero cells, while CV-A5-3487R3 / XY / CHN / 2017 could infect Vero cells;

[0014] The virus infected with Vero cells was harvested and named CV-A5-3487V1 / XY / CHN / 2017, with a growth phenotype of RD+ / Vero+.

[0015] Compared with the prior art, the advantages of the present invention are:

[0016] The present invention provides for the first time a CV-A5 domesticated strain that can proliferate in RD and Vero cells, and clarifies the nucleotide and amino acid sites that affect the growth of the CV-A5 virus strain in Vero cells, thereby providing a research basis for studies on CV-A5-related cell tropism, cell growth characteristics, pathogenic mechanism, gene-directed modification, establishment of animal models in vaccine development, immunogenicity studies, and screening of antiviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are pathological changes detection diagrams and fluorescence test diagrams of Vero cells inoculated with the two virus strains in Example 3; wherein, a is the cytopathic state of Vero cells inoculated with CV-A5-3487R1 / XY / CHN / 2017 for 48 hours, b is the cytopathic state of Vero cells inoculated with CV-A5-3487V1 / XY / CHN / 2017 for 48 hours, c is the cytopathic state of Vero cells inoculated with CV-A5-3487R1 / XY / CHN / 2017 for 72 hours, d is the cytopathic state of Vero cells inoculated with CV-A5-3487V1 / XY / CHN / 2017 for 72 hours, e is the immunofluorescence experimental result of Vero cells inoculated with CV-A5-3487R1 / XY / CHN / 2017 for 72 hours, and f is the immunofluorescence experimental result of Vero cells inoculated with CV-A5-3487V1 / XY / CHN / 2017 for 72 hours.

[0018] Figure 2 Growth curves of CV-A5-3487R1 / XY / CHN / 2017 and CV-A5-3487V1 / XY / CHN / 2017 on Vero cells. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0020] The following embodiments are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0021] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0022] Example 1

[0023] This example provides a method for isolating and culturing the CV-A5-3487R1 / XY / CHN / 2017 virus mother strain, comprising the following steps:

[0024] S1. An anal swab sample (human enterovirus 3487, HEV3487) from a patient clinically diagnosed with hand, foot and mouth disease in Xiangyang City, Hubei Province was centrifuged (3000×g, 15 min), the sample was placed in a sterile EP tube, and antibiotics (final concentration of penicillin: 100 U / mL, final concentration of streptomycin: 100 μg / mL) were added and treated at 4°C overnight to obtain the sample solution.

[0025] S2: Human rhabdoid tumor cells (RD cells) were cultured in 24-well plates. When the cell confluence reached 85-95%, the culture medium was discarded and 1 ml of cell maintenance medium (DMEM, serum-free, Thermo Fisher Scientific) was added. 100 μL of the antibiotic-treated sample solution was inoculated onto the RD cells and incubated at 37°C in a 5% CO2 incubator. When the cytopathic effect reached 80-90%, the cells were frozen and thawed three times, and the cell debris was removed by centrifugation. The resulting virus sample was the first-generation RD cell isolate, designated CV-A5-3487R1 / XY / CHN / 2017.

[0026] Example 2

[0027] This example provides a subculture method for the CV-A5-3487R1 / XY / CHN / 2017 virus strain, comprising the following steps:

[0028] (1) Take RD cells (T25 flask) with good growth status and 100% confluence, discard the cell culture medium, wash three times with PBS buffer, inoculate 1 mL of CV-A5-3487R1 / XY / CHN / 2017 virus solution, place in a 37°C, 5% carbon dioxide incubator for 2 h, and add 4 ml of cell maintenance medium after virus adsorption.

[0029] (2) When the cytopathic effect reached 80-90%, the virus solution was harvested by repeated freezing and thawing three times and then centrifuged and stored in aliquots to obtain the CV-A5-3487R2 / XY / CHN / 2017 virus strain.

[0030] (3) Repeat the above steps once to obtain the CV-A5-3487R3 / XY / CHN / 2017 virus strain.

[0031] Example 3

[0032] This example provides a method for adaptive subculture of the CV-A5-3487R3 / XY / CHN / 2017 virus strain in African green monkey kidney cells (Vero cells), comprising the following steps: incubating CV-A5-3487R1 / XY / CHN / 2017 virus solution and CV-A5-3487R3 / XY / CHN / 2017 virus solution on Vero cells in a 12-well cell culture plate at the same multiplicity of infection (MOI), culturing at 37°C in a 5% carbon dioxide incubator for 7 days, and observing every day for the appearance of CPE. A virus strain adapted to Vero cells (named (CV-A5-3487V1 / XY / CHN / 2017) and a virus strain that cannot adapt to Vero cells (CV-A5-3487R1 / XY / CHN / 2017) were obtained.

[0033] Vero cell pathological changes of the two strains were observed, indirect immunofluorescence assay was performed, and Vero cell growth curves were used to verify their cell tropism. Figure 1 and Figure 2 .

[0034] Depend on Figure 1 It can be seen that after CV-A5-3487R1 / XY / CHN / 2017 was inoculated into Vero cells, no CPE and green fluorescent spots were observed at 48 h and 72 h; after CV-A5-3487V1 / XY / CHN / 2017 was inoculated into Vero cells, obvious CPE and green fluorescent spots appeared at 48 h and 72 h, proving that CV-A5-3487V1 / XY / CHN / 2017 can adapt to and replicate on Vero cells.

[0035] In this example, CV-A5-3487R1 / XY / CHN / 2017 could not be proliferated in Vero cells, and the viral titer could not be detected. This does not mean that there was no residual virus after adsorption, so the titer at each time point was assigned a value of 1.

[0036] Depend on Figure 2 It can be seen that the CV-A5-3487V1 / XY / CHN / 2017 virus has a proliferation cycle, including adsorption, invasion and uncoating, biosynthesis, assembly and release. The virus proliferation cycle is 2-4 hours.

[0037] Example 4

[0038] This embodiment performs full genome sequencing on the two virus strains (CV-A5-3487R1 / XY / CHN / 2017 and CV-A5-3487V1 / XY / CHN / 2017) obtained in Example 3, including the following steps:

[0039] S1. Viral RNA extraction

[0040] Viral RNA was extracted and purified using the column-type viral RNA extraction and purification kit (catalog number: B518667) from Sangon Biotech (Shanghai) Co., Ltd.

[0041] S2, reverse transcription and cDNA synthesis

[0042] The reverse transcription cDNA synthesis reaction system is as follows Tables 1 and 2:

[0043] Table 1 Reverse transcription cDNA synthesis reaction system

[0044] Reagents Volume (μL) <![CDATA[RNase Free H2O]]> 5.0 Oligo dT (50μM) 1.0 dNTP Mixture (10mM each) 1.0 RNA 5.0 Total Volume 10.0

[0045] Table 2 Reverse transcription cDNA synthesis reaction system

[0046] Reagents Volume (μL) 5×Prime Script Buffer 4.0 RNase Inhibitor (40U / μL) 0.5 Prime Script RTase (200U / μL) 1.0 <![CDATA[RNase Free H2O]]> 4.5 Total Volume 10.0

[0047] (1) Add the reagents as shown in Table 1 above to the PCR tube, mix well, and place in a PCR instrument. React at 65°C for 5 minutes, then quickly place on ice to quench.

[0048] (2) Add the following reagents as shown in Table 2 to the above reaction solution, mix well, and place in a PCR instrument at 30°C for 15 minutes; 42°C for 60 minutes; and 70°C for 15 minutes.

[0049] S3, PCR amplification

[0050] (1) Amplification primers: CV-A5 full sequence amplification and determination related primers are shown in Table 3 below:

[0051] Table 3 Primers for CV-A5 whole genome sequencing

[0052]

[0053]

[0054] (2) PCR amplification was performed using KOD-Plus-Neo (Code No. KOD-401) produced by Toyobo, 98°C for 10 min → 98°C for 30 sec, 57°C for 3 min 30 sec, 68°C (extension rate 30 sec. / kb), and the number of cycles was 30 to 35 cycles.

[0055] S4, agarose gel electrophoresis

[0056] After the reaction is complete, 1% agarose gel electrophoresis is performed to identify the PCR amplified bands. Once the PCR product is correctly identified, the remaining amplified product is sent to Sangon Bioengineering Technology Service Co., Ltd. for sequencing, and the sequenced fragments are spliced ​​to obtain the full genome sequence.

[0057] Sequencing results showed that the full genome sequence of CV-A5-3487R1 / XY / CHN / 2017 is as follows:

[0058]

[0059]

[0060]

[0061]

[0062] Italic bold These are primer sequences, not exact viral sequences.

[0063] The amino acid sequence of the polyprotein of CV-A5-3487R1 / XY / CHN / 2017 is as follows:

[0064]

[0065]

[0066] The sequence of the CV-A5-3487V1 / XY / CHN / 2017 full genome sequence is as follows:

[0067]

[0068]

[0069]

[0070]

[0071] The amino acid sequence of the polyprotein of CV-A5-3487V1 / XY / CHN / 2017 is as follows:

[0072]

[0073] The genome of the domesticated strain is 7402 nucleotides long and consists of a 5'-UTR (1-745), a 3'-UTR (7322-7402), and a viral protein coding region. The virus encodes 11 proteins, namely the structural proteins VP4, VP2, VP3, and VP1, and the non-structural proteins 2A, 2B, 2C, and 3A, 3B, 3C, and 3D. The non-coding regions 5'-UTR and 3'-UTR are located at nucleotide positions 1-745 and 7322-7402, respectively, after which the 3'-UTR is followed by a poly A tail of undetermined length. The encoded polyprotein is 2191 amino acid residues long, of which the amino acid sequence and position of the CV-A5 structural protein are as follows:

[0074] The VP4 protein is the amino acid sequence from position 1 to 69;

[0075] The VP2 protein is the amino acid sequence from position 70 to 324;

[0076] The VP3 protein is the amino acid sequence from position 325 to 564;

[0077] The VP1 protein is the amino acid sequence from position 565 to 860;

[0078] The 2A protein is the amino acid sequence from position 861 to 1010;

[0079] The 2B protein is the amino acid sequence from positions 1011 to 1109;

[0080] The 2C protein is the amino acid sequence from position 1110 to 1438;

[0081] The 3A protein is the amino acid sequence from position 1439 to 1524;

[0082] The 3B protein is the amino acid sequence from position 1525 to 1546;

[0083] The 3C protein is the amino acid sequence from position 1547 to 1729;

[0084] The 3D protein is the amino acid sequence from position 1730 to 2191.

[0085] Example 5

[0086] The whole genome sequences of CV-A5-3487R1 / XY / CHN / 2017 and CV-A5-3487V1 / XY / CHN / 2017 were aligned using SnapGene software.

[0087] The nucleotide at position 102 of the CV-A5-3487R1 / XY / CHN / 2017 virus strain is guanine (G); the amino acid at position 30 of the VP2 protein is asparagine (N); the amino acid at position 144 of the VP1 protein is asparagine (N), and the amino acid at position 288 is serine (Ser); the nucleotide at position 3418 of the 2A protein region is guanine (G); the nucleotide at position 4873 of the 2C protein region is adenine (A), and the amino acid at position 268 is alanine (A); the nucleotide at position 5202 of the 3A protein region is cytosine (C); the nucleotide at position 6772 of the 3D protein region is cytosine (C), and the amino acid at position 308 is alanine (A).

[0088] The nucleotide at position 102 of the CV-A5-3487V1 / XY / CHN / 2017 virus strain is cytosine (C); the amino acid at position 30 of the VP2 protein is tyrosine (Y); the amino acid at position 144 of the VP1 protein is lysine (K), and the amino acid at position 288 is phenylalanine (F); the nucleotide at position 3418 of the 2A protein region is adenine (A); the nucleotide at position 4873 of the 2C protein region is adenine (A), and the amino acid at position 268 is lysine (K); the nucleotide at position 5202 of the 3A protein region is thymine (T); the nucleotide at position 6772 of the 3D protein region is thymine (T), and the amino acid at position 308 is threonine (T).

[0089] The key sites that affect the proliferation of the strain in Vero cells are shown in the following table:

[0090] Table 4 Key sites affecting the proliferation of strains in Vero cells

[0091]

[0092] The acclimated strain, adapted to Vero cells by RD and subsequently passaged and plaque-purified, underwent multiple mutations. The following nucleotide and amino acid mutations occurred in the 5'-UTR and coding regions: 5'-UTR-T102C, 2A-A3418G, 2C-C4873A, 3A-T5203C, and 3D-T6772C. The following nucleotide and amino acid mutations occurred in the coding region: VP2-T1040A / Y30N, VP1-A2869T / L144N, VP1-T3300C / F288S, 2C-A4875G / K268R, and 3D-A6854G / T308A.

[0093] Example 6

[0094] Based on the titration results of the two viruses on RD cells, CV-A5-3487R1 / XY / CHN / 2017 and CV-A5-3487V1 / XY / CHN / 2017 were inoculated into Vero cells in 16-well plates at an MOI of 0.01. The virus adsorption volume was 1 mL. The virus solution was washed away after 2 hours, and 2 mL of fresh virus maintenance medium was added. Subsequently, samples were collected and frozen three times at 4, 8, 12, 20, 24, 36, 72, and 96 hours of infection to complete virus titration.

[0095] Calculate the viral titer using the Reed-Muench formula:

[0096] 1 gCCID50 / 1.0 mL=d(lesion rate higher than 50%-50%) / (lesion rate higher than 50%-lesion rate less than 50%)+XN.

[0097] Where: d = logarithm of dilution coefficient (10-fold dilution is 1),

[0098] Distance ratio = (lesion rate above 50% - 50%) (lesion rate above / 50% - less than 50%), XN = negative logarithm of the virus dilution that gave a lesion rate above 50%.

[0099] Virus titer results statistics:

[0100] Virus titer results after infection (unit: CCID 50 / ml) were 1.5 (2h), 2.25 (4h), 2.75 (8h), 3 (12h), 4.5 (20h), 4.83 (24h), 5.5 (36h), 4.75 (72h) and 4 (96h), respectively.

[0101] The present invention obtains a CV-A5 domesticated strain (cell growth phenotype is RD+ / Vero+) by using RD and Vero cells as hosts for isolation, passage and adaptation culture, and analyzes the key nucleotide and amino acid site variations, which can be applied to the genetic modification of multiple gene sites directed by vaccine strains to construct a CV-A5 (Vero+) library.

[0102] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A coxsackievirus group A type 5 domesticated strain, characterized in that The nucleotide sequence is shown in SEQ ID NO.17, the amino acid sequence is shown in SEQ ID NO.18, the cell growth phenotype is RD+ / Vero+, and some of the variant sites are as follows: Nucleotide 102 is cytosine; The 30th amino acid of the VP2 protein is tyrosine; The 144th amino acid of the VP1 protein is lysine, and the 288th amino acid is phenylalanine; Nucleotide 3418 in the 2A protein region is adenine; In the 2C protein region, nucleotide position 4873 is adenine, and amino acid position 268 is lysine; Nucleotide 5202 in the 3A protein region is thymine; The 6772nd nucleotide of the 3D protein region is thymine, and the 308th amino acid is threonine.

2. The domesticated strain of Coxsackievirus group A type 5 according to claim 1, characterized in that The deposit number is CCTCCNO: V202234.

3. The domesticated strain of Coxsackievirus group A type 5 according to any one of claims 1 or 2, characterized in that The virus titer was 1×10 6.5 CCID 50 / mL.

4. The domesticated strain of Coxsackievirus group A type 5 according to claim 1 or 2, characterized in that The cell growth phenotype of the mother strain of the Coxsackievirus group A type 5 domesticated strain is RD+ / Vero-, the 102nd nucleotide is guanine, the 30th amino acid of the VP2 protein is asparagine; the 144th amino acid of the VP1 protein is asparagine, and the 288th amino acid is serine; the 3418th nucleotide of the 2A protein region is guanine; the 4873rd nucleotide of the 2C protein region is adenine, and the 268th amino acid is alanine; the 5202nd nucleotide of the 3A protein region is cytosine; the 6772nd nucleotide of the 3D protein region is cytosine, and the 308th amino acid is alanine.

5. Use of the domesticated strain of Coxsackievirus group A type 5 according to any one of claims 1 to 4 in constructing a Coxsackievirus group A type 5 gene library.

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