Chimeric virus-like particle vaccine based on echovirus 30 type key epitope as well as preparation method and application of chimeric virus-like particle vaccine

Abstract

The invention relates to a vaccine, in particular to a chimeric virus-like particle vaccine based on an echovirus 30 type key epitope as well as a preparation method and application of the chimeric virus-like particle vaccine. The invention provides a method for screening antigen epitopes, which comprises the following steps: firstly, predicting the antigen epitope of capsid protein VP1 through bioinformatics analysis, and then screening epitopes which can be effectively combined with anti-VP1 complete sequence serum through enzyme-linked immunosorbent assay. Results of an enzyme-linked immunosorbent assay show that the expressed antigen epitope peptide E30-6 (SEQ ID NO: 2) can be strongly combined with E30 VP1 antiserum, so that one high-immunogenicity antigen epitope peptide is screened based on the complete sequence of the echovirus 30 VP1, which has important guiding significance for research and development of echovirus 30 vaccines. Besides, the HBc-E30-6 vaccine is designed and prepared based on the epitope, the HBc-E30-6 vaccine is high in purity and can be self-assembled into VLP nanoparticles, and more importantly, serum immunized by the vaccine can effectively neutralize the echovirus 30.

Description

Technical Field

[0001] This invention relates to vaccines, specifically to chimeric virus-like particle vaccines based on key epitopes of echovirus type 30, their preparation methods, and applications. Background Technology

[0002] Human enteroviruses are widespread globally, comprising four groups: A, B, C, and D. The largest group is enterovirus B (EV-B), which includes over 60 serotypes. Echoviruses are a major group of EV-B, belonging to the genus Enterovirus in the family Picornaviridae. First discovered in 1951, they were isolated from fecal samples of asymptomatic individuals. Echovirus infection is associated with a variety of diseases; mild infections can cause febrile rashes and hand-foot-and-mouth disease, while severe infections can lead to fatal illnesses such as aseptic meningitis, myocarditis, and acute flaccid paralysis. Echoviruses can cause illness in all age groups, with significantly higher incidence in infants and young children, and relatively higher incidence in summer and autumn. Currently, there are no approved vaccines or effective antiviral drugs; clinical treatment primarily focuses on symptom relief. Twenty-eight different types of echoviruses have been reported, with echovirus 30 (E30) being a common subtype and one of the most common pathogens causing outbreaks of aseptic meningitis.

[0003] VP1 is one of the four structural proteins (VP1-VP4) of the enterovirus capsid, located on the outermost layer of the capsid and in direct contact with the host environment. Together with other capsid proteins (VP2, VP3, and VP4), it forms the icosahedral symmetry structure of the viral particle. In recent years, numerous studies have demonstrated that the VP1 protein of enteroviruses plays a crucial role in viral typing and mediating host target cell infection. VP1 contains a receptor-binding domain and a neutralizing antigen site, making it the main neutralizing antigen of the virus. Currently, antibodies targeting the VP1 protein of different echovirus serotypes are under development, indicating that the VP1 protein is the most promising target for echovirus vaccine development.

[0004] While predicting and screening highly immunogenic epitopes from vaccine target proteins is a crucial research direction in modern vaccinology, the obtained epitopes are often insufficient to elicit a strong specific immune response in vivo due to their small molecular weight and weak immunogenicity. Virus-like particles (VLPs) are nanoscale structures formed by the self-assembly of viral structural proteins. Lacking viral genetic material, they are non-infectious. VLP-based vaccines can display exogenous antigenic epitopes with high abundance on their surface, stimulating the human immune system through a virus-like infection pathway and efficiently inducing a protective immune response. They have become a preferred platform for vaccine design and are widely used in various vaccine fields.

[0005] Currently, clinical treatment for echovirus infection mainly focuses on symptomatic relief, and no effective therapeutic drugs or preventative vaccines have been developed. No studies have reported highly immunogenic epitope sequences of echovirus 30, and no vaccines based on these sequences have been developed. Summary of the Invention

[0006] The first objective of this invention is to provide the amino acid sequence of an antigenic epitope peptide from the echovirus 30 VP1 protein. Another objective of this invention is to provide a virus-like particle vaccine based on this epitope.

[0007] A chimeric virus-like particle vaccine HBc-E30-6 based on a key epitope of echovirus type 30, wherein the vaccine expresses the antigenic epitope peptide E30-6, the amino acid sequence of which is shown in SEQ ID NO: 2, and the amino acid sequence of HBc-E30-6 is shown in SEQ ID NO: 3.

[0008] The method for constructing the chimeric virus-like particle vaccine HBc-E30-6 based on the key epitope of echovirus type 30 is implemented according to the following steps: Echovirus E30-VP1 was obtained through genetic engineering or peptide synthesis, and its amino acid sequence is shown in SEQ ID NO: 1. Serum antibodies were obtained by using it as an immunogen, and the antigenic epitope peptide E30-6 was screened out using the serum antibodies, and its amino acid sequence is shown in SEQ ID NO: 2. Chimeric protein HBc-E30-6 was designed by using a truncated HBc VLP-presenting antigenic epitope, and its amino acid sequence is shown in SEQ ID NO: 3. HBc-E30-6 protein was obtained through genetic engineering and self-assembled into VLP nanoparticles to obtain the chimeric virus-like particle vaccine HBc-E30-6.

[0009] The method for constructing the chimeric virus-like particle vaccine HBc-E30-6 based on the key epitope of echovirus type 30 is implemented according to the following steps: Echovirus E30-VP1 was obtained through genetic engineering or peptide synthesis, and its amino acid sequence is shown in SEQ ID NO: 1. Mice were immunized with this virus to obtain serum antibodies. The serum antibodies were then used to further screen for the antigenic epitope peptide E30-6, whose amino acid sequence is shown in SEQ ID NO: 2. A chimeric protein HBc-E30-6 was designed using a truncated HBc VLP-presenting epitope, and its amino acid sequence is shown in SEQ ID NO: 3. A recombinant plasmid containing the HBc-E30-6 gene sequence was constructed, and the HBc-E30-6 protein was expressed and self-assembled to form VLP nanoparticles, thus obtaining the chimeric virus-like particle vaccine HBc-E30-6.

[0010] The application of the chimeric virus-like particle vaccine HBc-E30-6 based on the key epitope of echovirus type 30 in the preparation of drugs for treating diseases caused by echovirus or its infection.

[0011] The application described herein refers to echovirus type 30.

[0012] The aforementioned applications, and the diseases caused by echovirus infection, include febrile rashes, hand-foot-mouth disease, aseptic meningitis, myocarditis, or acute flaccid paralysis.

[0013] Specifically: A chimeric virus-like particle vaccine based on a key epitope of echovirus type 30, wherein the vaccine expresses the antigenic epitope peptide E30-6, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0014] The chimeric virus-like particle vaccine is obtained through the following steps: This invention first obtains VP1 of echovirus 30 (its amino acid sequence is shown in SEQ ID NO: 1, GenBank accession number: AFH20827.1) through genetic engineering or polypeptide synthesis. It is then used as an immunogen to immunize mice to obtain serum antibodies. The serum antibodies are then used to further screen for the antigenic epitope peptide E30-6, whose amino acid sequence is shown in SEQ ID NO: 2.

[0015] The chimeric protein HBc-E30-6 was designed by using a truncated HBc VLP-presenting epitope, and the HBc-E30-6 protein (its amino acid sequence is shown in SEQ ID NO.3) was synthesized. It was then self-assembled in vitro into uniform VLP nanoparticles of 30-40 nm and characterized. Finally, the virus neutralizing activity of the serum after vaccine immunization was verified.

[0016] Specifically, the technical solution of this invention is to isolate a live E30 strain from clinical samples infected with enteroviruses, perform whole-genome sequencing analysis to obtain the VP1 nucleotide sequence, and construct a recombinant plasmid pET-28a(+)-E30-VP1 containing the E30-VP1 gene sequence using pET-28a(+) as a prokaryotic expression vector (see [link to relevant documentation]). Figure 1 ), E. coliBL21(DE3) was used as the expression strain to express the E30-VP1 protein. After purification, the target protein with a purity greater than 90% was obtained. C57BL / 6 mice were immunized with the E30-VP1 protein as an immunogen, and serum was collected. Bioinformatics analysis was used to predict the antigenic epitopes in the full-length E30-VP1 sequence. Epitopes exposed near FcRn were selected for synthesis, and enzyme-linked immunosorbent assay (ELISA) was used to screen for epitopes that could bind to E30-VP1 antiserum. Finally, the antigenic epitope sequences from the E30-VP1 protein were obtained (amino acid sequences are shown in SEQ ID NO: 2 in the sequence listing). Next, pET-28a(+) was used as the prokaryotic expression vector to construct the recombinant plasmid pET-28a(+)-HBc-E30-6 containing the HBc-E30-6 gene sequence (see [link to pET-28a(+)-HBc-E30-6]). Figure 2 ), E. coli BL21(DE3) was used as the expression strain to express HBc-E30-6 protein. After purification, the target protein with a purity greater than 90% was obtained and characterized using nanoparticle characterization. Finally, C57BL / 6 mice were immunized with HBc-E30-6 protein as an immunogen, and serum was collected. The E30 virus neutralization capacity of the serum was evaluated using an in vitro virus neutralization assay.

[0017] Research process of this invention: 1. E30-VP1 protein was obtained through prokaryotic expression; 2. E30-VP1 antiserum was obtained by immunizing C57BL / 6 mice; 3. Prediction of E30-VP1 protein antigenic epitopes; 4. Screening of E30-VP1 protein antigenic epitopes; 5. Preparation and characterization of HBc-E30-6 VLP; 6. Obtain HBc-E30-6 antiserum by immunizing C57BL / 6 mice; 7. Evaluation of the virus neutralizing activity of HBc-E30-6 antiserum.

[0018] principle: HBc-E30-6 utilizes the self-assembly properties of HBc VLPs to form VLP nanoparticles with high surface abundance displaying E30-6.

[0019] Beneficial Effects: This invention provides a method for screening antigenic epitopes. First, bioinformatics analysis is used to predict the VP1 antigenic epitope of the capsid protein. Then, an enzyme-linked immunosorbent assay (ELISA) is used to screen for epitopes that can effectively bind to the full-sequence anti-VP1 serum. The ELISA results show that the expressed antigenic epitope peptide E30-6 (SEQ ID NO:2) can strongly bind to E30 VP1 antiserum. Therefore, this invention screens a highly immunogenic antigenic epitope peptide based on the full-sequence of echovirus 30 VP1, which has important guiding significance for the development of echovirus 30 vaccines.

[0020] Furthermore, this invention also designed and prepared an HBc-E30-6 vaccine based on epitope, which has high purity and can self-assemble into VLP nanoparticles. More importantly, the serum after immunization with this vaccine can effectively neutralize echovirus 30.

[0021] Attached image description: Figure 1 Image of E30-VP1_pET-28a(+) plasmid; Figure 2 Purified E30-VP1 protein; lanes: M: Protein Marker 15-250 kD, 1: Unpurified sample, 2: Protein flow-through buffer, 3: 120 mM imidazole (30-fold concentrated) elution, 4: 200 mM imidazole elution. Figure 3 Trends in serum antibody changes after E30-VP1 immunization; Figure 4 Screening for antigenic epitope peptides, Epitope 6: SEQ ID NO: 2; Figure 5 HBc-E30-6_pET-28a(+) plasmid map; Figure 6 Purified HBc-E30-6 protein; lanes: M: Protein Marker 15-250 kD, 1: Unpurified sample, 2: Protein flow-through, 3: 100 mM imidazole elution, 4: 200 mM imidazole elution. Figure 7 Electron microscopy analysis of HBc-E30-6; Figure 8 Trends in serum antibody changes after HBc-E30-6 immunization; Figure 9 The number of E30 virus RNA copies in RD cells of HBc-E30-6 mouse antiserum at different dilutions. Detailed Implementation

[0022] Table 1 shows the antigenic epitope sequences involved in this invention. .

[0023] Example 1, Construction and transformation of pET-28a(+)-E30-VP1: Using pET-28a(+) as a prokaryotic expression vector, a restriction endonuclease NcoI sequence was added to the 5' end of the E30-VP1 gene (GenBank accession number: AFH20827.1, its amino acid sequence is as shown in SEQ ID NO: 1), and a restriction endonuclease XhoI sequence was added to the 3' end. Codons preferred by *E. coli* were selected, and the recombinant plasmid pET-28a(+)-E30-VP1 (see Example 1) was chemically synthesized and designed by GenScript. Figure 1 The recombinant plasmid was dissolved to 100 ng / L, and 2 μL of plasmid was used to transform 50 μL of the solution. E. coli Positive monoclonal cells were selected from DH5α competent cells using LB solid medium containing kanamycin; the monoclonal cells were then inoculated into LB liquid medium and cultured for 12 h. After collecting the cells, plasmids were extracted using a plasmid extraction kit. XhoI After single enzyme digestion, the band size was confirmed to be correct by 1% agarose gel electrophoresis.

[0024] Example 2, Induction of pET-28a(+)-E30-VP1 expression: 2 μL of plasmid was converted to 50 μL of... E. coli In BL21 competent cells, positive monoclonal antibodies were screened using LB solid medium containing kanamycin. These monoclonal antibodies were then inoculated into LB liquid medium and cultured for 6 h. PCR identification using 1 μL of the bacterial culture confirmed all were positive monoclonal antibodies. The monoclonal antibodies were then inoculated into LB liquid medium and cultured at 37°C for 4 h on a shaker at 200 rpm until OD (dose retardation). 600 When the concentration reaches 0.8-1.0, lactose is added as an inducer to a final concentration of 5 mmol / L, and the mixture is cultured at 18℃ and 200 rpm for 12 h in a shaker to induce recombinant protein expression.

[0025] Example 3, Purification of E30-VP1: After centrifugation, inducible bacterial cells were collected. A certain weight of wet bacterial cells was weighed, and lysis buffer (0.5 g / L lysozyme, PBS buffer, 1% Triton-100) was added, followed by resuspending the cells. The cells were then sonicated in an ice-water bath for 30 min, and the supernatant was discarded after centrifugation. The precipitate was resuspended in washing buffer (PBS buffer, 1% Triton-100), stirred thoroughly, centrifuged, and the supernatant was discarded. This step was repeated 3 times. The precipitate was resuspended in low-urea washing buffer (1.5 M urea, PBS buffer, 1% Triton-100), stirred thoroughly, centrifuged, and the supernatant was discarded. The precipitate was resuspended in denaturing buffer (8 M urea, PBS buffer), sonicated in an ice-water bath for 30 min, centrifuged, and the supernatant was collected. The supernatant was filtered through a 0.45 μm organic filter membrane to obtain a sample solution containing recombinant protein. After thoroughly mixing the sample solution with 4 volumes of equilibration buffer (8 M urea, PBS buffer, 10 mM imidazole), add the solution to NiNTA Beads. After loading, rinse with equilibration buffer until the baseline stabilizes. Rinse the column with 120 mM imidazole elution buffer (8 M urea, PBS buffer, 120 mM imidazole) to remove most of the contaminating proteins. After the baseline stabilizes, rinse with equilibration buffer until the baseline stabilizes again. Rinse the column with 200 mM imidazole elution buffer (8 M urea, PBS buffer, 200 mM imidazole) to collect the elution buffer containing the target protein until the baseline stabilizes. Rinse the column with 500 mM imidazole elution buffer (8 M urea, PBS buffer, 500 mM imidazole) to elute all proteins. The sample solution was dialyzed using a gradient dialysis method (50 mM NaH2PO4, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 6 / 4 / 2 / 1 / 0 M urea) to remove urea and imidazole. After lyophilization, the target protein powder was obtained and stored at -20℃ for later use. The purified E30-VP1 protein was verified by SDS-PAGE electrophoresis. See results Figure 2 As shown, the final E30-VP1 band is single, which meets the requirements of subsequent experiments.

[0026] Example 4, Preparation of E30-VP1 mouse antiserum: E30-VP1 protein was dissolved in injectable physiological saline to a concentration of 2 mg / mL, and emulsified with an equal volume of complete / incomplete Freund's adjuvant. Five-week-old male C57BL / 6 mice were immunized subcutaneously at a dose of 0.1 mg / mouse, with three immunizations at weeks 0, 2, and 4. The initial immunization used complete Freund's adjuvant, and booster immunizations used incomplete Freund's adjuvant. One week after each immunization, blood was collected from the orbital cavity, centrifuged at 4000 r / min for 15 min at 4°C, and the serum was collected and stored at -20°C. One week after the final immunization (day 35), whole blood was collected from the mice by enucleation, centrifugation, and serum collection.

[0027] Example 5, Detection of E30-VP1 mouse serum antibody levels: E30-VP1 protein was dissolved in coating diluent to prepare a 0.1 mg / mL coating buffer. 100 μL of coating buffer was added to each well of a 96-well plate, and the plate was coated at 4°C for 12 h. After discarding the coating buffer, the plate was washed, and each well was filled with PBST washing buffer. After standing for 2 min, the washing buffer was discarded. This process was repeated three times. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 h. After discarding the blocking buffer, the plate was washed three times. Serum was diluted with sample diluent at ratios of 1:5000, 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:64000, and 1:128000. 100 μL of serum diluent was added to each well, and the plate was incubated at 37°C for 2 h. h; After discarding the serum diluent, wash the plate three times, dilute the HRP-conjugated goat anti-mouse IgG antibody with sample diluent, add 100 μL of antibody diluent to each well, and incubate at 37°C for 1 h; after discarding the antibody diluent, wash the plate three times, add 100 μL of TMB chromogenic solution to each well, and incubate at 37°C for 0.5 h; add 50 μL of reaction stop solution to each well, mix well, and read the OD. 450 .

[0028] Results: The trend of changes in mouse antiserum is shown in the figure. Figure 3 After the first booster immunization, the level of E30-VP1 specific antibodies in mice was significantly increased, and the level of specific antibodies continued to increase with the number of booster immunizations, indicating that after two booster immunizations, E30-VP1 induced the production of high levels of specific antibodies in mice.

[0029] Example 6, Prediction and synthesis of E30-VP1 antigenic epitopes: E30-VP1 antigenic epitopes were predicted using ABCPreds, BCPreds and IEDB websites respectively. Sequences that could be predicted by all three methods were summarized. Epitopes exposed near the FcRn binding site were selected, and GenScript was commissioned to synthesize 6 antigenic epitope peptides.

[0030] Example 7, Screening of E30-VP1 antigenic epitope peptides: The antigenic epitope peptides were dissolved in coating diluent to prepare a 0.1 mg / mL coating buffer. 100 μL of coating buffer was added to each well of a 96-well plate, and the plate was coated at 4°C for 12 h. After discarding the coating buffer, the plate was washed, and each well was filled with PBST washing buffer. After standing for 2 min, the washing buffer was discarded. This process was repeated three times. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 h. After discarding the blocking buffer, the plate was washed three times. The serum was diluted with sample diluent at a ratio of 1:40000, and 100 μL of serum diluent was added to each well, and the plate was incubated at 37°C for 2 h. After discarding the serum diluent, the plate was washed three times. The HRP-conjugated goat anti-mouse IgG antibody was diluted with sample diluent, and 100 μL of antibody diluent was added to each well, and the plate was incubated at 37°C for 1 h. After discarding the antibody diluent, the plate was washed three times. 100 μL of TMB chromogenic solution was added to each well, and the plate was incubated at 37°C for 0.5 h. 50 μL of TMB chromogenic solution was added to each well. The reaction termination solution was mixed thoroughly and the OD was read. 450 .

[0031] Results: The screening results for antigenic epitope peptides are shown in the table below. Figure 4 Among them, epitope peptides 1-5 were inferior to the full E30 sequence, while epitope peptide 6 (named E30-6) was superior to the full E30 sequence. Therefore, E30-6 will be further studied.

[0032] Example 8: Construction, transformation, protein induction, expression, and purification of pET-28a(+)-HBc-E30-6: The specific methods and steps are the same as in Examples 1-3. The recombinant plasmid pET-28a(+)-HBc-E30-6 (constructed by GenScript) is shown in the image. Figure 5 As shown, the purified HBc-E30-6 protein was also verified by SDS-PAGE electrophoresis. Result: See Figure 6 The obtained HBc-E30-6 protein met the requirements for subsequent experiments.

[0033] Example 9, Characterization of HBc-E30-6 vaccine: HBc-E30-6 was analyzed by transmission electron microscopy. First, 10 μL of sample solution was evenly dropped onto the center of a copper grid. After standing for 8 min, the liquid on the copper grid was absorbed with filter paper. Then, 10 μL of 1% phosphotungstic acid staining solution was evenly dropped onto the center of the copper grid. After standing for 5 min, the liquid on the copper grid was absorbed with filter paper. The copper grid was then placed under an infrared lamp for 1 min to bake, and then the image was taken by adjusting the magnification.

[0034] Result: See Figure 7 The HBc-E30-6 protein can self-assemble into 30-40 nm uniform VLP nanoparticles in vitro.

[0035] Example 10, Preparation of HBc-E30-6 mouse antiserum and detection of serum antibody levels: The specific methods and steps are the same as in Examples 4-5.

[0036] Result: See Figure 8 After immunization with HBc-E30-6, specific antibodies were produced in mice, and the antibody level continued to rise with the number of booster shots.

[0037] Example 11, Detection of virus neutralizing activity of HBc-E30-6 mouse antiserum: 1×10 6 RD cells were seeded into 6-well plates and cultured to a cell density of 80%. Diluted E30 virus solution and vaccine antiserum at different dilutions were mixed in equal proportions and incubated at 37°C for 2 hours. The supernatant culture medium of the RD cells was discarded, and the cells were washed twice with PBS. 1 mL of incubation medium was added to each well. A blank control (NC) group without virus and a positive control group (E30) with only diluted virus solution were also set up. The cells were incubated at 37°C for 30 hours.

[0038] The upper viral suspension was discarded, and the cells were washed three times with PBS solution. RNA from each group was collected, and qPCR was used to analyze the viral replication in RD cells of different treatment groups.

[0039] Result: See Figure 9 As serum dilution decreased, the number of viruses entering the cells continuously decreased. Notably, the viral RNA copy number in the 1:100 dilution group was similar to that in uninfected RD cells, suggesting good virus neutralization capacity. These results indicate that HBc-E30-6 VLP vaccine antiserum can effectively neutralize E30, providing crucial experimental evidence for subsequent vaccine efficacy evaluation.

Claims

1. A chimeric virus-like particle vaccine HBc-E30-6 based on a key epitope of echovirus type 30, characterized in that, The vaccine expresses the antigenic epitope peptide E30-6, the amino acid sequence of which is shown in SEQ ID NO: 2, and the amino acid sequence of HBc-E30-6 is shown in SEQ ID NO:

3.

2. The method for constructing the chimeric virus-like particle vaccine HBc-E30-6 based on the key epitope of echovirus type 30 according to claim , characterized in that, To achieve this, follow these steps: The echovirus protein E30-VP1 was obtained through genetic engineering or peptide synthesis, and its amino acid sequence is shown in SEQ ID NO:

1. Serum antibodies were obtained by using it as an immunogen, and the antigenic epitope peptide E30-6 was screened out using the serum antibodies, and its amino acid sequence is shown in SEQ ID NO:

2. The chimeric protein HBc-E30-6 was designed by using a truncated HBc VLP-presenting antigenic epitope, and its amino acid sequence is shown in SEQ ID NO:

3. The HBc-E30-6 protein was obtained by genetic engineering and self-assembled into VLP nanoparticles to obtain the chimeric virus-like particle vaccine HBc-E30-6.

3. The method according to claim 2, characterized in that, Echovirus E30-VP1 was expressed and purified in E. coli prokaryotes, and its amino acid sequence is shown in SEQ ID NO:

1. Mice were immunized with this virus to obtain serum antibodies. The serum antibodies were then used to further screen for the antigenic epitope peptide E30-6, whose amino acid sequence is shown in SEQ ID NO:

2. A chimeric protein HBc-E30-6 was designed using a truncated HBc VLP-presenting epitope, and its amino acid sequence is shown in SEQ ID NO:

3. A recombinant plasmid containing the HBc-E30-6 gene sequence was constructed, and the HBc-E30-6 protein was expressed and self-assembled to form VLP nanoparticles, thus obtaining the chimeric virus-like particle vaccine HBc-E30-6.

4. The use of the chimeric virus-like particle vaccine HBc-E30-6 based on the key epitope of echovirus type 30 according to claim 1 in the preparation of drugs for treating diseases caused by echovirus infection.

5. The application according to claim 4, characterized in that, The echovirus in question is echovirus type 30.

6. The application according to claim 4, characterized in that, The diseases caused by echovirus infection include febrile rash, hand-foot-mouth disease, aseptic meningitis, myocarditis, or acute flaccid paralysis.

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