Coxsackie virus group A type 4 virus strain, and preparation method, biological product and application thereof

Abstract

The invention provides a coxsackie virus group A type 4 virus strain as well as a preparation method, a biological product and application thereof, and belongs to the technical field of biological medicines. The new coxsackie virus group A type 4 strain with excellent immunogenicity and hereditary stability is successfully separated and obtained, can be efficiently proliferated in human diploid cells, and is suitable for being used as a substrate for vaccine production; after being prepared into an experimental inactivated vaccine, the vaccine can induce an organism to generate high-level neutralizing antibody and cellular immune response, can induce good immunogenicity in an adult animal body, provides effective immune protection for a newborn animal through a mother-borne antibody, and remarkably reduces morbidity and death caused by virus infection; the invention provides a key core material and technical basis for developing safe and effective CVA4 monovalent or multivalent hand-foot-and-mouth disease vaccines, and also has important value for developing diagnostic preparations for related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to Coxsackievirus A4 strain, its preparation method, biological products, and applications. Background Technology

[0002] Enteroviruses are a large group of common viruses belonging to the Picornaviridae family. Members include polioviruses, Coxsackieviruses, echoviruses, and novel enteroviruses. These viruses are primarily transmitted via the fecal-oral route, have a wide range of infective ranges, and can cause a variety of human diseases, from the common cold and hand-foot-and-mouth disease to severe aseptic meningitis, encephalitis, acute flaccid paralysis, and myocarditis. Coxsackieviruses, based on their pathogenicity in suckling mice and their histopathological differences, are divided into group A and group B. Coxsackievirus A (CVA) is one of the main pathogens causing hand-foot-and-mouth disease and herpetic pharyngitis in children.

[0003] Hand, foot, and mouth disease (HFMD) is a common infectious disease in children, characterized by rashes and blisters on the hands, feet, and mouth. In recent years, in addition to the common enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), other serotypes of Coxsackievirus, such as CVA4, CVA6, and CVA10, have shown an increasing detection rate in HFMD cases, attracting widespread public health attention. CVA4 virus infection not only causes typical HFMD symptoms but is also closely related to outbreaks of herpetic pharyngitis, and in some cases may even lead to serious neurological complications such as encephalitis, posing a threat to children's health.

[0004] Currently, inactivated vaccines against hand-foot-and-mouth disease caused by EV71 are available and used in the population, playing a crucial role in controlling EV71 outbreaks. However, existing EV71 vaccines do not provide effective cross-protection against infections caused by Coxsackieviruses (such as CVA16, CVA4, and CVA6). Furthermore, no specific vaccine against CVA4 has been approved on the market. Therefore, given the increasingly active prevalence of CVA4, developing a vaccine that can effectively prevent CVA4 infection is particularly urgent. The primary prerequisite for vaccine development is obtaining a virus strain with good immunogenicity, stable replication, and suitability for vaccine production; this is the crucial foundation for all subsequent vaccine development work. Summary of the Invention

[0005] The purpose of this invention is to provide a Coxsackievirus A4 strain, its preparation method, biological products, and applications, aiming to solve the technical problem of the lack of effective vaccine candidate strains against Coxsackievirus A4 (CVA4) in the prior art, which leads to the lack of effective immunization against diseases caused by this virus (such as hand-foot-mouth disease, herpetic pharyngitis, and neurological complications).

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Coxsackievirus A4 strain, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: V202525.

[0007] The present invention also provides a Coxsackievirus A4 strain, the strain comprising structural proteins as shown in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.

[0008] The present invention also provides an immune composition comprising the above-described Coxsackievirus A4 strain and a pharmaceutically acceptable vector.

[0009] Preferably, the immune composition is an inactivated vaccine containing an adjuvant.

[0010] The present invention also provides an antibody against Coxsackievirus A4, which is prepared by immunization with the Coxsackievirus A4 strain as described in claim 1 or 2.

[0011] The present invention also provides a polynucleotide that encodes the VP1, VP2 or VP3 structural proteins of the above-mentioned Coxsackievirus Group A4 strain.

[0012] The present invention also provides that the DNA sequence of the polynucleotide is any one or more of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.

[0013] The present invention also provides a detection kit for Coxsackievirus A4, comprising a detection unit selected from one of the following: a) The viral antigens of the above-mentioned Coxsackievirus A4 strain; b) The above-mentioned antibodies; c) The above-mentioned polynucleotides.

[0014] The present invention also provides the application of the above-mentioned Coxsackievirus A4 strain in the preparation of a vaccine for the prevention of Coxsackievirus A4 infection.

[0015] The present invention also provides the application of the above-mentioned Coxsackievirus A4 strain in the preparation of diagnostic reagents for detecting Coxsackievirus A4 infection.

[0016] The beneficial effects of this invention are: This invention successfully isolated and obtained a novel Coxsackievirus A4 strain with excellent immunogenicity and genetic stability. This strain can proliferate efficiently in human diploid cells, making it suitable as a substrate for vaccine production. When prepared into an experimental inactivated vaccine, it can induce high levels of neutralizing antibodies and cellular immune responses in the body. It can induce good immunogenicity in adult animals and provide effective immune protection to newborn animals through maternal antibodies, significantly reducing morbidity and mortality caused by viral infection. This invention provides key core materials and technical foundations for the development of safe and effective monovalent or multivalent CVA4 hand-foot-mouth disease vaccines, and also has important value for the development of diagnostic agents for related diseases. Attached Figure Description

[0017] Figure 1 The images show the preliminary PCR identification results of the viral gene sequence and viral plaque cloning, including: A) PCR amplification of the viral gene sequence using 5'UTR universal primers; B) amplification of the viral gene sequence using CV-A4 VP1 specific primers; and C) analysis of the evolutionary lineage characteristics of the viral VP1 gene.

[0018] Figure 2 Figures show the results of morphological observation and identification of major proteins of CV-A4 virus particles, including: A) CV-A4 virus particle morphology observed under an electron microscope after density gradient ultracentrifugation; B) Major viral proteins of CVA4 virus solution purified by silver staining after SDS-PAGE gel electrophoresis; C) Western blotting identification of major viral proteins of CVA4 virus solution purified by Western blotting.

[0019] Figure 3 The graph shows the viral titer detection results at different time points, including: A) CVA4 E-2, E-4, and B-2 viral plaque clones; B) Viral titers of different CV-A4 strains infecting K17 cells under different MOI conditions, in units of CCID50 / mL; C) Clinical symptoms of CVA4 strains infecting suckling mice, with representative symptoms shown in the graph; D) Survival analysis and survival curves of different CVA4 virus strains infecting suckling mice. Different colored curves represent the infection of suckling mice by different virus strains.

[0020] Figure 4Figure 1 shows the results of neutralizing antibody level detection and cellular immune response analysis after immunizing mice with different virus strains: A) Neutralizing antibody levels, expressed as GMT geometric mean titer; B) ELISApot detection of the secretion capacity of mouse spleen cytokines IL4 and IFN-γ after CVA4 virus antigen stimulation, with the ordinate arranged according to 10... 6 The number of SFCs (SpotForming Cells) is calculated from each spleen cell.

[0021] Figure 5 Figure 1: VP1 amino acid sequence analysis results of CVA4 from different generations. The red boxes in the image indicate the mutation sites of the amino acids.

[0022] Figure 6 Images show the results of electron microscopy observation and major structural protein analysis of virus particles after purification of CVA4 cell factory harvest fluid. A) Electron microscopy observation of virus particle morphology; B) Images of Western blotting identification of major structural proteins VP1, VP2, and VP3.

[0023] Figure 7 Figure 1 shows the results of specific immune response induced by the CVA4 experimental vaccine. A) Neutralizing antibody level detection; B) Protective effect of immunized suckling mice against viral challenge; C) Detection of IFN-γ and IL4 cytokine secretion levels induced by specific cellular immune response. Biological Preservation

[0024] The Coxsackievirus A4 strain involved in this invention, named Coxsackievirus A4 CVA4 HW67 / 08 / YN / 2022, was deposited on April 10, 2025, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: V202525. The address of the collection center is: Wuhan University, Wuhan, China, postcode 430072. Detailed Implementation

[0025] The core of this invention lies in providing a novel Coxsackievirus A4 (CVA4) strain. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: V202525. According to the International Committee on Taxonomy of Viruses (ICTV), Coxsackievirus A4 belongs to the genus Enterovirus in the family Picornaviridae. Viruses in this genus are non-enveloped, positive-sense, single-stranded RNA viruses with icosahedral symmetry and a diameter of approximately 20-30 nanometers. The virus strain of this invention was isolated from clinical samples and purified and amplified using human diploid K17 cells through plaque phage purification, ensuring the purity and genetic consistency of the strain. This deposit ensures the reproducibility of the biological material, providing material support for subsequent scientific research and industrial development.

[0026] In one specific embodiment of the present invention, the provided Coxsackievirus A4 strain contains structural proteins composed of specific amino acid sequences. Specifically, the amino acid sequences of its capsid proteins VP1, VP2, and VP3 are shown in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, respectively. Viral capsid proteins are key components determining viral antigenicity and host immune responses. VP1 protein is the most abundant antigenic protein on the viral surface, often containing neutralizing epitopes, and is the main target for stimulating the body to produce protective neutralizing antibodies. VP2 and VP3 proteins are also exposed on the viral surface, and together with VP1, constitute the spatial conformation of the virus, playing an important role in viral cell tropism, receptor binding, and immune recognition. The specific structural protein sequences possessed by the viral strain of the present invention constitute its unique antigenic basis and are the core of its ability to induce highly efficient and specific immune protection.

[0027] Based on the aforementioned viral strain, this invention further provides an immune composition. This immune composition comprises the Coxsackievirus A4 strain described in this invention and a pharmaceutically acceptable vector. The term "pharmaceutically acceptable vector" as used herein is well-known in the art and refers to various excipients that are pharmacologically and physiologically compatible with the active ingredient (i.e., viral antigen) and do not cause excessive adverse reactions in the host. These excipients are diverse and can be selected according to dosage form requirements, including but not limited to: diluents (such as phosphate-buffered saline PBS, physiological saline), stabilizers (such as sugars, amino acids), preservatives (such as thimerosal, 2-phenoxyethanol), and salts for adjusting osmotic pressure. This immune composition can be in the form of a liquid formulation, a lyophilized formulation, etc., designed to safely and effectively deliver the viral antigen into the host.

[0028] In a preferred embodiment, the immune composition of the present invention is prepared as an inactivated vaccine. An inactivated vaccine is a type of vaccine in which a virus loses its infectivity and replication ability through physical or chemical methods, but retains its immunogenicity. Commonly used virus inactivating agents include formaldehyde, β-propiolactone (BPL), and diethyleneimine (BEI). The virus strains described in this invention, after inactivation treatment, can be mixed with adjuvants to further enhance their immunogenicity. An adjuvant is a substance that can non-specifically enhance the body's immune response to an antigen or alter the type of immune response. Adjuvants that can be used in this invention include, but are not limited to: aluminum adjuvants (such as aluminum hydroxide and aluminum phosphate), oil emulsion adjuvants (such as MF59 and ASO3), and other novel adjuvant systems. Adding adjuvants can generally reduce the amount of antigen used, increase antibody titers, and may prolong the duration of immune protection.

[0029] This invention also provides a specific antibody against Coxsackievirus A4. This antibody is prepared by immunizing an appropriate host animal (such as a mouse, rat, rabbit, goat, etc.) with the viral strain of this invention (whether intact viral particles, lysis products, or their structural proteins) as an immunogen. Depending on the preparation method, the antibody can be a polyclonal antibody or a monoclonal antibody. Polyclonal antibodies are mixtures of antibodies produced by multiple B cell clones in the host body, targeting different antigenic epitopes of the virus, and can be obtained by collecting and purifying the serum of immunized animals. Monoclonal antibodies are homogeneous antibodies produced by a single B cell clone, targeting only a specific antigenic epitope, and are usually prepared using hybridoma technology. These antibodies can be used in scientific research, disease diagnosis, and even passive immunotherapy.

[0030] This invention further provides a polynucleotide molecule. This polynucleotide encodes the VP1, VP2, or VP3 structural proteins of the Coxsackievirus A4 strain described in this invention. The polynucleotide can be in DNA or RNA form, including but not limited to genomic DNA, cDNA, mRNA, and chemically synthesized oligonucleotides. These polynucleotide sequences contain genetic information encoding proteins with specific immunogenicity, and their DNA sequences may be, for example, as shown in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. Such polynucleotides have wide applications in the field of biotechnology, for example, in constructing expression vectors to recombinantly express viral proteins in prokaryotic or eukaryotic cells for the development of subunit vaccines, the preparation of diagnostic antigens, or structural and functional studies.

[0031] Based on the above-described invention, this invention also provides a detection kit for detecting Coxsackievirus A4. The core of this kit is a detection unit, which can be selected from one of the following three forms or a combination thereof: a) Viral antigens: namely, the viral strains described in this invention or their lysed and purified antigenic components. These antigens can be used to coat enzyme-linked immunosorbent assay (ELISA) plates or as the detection lines of lateral chromatography test strips to capture specific antibodies that may be present in the sample.

[0032] b) Specific antibodies: namely, the antibodies provided in this invention. These antibodies can be used to establish antigen capture detection methods, for example as capture antibodies or detection antibodies in ELISA, or for detection techniques such as immunofluorescence and immunohistochemistry, to directly identify viral antigens in samples.

[0033] c) Specific polynucleotides: namely, the polynucleotide sequences or their complementary sequences provided in this invention. These sequences can be used as primers for polymerase chain reaction (PCR) or real-time quantitative PCR (qPCR), or as probes for nucleic acid hybridization (such as gene chips) to detect the presence of viral nucleic acids in samples with high sensitivity.

[0034] The kit may also contain other standard components required to complete the test, such as reaction buffer, substrate chromogenic solution, control, and instructions.

[0035] This invention also provides the application of the Coxsackievirus A4 (CVA4) strain in the preparation of a vaccine for the prevention of Coxsackievirus A4 (CVA4) infection. Hand-foot-and-mouth disease, herpetic pharyngitis, and other diseases can be caused by CVA4, and currently there are no specific preventative measures. Vaccines prepared using this virus strain, especially inactivated vaccines, can stimulate the body to produce specific active immunity against CVA4, thereby effectively preventing the occurrence and spread of related diseases and possessing significant public health value.

[0036] This invention provides the application of the Coxsackievirus A4 strain in the preparation of diagnostic reagents for detecting Coxsackievirus A4 infection. This strain can be used as a positive control, calibrator, or directly as a coating antigen to develop various immunological or molecular biological diagnostic reagents mentioned above, providing clinicians with rapid and accurate diagnostic tools for CVA4 infection, thus facilitating timely monitoring and treatment of the disease.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] SEQ ID NO: 1 corresponds to the DNA sequence encoding the VP1 structural protein: GGTGATGCAATAGCTGATGCTATACAAAACACAGTCACATCCACCATACAGAGGGTCACCACTAATGTTGTCGGGCAAGACGCAACAGCCGCTAATACCACACCTAGCTCCCATAGTCTGAACACTGGCCTAGTTCCTGCCCTCCAGGCTGCGGAGACAGGAGCTTCATCTACAGCTACAGATGGTAATTTGATAGAGACGAGATGTGTTGTGAACTCCAATGGCACTCGTGAAACACACATTGAACATTTCTTCTCTAGGTCAGGACTAGTGGGTGTTATGGAGGTGGATGATACGGGCACTGCTGGAAAGGGGTTCTCAAACTGGGATATTGACATTATGGCGTTTGTGCAACTACGCCGCAAGCTTGAGGCATTCACATATATGCGGTTTGATGCAGAGTTCACCTTTGTTACCAATCTAGAAAATGGGCTCACAAACAACAGTGTGATCCAATACATGTATGTGCCACCAGGGGCCCCTAAACCCGATGCTCGGGAGTCATTCCAGTGGCAAACCGCGACCAATCCGTCAGTTTTCCAGAAAATGGATAGCCCACCACCGCAAGTGTCGGTGCCCTTTATGTCACCAGCTAGCACCTACCAATGGTTCTATGATGGTTATCCCACTTTTGGACCTCACTCAGAGACATCTAACCTATCTTATGGGCAGTGTCCTAACAACATGTTGGGGACGTTTTCAGCTAGAGTTGTGAGTAAGCAAATCACCAATCAGAAGTTTCAGATTCGCATTTATTTGCGGCTTAAGAGAGTAAGGGCATGGGTCCCTAGACCTTTGAGATCACAACCGTACATTTACAAAAACTACCCTACTTATGGTACTACTATTCAACACCTGGCCAAGGATAGGCGCAAGATCACTGAAACTGACTATAACGCTGAACAACGCACGCAC。

[0039] Amino acid sequence corresponding to the VP1 structural protein of SEQ ID NO: 2: GDAIADAIQNTVTSTIQRVTTNVVGQDATAANTTPSSHSLNTGLVPALQAAETGASSTATDGNLIETRCVVNSNGTRETHIEHFFSRSGLVGVMEVDDTGTAGKGFSNWDIDIMAFVQLRRKLEAFTYMRFDAEFTFVTNLENGLTNNSVIQYMYVPPGAPKPDARESFQWQTATNPSVFQKMDSPPPQVSVPFMSPASTYQWFYDGYPTFGPHSETSNLSYGQCPNNMLGTFSARVVSKQITNQKFQIRIYLRLKRVRAWVPRPLRSQPYIYKNYPTYGTTIQHLAKDRRKITETDYNAEQRTH。

[0040] SEQ ID NO: 3 corresponds to the DNA sequence encoding the VP2 structural protein: TCTCCTTCAGCTGAAGCATGTGGGTATAGTGATAGGGTTGCCCAATTAACTGTGGGAAACTCTACTATAACAACACAAGAAGCTGCAAACATAGTAGTGGGCTATGGCGAGTGGCCAAATTACTGTCCCGACACAGATGCCACTGCAGTGGATAAACCTACCAGACCTGATGTTTCCGTGAATAGGTTTTATACATTGTCTACTAAGTCTTGGACCCAAAACTCGAAAGGCTGGTATTGGAAATTCCCTGATGTCCTTAACGACGTTGGAGTTTTTGGCCAGAATGCGCAATACCACTATCTGTACAGATCAGGATTTTGCGTTCACGTGCAATGTAATGCCAGCAAGTTTCATCAGGGAGCACTCTTAGTTGCCATGGTCCCCGAATTTGTCATCGCTTCTAAGAGTAAACGTTCAAAACCCAATGACCCCTCACAATATCCGGCTTTTTCAGTGACTAATCCAGGAAAGAATGGGAGGGAATTTACAGATCCATATGTTTTGGATGCAGGGATACCGTTAAGTCAAGCATTGATCTTCCCACACCAGTGGATCAACCTTAGGACCAACAATTGTGCCACTATTATTATGCCTTACATTAACGCGGTCCCTTTTGATTCAGCCATAAACCACAACAATTGTGGGTTGGTAGTGGTGCCTGTGGCACCCCTCAAGTACAGTGCCGGTGCCACCACTGAGATCCCAATCACTATTACAATAGCTCCTTTAAATTCAGAGTTCAGCGGTCTCAGGCAAGCCGTGAAACAA。

[0041] SEQ ID NO: 4 corresponds to the amino acid sequence of the VP2 structural protein: SPSAEACGYSDRVAQLTVGNSTITTQEAANIVVGYGEWPNYCPDTDATAVDKPTRPDVSVNRFYTLSTKSWTQNSKGWYWKFPDVLNDVGVFGQNAQYHYLYRSGFCVHVQCNASKFHQGALLVAMVPEFVIASKSKRSKPNDPSQYPAFSVTNPGKNGREFTDPYVLDAGIPLSQALIFPHQWINLRTNNCATIIMPYINAVPFDSAINHNNCGLVVVPVAPLKYSAGATTEIPITITIAPLNSEFSGLRQAVKQ.

[0042] SEQ ID NO: 5 corresponds to the DNA sequence encoding the VP3 structural protein: GGTATCCCTGTTGAGATGAAACCCGGAACCAACCAGTTTCTGACCACAGATGATGGAGTGTCTGCGCCCATACTACCTGGATTTGACCCTACCCCAGTAATACACATCCCTGGGGAAGTTAGTAGTATACTTGAATTATGTCAAGTGGAAACTATACTTGAAGTGAACAATGTGACTGATGTGCAGGGCGTAAGTCGGTTGCTCATACCAGTAAAAGCTCAGGAGACAGTGGATACACTTTGTGCATCATTCCAGGTCGACCCTGGACGCGACGGACCCTGGCAGTCAACCTTGATTGGGCAGTTATGTAGATACTACACTCAGTGGTCAGGTTCTTTAGAGATTACTTTTATGTTTACGGGCTCATTCATGGCAACAGGCAAGATGCTTATAGCATACACCCCTCCAGGCAGCGCACAACCCACAACACGTGAGTTGGCAATGCTCGGCACACACATAATCTGGGATTTTGGGTTGCAGTCATCAGTCACCCTTGTGGTACCCTGGATAAGTAACACACACTTCAGAACTGTGAAAACAGGGGGTTACTTAGATTATTATGCCACAGGTGTGGTTACTATATGGTACCAGACAAACTTCGTAGTGCCACCAGACACGCCAAACGAAGCCAACATTATAGCAATGGGAGCAGGGCAAAAGAATTTTACGTTAAAATTGTGCAAAGATACACCTGAGATAACCCAGCAGGCTGTGCTGCAG。

[0043] SEQ ID NO: 6 Amino acid sequence corresponding to VP3 structural protein: GIPVEMKPGTNQFLTTDDGVSAPILPGFDPTPVIHIPGEVSSILELCQVETILEVNNVTDVQGVSRLLIPVKAQETVDTLCASFQVDPGRDGPWQSTLIGQLCRYYTQWSGS LEITFMFTGSFMATGKMLIAYTPPGSAQPTTRELAMLGTHIIWDFGLQSSVTLVVPWISNTHFRTVKTGGYLDYYATGVVTIWYQTNFVVPPDTPNEANIIAMGAGQKNFTLKLCKDTPEITQQAVLQ. Example

[0044] Experimental materials and methods: 1. Viral genotyping: Total RNA was extracted from human fecal samples according to the manufacturer's instructions (Tiangen Biotech Co., Ltd., DP424). The main steps include: 1) Mix the sample solution and RNAiso Plus at a ratio of 1:4 and shake vigorously using a vortex mixer; 2) Add 200 μl of chloroform, vortex to mix, extract RNA, place at room temperature for 3 minutes, centrifuge at 12,000 rpm for 15 minutes (keeping at 4°C throughout). 3) Take the supernatant and mix it with an equal volume of isopropanol. Invert the mixture to mix thoroughly and let it stand for 10 minutes at room temperature.

[0045] 4) Centrifuge at 12000 rpm for 10 minutes and discard the supernatant; then add 1 ml of 75% ethanol, centrifuge at 12500 rpm for 5 minutes and discard the supernatant; all the above steps are performed at 4℃.

[0046] 5) After air drying, the RNA was dissolved in enzyme-free water, and the nucleic acid concentration and quality were detected using nanodrop.

[0047] 2. Viral plaque purification: K17 cell suspension was seeded at an appropriate density into 6-well culture plates to form a uniform monolayer. Viral samples were purified by 10... -1 Up to 10 -7After serial dilution, the cells were inoculated into a cell monolayer and incubated at 37°C and 5% CO2 for 1 hour. Subsequently, an agar mixture containing nutrients was heated and cooled to 45–50°C, covering the cell surface to form an agar layer. The culture plate remained in the 37°C, 5% CO2 environment until plaques appeared. After plaque formation, they were stained with neutral red and observed under a microscope; clear and regularly shaped plaques were selected. These plaques were removed using a micropipette, eluted, freeze-thawed, and centrifuged. The supernatant was collected as purified virus.

[0048] 3. Construction of the Viral Gene Sequence Phylogenetic Tree: First, existing literature on CV-A4 genotyping was consulted to obtain the NCBI accession numbers for different types, and the corresponding VP1 gene sequences were downloaded from the public database (NCBI GenBank). Simultaneously, based on the VP1 gene sequences of the three CV-A4 strains obtained in this experiment, all sequences were uniformly converted to FASTA format and quality-controlled to remove low-quality or incomplete sequences. Subsequently, the ClustalW algorithm in MEGA12 software was used to align the sequences, removing redundant and missing parts, and the results were saved in MEGA format. Finally, maximum likelihood (ML) was used for phylogenetic analysis to construct and visualize the phylogenetic tree.

[0049] 4. Comparison of Viral Replication Kinetics: The replication kinetics of three CVA4 viruses were studied in K17 cells, with MOIs set at 0.01 and 0.05. Samples were collected at different infection time points (3h, 6h, 9h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 60h, 72h, 84h, 96h, 120h, 144h, and 168h), and the number of infectious particles was determined by viral titration. Viral titers (LogCCID) were calculated using the Reed-Muench method. 50 And the proliferation curves under different MOI conditions were plotted using GraphPad software.

[0050] 5. CVA4 Suckling Mouse Survival Analysis: Two-day-old BALB / c suckling mice were infected intracranially with a viral load of 2000 CCID50. Three virus strains were used in the infection group, along with a PBS control group. Each group contained at least 10 suckling mice to ensure data reliability. From the start of infection, the survival status of the suckling mice was observed daily, and the time of death was recorded. The number of surviving mice in each group was recorded at different time points, and the survival rate at each time point was calculated (surviving number / initial number × 100%). In GraphPad Prism, the "Survival Analysis" function was selected, and the survival rate data recorded after infecting the suckling mice with the three virus strains were input to generate survival curves for the infected suckling mice. Statistical analysis of the survival curves, such as the Log-rank test, can also be performed to compare survival differences between different groups.

[0051] 6. Neutralization assay to analyze antibody levels: Serum was separated from mice after intravenous blood collection, inactivated by water bath at 56℃ for 30 minutes, and stored at -80℃. The serum was serially diluted 2-fold (50 μL / well) in 96-well plates and mixed with an equal volume containing 100 CCID. 50 The virus mixture was incubated with control wells (serum-free and virus-free) and virus re-spill wells, and neutralized at 37°C for 2 hours. Subsequently, healthy RD cells were digested and their concentration adjusted, and 100 μL was added to each well of the mixture. The wells were then incubated at 37°C with 5% CO2 for 7 days, during which the cytopathic effect in both control and experimental wells was observed daily. After incubation, assuming both control and virus re-spill were effective, the neutralizing antibody titer was calculated using the Reed-Muench method.

[0052] 7. ELISpot assay for cytokine secretion levels: PVDF plates were pre-coated with IFN-γ or IL-4 and washed three times with sterile PBS. 200 μL of DMEM blocking buffer containing 10% FCS was added to each well, and the plates were incubated at room temperature for 1 hour. Subsequently, mouse spleen single-cell suspensions (2 × 10⁻⁶ cells) were... 5 Add cells (number of cells / well) to each well, along with inactivated whole virus or viral protein as a stimulus. Incubate the ELISpot plate at 37°C with 5% CO2 for 36 hours. After incubation, discard the culture. Add 200 μL of ice-cold deionized water to each well, then wash five times with PBS containing 0.05% Tween (PBST), blotting dry with absorbent paper after each wash to remove cells and unbound factors. Add the detection antibody, incubate at room temperature for 1 hour, and repeat the PBST wash. Add 100 μL of enzyme-coupled streptavidin, incubate at room temperature for 1 hour, and wash again. Finally, add the substrate solution, develop at room temperature for 10–20 minutes, and air dry until the spots are clear. Analyze using an ELISpot plate reader and record the number of spots in each well.

[0053] 8. Analysis of the protective effect of maternal antibodies on suckling mice: Immunized mother mice (immunized twice with CVA4 vaccine according to a 0, 21 immunization schedule) and unimmunized mother mice (at least 3 mother mice in each group) were selected. On the second day after the mother mice gave birth to suckling mice (at least 10 suckling mice in each group), 2000 CCIDs were administered. 50 The virus was used to challenge suckling mice intracranially, and the survival of suckling mice in the vaccine group and the non-immunized group was recorded for 14 consecutive days. The survival rate of suckling mice in the experimental group and the control group was calculated, and the survival curve was plotted using the Kaplan-Meier survival analysis method. At the same time, the survival rate differences between different vaccine dose groups and the control group were compared.

[0054] Virus isolation and preliminary identification PCR identification of total RNA 5UTR in fecal samples from individuals with hand-foot-mouth disease revealed that three samples showed the expected target gene band of approximately 440 bp after gel electrophoresis. Figure 1 A), further sequencing analysis and comparison with existing gene sequences in Genebank, preliminarily identified as the CV-A4 virus strain. Subsequently, PCR identification of cDNA from the three samples was performed using the CV-A4-specific primer VP1, revealing distinct gene bands of approximately 900-1000 bp in size. Figure 1 B), sequence alignment analysis after VP1 sequencing revealed that all three clones belonged to the D2 genotype (B). Figure 1 C), after whole-genome sequencing of the three viruses, they were submitted to GeneBank with sequence numbers PQ304880.1, PQ304881.1, and PQ304882.1, respectively.

[0055] CVA4 virus particle morphology observation and major structural protein analysis The above-mentioned virus samples were amplified on K17 cells. The virus harvested solution was concentrated and purified by density gradient ultracentrifugation. The morphology of the purified virus particles was observed under an electron microscope, and complete solid CV-A4 virus particles with a size of about 20-30 nm were found. Figure 2 A). Silver staining SDS-PAGE showed that the purified CV-A4 antigen exhibited distinct major structural protein bands of VP1 and VP2 at the expected molecular weight. Figure 2 B). Western blotting analysis revealed that the purified CV-A4 antigen could be recognized by specific polyclonal antiserum, showing major structural protein bands of VP1 and VP2, with molecular weights of approximately 35 kDa and 26 kDa, respectively. Figure 2 C).

[0056] Analysis of CVA4 virus proliferation kinetics in K17 cells and infection characteristics in suckling mice The three CV-A4 virus strains were then purified by plaque phagocytosis in K17 cells to obtain E-4, E-2, and B-2 virus strains. Figure 3 A) Viral replication kinetics were analyzed in K17 cells. The results showed that, under MOI=0.01 infection conditions, the three viral strains E-4, E-2, and B-2 reached peak proliferation at 72, 48, and 60 hours after infection with K17 cells, respectively, with infectivity titers of 5.875 CCIDs. 50 / mL, 6.125 CCID 50 / mL, 8.563 CCID 50 / mL; Under MOI=0.05 infection conditions, the three viral strains E-4, E-2 and B-2 reached peak proliferation at 96 hours, 48 ​​hours and 36 hours after infection of K17 cells, respectively, with infectivity titers of 7.500 CCIDs. 50 / mL, 5.750 CCID 50 / mL, 7.750 CCID 50 / mL ( Figure 3 B). Intracranial challenge infection of suckling mice was performed using the three CV-A4 virus strains isolated above (2000 CCID). 50 / each), observed for 14 consecutive days and plotted survival curves for suckling mice. The results showed that all three groups of suckling mice developed symptoms such as arched back and limb paralysis 3 days after infection, and all died within 3-5 days after infection. Figure 3 C-3D) CV-A4 Virus Immunogenicity Analysis After amplifying the above three CV-A4 virus strains, mice were directly immunized intraperitoneally with the non-inactivated virus, with each mouse injected with 10 cisplatin. 6.0 CCID 50 The viral fluid was used to immunize mice twice according to the 0, 21 immunization schedule, and the serum neutralizing antibody levels were then measured. The results showed that the neutralizing antibody levels of the three viral strains, E-4, E-2, and B-2, reached 2622.2, 1456.1, and 5045.1, respectively, after two immunizations. Figure 4 A). Cellular immunoassay revealed that all three viral antigens induced the secretion of IL-4 and IFN-gamma cytokines in the mouse spleen after two immunizations. The average IL-4 cytokine secretion levels induced by E-4, E-2, and B-2 reached 93, 171, and 66 SFCs / 10, respectively. 6 The average secretion levels of IFN-gamma cytokines reached 311, 439, and 578 SFCs / 10 (spot-forming units per million cells). 6 cells ( Figure 4 B).

[0057] Based on the above analysis of the viral replication kinetics, infection characteristics in suckling mice, and immunogenicity results, we selected the E-4 virus strain as the candidate strain for the CVA4 vaccine and named it Coxsackievirus A4 HW67 / 08 / YN / 2022. We then evaluated the genetic stability, passage stability, immunogenicity in animals, and protective effect of this vaccine candidate strain.

[0058] Genetic stability analysis of CV-A4 virus strains The genetic stability of the HW67 / 08 / YN / 2022 CVA4 virus strain was then analyzed. Under conditions of 37℃ and MOI=0.05, the CVA4 virus was passaged continuously (P1-P20). Viruses from generations P1, P5, P10, P15, and P20 were then selected for sequencing analysis of the VP1 capsid protein coding region. The analysis showed that amino acid changes began to occur in the VP1 gene sequence from generation 15. Within the full-length 305 amino acid sequence, changes were observed at positions 102 and 305, with A changing to D (position 102) and H changing to R (position 305), respectively. The mutation rate at these amino acid positions was 0.007%, which is extremely low. Figure 5 Infectivity titer analysis of these generations of the virus revealed titers of 6.75 CCID. 50 / mL, 7.625 CCID 50 / mL, 6.875 CCID 50 / mL, 7.375 CCID 50 / mL, 7,000 CCID 50 / mL. In summary, this demonstrates that the Coxsackievirus A4 strain HW67 / 08 / YN / 2022 exhibits only an extremely low mutation frequency in its major structure VP1 after continuous passage, and this mutation has virtually no significant impact on its infectivity titer. Even after continuous passage to P20, it still achieves 7,000 CCID. 50 Virus titer of approximately / mL.

[0059] CV-A4 virus particle morphology observation and antigenicity identification Based on the preliminary identification of the main genes, viral replication kinetics, infection characteristics in suckling mice, and immunogenicity analysis in mice, the Coxsackievirus A4 strain HW67 / 08 / YN / 2022 (Coxsackievirus A4) selected in this invention was chosen as the subsequent vaccine strain. The CV-A4 virus obtained by amplifying this vaccine strain in K17 cells was ultrafiltered and concentrated, then purified using affinity chromatography. After purification, the shape and size of the virus particles were observed under an electron microscope. The results showed that the CV-A4 virus was approximately 20-30 nm in size, and most of the particles in the field of view were solid. Figure 6 A). The purified CV-A4 antigen was then identified, and Western blotting analysis revealed that the purified CV-A4 antigen could be recognized by specific antibodies, showing a band of the major structural protein VP1 (V1). Figure 6 B (left). SDS-page gel electrophoresis revealed distinct major structural protein bands of VP1, VP2, and VP3 at corresponding protein molecular weights in the CV-A4 purified antigen. Figure 6 B (right).

[0060] Immunogenicity and protective efficacy evaluation of the CV-A4 experimental vaccine The CV-A4 stock solution was prepared into an experimental vaccine containing aluminum adjuvant. After passing the tests, mice were immunized, and the immunogenicity and protective efficacy of the CV-A4 experimental vaccine were evaluated. Mice were divided into three doses: high (200U), medium (100U), and low (50U). After two immunizations, the geometric mean of neutralizing antibodies reached 61.1, 64, and 32, respectively. Figure 7 A). Subsequently, the protective effect of maternal antibodies in suckling mice was tested. The protective effect against intracranial challenge with CV-A4 virus was analyzed in suckling mice born to immunized mothers. The results showed that the 200U and 100U doses completely protected the suckling mice from viral attack, and the 50U dose also protected 80% of the suckling mice from CV-A4 virus attack. Figure 7 C). Following two immunizations, different doses of the CVA4 experimental vaccine induced the secretion of IL-4 and IFN-gamma cytokines in the mouse spleen. The average IL-4 cytokine secretion levels induced by the 200U, 100U, and 50U doses reached 1063, 950, and 743 SFCs / 10, respectively. 6 The average secretion levels of IFN-gamma cytokines reached 292, 418, and 206 SFCs / 10 (spot-forming units per million cells). 6 cells ( Figure 7 B).

[0061] As demonstrated by the above embodiments, this invention provides a novel Coxsackievirus A4 strain, CVA4 HW67 / 08 / YN / 2022, with promising application prospects. This strain can replicate stably and efficiently in human diploid K17 cells, exhibiting excellent proliferative capacity. Animal experiments show that this virus strain is clearly pathogenic to suckling mice, and immunization of adult mice induces high levels of neutralizing antibodies and specific cellular immune responses. More importantly, the experimental inactivated vaccine prepared using this strain not only elicits strong humoral immunity in immunized mice but also provides significant protective efficacy to its offspring suckling mice through maternal antibodies, effectively resisting viral attack. In summary, this virus strain demonstrates excellent overall performance and is an ideal candidate vaccine strain, laying a solid foundation for the subsequent development of related vaccine products for the prevention of CVA4 infection.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Coxsackievirus A4 strain, characterized in that, The virus strain is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: V202525.

2. A Coxsackievirus A4 strain, characterized in that, The viral strain includes structural proteins as shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO:

6.

3. An immune composition, characterized in that, It contains the Coxsackievirus A4 strain as described in claim 1 or 2, and a pharmaceutically acceptable vector.

4. The immune composition according to claim 3, characterized in that, The immune composition is an inactivated vaccine containing an adjuvant.

5. An antibody against Coxsackievirus A4, characterized in that, The antibody is prepared by immunization with the Coxsackievirus A4 strain as described in claim 1 or 2.

6. A polynucleotide, characterized in that, The polynucleotide encodes the VP1, VP2, or VP3 structural protein of the Coxsackievirus A4 strain as described in claim 2.

7. The polynucleotide of claim 6, characterized in that, The DNA sequence of the polynucleotide is any one or more of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:

5.

8. A detection kit for Coxsackievirus A4, characterized in that, Includes a detection unit, wherein the detection unit is selected from one of the following: a) The viral antigen of the Coxsackievirus A4 strain as described in claim 1 or 2; b) The antibody as described in claim 5; c) The polynucleotide as described in claim 6 or 7.

9. The use of the Coxsackievirus A4 strain as described in claim 1 or 2 in the preparation of a vaccine for the prevention of Coxsackievirus A4 infection.

10. The use of the Coxsackievirus A4 strain as described in claim 1 or 2 in the preparation of a diagnostic reagent for detecting Coxsackievirus A4 infection.

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