Coxsackie virus group B type 3 strain and application thereof

Abstract

The invention provides a coxsackie virus group B type 3 strain and application thereof, and relates to the technical field of biological medicine. The strain is named as KM50-W07, and is preserved in the China Center for Type Culture Collection on November 6, 2024, the preservation number is CCTCC NO: V202474, and the preservation address is Wuhan University, Wuhan, China. The virus strain is a human coxsackie virus group B type 3 wild type single purified virus strain, and the virus strain is high in virus replication capacity and good in hereditary stability and immunogenicity; the virus strain can be applied to development of a production virus seed of a human coxsackie virus group B type 3 vaccine, and is suitable for development of an attenuated coxsackie virus group B type 3 vaccine and an inactivated coxsackie virus group B type 3 vaccine; and an infection model can be established on Vero cells and suckling mice and is used for CVB3 infection and pathogenic mechanism research, CVB3 vaccine evaluation and antiviral drug screening.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to a Coxsackievirus B group 3 strain and an application thereof. Background Art

[0002] Coxsackievirus B3 (CVB3) belongs to the genus Enterovirus of the family Picornaviridae and is a major member of the Coxsackievirus B group (CVB). After infection with CVB3, patients mainly show flu-like symptoms or are asymptomatic. However, CVB3 can also cause serious diseases, including acute and chronic myocarditis, aseptic meningitis, pancreatitis, paralytic disease, rhabdomyolysis, pleurisy, hepatitis, insulin-dependent diabetes mellitus, and severe sepsis. It has also become an important pathogen of hand, foot and mouth disease (HFMD), thus bringing a huge burden to public health. Given that only a few HFMD pathogens are currently monitored in my country, such as enterovirus 71 (EV71), coxsackievirus A16 (CVA16), coxsackievirus A6 (CVA6), and coxsackievirus A10 (CVA10), the prevalence of CVB3 may be underestimated. Therefore, as one of the most economical and effective measures to prevent the disease, it is necessary to develop a monovalent CVB3-targeted or multivalent CVB preventive vaccine containing CVB3 antigen components as soon as possible.

[0003] With the development of molecular biology technology, a variety of new vaccine development technology routes have emerged, but there is no preventive vaccine or corresponding specific antiviral drug for CVB3 on the market. The current enterovirus vaccine development is still limited to whole virus particles, and the only enterovirus vaccines on the market are polio virus vaccine and EV71 vaccine. They are all developed and produced using traditional vaccine technology (attenuated or inactivated), and have good immune protection effects. There is no preventive vaccine or corresponding specific antiviral drug for CVB3 on the market. In addition, compared with live attenuated vaccines, inactivated vaccines do not have the disadvantages of virulence reversion and the threat of recombinant viruses derived from attenuated strains to the environment. Moreover, they have good immunogenicity and safety, and are currently the most feasible technical route.

[0004] Although there is only one serotype of CVB3 virus, it has genetic diversity and has evolved into five genotypes. According to existing literature reports, there are certain differences in the neutralization efficacy between different genotypes of the same enterovirus serotype. Therefore, it is imperative to develop an efficient CVB3 inactivated vaccine to prevent the epidemic strains (dominant genotypes) of CVB3 in China; it is crucial to screen CVB3 strains with strong proliferation ability in the cell matrix, good genetic stability, good immunogenicity and safety for the production of inactivated vaccines.

[0005] Therefore, screening a CVB3 strain that meets the conditions for vaccine development and applying it to the preparation of its vaccine is an urgent problem to be solved in this field. Summary of the invention

[0006] In view of this, the present invention provides a Coxsackievirus group B type 3 strain, which is used to prepare a Coxsackievirus group B type 3 vaccine and has good replication ability, genetic stability and immunogenicity.

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

[0008] A Coxsackievirus group B type 3 strain, the genome sequence of which is shown in SEQ ID NO: 1 or a completely complementary sequence of the sequence shown in SEQ ID NO: 1.

[0009] Preferably, the strain is named KM50-W07, and was deposited in the China Center for Type Culture Collection on November 6, 2024, with a preservation number of CCTCC NO: V202474, and a classification name: Coxsackievirus group B type 3 KM50-W07Coxsackievirus B3. The storage address is Wuhan University, Wuhan, China.

[0010] Another object of the present invention is to provide a biomaterial, which is any one of the following:

[0011] A. A nucleic acid molecule with a sequence as shown in SEQ ID NO: 1 or a completely complementary sequence to the sequence shown in SEQ ID NO: 1;

[0012] B. an expression cassette containing the nucleic acid molecule described in A;

[0013] C. a recombinant vector containing the nucleic acid molecule described in A;

[0014] D. a recombinant microorganism containing the nucleic acid molecule described in A, the expression cassette described in B, or the recombinant vector described in C;

[0015] E. A cell line containing the nucleic acid molecule described in A, the expression cassette described in B, or the recombinant vector described in C.

[0016] Another object of the present invention is to provide an immunogenic composition, which contains the above-mentioned Coxsackievirus group B type 3 strain or the above-mentioned biological material.

[0017] Another object of the present invention is to provide the use of the above-mentioned Coxsackievirus Group B Type 3 strain or the above-mentioned biological material or the above-mentioned immunogenic composition, which is any one of the following:

[0018] a. Use in the preparation of drugs for preventing and / or treating coxsackievirus infection or diseases caused by coxsackievirus infection;

[0019] b. Use in the preparation of vaccines for the prevention and / or treatment of coxsackievirus infection or diseases caused by coxsackievirus infection;

[0020] c. Use in the preparation of antibodies for preventing and / or treating coxsackievirus infection or diseases caused by coxsackievirus infection;

[0021] d. Use in the preparation of antiserum for preventing and / or treating coxsackievirus infection or diseases caused by coxsackievirus infection;

[0022] e. Application in the preparation of reagents or kits for detecting Coxsackie virus;

[0023] f. Application in the evaluation of immunogenicity of coxsackievirus vaccines for non-diagnostic and therapeutic purposes;

[0024] g. Application in the evaluation of the protective efficacy of coxsackievirus vaccines for non-diagnostic and therapeutic purposes;

[0025] h. Application in the preparation of Coxsackie virus infected cell models;

[0026] i. Application in the preparation of Coxsackie virus infection animal models;

[0027] j. Application in drug screening or efficacy evaluation for the prevention and / or treatment of coxsackievirus infection or diseases caused by coxsackievirus infection.

[0028] Another object of the present invention is to provide a vaccine, which contains the above-mentioned Coxsackievirus Group B Type 3 strain or an inactivated vaccine or attenuated vaccine prepared from the above-mentioned biological material.

[0029] Another object of the present invention is to provide a method for preparing the vaccine, wherein the above-mentioned Coxsackievirus B group 3 strain or the above-mentioned biological material is subjected to cell culture, the virus liquid is harvested, and after inactivation and purification, an adjuvant is added to prepare an inactivated vaccine;

[0030] Alternatively, an attenuated strain with weakened virulence is obtained through adaptive passage or reverse genetics methods, and the virus liquid is harvested through cell culture. After purification, stabilizers and adjuvants are added to prepare an attenuated vaccine.

[0031] Another object of the present invention is to provide the above-mentioned Coxsackievirus group B type 3 strain or the above-mentioned biological material, which is obtained by adaptive passage or transfection of cell strains or cell lines to obtain a virus-adapted strain or clone.

[0032] Preferably, the cell strain or cell line is a diploid MRC-5 cell, KMB-17 cell or 2BS cell.

[0033] Beneficial effects: The present invention screens a representative Coxsackievirus B group 3 strain KM50-W07, and the isolation and culture of the strain are both carried out using Vero cells. This cell matrix is ​​safe and reliable as a human vaccine cell matrix. The strain has strong proliferation ability, good genetic stability and good immunogenicity. The genotype E to which it belongs is the CVB3 genotype that is dominant in China. After immunizing animals with an inactivated vaccine prepared from the virus seed, antiserum with high neutralizing efficacy against CVB3 isolates from different sources in China can be obtained. In addition, the present invention provides the preparation and immunological method of an inactivated vaccine using KM50-W07 as the virus seed, which lays a solid foundation for the development of a safe, effective and economical CVB3 inactivated vaccine. In addition, the KM50-W07 strain of the present invention can also be used to construct a stable animal model of CVB3 intracellular proliferation and acute infection, which can provide a research tool for developing the pathogenic mechanism of CVB3 infection, screening antiviral drugs and vaccine evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0035] Figure 1 Schematic diagram of the adaptive proliferation of the CVB3 strain on Vero cells in the present invention, wherein Figure 1 The left is a normal Vero cell. Figure 1 The right side shows the cytopathic effect of the KM50-W07 strain;

[0036] Figure 2 It is the plaque morphology of the KM50-W07 strain in the present invention; wherein Figure 2 The left is a normal Vero cell. Figure 2 The right side shows the cytopathic effect of the KM50-W07 strain. The right side → indicates the plaques that appear on Vero cells caused by the KM50-W07 strain.

[0037] Figure 3 It is the phylogenetic tree of CVB3 based on the full-length VP1 sequence in the present invention; wherein ● represents the CVB3 isolates of the D and E genotypes involved in this study; ▲ represents the CVB3 prototype strain;

[0038] Figure 4 This is the peak diagram of the chromatography purification of the CVB3 virus concentrate in the present invention;

[0039] Figure 5The immunogenicity of the CVB3 inactivated vaccine of the present invention; Figure A shows the neutralization titer of each group of BALB / c female mice before the first immunization; Figure B shows the neutralization titer of each group of BALB / c female mice before the second immunization; Figure C shows the neutralization titer of each group of BALB / c female mice before the third immunization; Figure D shows the neutralization titer of each group of BALB / c female mice 21 days after the third immunization;

[0040] Figure 6 The clinical manifestations of neonatal mice infected with the CVB3 strain of the present invention; wherein Figure A is a weight curve of neonatal mice infected with the CVB3 strain; Figure B is a clinical score curve of neonatal mice infected with the CVB3 strain; Figure C is a survival curve of neonatal mice infected with the CVB3 strain;

[0041] Figure 7 This is a HE-stained section of a newborn suckling mouse infected with the CVB3 strain of the present invention;

[0042] Figure 8 It is the viral load of the tissue of newborn mice infected with the CVB3 strain of the present invention. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1: Isolation and identification of Coxsackievirus group B type 3 (CVB3) strain KM50-W07

[0045] (1) Sample processing

[0046] Feces from children with hand, foot and mouth disease in Yunnan Province were collected, 1 g was suspended in PBS to 5 ml, mixed, centrifuged at 1500 g for 20 min, and the supernatant was filtered and sterilized with a 0.22 μm filter membrane.

[0047] (2) Virus adaptation and isolation on Vero

[0048] Take 100 μL of the filtrate and inoculate it into Vero cells that have grown into a monolayer, add MEM medium without bovine serum, incubate at 35°C, 5% CO2 and observe the cell lesions. After the lesions appear, harvest the culture and continue to inoculate Vero cells twice in the same way. Harvest the culture and freeze it for later use. See the attached Figure 1 .

[0049] (3) Plaque purification

[0050] The virus harvested from the Vero cells that had been adapted to the third generation was serially diluted 10-fold, and 10 -2 , 10 -3 , 10 -4 , 10 -5、 10 -6 The five dilutions were inoculated into a monolayer of Vero cells respectively, and after adsorption at 37°C and 5% CO2 for 1 hour, 3% methylcellulose-MEM was added, and the cells were cultured at 35°C and 5% CO2, and the cytopathic effect was observed under a microscope. After plaque formation, the plaques were sucked out with a pipette tip and then inoculated into a monolayer of Vero cells, cultured at 37°C and 5% CO2, and the virus was harvested after the cytopathic effect reached more than 95%. The virus was then inoculated into Vero cells for plaque purification twice in the same way. After three rounds of plaque purification, a single plaque was picked out, which was the monoclonal purified strain, inoculated into Vero cells and the virus liquid was collected to make a single purified strain of Coxsackievirus group B type 3 wild type, see attached. Figure 2 .

[0051] (4) Virus identification

[0052] Take 200 μL of virus solution, extract viral RNA according to the instructions of Axygen Body Fluid Viral DNA / RNA Miniprep Kit (Axygen, USA), and store it in a -80°C refrigerator.

[0053] According to Primescript TMThe operating instructions of One Step RT PCR Kit Ver.2 (Takara, Dalian) were followed and the primers "BOS-BOAS" (see Table 1, where Y represents C or T, N represents A or C or G or T, and R represents A or G) were used to perform RT-PCR on the viral RNA extracted above. Reaction system: total volume 20 μL, 0.5 μL each of BOS and BOAS; 10 μL of 2× one-step buffer; 8.2 μL of viral RNA; 0.8 μL of PrimeScript 1 Step Enzyme Mix. RT-PCR reaction conditions: reverse transcription at 50°C for 30 min; pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min 10 s, for a total of 35 cycles; further extension at 72°C for 7 min. Take 5 μL of the PCR product, identify it by 1% agarose gel electrophoresis, and obtain the VP1 sequence by sequencing (Sanger sequencing method, ABI373XL sequencer) by Beijing Qingke Biotechnology Co., Ltd. (Kunming, China). The virus serotype is determined to be CVB3 by Blast alignment in NCBI. Afterwards, MEGA 7.0 software was used to align the CVB3 VP1 sequence obtained in the GenBank database, and the phylogenetic tree was constructed using the neighbor-joining method (NJ) of MEGA 6.0 and the Kimura 2-parameter model. The Bootstrap replication number was set to 1000 to evaluate the reliability and repeatability of the evolutionary tree, and a phylogenetic analysis was performed on the CVB3 strains isolated this time. Strain KM50-W07 belongs to the E genotype, which is one of the main CVB3 genotypes in China, see the attached. Figure 3 .

[0054] Table 1 Whole genome sequence amplification and sequencing primers

[0055]

[0056] The primers described in Table 1 were used to amplify the fragments of the virus genome in segments, and the full-length genome nucleotide sequence was obtained by splicing. The full-length genome nucleotide sequence of CVB3 isolate KM50-W07 obtained in this example is shown in SEQ ID NO:1.

[0057]

[0058] The CVB3 strain KM50-W07 was deposited in the China Center for Type Culture Collection on November 6, 2024, with the deposit number CCTCC NO: V202474, classification name: Coxsackievirus group B type 3 KM50-W07 Coxsackievirus B3, and the deposit address is Wuhan University, Wuhan, China.

[0059] (5) Virus infectivity titer detection

[0060] The titer of CVB3 strain KM50-W07 was determined using the microcytopathic assay. The CVB3 virus solution was serially diluted 10-fold using virus diluent (MEM medium without serum) to obtain 10 -3 ~10 -8 The virus solution was added to 8 wells of each dilution in a 96-well plate, with 100 μL of virus solution of different dilutions added to each well. At least 8 wells were reserved as controls, and 100 μL of virus dilution was added. The digested Vero cells were inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well. 4 cells. Cultured at 35°C, 5% CO2 for 7 days, observed the cell lesion under a microscope. Calculated the cell culture infectious dose (CCID50) by Karber method. The calculation formula is as follows: LgCCID50 = L + d (S-0.5), L is the logarithm of the lowest dilution of the virus, d is the dilution factor, and S is the total percentage of cell lesion wells.

[0061] Results: The virus titer of strain KM50-W07 was 7.25lgCCID 50 / ml.

[0062] (6) Genetic stability analysis

[0063] Virus harvest fluids from P1 to P20 were harvested by multiple subculturing on Vero cells. 200 μL of virus harvest fluids from P1, P5, P10, P15 and P20 were taken and viral RNA was extracted according to the instructions of Axygen Body Fluid Viral DNA / RNA Miniprep Kit (Axygen, USA). TMThe operation instructions of One Step RTPCR Kit Ver.2 (Takara, Dalian) were followed and the whole genome of the virus was amplified in segments using the primers described in Table 1. RT-PCR reaction conditions: reverse transcription at 50°C for 30min; pre-denaturation at 94°C for 2min; denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 50s, for a total of 35 cycles; further extension at 72°C for 7min. The PCR product was sent to Kunming Qingke Biotechnology Co., Ltd. for sequencing (Sanger sequencing, ABI373XL sequencer). The sequencing primers are shown in Table 1. SeqMan in DNAStar7.1 software was used to align and splice the whole genome sequence to obtain the whole genome sequence of each generation of the virus. MEGA 6.0 software was used to analyze the changes in nucleotides and amino acids in the whole gene sequences of different generations.

[0064] After KM50-W07 was adapted and passaged on Vero cells, the titer of P1, P5, P10, P15 and P20 viruses was measured and the whole genome was sequenced, and nucleotide and amino acid mutation analysis was performed (Table 2). There was no significant change in the titer of KM50-W07 P1, P5, P10, P15 and P20 viruses. The titer of the P1 virus of the KM50-W07 strain was 7.25LgCCID50 / mL, and the titer of the P5, P10, P15 and P20 viruses was 7.75LgCCID50 / mL; the KM50-W07 strain mutated at the 522 amino acid position in the VP2 region at the 20th generation, R→V. There were three mutations in the VP1 region: the 946 amino acid position mutated at the 15th generation, Q→K; the 1079 amino acid position mutated at the 5th generation, V→L. Two sites in the 3D region mutated: the 1982 amino acid site mutated in the 5th generation, K→E, but mutated again in the 10th generation, E→K; the 2177 amino acid site mutated in the 5th generation, R→Q. Therefore, the KM50-W07 strain has a higher titer and better genetic stability.

[0065] Table 2 Changes in nucleotide and amino acid sequences of the complete gene sequence of KM50-W07 strains at each generation

[0066]

[0067] Example 2: Preparation of CVB3 inactivated vaccine using KM50-W07 strain as virus seed

[0068] 1. Preparation of CVB3 inactivated vaccine

[0069] (1) Virus culture

[0070] Vero cells are cultured to the required number using a 10-layer cell factory or a T225 cell culture flask. The culture conditions are: MEM medium containing 10% newborn calf serum, 37°C, 5% CO2 static culture. Until the cells grow into a monolayer. Coxsackievirus group B type 3 strain KM50-W07 is inoculated onto a monolayer Vero at a multiplicity of infection of 0.1, and MEM medium without calf serum is used. The cells are cultured at 35°C, 5% CO2 for 2-4 days until the cell lesion rate reaches more than 90%, and the virus liquid is harvested, which is the virus harvest liquid.

[0071] (2) Virus inactivation

[0072] After the virus harvest liquid was repeatedly frozen and thawed three times at room temperature / -20℃, it was inactivated with formaldehyde at a final concentration of 100μg / ml at 37℃ to obtain the inactivated virus liquid. Take a sample of the virus liquid inactivated for 144h, neutralize it with sodium bisulfite, inoculate Vero cells and blindly pass it for 3 generations to verify the inactivation effect. No cytopathic effect occurred, indicating that the above concentration of formaldehyde can completely inactivate the virus within 144h (6 days). In order to better ensure the thoroughness of virus inactivation, the inactivation time is set to 7 days.

[0073] (3) Clarification, concentration and purification of inactivated virus solution

[0074] The inactivated virus solution was centrifuged at 8000rpm for 30min to remove cell debris; the clarified virus solution was tangentially filtered using an ultrafiltration membrane with a pore size of 100kD and concentrated 30 times to obtain a virus concentrate; the virus concentrate was purified by Capto Core 400 chromatography column and the flow-through was collected as the purified virus solution, see attached Figure 4 The purified virus solution is sterilized by filtration through a 0.22um filter to become the original virus solution.

[0075] (4) Preparation of inactivated vaccines

[0076] According to the designed volume, antigen dose and adjuvant concentration, add the corresponding volume of virus stock solution, aluminum adjuvant and diluent (PBS), and mix them to form CVB3 inactivated vaccine. In the experiment to evaluate the immunogenicity of the vaccine, the vaccine volume was 0.1ml / dose, the antigen content was 1-10ug / 0.1ml, and the aluminum content concentration was 0.35mg / ml.

[0077] 2. CVB3 virus neutralizing antibody detection

[0078] Neutralization experimental method: After the virus infects sensitive cells, it causes cell morphological changes and cytopathic effect (CPE). After the specific neutralizing antibodies bind to the virus, the virus particles lose their infectivity and inhibit the occurrence of CPE.

[0079] (1) Attack virus CCID 50 Titration and Titer Gradient Preparation

[0080] Titrate the virus 2-3 times according to the method in step 1 above, take the average value, and calculate the content of 100 CCID in each 0.05 ml. 50 The viral load was calculated by diluting the attack virus according to the calculated dilution ratio to obtain the total amount of virus required for the experiment (100 CCID 50 / 0.05ml). Take 3 small tubes, add 0.9ml of virus diluent to each tube, and pipette 0.1ml of the diluted attack virus solution into the first small tube (i.e. 10CCID 50 / 0.05ml), change the pipette tip and mix well, and dilute to 1CCID in this way 50 / 0.05ml and 0.1CCID 50 / 0.05ml, and conduct virus back titration test.

[0081] (2) Dilute the serum to be tested

[0082] The serum to be tested is frozen at -20℃ for testing. Take several sterile tubes and place them on a test tube rack. Add 0.3ml of diluent to each tube, add 0.1ml of serum to be tested, cover tightly, shake and mix, and the result is 1:4 diluted serum. Inactivate at 56℃ for 30min. Open the sterile packaged 96-well tissue culture plate, add 0.05ml of diluent to each well, perform a 2-fold serial dilution on each serum specimen, and make two replicate wells for each dilution of each serum specimen. At the same time, set two wells for each serum control well and cell control well for each serum to be tested.

[0083] (3) Operation steps for virus neutralizing antibody assay:

[0084] Add 0.05 ml of the attack virus (containing 100 CCID 50 / 0.05ml) and incubate at 37℃ for 2h. Take a new 96-well plate and perform a virus titration test (must be done for each experiment). After the incubation is completed, digest the cells with trypsin digestion solution to prepare a cell suspension. Adjust the concentration of the cell suspension to 2×10 5 Add 0.1 ml of cell suspension to each well of the specimen plate and virus titration plate, incubate at 35°C, 5% CO2, determine the final result after 7 days, and record the virus titration result. Note: If the virus titration result is not between 32-320CCID 50 / 0.05ml, the test is invalid and needs to be retried.

[0085] (4) Result determination:

[0086] When cytopathic effect is shown in one of the two wells of the highest dilution serum and not in the other well, the reciprocal of the dilution is the neutralizing antibody titer of the serum specimen; when 2 wells of the high dilution are completely diseased and the 2 adjacent wells of the low dilution are completely diseased, the reciprocal of the average dilution of the two is the neutralizing antibody titer of the serum specimen; when cytopathic effect is shown in one well of the two adjacent dilution serum and not in the other well, the reciprocal of the average dilution of the two is the neutralizing antibody titer of the serum specimen.

[0087] 3. Evaluation of immunogenicity of CVB3 inactivated vaccine

[0088] (1) Immunize BALB / c mice with the vaccine antigen prepared according to the method of step 1 above, and analyze the immune effects of different doses of antigen. The content of vaccine antigen is 0.1ml containing 1ug, 3μg, 5μg, and 10μg. Immunization procedure: 3 subcutaneous injections, 0.1ml / mouse, blood is collected after 1, 2, and 3 immunizations, serum is separated, and neutralizing antibodies are detected. During the experiment, BALB / c mice were divided into 6 groups, each with 10 mice, and the controls were aluminum adjuvant group and PBS buffer group.

[0089] The neutralizing antibody assay protocol is as follows:

[0090] Serum was diluted at 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:1024, 1:2048, 1:4096; positive control: rabbit polyclonal antibody serum prepared in this laboratory; negative control: rabbit negative serum prepared in this laboratory. Neutralizing antibody titer of serum after immunization in each experimental group and control group was tested according to the neutralizing antibody detection method in step 2

[0091] The results showed that the dose-effect relationship of the vaccine was significant. As the dose increased, the neutralizing titer increased accordingly. After 3 immunizations, the neutralizing antibody titer against KM50-W07 was ≥1:384, reaching the protective antibody level. See attached Figure 5 .

[0092] (2) Two other D genotype CVB3 isolates, one E genotype CVB3, the CVB3 prototype strain, and sera from the 10ug dose group 3 were used to test the immune effect of the vaccine prepared by KM50-W07 against other isolates of the same D genotype and CVB3 strains of genotypes A and E.

[0093] The results showed that the neutralizing antibody titers of the serum in the 10ug dose group 3 after immunization against other A, D and E genotype CVB3 strains were ≥1:128, indicating a protective effect.

[0094] 4. Evaluation of the protective efficacy of CVB3 inactivated vaccine

[0095] In order to study the immune protection of maternally transmitted antibodies on newborn mice, the experimental group was set up with 20μg / 100μL CVB3 vaccine, and the control group was set up with PBS group and aluminum adjuvant group. 8-week-old BALB / c female mice were immunized, with 5 animals immunized in each group. After the first immunization of the female mice, they were placed in the same cage with male mice. After the female mice became pregnant, they were immunized for the second time. The female mice were pregnant about two weeks after the second immunization. 24h after delivery, the virus challenge experiment was carried out on 1-day-old mice. Each mouse was intraperitoneally injected with a lethal dose (30μL, virus content 10 5 CCID 50 ) were tested in step 3 of the present invention. The other two D genotype CVB3 isolates and one E genotype CVB3 isolate virus fluid were used to observe the weight changes and clinical scores of each group of suckling mice daily to evaluate the protectiveness of their maternal antibodies.

[0096] The results showed that the mice in the experimental mother group gained weight normally after the virus was inoculated, had no clinical symptoms, were healthy, and had a final survival rate of 100%. The mice in the control group had almost no weight gain after the virus was inoculated, had obvious clinical symptoms, began to die on the 2nd to 3rd day, and all died on the 3rd to 6th day, with a final survival rate of 0%. The above data show that the maternal antibodies in the experimental group can provide complete protection for the mice against a lethal dose of CVB3.

[0097] Example 3: Preparation of an infected animal model using CVB3 virus strain KM50-W07

[0098] The KM50-W07 strain was injected intraperitoneally (IP) at a rate of 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 CCID 50 The six groups of doses were used to infect 1-day-old BALB / c suckling mice, respectively, to explore the optimal infection dose for establishing the CVB3 infection model of 1-day-old BALB / c suckling mice. The clinical scores of suckling mice were calculated according to Table 3.

[0099] Table 3 Clinical scores

[0100] Clinical scoring Clinical manifestations 0 points healthy 1 point Lethargy, mental depression 2 points thin 3 points Weakness in the limbs, thinning hair, hunchback or weakness 4 points Dying and Death

[0101] The results showed that 1-day-old BALB / c suckling mice were challenged with intraperitoneal injection of the drug, with the dose group being 10 6 CCID50 / mouse group, 10 5 CCID50 / mouse group, 10 4 CCID50 / mouse group, 10 3 CCID50 / mouse group, 102 CCID50 / mouse group, 10 1 CCID50 / mouse group; control group intraperitoneally injected 30uL Vero cell culture supernatant, and their body weight, clinical score and survival status were recorded. The results are shown in the attached Figure 6 The results show that 10 6 CCID50 / mouse group, and 10 5 The BALB / c suckling mice in the CCID50 / mouse group showed a tendency to lose weight, and the growth of the other dose groups was slower than that of the control group from 2 to 6 days after the challenge. The subsequent weight gain may be due to the enhanced immunity; 6 The CCID50 / mouse group showed hunchback symptoms 24 hours after the challenge, and all died within 48 hours. 5 The CCID50 / mouse dose group showed symptoms of mental depression 48 hours after the challenge, some showed hunchback or limb weakness at 72 hours, and all died at 96 hours. Some deaths occurred in other dose groups, but not completely. Therefore, the 10 dose group was determined through dose screening. 5 CCID50 / mouse is the optimal infectious dose.

[0102] The KM50-W07 strain was selected and the dose was 10 5 CCID 50 / mice were used to carry out infection experiments on newborn mice. When the experimental mice appeared to be in a dying state, they were killed and their heart, liver, spleen, lung, kidney, pancreas, small intestine, brain, upper limb muscles and lower limb muscles were collected. All organs were fixed in 10% neutral formalin for 48 hours, and then embedded in paraffin to make paraffin sections for HE staining and immunohistochemistry (IHC) detection. For immunohistochemistry detection, paraffin sections were baked at 60℃ for 1 hour, dewaxed with alcohol gradient, EDTA antigen repair, dehydrated, and incubated with CVB3 polyclonal antibody prepared in this laboratory at 4℃ overnight. After incubation at room temperature for 30 minutes, DAB was added for color development and hematoxylin was used for blueing. Negative serum was used for the negative control group section antibody. Results were detected under a microscope.

[0103] HE staining showed obvious histopathological changes in the liver and pancreas in the experimental group: inflammatory reactions in the liver and pancreatic cells; hepatocyte edema, punctate necrosis of hepatocytes with infiltration of inflammatory cells; unclear serous glandular structure in the exocrine pancreas, swollen and degenerated cells, and infiltration of inflammatory cells, see attached Figure 7 .

[0104] Collect 10 5 CCID 50RNA was extracted from various tissues of suckling mice in the / dose group, and the viral load of each tissue of suckling mice was measured by one-step real-time fluorescence quantitative RT-PCR reaction. The primers CVB3vp1-qPF (GGC AGA CAT CCA CTA ACC CT, SEQ ID NO: 16) and CVB3vp1-qPR (TTC TGG CAAATT CTG ACC AAC C, SEQ ID NO: 17) designed according to the conservative sequence of CVB3 virus VP1 and Taqman probe CVB3vp1-Probe (ATG CAC CGC CAC GTATGT CC 5'FAM 3'MGB, SEQ ID NO: 18) and CVB3 RNA standards prepared in this laboratory were used to detect the number of viral copies by RT-qPCR. The reaction system and reaction conditions are shown in Tables 4 and 5. The results showed that in the experimental group, the viral load levels of the brain and liver were significantly higher than those of other tissues, see Appendix Figure 8 .

[0105] Table 4 RT-qPCR reaction system

[0106]

[0107]

[0108] Table 5 RT-qPCR reaction conditions

[0109]

[0110] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0111] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A coxsackievirus group B type 3 strain, characterized in that: The genome sequence of the strain is shown in SEQ ID NO: 1 or a completely complementary sequence of the sequence shown in SEQ ID NO:

1.

2. A Coxsackievirus group B type 3 strain according to claim 1, characterized in that: The strain was named KM50-W07 and was deposited in the China Center for Type Culture Collection on November 6, 2024. The collection number is CCTCC NO: V202474. The classification name is Coxsackievirus Group B Type 3 KM50-W07 Coxsackievirus B3. The collection address is Wuhan University, Wuhan, China.

3. A biomaterial, characterized in that: Any of the following: A. A nucleic acid molecule with a sequence as shown in SEQ ID NO: 1 or a completely complementary sequence to the sequence shown in SEQ ID NO: 1; B. an expression cassette containing the nucleic acid molecule described in A; C. a recombinant vector containing the nucleic acid molecule described in A; D. a recombinant microorganism containing the nucleic acid molecule described in A, the expression cassette described in B, or the recombinant vector described in C; E. A cell line containing the nucleic acid molecule described in A, the expression cassette described in B, or the recombinant vector described in C.

4. An immunogenic composition, characterized in that The immunogenic composition contains the Coxsackievirus group B type 3 strain according to claim 1 or 2 or contains the biological material according to claim 3.

5. Use of the Coxsackievirus group B type 3 strain according to claim 1 or 2, or the biological material according to claim 3, or the immunogenic composition according to claim 4, characterized in that: Any of the following: a. Use in the preparation of drugs for preventing and / or treating coxsackievirus infection or diseases caused by coxsackievirus infection; b. Use in the preparation of vaccines for the prevention and / or treatment of coxsackievirus infection or diseases caused by coxsackievirus infection; c. Use in the preparation of antibodies for preventing and / or treating coxsackievirus infection or diseases caused by coxsackievirus infection; d. Use in the preparation of antiserum for preventing and / or treating coxsackievirus infection or diseases caused by coxsackievirus infection; e. Application in the preparation of reagents or kits for detecting Coxsackie virus; f. Application in the evaluation of immunogenicity of coxsackievirus vaccines for non-diagnostic and therapeutic purposes; g. Application in the evaluation of the protective efficacy of coxsackievirus vaccines for non-diagnostic and therapeutic purposes; h. Application in the preparation of Coxsackie virus infected cell models; i. Application in the preparation of Coxsackie virus infection animal models; j. Application in drug screening or efficacy evaluation for the prevention and / or treatment of coxsackievirus infection or diseases caused by coxsackievirus infection.

6. A vaccine, characterized in that The vaccine contains the Coxsackievirus group B type 3 strain according to claim 1 or 2, or an inactivated vaccine or attenuated vaccine prepared from the biological material according to claim 3.

7. The method for preparing the vaccine according to claim 6, characterized in that: The Coxsackievirus B group 3 strain according to claim 1 or 2 or the biological material according to claim 3 is subjected to cell culture, the virus liquid is harvested, inactivated and purified, and then an adjuvant is added to prepare an inactivated vaccine; Alternatively, an attenuated strain with weakened virulence is obtained through adaptive passage or reverse genetics methods, and the virus liquid is harvested through cell culture. After purification, stabilizers and adjuvants are added to prepare an attenuated vaccine.

8. The method for preparing a vaccine according to claim 7, characterized in that: The cells are Vero cells, diploid MRC-5 cells, KMB-17 cells or 2BS cells.

9. A coxsackievirus group B type 3 strain, characterized in that: The strain is a virus adapted strain or clone obtained by adaptive passage or transfection of a cell strain or cell line using the Coxsackievirus group B type 3 strain described in claim 1 or 2 or containing the biological material described in claim 3.

10. The Coxsackievirus group B type 3 strain according to claim 9, characterized in that: The cell strain or cell line is a diploid MRC-5 cell, a KMB-17 cell or a 2BS cell.

Images