A G9P[23] genotype porcine rotavirus strain and its application

Abstract

The present invention discloses a G9P[23] genotype porcine rotavirus strain and its application; the G9P[23] genotype porcine rotavirus is isolated and passaged from a piglet diarrhea sample in a pig farm in Guangdong Province, named RVA / pig / CH / U945 / 2022 / G9P[23], and the deposit number is CCTCC NO: V202454. The isolated strain can stably proliferate in MA104 cells and produce typical cytopathic effects. The G9P[23] genotype porcine rotavirus strain isolated by the present invention can be used for the preparation of virus strains for testing and inactivated vaccines. As a vaccine strain, it has good immunogenicity and can produce a high level of antibodies after immunizing animals, providing the necessary material and theoretical basis for the subsequent research and development of rotavirus vaccines.

Description

Technical Field

[0001] The present invention relates to the field of rotavirus, and in particular to a G9P

[23] genotype porcine rotavirus strain and its application. Background Art

[0002] Rotavirus belongs to the genus Rotavirus of the family Reoviridae and is an 11-segment double-stranded RNA virus. The diameter of rotavirus is about 65-75 nm, and the full length of the genome is about 18.5 kb.

[0003] Rotavirus is a zoonotic pathogen, and its main clinical manifestations are vomiting, diarrhea and dehydration. It is the main cause of severe gastroenteritis in infants and young animals. Rotavirus infection has great harm to human health and the development of the breeding industry, and often causes huge social burden and economic losses.

[0004] According to the difference in VP6 protein antigenicity, rotavirus is divided into 9 different serogroups (A~D, FJ), among which Group A Rotavirus (RVA) has the highest prevalence and is the main cause of acute dehydration diarrhea in humans and a variety of animals. A binary typing system is established through the two main capsid proteins VP7 and VP4 of rotavirus, and rotavirus is divided into different G serotypes and P serotypes. Currently, there are 58 P (VP4 gene) and 42 G (VP7 gene) genotypes of RVA found in human and animal populations.

[0005] Rotavirus infection is very common in pigs and is often mixed with other diarrheal viruses, such as PEDV, PDCoV, etc., which seriously hinders the development of the pig industry. At present, there is no other good treatment for porcine rotavirus infection except disease prevention through vaccination. Early studies found that porcine rotavirus is difficult to adapt to in vitro cell culture. With further research, it was found that the outer capsid protein VP4 on the surface of rotavirus can inhibit the growth of the virus on cells. Trypsin treatment will cleave the VP4 protein into VP5 and VP8*, allowing the virus to bind to cell surface receptors and improve the adaptability of rotavirus to cells.

[0006] Porcine rotavirus can be detected in both diarrheal and non-diarrhea pigs. Based on literature reports before 2011, porcine group A rotavirus G5 is the main prevalent genotype in the world. In recent years, the detection rate of G9 genotype RVA has become increasingly higher. The reason may be that the herd immunity of the G5 genotype vaccine has caused the G5 genotype to gradually decrease under selection pressure, resulting in the spread of new strains. Therefore, the current vaccine's protective effect against rotavirus infection is limited. Epidemiological surveys on porcine viral diarrhea in recent years in my country have shown that G9, G5, G4, G3 and G11 genotypes currently exist simultaneously, among which G9 is the main prevalent genotype in my country, and is usually prevalent in combination with P

[23] .

[0007] Vaccination is an effective measure to prevent and control porcine rotavirus infection. Therefore, the development of inactivated vaccines against the epidemic genotypes of porcine rotavirus is of great significance for the prevention and control of the prevalence and outbreak of porcine rotavirus. Summary of the invention

[0008] The first object of the present invention is to provide a porcine rotavirus strain.

[0009] The second objective of the present invention is to provide the application of the aforementioned porcine rotavirus strain.

[0010] The third object of the present invention is to provide a vaccine containing the aforementioned porcine rotavirus strain.

[0011] To achieve the above object, the technical solution adopted by the present invention is:

[0012] In the first aspect, the present invention first provides a G9P

[23] genotype porcine rotavirus strain, named RVA / pig / CH / U945 / 2022 / G9P

[23] , which was deposited in the China Center for Type Culture Collection on May 22, 2024, with the deposit number CCTCC NO: V202454, and the deposit address is: Wuhan University, Wuhan, China.

[0013] The G9P

[23] genotype porcine rotavirus strain of the present invention is the current epidemic strain of porcine rotavirus, isolated from a pig farm in Guangdong Province. The strain can produce stable lesions during the passage process and has a strong reproduction ability on MA104 cells, and can be used to produce virus seeds for vaccine research and development.

[0014] In a second aspect, the present invention provides the use of the porcine rotavirus isolated strain in the preparation of a porcine rotavirus diagnostic reagent; the diagnostic reagent is an indirect ELISA kit coated with the porcine rotavirus whole virus, which can better detect the RVA antibody level in serum.

[0015] In a third aspect, the present invention provides the use of the aforementioned G9P

[23] genotype porcine rotavirus in the preparation of an inactivated rotavirus vaccine.

[0016] In a specific embodiment, the G9P

[23] genotype porcine rotavirus is the only active ingredient or one of the active ingredients.

[0017] In a fourth aspect, the present invention protects the use of the aforementioned G9P

[23] genotype porcine rotavirus in the preparation of a drug, wherein the drug is used to prevent diseases caused by rotavirus.

[0018] In a fifth aspect, the present invention protects a vaccine comprising the porcine rotavirus of the G9P

[23] genotype as described above.

[0019] In a specific embodiment, the vaccine further contains a pharmaceutically acceptable auxiliary material or adjuvant.

[0020] In a fifth aspect, the present invention provides a method for preparing an inactivated vaccine using the isolated strain RVA / pig / CH / U945 / 2022 / G9P

[23] . The porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P

[23] described above is used as a virus seed to prepare a virus solution, which is inactivated, diluted, and then mixed with an adjuvant to obtain an inactivated vaccine.

[0021] In a specific embodiment, the adjuvant is ISA 15VG.

[0022] In a more specific embodiment, the method comprises the following steps:

[0023] (1) The porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P

[23] described above was inoculated into MA104 cells for culture and the virus fluid was harvested;

[0024] (2) The virus solution was repeatedly frozen and thawed three times and then centrifuged to obtain the supernatant. The titer of the harvested supernatant was measured. The titer of the required virus solution must be greater than 10 7.5 CCID 50 / mL (CCID50 was measured by CPE (cytopathic effect) combined with FITC-labeled immunofluorescence method to determine the viral infectivity titer);

[0025] (3) Add 0.2% formaldehyde to inactivate the virus to obtain an inactivated virus solution;

[0026] (4) Mix inactivated porcine rotavirus liquid and ISA 15VG adjuvant in a 1:1 ratio, emulsify, and prepare an inactivated rotavirus vaccine.

[0027] The inactivated vaccine immunization route is preferably intramuscular injection.

[0028] The positive beneficial effects of the present invention are:

[0029] (1) The porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P

[23] isolated in the present invention is a currently prevalent strain in China and has a good strain background. It can be used as an inactivated vaccine production strain and a test strain, providing materials for subsequent related experimental research and laying a material foundation.

[0030] (2) The indirect ELISA method established by the present invention using the whole virus of the isolated porcine rotavirus strain as the coating antigen has the characteristics of good specificity, high sensitivity and batch operation, and can be used to evaluate the immune effect of inactivated vaccines.

[0031] (3) The inactivated vaccine of the present invention has good immunogenicity and can induce animals to produce strong neutralizing antibodies after immunization; the strain used in the inactivated vaccine is a strain newly isolated in 2022, which is the main genotype strain prevalent in pig herds in recent years. It can effectively prevent the spread and spread of porcine rotavirus in pig herds and has broad application prospects.

[0032] Collection Information

[0033] Strain name: Porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P

[23] Porcine rotavirus A;

[0034] Depository: China Center for Type Culture Collection;

[0035] Abbreviation of depository institution: CCTCC;

[0036] Deposit address: Wuhan University, Wuhan, China;

[0037] Deposit date: May 22, 2024;

[0038] The registration number of the Collection Center is: CCTCC NO: V202454. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The cytopathic effect is caused by porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P

[23] infecting MA104 cells; Figure 1 Figure A shows normal MA104 cells. Figure 1 Figure B shows the infection lesions of the isolated strain.

[0040] Figure 2 Indirect immunofluorescence identification of porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P

[23] .

[0041] Figure 3The following are the results of RT-PCR identification of different generations of porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P

[23] ; M: DL2000 Marker; 1-6: the amplification results of the primers for detecting the P3, P5, P10, P15, P20, and P25 generations of RVA / pig / CH / U945 / 2022 / G9P

[23] ; 7: negative control.

[0042] Figure 4 This is the growth kinetic curve of porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P

[23] in MA104 cells.

[0043] Figure 5 The indirect ELISA method was used to detect the level of rotavirus-specific IgG antibodies in serum; the data are the mean ± standard deviation (SD) of each group of mice.

[0044] Figure 6 The rotavirus antibody titers in serum were measured for virus neutralization assay; data are mean ± standard deviation (SD) of mice in each group. DETAILED DESCRIPTION

[0045] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The reagents or instruments used without indicating the manufacturer are considered to be conventional products that can be purchased on the market.

[0046] Example 1 Isolation, culture, identification and passage of Group A porcine rotavirus (RVA)

[0047] 1.1 Pathological materials and cell lines

[0048] Pathological materials: Feces, intestines and contents of diarrheal piglets were collected from pig farms in various provinces of my country from 2022 to 2024. The pathological materials from which porcine rotavirus was isolated came from a pig farm in Zhanjiang City, Guangdong Province in August 2022. After sampling, the samples were stored in a -80 ℃ refrigerator. During the sampling process, the collection location, time and sample information of the samples were recorded in detail.

[0049] Cell line: African green monkey embryonic kidney MA104 cells.

[0050] 1.2 Treatment of diseased materials

[0051] Add 1 ml of PBS to the feces, anal swabs, intestines and their contents, add steel beads to the intestines and their contents and grind them into a paste in a grinder to make a tissue suspension. The feces and anal swabs are vortexed, and then all samples are centrifuged at 12000 rpm and 4 ℃ for 5 min. The supernatant is aspirated and filtered with a 0.22 µm filter. The filtered liquid is packaged and stored at -80 ℃ for later use.

[0052] 1.3 Virus inoculation

[0053] Trypsin was added to the filtered virus solution to make the final concentration reach 10 µg / ml. Mix well and let it stand at 37 °C for 2 h. Inoculate the cells when the cell density reaches 80%. Discard the original culture medium and wash three times with PBS. Inoculate the virus solution at 15% of the original culture medium. Incubate at 37 °C, 5% CO2 incubator for 1 h. Discard the virus solution, add serum-free DMEM culture medium containing 2.5 µg / ml trypsin, and let it stand at 37 °C, 5% CO2 incubator. Observe the cell lesions every 12 h. 24 h after the first generation of infection, the cells showed obvious CPE, and CPE appeared stably after 7 generations of continuous transmission. In contrast, there was no obvious change in the cells of the control group. With the increase in the number of passages, CPE can be seen as early as 12 h, manifested as cell rounding, aggregation, and net pulling, and finally cell collapse and detachment. Cells not inoculated with RVA are in good growth condition ( Figure 1 ).

[0054] 1.4 Plaque purification of viruses

[0055] After the MA104 cells in the 12-well plate have grown into a monolayer, the culture medium was discarded, the cells were washed three times with PBS, and a ten-fold diluted virus solution (10 -1 -10 -6 ) was inoculated in a 12-well plate, gently shaken to mix, and adsorbed at 37 °C for 1 h. 2×DMEM containing 6 μg / mL trypsin and 2% low-melting point agarose were mixed in equal volumes and placed at 37 °C for use. The virus solution in the 12-well plate was discarded and washed once, and 1 ml of the mixture of low-melting point agarose and culture medium prepared above was added. After complete solidification, the cell culture plate was inverted for culture. After 48 h of inoculation and culture, a single plaque was picked up with a 10 μl pipette tip and inoculated into new MA104 cells. After obvious lesions appeared, the sample was collected and plaque purification was performed three times in a row.

[0056] 1.5 Virus identification

[0057] (1) Indirect immunofluorescence detection

[0058] MA104 cells were plated in a 96-well plate, and when the cell density reached 80%, the virus was inoculated as described above, and a negative control was set up at the same time. 24 hours after infection, the culture medium was discarded, the cells were washed once with PBS, and an acetone-methanol fixative (a commercial propanol solution and a methanol solution mixed in a ratio of 1:1) was added, and the cells were allowed to stand at room temperature for 30 min. The fixative was discarded and washed twice. A polyclonal antibody to the rotavirus VP4 protein was added, and the cells were placed at 37°C for 1 hour. The liquid was discarded and washed five times with PBS, and FITC-labeled goat anti-mouse secondary antibody was added, and the cells were placed at 37°C for 1 hour. After washing five times, DAPI was added, and the cells were gently shaken at room temperature for 10 minutes before being observed under a fluorescence microscope. The results showed that specific green fluorescence was visible in the cytoplasm of cells infected with the isolated strain, and no fluorescence was seen in the wells of normal MA104 cells ( Figure 2 ). The results showed that porcine rotavirus was successfully isolated from piglet feces.

[0059] (2) RT-PCR identification of viruses of different generations

[0060] RNA of different generations of viruses was extracted and reverse transcribed, and the RVA NSP5 gene was amplified for virus identification. The detection primers are shown in Table 1. The results showed that the RT-PCR test results of the 3rd, 5th, 10th, 15th, 20th and 25th generation virus fluids were all RVA positive ( Figure 3 ), confirming that porcine rotavirus has stably proliferated on MA104 cells.

[0061] Table 1 Primers for porcine rotavirus detection

[0062]

[0063] 1.6 Whole genome sequencing and sequence analysis of isolated porcine rotavirus strains

[0064] The whole genome of the isolated strain was amplified by high-fidelity enzyme. The primers for amplifying the whole gene sequence of rotavirus are shown in Table 2. The tail was added and connected to the pMD-18T vector. After transformation, the bacterial solution was identified and sequenced. According to the latest RCWG rotavirus 11-gene classification system, each of the 11 gene fragments can determine a genotype. Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx represents the VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3 -NSP4-NSP5 / NSP6 coding genes, respectively. The genotype of each gene of the isolated strain was determined by sequencing the complete ORF of each gene. The sequencing results showed that the homology of the 11 gene fragments with the representative strains was 94.06% to 98.82%. The NSP1 (SEQ ID NO: 7) and NSP2 (SEQ ID NO: 8) genes had the highest nucleotide homology with human rotavirus, and the VP1 (SEQ ID NO: 1), VP2 (SEQ ID NO: 2), VP3 (SEQ ID NO: 3), VP4 (SEQ ID NO: 4), VP6 (SEQ ID NO: 5), VP7 (SEQ ID NO: 6), NSP3 (SEQ ID NO: 9), NSP4 (SEQ ID NO: 10) and NSP5 (SEQ ID NO: 11) genes had the closest nucleotide relationship with porcine rotavirus (Table 3). The genome sequence of RVA / pig / CH / U945 / 2022 / G9P

[23] was G9-P

[23] -I5-R1-C1-M1-A8-N1-T1-E1-H1. The isolated strain contains genetic fragments that are highly homologous to human samples reported in previous studies, suggesting zoonotic transmission.

[0065] Table 2 Primers for amplification of the whole genome of isolated porcine rotavirus strains

[0066]

[0067] Table 3 The consistency of nucleotide sequences between RVA / pig-wt / CH / U945 / 2022 / G9P

[23] strain and closely related strains

[0068]

[0069] 1.7 Growth characteristics of RVA isolates

[0070] (1) CCID of RVA isolated strains 50 Determination of

[0071] The virus was diluted 10 times in DMEM culture medium containing 3.0 µg / mL pancreatin, and different dilutions of virus solution were inoculated in 96-well plates (100 µL / well), with 6 wells for each dilution, and a negative control was set up at the same time. The plate was placed in a cell culture incubator, and the wells that produced cytopathic effects after 3-4 days were positive wells. The positive wells with inconspicuous lesions were determined by indirect immunofluorescence, and the virus CCID was calculated according to the Reed-Muench method. 50 value.

[0072] (2) Growth curve determination of RVA isolated strains

[0073] The isolated strain RVA / pig / CH / U945 / 2022 / G9P

[23] was inoculated at an MOI of 0.1, and its growth curve on MA104 cells was determined. The specific steps are as follows: After culturing MA104 cells for 24 h, the culture medium was discarded and washed three times with PBS. The virus solution was diluted to MOI = 0.1 and then inoculated. 500 µL was inoculated into each well and adsorbed in a cell culture incubator for 1 h. After adsorption, it was washed three times with PBS solution and replaced with DMEM containing 2.5 µg / mL trypsin (500 µL / well). Then, it was placed in a cell culture incubator for continuous culture. The supernatant was aspirated at 12 h, 24 h, 36 h, 48 h and 60 h after inoculation, and 3 wells were taken as replicates at each time point. CCID 50 The virus titer of the supernatant at different time points was determined by the method. The different infection time was used as the horizontal axis and the CCID 50 The value is the vertical axis, and the growth curve of the RVA isolated strain on MA104 cells is plotted ( Figure 4 ).

[0074] Example 2 Preparation of inactivated vaccine

[0075] The isolated porcine rotavirus strains were propagated and the virus titer was higher than 10 7.5 CCID 50 / ml virus liquid was used to prepare inactivated vaccine, which was sterilized by filtering with a 0.22 µm filter, and inactivated at 37 °C for 48h by adding formaldehyde solution with a final concentration of 0.2%. After inactivation, sodium bisulfite was added to neutralize the residual formaldehyde. The inactivated virus liquid was taken for verification and detection; three consecutive generations were blindly propagated on MA104 cells, and positive and negative controls were set up at the same time. Each generation was cultured in a 37 °C, 5% CO2 incubator for 5 days, and the cell pathological changes were observed daily. The results showed that the positive control showed obvious pathological changes 12 hours after infection, while the negative control and the inactivated virus liquid had no pathological changes, confirming that the virus liquid was completely inactivated.

[0076] The formaldehyde-inactivated porcine rotavirus vaccine was mixed with ISA 15VG adjuvant in a ratio of 1:1 and fully emulsified to prepare an inactivated vaccine. The inactivated vaccine was a milky white suspension with uniform color. No stratification was observed after centrifugation at 5000 rpm / min for 5 min. The inactivated vaccine was inoculated into Balb / c mice for safety testing. The mice ate and drank normally after vaccination, and were in good mental state. There was no redness, swelling, nodules or other abnormalities at the injection site of the leg muscles, and no abnormal reactions were observed in any of the observed indicators. This indicates that the vaccine has good safety in the mouse model and can be used for further experiments.

[0077] Example 3

[0078] Five-week-old Balb / c female mice were selected, and blood samples were collected from each mouse before immunization to detect the antibody level of the female mice. The mice were randomly divided into two groups, namely the vaccine group and the control group. The female mice in the vaccine group were injected intramuscularly with the inactivated virus vaccine RVA / pig / CH / U945 / 2022 / G9P

[23] , 200 μL / mouse. After an interval of 14 days, the second injection was given with the same dose. The control group was injected with PBS 200 μL / mouse in the same way. Blood samples were collected every 7 days after immunization, incubated at 37 °C for 2 h, and centrifuged at 3000 rpm for 15 min to collect serum.

[0079] (1) Serum rotavirus-specific IgG antibody levels after immunization of mice

[0080] The whole virus was used as the coating antigen, and the antigen and serum were diluted in multiples. The optimal virus coating concentration and the optimal serum dilution were determined by the chessboard method. The optimal virus coating concentration was 3 µg / ml, and the optimal serum dilution was 1:100. The virus was diluted with the coating solution, 100 µL per well, and coated overnight at 4 °C. The coating solution was discarded, washed 3 times with PBS, and blocked with 5% skim milk at 37 °C for 2 h. Discarded, washed 5 times with PBST. Dilute the serum to be tested, 100 μL per well, incubated at 37 °C for 1 h, and set up negative and positive serum controls. Add 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG, 100 μL per well, incubated at 37 °C for 1 h. Discard the secondary antibody and wash 5 times with PBST. Add 100 μL TMB colorimetric solution and color for 15 min in the dark. Add 50 μL of stop solution 2 M H2SO4 to each well, OD 450 Read the value.

[0081] (2) Serum rotavirus neutralizing antibody levels after immunization of mice

[0082] Serum antibody neutralization levels were measured in 96-well cell culture plates. 50The isolated porcine rotavirus strain was treated with 20 μg / mL pancreatin at 37°C for 1 h and then mixed with an equal amount of twice-diluted immune serum (the lowest serum dilution gradient was 1:2 4 ), add it to the MA104 cells that have grown monolayers after incubation at 37 ℃ for 1 h, 100 μL / well, incubate at 37 ℃ for 1 h, discard the solution, and add DMEM maintenance solution containing trypsin at a final concentration of 3.0 μg / mL, 100 μL / well. After culturing at 37 ℃ and 5% CO2 for 72 h, observe the pathological changes of the cells. For the uncertain cell wells, the indirect immunofluorescence method is used for determination. The highest dilution of cells without fluorescent signal labeling is the maximum dilution of the serum that can neutralize the virus after dilution. The titer of neutralizing antibodies is expressed as the reciprocal of the highest dilution of serum.

[0083] Indirect ELISA results showed that the inactivated vaccine induced a weak immune response 14 days after the first vaccination, while the IgG level increased significantly after booster immunization. Neutralization test results showed that mice produced high levels of neutralizing antibodies 7 days after the second immunization, with the highest neutralizing antibody titer reaching 1:1250, while no antibody was produced in the negative control, indicating that the rotavirus isolate has good immunogenicity and can be used as a candidate strain for porcine rotavirus vaccine.

[0084] The above embodiments are only preferred modes of the present invention, but the implementation modes of the present invention are not limited by the embodiments. Any other changes and modifications that do not deviate from the principles of the present invention should be regarded as within the protection scope of the present invention.

Claims

1. A strain of porcine rotavirus of genotype G9P[23], named RVA / pig / CH / U945 / 2022 / G9P[23], was deposited in the China Center for Type Culture Collection on May 22, 2024, with the deposit number CCTCC NO: V202454. The deposit address is: Wuhan University, Wuhan, China.

2. Use of the G9P[23] genotype porcine rotavirus according to claim 1 in the preparation of porcine rotavirus diagnostic reagents.

3. Use of the G9P[23] genotype porcine rotavirus according to claim 1 in the preparation of an inactivated rotavirus vaccine.

4. The use according to claim 3, characterized in that: The G9P[23] genotype porcine rotavirus is the only effective ingredient or one of the effective ingredients.

5. Use of the G9P[23] genotype porcine rotavirus according to claim 1 in the preparation of a medicament for preventing diseases caused by rotavirus.

6. A vaccine comprising the porcine rotavirus of the G9P[23] genotype according to claim 1.

7. The vaccine according to claim 6, characterized in that The vaccine also contains pharmaceutically acceptable auxiliary materials or adjuvants.

8. A method for preparing an inactivated vaccine using the G9P[23] genotype porcine rotavirus according to claim 1, wherein the porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P[23] according to claim 1 is used as a virus seed to prepare a virus solution, and the virus solution is inactivated, diluted, and then mixed with an adjuvant to obtain an inactivated vaccine.

9. The method according to claim 8, characterized in that The adjuvant was ISA 15VG.

10. The method according to claim 8 or 9, characterized in that: The method comprises the following specific steps: (1) inoculating the porcine rotavirus RVA / pig / CH / U945 / 2022 / G9P[23] described in claim 1 into MA104 cells for culture and harvesting the virus liquid; (2) The virus solution was repeatedly frozen and thawed three times and then centrifuged to obtain the supernatant. The titer of the harvested supernatant was measured. The titer of the required virus solution must be greater than 10 7.5 CCID 50 / mL; (3) Add 0.2% formaldehyde to inactivate the virus to obtain an inactivated virus solution; (4) Mix inactivated porcine rotavirus liquid and ISA 15VG adjuvant in a 1:1 ratio, emulsify, and prepare an inactivated rotavirus vaccine.

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