Porcine rotavirus SCLS3 strain and application thereof

Abstract

The invention relates to the field of research and development of veterinary biological products, and particularly provides a porcine rotavirus SCLS3 strain and application thereof. The invention discloses a porcine rotavirus SCLS3 strain which is preserved in China Center for Type Culture Collection on October 29, 2024, the preservation number is CCTCC NO: V202494, and the preservation address is Wuhan University, Wuhan, China. The vaccine composition prepared from the strain can prevent spreading of porcine rotavirus epidemic situation, and after the vaccine composition containing the strain is used for immunizing animals, the animal bodies can quickly generate antibodies, so that the vaccine composition has good prevention and control effects on current epidemic porcine rotavirus infection, and is good in biological safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of development of veterinary biological products, and specifically provides a porcine rotavirus SCLS3 strain and application thereof. BACKGROUND

[0002] Rotavirus (RV) belongs to the Reoviridae family and is a non-enveloped double-stranded RNA virus. According to serology, rotavirus can be divided into 10 groups (RVA~RVJ), among which A, B, C, H and E groups can be detected in pig groups, and the clinically prevalent porcine rotavirus (PoRV) strain refers to the A group porcine rotavirus. Porcine rotavirus is one of the main pathogens causing diarrhea in piglets, and pigs of all ages can be infected with rotavirus. After infection, young piglets mainly show symptoms such as diarrhea, dehydration and vomiting, and adult pigs show latent infection. After infection, pigs continuously shed the virus, which causes great difficulty in preventing and controlling diseases in pig farms.

[0003] The full-length genome of porcine rotavirus is about 18.5 kb, which is composed of 11 segmented RNA fragments, encoding 6 structural proteins (VP1~VP4, VP6 and VP7) and 5 / 6 non-structural proteins (NSP1~NSP5 / 6). The outer capsid proteins VP7 (glycoprotein) and VP4 (protease-sensitive protein) contain neutralizing epitopes that can induce protective immunity in the body and form a G and P double-genotype typing system. In pigs, at least 12 G types (G1~G6, G8~G12 and G26) and 16 P types (P[1]~P[8], P

[13] , P

[19] , P

[23] , P

[26] ~P

[28] , P

[32] and P

[34] ) have been identified. Research reports show that the main G types prevalent in the clinic are G9, G5, G4 and G3, and the main P types are P

[23] , P

[13] and P[7].

[0004] At present, there is no effective treatment for porcine rotavirus disease, and vaccination is an effective means to prevent and control the incidence of porcine rotavirus. The existing RVA vaccine is mainly attenuated live vaccine, which belongs to the G5P[7] strain, and has limited cross-neutralization ability to epidemic strains. Therefore, it is urgent to isolate porcine rotavirus wild strains with good immunogenicity and broad-spectrum neutralization ability.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide a porcine rotavirus SCLS3 strain and application thereof, which can effectively prevent the infection of porcine rotavirus.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A porcine rotavirus SCLS3 strain, preserved in the China Center for Type Culture Collection on October 29, 2024, with a preservation number of CCTCC NO:V202494, and a preservation address of Wuhan, Wuhan University, China.

[0009] The above-mentioned SCLS3 strain is applied to the preparation of a vaccine for preventing and / or treating porcine rotavirus disease.

[0010] A vaccine composition for preventing porcine rotavirus disease, comprising an immunizing amount of porcine rotavirus antigen and a pharmaceutically acceptable carrier, wherein the porcine rotavirus antigen comprises inactivated antigen of the porcine rotavirus SCLS3 strain or culture thereof.

[0011] Further, the SCLS3 strain is ≥10 5.0 TCID 50 / ml, preferably 10 5.0 -10 7.0 TCID 50 / ml, further preferably 10 6.0 TCID 50 / ml.

[0012] Further, the pharmaceutically acceptable carrier comprises at least one of an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative.

[0013] Further, the adjuvant comprises one or more of aluminum hydroxide adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, Montanide ISA 206, Montanide ISA 201, or Gel adjuvant;

[0014] Preferably, the adjuvant is a biphasic adjuvant for preparing a water-in-oil-in-water emulsion.

[0015] Preferably, the concentration of the adjuvant ranges from 5Wt% to 50Wt%, preferably 30Wt% to 50Wt%, more preferably 50Wt%.

[0016] Further, the lyoprotectant is selected from sugar, polyol, polymer, surfactant, salt, amine, or amino acid.

[0017] Further, the immunostimulant includes alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin 2.

[0018] Compared with the prior art, the technical effects of the present application are:

[0019] The porcine rotavirus strain of the present application is a prevalent porcine rotavirus wild strain, and the vaccine composition prepared from the strain can prevent the spread of porcine rotavirus epidemic, and the vaccine composition containing the strain can enable the animal body to rapidly produce antibodies after immunizing the animal, and has good prevention and control effect on the current prevalent porcine rotavirus infection, and has good biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0020] The various technical features of the present application and the relationship between them will be further described below with reference to the accompanying drawings. The drawings are exemplary, some technical features are not shown in actual proportion, and some technical features in the drawings can be omitted, which are conventional in the technical field to which the present application belongs and are not essential for understanding and implementing the present application, or additional technical features are shown, which are not essential for understanding and implementing the present application, that is, the combination of various technical features shown in the drawings is not used to limit the present application. In addition, the same reference signs refer to the same contents throughout the present application. The specific drawings are as follows:

[0021] Figure 1 is the IFA identification result of 1.2 in Example 1 of the present application. DETAILED DESCRIPTION

[0022] In order for those skilled in the art to better understand the technical solutions of the present application, the technical solutions described in the present application will be further described in detail below in combination with specific embodiments.

[0023] In the present application, "further", "still further", "in particular" and the like are used for description purposes, indicating differences in content, but should not be understood as limiting the scope of protection of the present application.

[0024] In the present application, "optionally", "optional" and "optional" mean optional, that is, selected from either of the two parallel schemes of "yes" or "no". If there are multiple "optional" in a technical solution, and there is no contradictory or mutually restrictive relationship, each "optional" is independent.

[0025] In the present application, "multiple", "various", "multiple times", "multiple" and the like refer to more than two or equal to two in quantity, unless otherwise specified. For example, "one or more" means one or more than two.

[0026] The embodiments of the present application are described below.

[0027] The term "Porcine Rotavirus" (PoRV) is a member of the Reoviridae family, Rotavirus genus, consisting of 11 double-stranded RNA segments, and the clinical symptoms caused by it include diarrhea, accompanied by anorexia, vomiting, dehydration, etc. The pathological changes are characterized by thin and watery intestinal wall, watery contents, and atrophy of small intestinal villi.

[0028] The term "Porcine Rotavirus strain" (PoRV strain) is used interchangeably with "Porcine Rotavirus wild strain" (PoRV wild strain), "Porcine Rotavirus virulent strain" (PoRV virulent strain), and "Wild-type PoRV strain" in the present application unless otherwise specified.

[0029] The present application relates to the Porcine Rotavirus SCLS3 strain, which is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: V202494, and the preservation address Wuhan, Wuhan University, China, and the preservation date October 29, 2024.

[0030] The strain has strong virulence and good immunogenicity.

[0031] The present application relates to a vaccine composition, wherein the vaccine composition comprises an immunizing amount of a Porcine Rotavirus antigen and a pharmaceutically acceptable carrier, and the Porcine Rotavirus antigen comprises the inactivated antigen of the Porcine Rotavirus SCLS3 strain of the present application or the culture thereof. The vaccine composition is prepared from a virulent strain and has good immunogenicity, which can provide complete protection for pigs.

[0032] The term "vaccine composition" is also referred to as "immunogenic composition", which refers to a preparation containing an immunogen, including whole cells, inactivated or attenuated, live viruses or bacteria, or polysaccharides, or combinations thereof, which is administered to stimulate the humoral and cellular immune responses of the recipient to one or more antigens present in the immunogenic composition. Immunization is the process of administering a vaccine composition and stimulating an immune or immunogenic response to an antigen in a host, preferably an animal such as a pig.

[0033] The term "immunizing amount" shall be understood as "immunologically effective amount", also known as immunoprotective amount or effective amount to generate an immune response, is an amount of antigen effective to induce an immune response in the recipient, which is sufficient to prevent or ameliorate the signs or symptoms of disease, including adverse health effects or complications thereof. The immune response can be sufficient for diagnostic purposes or other assays, or can be suitable for preventing signs or symptoms of disease, including adverse health outcomes or complications thereof caused by infection with a pathogen. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example, by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms following challenge with a wild-type strain, while the protective immunity provided by the vaccine can be assessed by measuring, for example, a reduction in clinical signs such as mortality, morbidity, temperature values, overall physiological condition and general health and performance of the subjects. The immune response can include, but is not limited to, the induction of cellular and / or humoral immunity.

[0034] The term "porcine rotavirus antigen" refers to any composition containing at least one form of porcine rotavirus antigen that induces, stimulates or is capable of an immune response against a porcine rotavirus infection, including but not limited to inactivated, attenuated or subunit antigens. Among them, inactivated antigens can be inactivated while maintaining their immunogenicity by various methods including chemical treatment, physical treatment (such as sonication, irradiation, heat) or any other common method sufficient to stop the replication or growth of the organism, preferably the pathogen is inactivated after collection and optionally subjected to clarification purification, by chemical treatment using, for example, formalin or formaldehyde, beta-propiolactone, ethyleneimine, binary ethyleneimine BEI, thiomersal, etc.; methods of inactivation are well known to those skilled in the art, such as by beta-propiolactone (Plana-Duran et al., Vet. Microbiol., 1997, 55: 361-370) or by BEI treatment (US5587164).

[0035] The term "pharmaceutically acceptable carrier" refers to all the components other than the porcine rotavirus antigen in the vaccine composition of the application, a carrier or diluent that does not stimulate the organism and does not hinder the biological activity and properties of the compound used, preferably an adjuvant.

[0036] The term "adjuvant" can include aluminium hydrogel adjuvants; saponins such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic or methacrylic acid; co-polymers of maleic anhydride and alkenyl derivatives selected from the group consisting of compounds.

[0037] The term "emulsion" can be based on, inter alia, light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from the oligomerisation of isoolefins, such as squalane or squalene oil, in particular iso-butene or eucalyptene; linear alkyl esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(octanoate / euric)ate), glycerol tri-(octanoate / euric)ate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearate. The oil is used in combination with an emulsifying agent in order to form the emulsion. The emulsifying agent is preferably a non-ionic surfactant, in particular an ester of sorbitan, an ester of mannide (such as anhydrous mannide oleate), an ester of a fatty glycol, an ester of polyglycerol, an ester of propylene glycol and an ester of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular the Pluronic products, in particular L121. See Hunter et al. (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and Todd et al. (Vaccine, 1997, 15: 564-570). For example, the SPT emulsion described on page 147 and the MF59 emulsion described on page 183 of "Vaccine design, the Subunit and adjuvant approach" by Powell M and Newman M (Plenum Press, 1995) can be used.

[0038] The term "polymers of acrylic or methacrylic acid" are preferably cross-linked polymers of acrylic or methacrylic acid, in particular cross-linked with polyalkenyl ethers of sugars or polyalcohols, which compounds are known under the name Carbomer (Carbopol, trade name) (Pharm. Europa, 1996, 8(2)). The skilled person can also refer to US patent 2 909 462, which describes such acrylic acid polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, in which at least 3 of the hydroxyl groups have their hydrogen atoms replaced by an unsaturated aliphatic radical having at least 2 carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups themselves can contain further substituents, such as methyl groups. These products are sold under the name Carbopol (BF Goodrich, Ohio, USA) and are particularly suitable. They are cross-linked with allyl sucrose or with allyl pentaerythritol. Among these, Carbopol 974P, 934P and 971P can be mentioned, the most preferred being Carbopol 971P.

[0039] The term "co-polymers of maleic anhydride and alkenyl derivatives" also contemplates co-polymers of maleic anhydride with ethylene, EMA (Monsanto), which polymers dissolve in water to produce an acidic solution, which is neutralized, preferably to physiological pH, in order to produce an adjuvant solution into which the immunogenic, immunizing or vaccinal composition itself can be incorporated.

[0040] The term "adjuvant" also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvant, etc.

[0041] In preferred embodiments, the adjuvant comprises one or more of mineral oil, aluminum hydrogel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymers of acrylic or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, Montanide ISA 201 or Gel adjuvant.

[0042] Preferably, the adjuvant is a biphasic adjuvant used to prepare a water-in-oil-in-water emulsion.

[0043] In some embodiments, the concentration of the adjuvant ranges from 5 Wt% to 50 Wt%, preferably 30 Wt% to 50 Wt%, more preferably 50 Wt%.

[0044] The concentration of the adjuvant can range, but is not limited to, 5 Wt%, 10 Wt%, 15 Wt%, 20 Wt%, 25 Wt%, 30 Wt%, 35 Wt%, 40 Wt%, 45 Wt% or 50 Wt%.

[0045] The term "lyoprotectant" refers to an ingredient other than an excipient that protects the pharmaceutical active during the process of freeze-drying and during the storage phase after lyophilization. Lyoprotectants can be selected from sugars, polyols, polymers, surfactants, salts, amines or amino acids.

[0046] In some embodiments, the immunostimulant comprises alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor or interleukin 2.

[0047] As a preferred embodiment of the present application, the inactivated antigen content of the porcine rotavirus SCLS3 strain or culture thereof in the vaccine composition described in the present application is ≥ 10 5.0 TCID 50 / ml before inactivation.

[0048] As a more preferred embodiment of the present application, the inactivated antigen content of the porcine rotavirus SCLS3 strain or culture thereof in the vaccine composition described in the present application is 10 5.0 -10 7.0 TCID 50 / ml before inactivation.

[0049] As a most preferred embodiment of the present application, the inactivated antigen content of the porcine rotavirus SCLS3 strain or culture thereof in the vaccine composition described in the present application is 106.0 TCID 50 / ml.

[0050] The term "preventing and / or treating" in relation to porcine rotavirus infection means inhibiting the replication of porcine rotavirus, inhibiting the transmission of porcine rotavirus or preventing porcine rotavirus from inhabiting in its host, and alleviating the symptoms of the disease or condition caused by porcine rotavirus infection. If the viral load is reduced, the condition is alleviated, and / or the food intake and / or growth is increased, then the treatment is considered to have achieved a therapeutic effect.

[0051] The present application also relates to the use of the vaccine composition described in the preparation of a medicament for preventing the diseases associated with porcine rotavirus infection.

[0052] The advantages and features of the present application will become more apparent with the description of the specific embodiments. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present application without departing from the spirit and scope of the present application, and such modifications and substitutions shall fall within the protection scope of the present application.

[0053] The chemical reagents used in the embodiments of the present application are all of analytical purity and purchased from the National Pharmaceutical Group.

[0054] In order to make the present application easier to understand, the present application will be further described below in conjunction with specific embodiments. The experimental methods described in the present application are all conventional methods if not otherwise specified, and the biological materials described in the present application can be obtained from commercial channels if not otherwise specified.

[0055] Example 1 Isolation and identification of porcine rotavirus

[0056] 1.1 Virus isolation The intestines of the diarrhea piglets identified as PoRV positive by the RT-qPCR method were taken, and the intestinal contents were prepared into a suspension with α-MEM medium at a volume ratio of 1:5, frozen-thawed once at -80℃, centrifuged at 10000 rpm for 10 min at 4℃, and the supernatant was filtered with a 0.22 μm filter. The filtered virus solution was added with trypsin at a final concentration of 10 μg / ml, and incubated at 37℃ in a 5% CO2 incubator for 1 h. A T25 cell bottle with long single-layer MA104 cells was taken, the liquid was discarded, and the cells were washed once with PBS, 1 ml of the virus suspension incubated for 1 h was added, and the cells were adsorbed in a 37℃, 5% CO2 incubator for 1 h, then the virus solution was discarded, and 5 ml of α-MEM medium containing trypsin at a final concentration of 0.5 μg / ml was added. The cells were observed daily for whether cytopathic effect was produced, and the cells were frozen-thawed 3 times and continued to be blind-transmitted for 5 days. On the third day of culturing the F2 generation, it was observed that the cells produced obvious cytopathic effect (cell rounding, aggregation, filamentation, and shedding, etc.).

[0057] 1.2 Identification of IFA The harvested F5 virus liquid was diluted 100 times and inoculated into 96-well cell culture plates with single layer of MA104 cells, and a negative control was set. After 36 hours of inoculation, the culture medium was discarded, and the cells were washed once with PBS. 80% pre-cooled acetone was added to each well, and the cells were incubated at 2-8°C for 30 minutes. After the fixation solution was discarded, the cells were washed twice. Porcine rotavirus polyclonal antibody was added, and the cells were incubated at 37°C for 1 hour. The solution was discarded, and the cells were washed three times with PBS. FITC-labeled goat anti-mouse secondary antibody was added, and the cells were incubated at 37°C for 1 hour. The cells were washed three times with PBS. After 50 μl of PBS was added to each well, the cells were observed under a fluorescence microscope. The results showed that specific green fluorescence was observed in the inoculated cell wells, and no specific green fluorescence was observed in the normal cell control wells. Figure 1

[0058] 1.3 RT-qPCR identification and sequence analysis In this experiment, the A group rotavirus detection method ordinary PCR and real-time fluorescent PCR method in the industry standard SN / T 2520-2010 classification were used to identify the proliferation of F0-F3 cell cultures. The results showed that the CT value decreased with the increase of the generation, which proved that the porcine rotavirus was successfully isolated.

[0059] In this experiment, the porcine rotavirus VP4, VP7, VP6 and NSP4 gene sequences collected in GenBank were used to design amplification primers for each gene. The nucleotide sequences of the primers are shown in Table 1.

[0060] Table 1 Information of PoRV VP4 and VP7 primers

[0061] The above specific sequencing primers were used to amplify the isolated strain by PCR. The product was sent to Shanghai Shengong Company for sequencing and the results were spliced. The nucleotide homology was compared with the published porcine rotavirus VP4, VP7, VP6 and NSP4 gene sequences in NCBI. The results showed that the nucleotide identity of the four gene fragments of the strain with the representative strain was 97.20%-98.88%, the homology of VP6 gene with human rotavirus was the highest, and the nucleotide identity was 98.27%. The VP4, VP7 and NSP4 genes were closely related to porcine rotavirus. The virus strain belongs to G9-P[7]-I1-E1 type, which suggests that it may exist in human-animal co-infection transmission. Table 2 is the nucleotide identity analysis of SCLS3 strain with closely related strains.

[0062] Table 2 Nucleotide identity analysis of SCLS3 strain with closely related strains

[0063] ​1.4 Toxicity determination Take 96-well plates with monolayer MA104 cells, discard the culture solution, and wash once with PBS. Serially pass the virus solution with 0.5 μg / ml trypsin in α-MEM medium to make 10-fold serial dilutions, and inoculate 10 μl of each dilution to 4 wells of the 96-well plate. Set up negative and positive control wells. Place the culture plate in a 37°C, 5% CO2 incubator for 3-5 days, and observe the CPE and record the results daily. Calculate the virus titer according to the Reed-Muench method. The titer of the porcine rotavirus strain is 10 -3 ~10 -4 Dilution Virus solution is inoculated to the 96-well plate, 4 wells for each dilution, and negative and positive control wells are set up. Place the culture plate in a 37°C, 5% CO2 incubator for 3-5 days, and observe the CPE and record the results daily. Calculate the virus titer according to the Reed-Muench method. The titer of the porcine rotavirus strain is 10 8.11 . This indicates that the porcine rotavirus strain can be stably passaged on MA104 cells, and a high-titer virus solution can be obtained, which can be used as a candidate strain for a porcine rotavirus vaccine. The porcine rotavirus strain is named porcine rotavirus SCLS3 strain, and the porcine rotavirus SCLS3 strain is preserved.

[0064] Example 2 Pathogenicity test of porcine rotavirus SCLS3 strain

[0065] Pregnant sows are bled for detection of porcine reproductive and respiratory syndrome (PRRSV), porcine circovirus (PCV2) antigens, and porcine rotavirus (PoRV) IFA antibodies, and anal swabs are collected for detection of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and PoRV antigens. Piglets born from sows that are negative for PoRV antigens and antibodies and negative for PRRSV, PCV2, PEDV, and TGEV antigens are selected for the test, and the piglets do not eat colostrum. Ten 3-day-old piglets that do not eat colostrum are selected and randomly divided into two groups, 5 piglets in each group. The first group is orally inoculated with 1 ml of porcine rotavirus SCLS3 strain virus solution (virus content is 10 7.0 TCID 50 / ml), and the second group is a blank control group that is orally inoculated with 1 ml of α-MEM culture solution. Clinical symptoms are observed daily. The second group of blank control pigs are normal in spirit and diet, and the feces are normal, and no diarrhea is observed. All 5 pigs in the first group of challenge group are sick, and clinical symptoms of vomiting and watery diarrhea are observed. The pigs are decreased in spirit and appetite, and are emaciated. The results are shown in Table 3.

[0066] Table 3 Pathogenicity test results of porcine rotavirus SCLS3 strain Group Number of animals Inoculation dose Number of cases 1 5 5ml / animal 5 / 5 2 5 5ml of culture solution / animal 0 / 5

[0067] After the pigs in the challenge group show clinical symptoms, necropsy is performed to observe the pathological changes of the organs and tissues. The small intestinal contents of the sick pigs are taken, and viral RNA is extracted for RT-PCR detection. The virus is isolated, inoculated to MA104 cells, and the cytopathic effect is observed.

[0068] Autopsy lesions: The blank control group pigs had no abnormalities in the small intestine and other organs. The challenged group pigs had thin and transparent small intestinal walls filled with yellow contents.

[0069] RT-PCR analysis of the small intestinal contents collected from the autopsied pigs showed PoRV positive and porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) negative. The small intestinal contents were also inoculated into MA104 cells, and the same cytopathic effect was observed. This indicated that the diarrhea in piglets in this experiment was indeed caused by porcine rotavirus. The isolate SCLS3 strain described in this application is a virulent strain of porcine rotavirus.

[0070] Example 3 Virus culture of porcine rotavirus SCLS3 strain

[0071] MA104 cells were grown into a good monolayer, the cell culture medium was discarded, and the cells were washed once with 37°C preheated PBS (0.02 mol / L, pH 7.4). The virus was diluted 1000-fold and incubated at 37°C for 1 hour, with mixing every 20 minutes. Then the virus solution was discarded, and the cells were washed once with 37°C preheated PBS (0.02 mol / L, pH 7.4). Cell maintenance solution (α-MEM + 0.5 μg / ml trypsin) was added, and the cells were cultured at 37°C in 5% CO2 for 20-48 hours. When the CPE reached more than 80%, the virus solution was harvested, subjected to 2-3 freeze-thaw cycles, and stored at -20°C.

[0072] Example 4 Preparation of inactivated vaccine of porcine rotavirus SCLS3 strain

[0073] The virus solution obtained in Example 3 was centrifuged at 3000 rpm / min for 10 minutes to remove cell debris. Then 0.1%-0.2% formaldehyde solution was added for inactivation at 37°C for 24 hours. 0.1-0.2% neutralizing agent was added to neutralize the toxicity of formaldehyde. The inactivated porcine rotavirus was subjected to sterility test, mycoplasma test, and foreign virus test according to the method of Chinese Veterinary Pharmacopoeia (China Veterinary Drug Committee, 2010 edition, part 3, China Agriculture Press, 2010). The results showed that the inactivated porcine rotavirus SCLS3 strain was not contaminated by bacteria and molds, nor infected by mycoplasma and foreign viruses, and was of good purity.

[0074] The inactivated antigen is slowly added into the adjuvant under low speed condition, and mixed well, and then stirred at 250 r / min for 10 min. The adjuvant used in the present application can be known to those skilled in the art. In the present application, the adjuvant is selected to be a biphasic adjuvant (water-in-oil-in-water emulsion), for example, can be Montanide ISA 201 adjuvant. The specific ratio is shown in Table 4. After emulsification, the vaccine is subjected to dosage form test, which is operated as follows: a clean pipette is taken, and a small amount of vaccine is dropped on the surface of clean cold water, and the vaccine is diffused in the form of cloud, which shows that the dosage form of the vaccine is qualified. After emulsification, the vaccine is subjected to stability test, which is operated as follows: 10 ml of the vaccine is taken and added into a centrifugal tube, and then centrifuged at 3000 r / min for 15 min, and no delamination and demulsification is observed, which shows that the stability of the vaccine is qualified.

[0075] Table 4: Inactivated vaccine of porcine rotavirus SCLS3 strain

[0076] Example 5: Immunogenicity test of inactivated vaccine of porcine rotavirus SCLS3 strain

[0077] 20 piglets of 3-5 days old, which are negative for antigens and antibodies of porcine delta coronavirus, porcine rotavirus, porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus, are randomly divided into 4 groups, 5 piglets in each group. The piglets in the first group are injected with the vaccine 1 prepared in Example 4 (1 ml per piglet) in the neck muscle, the piglets in the second group are injected with the vaccine 2 prepared in Example 4 (1 ml per piglet) in the neck muscle, the piglets in the third group are injected with the vaccine 3 prepared in Example 4 (1 ml per piglet) in the neck muscle, and the piglets in the fourth group are injected with the same amount of sterile PBS in the neck muscle as the control group. The mental state, diet, body temperature and fecal condition of the piglets after immunization are observed.

[0078] On the 21st day after immunization, the blood is collected to separate serum, and the neutralizing antibody of the serum on the 21st day after immunization is determined by using the above-mentioned porcine rotavirus SCLS3 strain of P[7] type as the indicator virus. The results show that the neutralizing antibody titers of the serum of the control group are not higher than 1:8, and the neutralizing antibody titers of the serum of the immunized groups are not lower than 1:64. See Table 3 for details.

[0079] Table 3: Neutralizing antibody level of serum after immunization in different test groups

[0080] On the 21st day after immunization, all the piglets are orally administered with 5 ml of the porcine rotavirus SCLS3 strain of P[7] type virus liquid (containing 10 7.0 TCID 50 / ml), and the piglets are continuously observed for 7 days after the challenge. The results of the immunization and challenge protection are shown in Table 4.

[0081] Table 4 Results of challenge protection test of piglet immunized with inactivated porcine rotavirus vaccine

[0082] Note: " / " means that the piglet did not show mushy or watery stool after challenge.

[0083] The results showed that 21 days after immunization, the 5 piglets in the first group, the second group and the third group were normal in feeding, spirit, temperature and stool, and were all healthy. After challenge, 5 / 5 piglets in the fourth group showed typical symptoms of porcine rotavirus, such as vomiting and watery diarrhea. The protection rates of vaccine 1, vaccine 2 and vaccine 3 were all 100%, which indicated that the inactivated vaccine prepared by the strain had good immune protection effect on P[7] type porcine rotavirus infection.

[0084] Example 6 Evaluation of the protection of piglets by immunizing sows with inactivated porcine rotavirus vaccine

[0085] Twenty pregnant sows, which were negative for porcine delta coronavirus, porcine rotavirus, porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus antigen and antibody 4-6 weeks before delivery, were randomly divided into 4 groups, 5 sows in each group. The sows in the fifth group were injected with vaccine 1 prepared in Example 4 (2 ml per sow) in the neck muscle, the sows in the sixth group were injected with vaccine 2 prepared in Example 4 (2 ml per sow) in the neck muscle, the sows in the seventh group were injected with vaccine 3 prepared in Example 4 (2 ml per sow) in the neck muscle, and the sows in the eighth group were injected with the same amount of sterile PBS in the neck muscle as a control group. The mental state, diet and temperature of the sows after immunization were observed. The sows were boosted once again 2-4 weeks before delivery, and the mental state, diet and temperature of the sows after immunization were observed until the sows gave birth to piglets.

[0086] After the sows gave birth to piglets, 5 piglets of 3 days old from each sow in the fifth group to the eighth group were taken, and there were 25 piglets in each group, and a total of 100 piglets, which were all orally administered with 5 ml of P[7] type porcine rotavirus SCLS3 strain virus liquid (containing 10 7.0 TCID 50 / ml), and the clinical manifestations of the piglets after challenge were observed. The results are shown in Table 5.

[0087] Table 5 Results of evaluation of the protection of piglets by immunizing sows

[0088] The results show that: the piglets produced by the immunized sows, after 3 days of challenge, the 5th, 6th and 7th groups of immunized piglets have no abnormality in spirit, nursing, temperature and feces, and are all healthy; the 8th group of control piglets, after challenge, 25 / 25 piglets show typical porcine rotavirus symptoms, vomiting and watery diarrhea. The protection rates of the piglets produced by the immunized sows after immunization with the vaccine 1, the vaccine 2 and the vaccine 3 are all 100%, indicating that the inactivated vaccines 1, 2 and 3 have good protection effect, can induce high titer neutralizing antibodies and protect the nursing piglets against virus infection.

[0089] It should be noted that the above are only the preferred embodiments of the present application and the technical principles used. Those skilled in the art will understand that the present application is not limited to the specific embodiments described herein, and those skilled in the art can make various obvious changes, readjustments and substitutions without departing from the scope of the present application. Therefore, although the present application has been described in more detail through the above embodiments, the present application is not limited to the above embodiments, and can include more other equivalent embodiments without departing from the technical concept of the present application, all of which belong to the protection scope of the present application.

Claims

1. A strain of porcine rotavirus SCLS3 strain, characterized in that, The strain was deposited at China Center for Type Culture Collection on October 29, 2024, and the deposit number is CCTCC NO: V202494, and the deposit address is Wuhan, China, Wuhan University.

2. Use of the SCLS3 strain of claim 1 in the preparation of a vaccine for preventing and / or treating porcine rotavirus disease.

3. A vaccine composition for preventing porcine rotavirus disease, characterized in that, The vaccine comprises an immunizing amount of porcine rotavirus antigen comprising inactivated antigen of the porcine rotavirus SCLS3 strain of claim 1 or culture thereof and a pharmaceutically acceptable carrier.

4. The vaccine composition of claim 3, wherein, The porcine rotavirus SCLS3 strain is inactivated to a titer of > 10 5.0 TCID 50 / ml, preferably 10 5.0 - 10 7.0 TCID 50 / ml, further preferably 10 6.0 TCID 50 / ml.

5. The vaccine composition according to claim 3 or 4, characterized in that, The pharmaceutically acceptable carrier comprises at least one of an adjuvant, a lyoprotectant, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative.

6. The vaccine composition of claim 5, wherein, The adjuvant comprises one or more of aluminum hydroxide adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, E. coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, Montanide ISA 201, or Gel adjuvant.

7. The vaccine composition of claim 6, wherein, The adjuvant is a biphasic adjuvant for preparing a water-in-oil-in-water emulsion.

8. The vaccine composition of claim 5, wherein, The concentration of the adjuvant ranges from 5Wt% to 50Wt%, preferably 30Wt% to 50Wt%, and more preferably 50Wt%.

9. The vaccine composition of claim 5, wherein, The lyoprotectant is selected from a sugar, a polyol, a polymer, a surfactant, a salt, an amine, or an amino acid.

10. The vaccine composition of claim 5, wherein, The immunostimulant comprises α-interferon, β-interferon, γ-interferon, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin 2.

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