Porcine rotavirus LNSY5 strain and application thereof

Abstract

The invention relates to the field of research and development of veterinary biological products, in particular to a porcine rotavirus LNSY5 strain and application thereof. The porcine rotavirus strain LNSY5 provided by the invention is an epidemic porcine rotavirus wild strain, and the vaccine composition containing the porcine rotavirus strain can enable an animal body to quickly generate an antibody after immunizing an animal, and has good prevention and control effects on current epidemic porcine rotavirus infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of veterinary biological products research and development, in particular to a porcine rotavirus LNSY5 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 group A rotavirus is the main clinically prevalent porcine rotavirus. Porcine rotavirus is one of the main pathogenic agents causing diarrhea in piglets, and pigs of all ages can be infected with rotavirus. Young piglets mainly show symptoms such as diarrhea, dehydration, and vomiting after infection, and adult pigs show latent infection. After infection, pigs continue to shed the virus, which causes great difficulty in disease prevention and control 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. Literature reports that the main clinically prevalent G type is mainly G9, G5, G4, and G3, and the P type is mainly P

[23] , P

[13] , and P[7]. In 2020-2024, feces, intestinal samples, and other samples from suspected diarrhea piglets were detected, and it was found that P

[13] was the second most prevalent strain after P

[23] strain, and its harm in the clinic was also not negligible.

[0004] At present, there is no effective treatment for porcine rotavirus disease, and vaccination is an effective means of preventing and controlling porcine rotavirus disease. The existing RVA vaccine is mainly attenuated live vaccine, which belongs to G5P[7] strain, and has limited cross-neutralization ability to prevalent strains. Therefore, the development of inactivated vaccine against the prevalent genotype of porcine rotavirus is of great significance for the prevention and control of the prevalence and outbreak of porcine rotavirus.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide a porcine rotavirus LNSY5 strain and application thereof, which can effectively prevent the infection of porcine rotavirus.

[0007] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A porcine rotavirus LNSY5 strain, preserved in China Center for Type Culture Collection on October 29, 2024, with a preservation number of CCTCC NO: V202493, a preservation address of Wuhan, Wuhan University, China, and a classification name of porcine rotavirus, Strain LNSY5.

[0009] The LNSY5 strain is used in 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 the inactivated antigen of the porcine rotavirus LNSY5 strain or culture of claim 1.

[0011] In some embodiments, the porcine rotavirus LNSY5 strain is ≥ 10 5.0 TCID 50 / ml, preferably 10 5.0 ~ 10 6.0 TCID 50 / ml, further preferably 10 5.5 TCID 50 / ml.

[0012] In some embodiments, 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] In some embodiments, the adjuvant comprises one or more of 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.

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

[0015] In some embodiments, the concentration of the adjuvant ranges from 5 Wt% to 50 Wt%, preferably 30 Wt% to 50 Wt%, and more preferably 50 Wt%.

[0016] In some embodiments, the lyoprotectant is selected from a sugar, a polyol, a polymer, a surfactant, a salt, an amine, or an amino acid.

[0017] In some embodiments, the immunostimulant comprises 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 LNSY5 strain of the present application is a prevalent porcine rotavirus wild strain, and a 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 the purpose of description, 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 any one of the two parallel schemes of "yes" or "no". If there are multiple "optional" in a technical solution, and there is no special description, and there is no contradictory relationship or mutual restriction, each "optional" is independent.

[0025] In the present application, “a plurality of”, “a plurality of kinds”, “a plurality of times”, “a plurality of elements” and the like refer to more than two or equal to two in number, unless otherwise specified. For example, “one or more” means one or more than two.

[0026] The term “Porcine Rotavirus” (PoRV) is a member of the Reoviridae family, Rotavirus genus, which consists of 11 double-stranded RNA segments, and the clinical symptoms induced include diarrhea, accompanied by anorexia, vomiting, dehydration, etc. The pathological changes are characterized by thin and transparent small intestinal wall, watery contents, and atrophy of small intestinal villi.

[0027] 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.

[0028] The present application relates to the Porcine Rotavirus LNSY5 strain, which is deposited at the China Center for Type Culture Collection (CCTCC) and has the accession number CCTCC NO: V202493. The depositing address is Wuhan University, Wuhan, China, and the depositing date is October 29, 2024. The virus strain belongs to the G3-P

[13] -I5-M1-C1-A8-N1-T7-E1-H1 type.

[0029] The strain has strong virulence and good immunogenicity, and can be used for preparing a vaccine for preventing and / or treating Porcine Rotavirus disease.

[0030] The present application provides a vaccine composition for preventing Porcine Rotavirus disease, which comprises an immunizing amount of a Porcine Rotavirus antigen and a pharmaceutically acceptable carrier. The Porcine Rotavirus antigen comprises the inactivated antigen of the Porcine Rotavirus LNSY5 strain of the present application or a culture thereof. The vaccine composition is prepared from a virulent strain and has good immunogenicity, which can provide complete protection for pigs.

[0031] The term “vaccine composition” is also referred to as “immunogenic composition”, which refers to a preparation containing an immunogen, including, for example, whole cells, inactivated or attenuated, live viruses or bacteria, or polysaccharides, or a combination 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.

[0032] The term "immune dose" should be understood as "immunely effective dose," also known as immune protective dose or effective dose to elicit an immune response. It refers to the amount of antigen that can effectively induce an immune response in a recipient, sufficient to prevent or improve the signs or symptoms of disease, including adverse health effects or complications thereof. This immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of disease, including adverse health outcomes or complications of infection caused by a pathogen. Humoral immunity or cell-mediated immunity, or both, can be induced. An animal's immune response to an immunogenic composition can be indirectly assessed, for example, by measuring antibody titers, lymphocyte proliferation analysis, or directly assessed by monitoring signs or symptoms after challenge with a wild-type strain. The protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs in the subject such as mortality, reduction in morbidity, temperature values, overall physiological status, and overall health and performance. The immune response may include, but is not limited to, the induction of cellular and / or humoral immunity.

[0033] The term "porcine rotavirus antigen" refers to any composition containing at least one form of porcine rotavirus antigen that can induce, stimulate, or resist an immune response to porcine rotavirus infection. The antigen form includes, but is not limited to, inactivated, attenuated, or subunit antigens. Inactivated antigens can be inactivated and maintain their immunogenicity by various methods, including chemical treatment, physical treatment (such as acoustic treatment, irradiation, heat), or any other commonly used method sufficient to inhibit the replication or growth of the organism. Preferably, the pathogen is inactivated after collection and optionally subjected to clarification and purification. Chemical treatment uses, for example, formalin or formaldehyde, β-propiolactone, ethyleneimine, diethyleneimine BEI, thimerosal, etc. Inactivation methods are well known to those skilled in the art, such as treatment with β-propiolactone (Plana-Duran et al., Vet. Microbiol., 1997, 55:361-370) or with BEI (US5587164).

[0034] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent in the vaccine composition of this application that does not irritate the body or impede the biological activity and properties of the compound, except for the porcine rotavirus antigen, and is preferably an adjuvant.

[0035] In some implementations, the porcine rotavirus LNSY5 strain is ≥10 μmol / L before inactivation. 5.0 TCID 50 / ml, preferably 10 5.0 ~10 6.0 TCID 50 / ml, more preferably 10 5.5 TCID 50 / ml.

[0036] In some embodiments, the pharmaceutically acceptable carrier includes 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.

[0037] The term "adjuvant" can include an aluminum hydrogel adjuvant; a saponin, such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); a water-in-oil emulsion; an oil-in-water emulsion; a water-in-oil-in-water emulsion; a polymer of acrylic or methacrylic acid; a copolymer of maleic anhydride and an alkenyl derivative selected from the group consisting of compounds.

[0038] The term "emulsion" can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from the oligomerization of alkenes, such as squalane or squalene oil, in particular iso-octene or eucalyptene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(octanoate / eucalyptate), glycerol tri-(octanoate / eucalyptate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearyl esters. The oils are used in combination with emulsifiers in order to form emulsions. The emulsifiers are preferably non-ionic surfactants, in particular esters of sorbitan, esters of mannide (such as anhydrous mannitol oleate), esters of aliphatic glycols, esters of polyglycerol, esters of propylene glycol and esters 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] In some embodiments, the adjuvant is a biphasic adjuvant, which is used to prepare a water-in-oil-in-water emulsion.

[0044] 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%.

[0045] The concentration of the adjuvant can range from, 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%.

[0046] 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.

[0047] In some embodiments, the immunostimulant comprises alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor or interleukin 2.

[0048] The term "preventing and / or treating" in relation to porcine rotavirus infection refers to inhibiting the replication of porcine rotavirus, inhibiting the transmission of porcine rotavirus or preventing the establishment of porcine rotavirus in its host, as well as alleviating the symptoms of the disease or condition caused by porcine rotavirus infection. The treatment is considered to have therapeutic effect if the viral load is reduced, the condition is alleviated and / or the feed intake and / or growth is increased.

[0049] The application will be further described with reference to the following specific examples, which are intended to be purely exemplary and not limiting of the scope of the application. It will be understood by those skilled in the art that modifications or substitutions can be made to the details and forms of the application without departing from the spirit and scope of the application.

[0050] The chemical reagents used in the embodiments of the present application are all of analytical purity and purchased from the National Pharmaceutical Group.

[0051] 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 unless otherwise specified; the biological materials described in the present application can be obtained from commercial channels unless otherwise specified.

[0052] Example 1 Isolation and identification of porcine rotavirus

[0053] 1.1 Virus isolation The intestinal tract of a diarrhea piglet positive for PoRV identified by RT-qPCR method was taken, and the intestinal contents were used to prepare a suspension in α-MEM medium at a volume ratio of 1:5. The suspension was frozen-thawed once at -80℃, centrifuged at 10,000 rpm for 10 min at 4℃, and the supernatant was filtered with a 0.22 μm filter. The filtered supernatant 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 MA104 cells grown to a single layer was washed once with PBS, and 1 ml of the supernatant was added and adsorbed for 1 h in a 37℃, 5% CO2 incubator. The virus solution was then 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 cytopathic effect, and the culture was frozen-thawed 3 times and continued to be blind passaged for 5 days. On the third day of culture of the F3 generation, obvious cytopathic effect (cell rounding, aggregation, filamentation, and shedding) was observed.

[0054] 1.2 IFA identification of the isolated strain The harvested F6 generation virus solution was diluted 100 times and inoculated into MA104 cells grown to a single layer in a 96-well cell culture plate, with a negative control. After 36 h of inoculation, the culture medium was discarded, and the cells were washed once with PBS. Each well was added with 80% pre-cooled acetone and incubated at 2-8℃ for 30 min. After the pre-cooled acetone was discarded, the cells were washed twice. Porcine rotavirus polyclonal antibody was added and incubated at 37℃ for 1 h. The liquid was discarded, and the cells were washed 3 times with PBS. FITC-labeled goat anti-mouse secondary antibody was added and incubated at 37℃ for 1 h. The cells were washed 3 times with PBS. Each well was added with 50 μl of PBS and 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

[0055] 1.3 RT-qPCR identification and sequence analysis The F0-F3 generation cell cultures were identified by the RT-qPCR method in the industry standard SN / T 5196-2020 Technical Specifications for Porcine Rotavirus Infection Quarantine. The results showed that the CT value decreased with the increase of the generation, proving that the porcine rotavirus was successfully isolated.

[0056] ​This experiment used the gene sequences of various porcine rotavirus fragments collected in GenBank and referenced relevant literature to design primers for amplifying each gene. The nucleotide sequences of the primers are shown in Table 1.

[0057] Table 1 Primer information for the whole genome of PoRV

[0058] The isolated strain was amplified by PCR using the above-mentioned specific sequencing primers. The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. After the sequences were spliced ​​and cut, they were compared with the published rotavirus gene sequences in NCBI for homology. The results showed that five gene fragments (VP4, VP7, VP6, VP3, NSP4) of this strain had the highest homology with porcine rotavirus, with nucleotide identity of 94.30% to 97.06%. Four gene fragments (VP2, NSP1, NSP3, NSP5) had the highest homology with human rotavirus, with nucleotide identity of 94.74% to 98.82%. One gene fragment (NSP2) had the highest homology with canine rotavirus, with nucleotide identity of 97.06%. This virus strain belongs to the G3-P

[13] -I5-M1-C1-A8-N1-T7-E1-H1 type, suggesting that it may be transmitted between humans, pigs, and dogs. Table 2 shows the nucleotide consistency results with closely related strains.

[0059] Table 2. Nucleotide identity analysis between LNSY5 strain and closely related strains.

[0060] 1.4 Virus Titration: Take 96-well plates containing monolayers of MA104 cells, discard the culture medium, and wash once with PBS. Perform 10-fold serial dilutions of the continuously passaged virus solution in α-MEM medium containing 0.5 μg / ml trypsin. -3 ~10 -7 The diluted virus solution was inoculated into 96-well plates, with 4 wells for each dilution, and positive and negative control wells were also included. The plates were incubated at 37°C in a 5% CO2 incubator for 3–5 days, and CPE was observed and recorded daily. The viral titer was calculated using the Reed-Muench method; the titer of this porcine rotavirus strain was 10. 7.5 This indicates that the porcine rotavirus strain can be stably passaged in MA104 cells and yields high-titer viral fluid, making it a potential candidate strain for a porcine rotavirus vaccine. The porcine rotavirus strain was named Porcine Rotavirus LNSY5 and deposited with the accession number CCTCC NO: V202493.

[0061] Example 2 Pathogenicity test of porcine rotavirus LNSY5 strain

[0062] The pregnant sows were bled for detection of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) antigen, and porcine rotavirus (PoRV) IFA antibody. The anal swabs were collected for detection of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and PoRV antigen. The piglets born from sows negative for PoRV antigen and antibody, PRRSV, PCV2, PEDV, and TGEV antigen were selected for the test. The piglets did not suck colostrum. Ten 3-day-old piglets that did not suck colostrum were randomly divided into two groups, five in each group. The piglets in the first group were orally inoculated with 1 ml of porcine rotavirus LNSY5 strain virus solution (virus content: 10 6.0 TCID 50 / ml); the piglets in the second group were blank control and orally inoculated with 1 ml of α-MEM culture solution. The clinical symptoms were observed every day. The clinical symptoms: the piglets in the second group were normal in spirit and diet, and normal in feces, and no diarrhea was observed. All the piglets in the first group were sick, and the clinical symptoms of vomiting and watery diarrhea were observed. The piglets were decreased in spirit and appetite, and emaciated. The results are shown in Table 3.

[0063] Table 3 Results of pathogenicity test of porcine rotavirus LNSY5 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

[0064] After the piglets in the challenge group showed the clinical symptoms, necropsy was performed to observe the pathological changes of the organ tissues. The small intestinal contents of the sick piglets were taken, and the viral RNA was extracted for RT-PCR detection. The virus was isolated, inoculated into MA104 cells, and the cytopathic effect was observed.

[0065] Necropsy lesions: the piglets in the blank control group had no abnormal small intestine, and no abnormal other organs. The piglets in the challenge group had thin and transparent small intestinal wall, and the wall was filled with light yellow watery contents.

[0066] RT-PCR analysis of the small intestinal contents collected from the piglets subjected to necropsy showed PoRV positive and PEDV and TGEV negative. The small intestinal contents were inoculated into MA104 cells, and the same cytopathic effect was observed. This indicated that the porcine rotavirus was indeed the cause of diarrhea in the piglets in the test. The isolated strain LNSY5 strain described in the application is a strong strain of porcine rotavirus.

[0067] Example 3 Virus culture of porcine rotavirus LNSY5 strain

[0068] MA104 cells were taken and a good monolayer was formed. The cell culture solution was discarded and the cells were washed once with PBS (0.02 mol / L, pH 7.4) preheated at 37 °C. The cells were infected with the virus diluted 1000 times 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 PBS (0.02 mol / L, pH 7.4) preheated at 37 °C. Cell maintenance solution (α-MEM + 0.5 μg / ml trypsin) was added and the cells were cultured at 37 °C in a 5% CO2 environment 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.

[0069] Example 4 Preparation of the inactivated vaccine of porcine rotavirus LNSY5 strain

[0070] 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 and inactivated 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 LNSY5 strain was not contaminated by bacteria and molds, and was not infected by mycoplasma and foreign viruses, and was of good purity.

[0071] The inactivated antigen was slowly added to the adjuvant under low speed conditions, and then mixed and stirred at 250 r / min for 10 minutes. The adjuvant suitable for the present application can be known to those skilled in the art. In the present application, the adjuvant selected is a biphasic adjuvant (water-in-oil-in-water emulsion), which can be, for example, Montanide ISA 201 adjuvant. The specific ratio is shown in Table 4. After emulsification, the vaccine was subjected to dosage form test, which was performed as follows. A clean pipette was taken and a small amount of vaccine was dropped onto clean cold water, which spread in the form of mist, indicating that the vaccine dosage form was qualified. The emulsified vaccine was subjected to stability test, which was performed as follows. 10 ml of vaccine was added to a centrifuge tube and centrifuged at 3000 r / min for 15 minutes. No layering and demulsification were observed, indicating that the vaccine was stable.

[0072] Table 4 Ratio of the inactivated vaccine of porcine rotavirus LNSY5 strain

[0073] Example 5 Immunogenicity test of the inactivated vaccine of porcine rotavirus LNSY5 strain

[0074] Twenty piglets, 3-5 days old, negative for antibodies against porcine delta coronavirus, porcine rotavirus, porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus antigens, were randomly divided into four groups, 5 piglets in each group. The piglets in the first group were injected with the vaccine 1 prepared in Example 4 (1 ml per piglet) in the neck muscle, the piglets in the second group were injected with the vaccine 2 prepared in Example 4 (1 ml per piglet) in the neck muscle, the piglets in the third group were injected with the vaccine 3 prepared in Example 4 (1 ml per piglet) in the neck muscle, and the piglets in the fourth group were injected with the same amount of sterile PBS in the neck muscle as a control group. The mental state, diet, body temperature and stool condition of the piglets after immunization were observed.

[0075] Twenty-one days after immunization, blood was collected to separate serum, and the serum was subjected to neutralizing antibody determination using the porcine rotavirus LNSY5 strain of the P

[13] type as an indicator virus. The results showed that the neutralizing antibody titers of the serum in the control group were not higher than 1:8, and the neutralizing antibody titers of the serum in the immunized groups were not lower than 1:64. See Table 5 for details.

[0076] Table 5 Neutralizing antibody levels in serum after immunization in different test groups

[0077] Twenty-one days after immunization, all piglets were orally administered with 5 ml of the porcine rotavirus LNSY5 strain virus solution (containing 10 6.0 TCID 50 / ml). The piglets were continuously observed for 7 days after the challenge, 5 / 5 piglets in the immunized groups were protected, and 5 / 5 piglets in the challenge control group were sick. See Table 6 for the immunization and challenge protection results.

[0078] Table 6 Immunization of piglets with the inactivated vaccine of the porcine rotavirus LNSY5 strain and challenge protection test results

[0079] Note: " / " indicates that the piglets did not have mushy or watery stool after the challenge.

[0080] The test results showed that 21 days after immunization and challenge, the 5 piglets in the first group, the second group and the third group were healthy and showed no abnormalities in suckling, mental state, body temperature and stool; 5 / 5 piglets in the fourth group showed typical symptoms of porcine rotavirus, such as vomiting and watery diarrhea after the challenge. The protection rates of the vaccine 1, the vaccine 2 and the vaccine 3 were all 100%, indicating that the inactivated vaccine prepared from the strain had good immune protection effect on diarrhea caused by the infection of the porcine rotavirus of the P

[13] type.

[0081] Example 6 Evaluation of the protection of piglets by immunization of sows with the porcine rotavirus inactivated vaccine

[0082] Select 20 pregnant sows which are negative for porcine delta coronavirus, porcine rotavirus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus antigen antibody in prenatal 4-6 weeks, and randomly divide into 4 groups, 5 sows in each group. The 5th group is injected with the vaccine 1 prepared in Example 4 (2 ml per head) in the neck muscle, the 6th group is injected with the vaccine 2 prepared in Example 4 (2 ml per head) in the neck muscle, the 7th group is injected with the vaccine 3 prepared in Example 4 (2 ml per head) in the neck muscle, and the 8th group is injected with the same amount of sterile PBS in the neck muscle as a control group. The mental state, diet, and body temperature of the sows after immunization are observed. The sows are boosted once again in prenatal 2-4 weeks, and the mental state, diet, and body temperature of the sows after immunization are observed until the sows give birth to piglets.

[0083] After the sows give birth to piglets, 5 piglets of 5 days old are taken from each of the sows in the 5th group to the 8th group, and there are 25 piglets in each group, and a total of 100 piglets, which are all orally given 1 ml of P

[13] type porcine rotavirus LNSY5 strain virus liquid (containing 10 6.0 TCID 50 / ml), and the clinical manifestations of the piglets after challenge are observed. The results are shown in Table 7.

[0084] Table 7 Evaluation results of the protection of piglets immunized by sows

[0085] The results show that the piglets immunized by the sows are challenged at 5 days old, and the mental state, suckling, body temperature, and feces of the piglets in the 5th group, the 6th group, and the 7th group are normal, and the piglets are all healthy. The 25 / 25 piglets in the 8th group show typical porcine rotavirus symptoms, such as vomiting and watery diarrhea, after challenge. The protection rate of the piglets born by the sows immunized by the vaccine 1, the vaccine 2, and the vaccine 3 is 100%, which indicates that the inactivated vaccines 1, 2, and 3 have good protection effect, can induce high-titer neutralizing antibodies, and protect the suckling piglets from the disease.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. If there is a conflict between the definitions provided in this application and those provided in the art to which this application pertains, the definitions provided in this application shall control. In addition, the terminology used in the present specification is for the purpose of describing the embodiments of the present application only and is not intended to be limiting of the present application.

[0087] Note that the above only describes the preferred embodiments of the present application and the applied technical principles. Those skilled in the art will understand that the present application is not limited to the specific embodiments described herein, and that various obvious changes, reconfigurations and substitutions can be made by those skilled in the art without departing from the scope of the present application. Therefore, although the present application has been described in 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, and all fall within the protection scope of the present application.

Claims

1. A porcine rotavirus strain LNSY5, characterized in that, It was deposited at the China Center for Type Culture Collection on October 29, 2024, with accession number CCTCC NO: V202493, and the deposit address is Wuhan University, Wuhan, China.

2. The use of the LNSY5 strain according to claim 1 in the preparation of a vaccine for the prevention and / or treatment of porcine rotavirus disease.

3. A vaccine composition for the prevention of porcine rotavirus disease, characterized in that, It includes an immunizing dose of porcine rotavirus antigen and a pharmaceutically acceptable vector, wherein the porcine rotavirus antigen includes the inactivated antigen of the porcine rotavirus LNSY5 strain or its culture as described in claim 1.

4. The vaccine composition according to claim 3, characterized in that, The porcine rotavirus strain LNSY5 was ≥10 before inactivation. 5.0 TCID 50 / ml, preferably 10 5.0 ~10 6.0 TCID 50 / ml, more preferably 10 5.5 TCID 50 / ml.

5. The vaccine composition according to claim 3 or 4, characterized in that, The pharmaceutically acceptable carriers include at least one of adjuvants, lyophilization protectants, immunostimulants, antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.

6. The vaccine composition according to claim 5, characterized in that, The adjuvants include one or more of the following: aluminum gel adjuvant, saponins, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions, polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives, RIBI adjuvant systems, Block co-polymer, SAF-M, monophospholipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-sensitive enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, MontanideISA 206, MontanideISA 201, or gel adjuvant.

7. The vaccine composition according to claim 5, characterized in that, The adjuvant is a biphasic adjuvant used to prepare water-in-oil-in-water emulsions.

8. The vaccine composition according to claim 5, characterized in that, The concentration range of the adjuvant is from 5 wt% to 50 wt%, preferably from 30 wt% to 50 wt%, more preferably 50 wt%.

9. The vaccine composition according to claim 5, characterized in that, The freeze-drying protectant is selected from sugars, polyols, polymers, surfactants, salts, amines, or amino acids.

10. The vaccine composition according to claim 5, characterized in that, The immunostimulants include α-interferon, β-interferon, γ-interferon, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, or interleukin-2.

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