Cat feline calicivirus 100 strains and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a feline calicivirus 100 strain and application thereof. The feline calicivirus 100 strain is an artificially weakened virus strain, has stable biological characteristics and good immunogenicity, and a live attenuated vaccine prepared from the feline calicivirus 100 strain can obviously alleviate clinical symptoms caused by wild feline calicivirus, and can provide effective immunoprotection against epidemic strains in different regions.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically relating to 100 strains of feline calicivirus and its applications. Background Technology

[0002] Feline calicivirus disease is an upper respiratory tract infection caused by feline calicivirus (FCV). FCV primarily infects cats and wild cats such as tigers, cheetahs, and lions. Cats of all breeds and ages can be infected with FCV, with kittens being more susceptible. Affected cats mainly exhibit symptoms such as fever, nasal and ocular discharge, oral ulcers, and swollen or ulcerated paw pads. FCV was first isolated by Fastier et al. in 1957, and subsequent studies revealed its widespread prevalence globally. In 1999, Fan Quanshui et al. first isolated FCV in China.

[0003] Currently, there are no live vaccines related to feline calicivirus in China. Live vaccines can effectively stimulate the body to produce protective immunity. However, feline calicivirus has genetic diversity, and there are significant differences between the circulating strains in different regions. Therefore, it is particularly important and urgent to screen the dominant circulating strains in different regions of China and to develop and produce a safe and effective live vaccine related to feline calicivirus.

[0004] In view of the above, this application is hereby submitted. Summary of the Invention

[0005] One of the purposes of this application is to provide 100 strains of feline calicivirus to provide options for the prevention and detection of feline calicivirus.

[0006] To achieve the above objectives, this application adopts the following technical solution.

[0007] One hundred strains of feline calicivirus were deposited at the China Center for Type Culture Collection (CCTCC) on November 13, 2024, with accession number CCTCC NO:V2024109, at Wuhan University, Wuhan, China.

[0008] The use of 100 strains of feline calicivirus in any of the following (a)-(c):

[0009] (a) Preparation of products for detecting feline calicivirus or its antibodies;

[0010] (b) To prepare products for the prevention and / or treatment of feline calicivirus infection;

[0011] (c) Preparation of feline calicivirus antibodies.

[0012] Furthermore, the product in (b) includes a drug, which may be a vaccine.

[0013] A vaccine composition comprising an immunizing dose of feline calicivirus 100 antigen, wherein the feline calicivirus 100 antigen comprises live whole virus antigen of the aforementioned feline calicivirus 100 strain or a culture thereof.

[0014] Furthermore, the antigen content of 100 strains of feline calicivirus was ≥10. 6.5 TCID 50 / portion, can be set to 10 6.5 ~10 8.5 TCID 50 / portion, further optional 10 7.5 ~10 8.5 TCID 50 / Toufen.

[0015] Furthermore, the 100 strains of feline calicivirus culture are cultures that have undergone 1 to 110 passages.

[0016] Furthermore, it also includes freeze-drying protectants;

[0017] Optionally, the lyophilization protectant includes at least one of sugars, proteins, buffer solutions, and protein-containing substances.

[0018] Furthermore, the sugar is selected from sorbitol, mannitol, starch, sucrose, glucose or dextran, the protein is selected from albumin or casein, the protein-containing substance is bovine serum or skim milk, and the buffer solution is phosphate buffer.

[0019] Further, the freeze-drying protectant is an aqueous solution of 10 w / v%-30 w / v% sucrose and 1 w / v%-3 w / v% gelatin; more preferably, the freeze-drying protectant is an aqueous solution of 20 w / v% sucrose and 2 w / v% gelatin;

[0020] Optionally, the ratio of the freeze-drying protectant to feline calicivirus strain 100 antigen is 1:(0.5-1.5) by volume.

[0021] The application of this vaccine composition in the preparation of a medicament for preventing feline calicivirus infection, wherein the medicament may be a vaccine.

[0022] Compared with the prior art, the beneficial effects of this application are as follows:

[0023] The feline calicivirus strain 100 provided in this application exhibits significantly reduced pathogenicity compared to the parental virulent feline calicivirus strain 60, and is an artificially attenuated virus strain. Furthermore, the feline calicivirus strain 100 possesses stable biological characteristics and good immunogenicity, making it safe and reliable. The prepared attenuated live vaccine can significantly alleviate clinical symptoms caused by wild-type feline calicivirus and provides effective immune protection against prevalent strains in different regions.

[0024] The vaccine composition provided in this application can reduce clinical symptoms after cats are immunized with the virus, effectively prevent outbreaks caused by feline calicivirus, and provide effective immune protection against feline calicivirus infection in different regions. Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0026] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0027] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0028] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0029] The term "feline calicivirus" (FCV) belongs to the Caliciviridae family and is characterized by flu-like symptoms, such as an upper respiratory infection. Caliciviruses typically affect a cat's throat, eyes, nasal cavity, and mouth, but can sometimes also affect the lungs, musculoskeletal system, and intestines.

[0030] This application provides 100 strains of feline calicivirus (Strain 100), deposited at the China Center for Type Culture Collection (CCTCCNO.V2024109), Wuhan University, Wuhan, China, on November 13, 2024.

[0031] Pathogenicity tests showed that the pathogenicity of feline calicivirus strain 100, cultured between passage 1 and 110, was significantly reduced in cats. Cats inoculated at passage 110 and observed for 14 days showed no clinical symptoms, and no changes were observed in necropsy organs. Therefore, compared to the parent virulent feline calicivirus strain 60, this virus exhibits significantly reduced pathogenicity and is an artificially attenuated viral strain.

[0032] Immunogenicity tests showed that feline calicivirus strain 100, cultured to passage 110, still possessed good immunogenicity. Fourteen days after a single-dose repeat vaccination, cats vaccinated with feline calicivirus strain 60 showed significantly reduced clinical symptoms compared to cats vaccinated with strain 60.

[0033] Virulence reversion tests showed that viruses cultured from generation 1 to generation 110, after inoculation and repeated passages in a cat population, did not revert to virulent strains. Therefore, after inoculation into a cat population, the virus will not revert to a virulent strain and cause disease, ensuring its safety.

[0034] Compared with its parent strain 60 virulent feline calicivirus, the cultures of strain 100 from generation 1 to generation 110 all showed mutations at the amino acid sites E428K, S443N, S450G, S465G, I481V, V493L, E494N, and T519K in the amino acid sequence encoded by the major immunogenic protein VP1 gene.

[0035] The common characteristic changes in the amino acid encoding of the viral genes of 100 different passage cultures of feline calicivirus are likely the reason for the reduced virulence of its parent virulent strain 60, while these changes did not lead to changes in its immunogenicity.

[0036] The point mutation in the VP1 amino acid sequence of strain 100 of the attenuated feline calicivirus in this application reduces its virulence compared to strain 60 of the parent virulent strain, and also provides genetic stability.

[0037] This application also provides the use of the aforementioned 100 strains of feline calicivirus in the preparation of products for detecting feline calicivirus or its antibodies. The products can be reagents or kits, such as immunochromatographic test strips, ELISA kits, etc.

[0038] This application also provides the use of the aforementioned feline calicivirus strain 100 in the preparation of products for the prevention and / or treatment of feline calicivirus infection. The products may be pharmaceuticals, such as vaccines.

[0039] In some implementations, the drug is a live attenuated feline calicivirus vaccine.

[0040] This application also provides the use of the above-mentioned feline calicivirus strain 100 in the preparation of feline calicivirus antibodies.

[0041] This application provides a vaccine composition comprising an immunizing dose of feline calicivirus strain 100 antigen, wherein the feline calicivirus strain 100 antigen comprises the live whole virus antigen of the feline calicivirus strain 100 of this application or its culture.

[0042] The term "vaccine composition," also known as an immunogenic composition, refers to an immunogenic preparation, including whole cells, inactivated or attenuated, live viruses or bacteria, or polysaccharides, or combinations thereof, which are administered to stimulate a humoral and cellular immune response in a receptor 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 cat.

[0043] "Immunological dose" refers to the amount necessary for the composition to exert its immunological effects in the host to which it is administered without causing excessive side effects. The precise amounts of the ingredients used and the composition to be administered will vary depending on factors such as the type of disease being treated, the type and age of the animal being treated, the method of administration, and other components in the composition.

[0044] In a preferred embodiment, the antigen content of the feline calicivirus strain 100 is ≥10. 6.5 TCID 50 / portion, preferably 10 6.5 ~10 8.5 TCID 50 / portion, more preferably 10 7.5 ~10 8.5 TCID 50 / per dose. The antigen content of 100 strains of feline calicivirus can be selected from 10... 6.5 TCID 50 / First serving, 10 6.6 TCID 50 / First serving, 10 6.7 TCID 50 / First serving, 10 6.8 TCID 50 / First serving, 10 6.9 TCID 50 / First serving, 10 7.0 TCID 50 / First serving, 10 7.1 TCID 50 / First serving, 10 7.2 TCID 50 / First serving, 10 7.3 TCID 50 / First serving, 10 7.4 TCID 50 / First serving, 10 7.5 TCID 50 / First serving, 10 7.6 TCID 50 / First serving, 10 7.7 TCID 50 / First serving, 10 7.8 TCID 50 / First serving, 10 7.9 TCID 50 / First serving, 10 8.0 TCID 50 / First serving, 10 8.1 TCID 50 / First serving, 10 8.2 TCID 50 / First serving, 10 8.3 TCID 50 / First serving, 10 8.4 TCID 50 / First serving 10 8.5 TCID 50 / Toufen.

[0045] In a preferred embodiment, the 100 strains of feline calicivirus are cultures that have undergone 1 to 110 passages. Specifically, the 100 cultures of feline calicivirus can be selected from cultures of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, 35th, 40th, 45th, 50th, 55th, 60th, 65th, 70th, 75th, 80th, 85th, 90th, 95th, 100th, 105th, or 110th generation.

[0046] The term "culture of 100 strains of feline calicivirus from generation 1 to 110" refers to the culture obtained by continuously passaged naturally in F81 cells with 100 strains of feline calicivirus until they are adapted to grow and replicate in F81 cells (i.e., cytopathic effects such as rounding, aggregation, and shedding appear in the cells), and then continuing to passage naturally in F81 cells from generation 1 to 110.

[0047] In a preferred embodiment, the vaccine composition further includes a lyophilization protectant, which may be at least one of sugars, proteins, buffer solutions, and protein-containing substances. The sugars may be, for example, sorbitol, mannitol, starch, sucrose, glucose, or dextran; the proteins may be, for example, albumin or casein; the protein-containing substances may be, for example, bovine serum or skim milk; and the buffer solutions may be, for example, phosphate buffer. Preferably, the lyophilization protectant is an aqueous solution of 10 w / v%-30 w / v% sucrose and 1 w / v%-3 w / v% gelatin. Preferably, the lyophilization protectant is an aqueous solution of 20 w / v% sucrose and 2 w / v% gelatin. The sucrose concentration may be, but is not limited to, 10 w / v%, 11 w / v%, 12 w / v%, 13 w / v%, 14 w / v%, 15 w / v%, 16 w / v%, 17 w / v%, 18 w / v%, 19 w / v%, 20 w / v%, 21 w / v%, 22 w / v%, 23 w / v%, 24 w / v%, 25 w / v%, 26 w / v%, 27 w / v%, 28 w / v%, 29 w / v%, or 30 w / v, and the gelatin concentration may be, but is not limited to, 1 w / v%, 2 w / v%, or 3 w / v.

[0048] In a preferred embodiment, the ratio of the freeze-drying protectant to feline calicivirus 100 strain antigen is 1:(0.5-1.5) by volume, preferably 1:1.

[0049] This application provides a method for preparing a vaccine composition. According to the formulation amount, feline calicivirus strain 100 antigen can be used alone as the active ingredient of the vaccine composition, or it can be mixed with a freeze-drying protectant to obtain the vaccine composition.

[0050] This application provides for the use of a vaccine composition in the preparation of a medicament for the prevention of feline calicivirus infection. As used herein, the term "prevention" means the suppression of feline calicivirus infection or the reduction of disease symptoms by administration of the vaccine composition according to this application.

[0051] The present application is further described below with reference to specific embodiments. The advantages and features of the present application will become clear from the description. The embodiments described are merely 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 form of the technical solutions of the present application without departing from the spirit and scope of the present application, but such modifications and substitutions all fall within the protection scope of the present application.

[0052] All chemical reagents used in the embodiments of this application are of analytical grade and were purchased from Sinopharm Group.

[0053] To make this application easier to understand, specific embodiments are described below to further illustrate this application. Unless otherwise specified, the experimental methods described in this application are conventional methods; and unless otherwise specified, the biological materials described are all commercially available.

[0054] Example 1: Obtaining 100 strains of feline calicivirus

[0055] 1. Take well-grown F81 cells, digest them with trypsin, and inoculate them into cell culture flasks. Use cell growth medium containing 90%–98% (v / v) RPMI 1640 culture medium and 2%–10% (v / v) newborn calf serum (pH adjusted to 6.8–7.2) for virus inoculation.

[0056] 2. Adsorb 60 strains of feline calicivirus into F81 monolayer cells, and continue to culture in cell growth medium containing 90%–98% (v / v) RPMI 1640 medium and 2%–10% (v / v) newborn calf serum (pH adjusted to 6.8–7.2) at 33°C–37°C. When more than 80% of the cells show cytopathic effects, harvest the cell culture virus solution.

[0057] 3. Repeat the above steps for continuous subculturing to obtain a feline calicivirus attenuated strain. Sequencing of the obtained attenuated feline calicivirus strain showed stable amino acid variations in the VP1 protein at E428K, S443N, S450G, S465G, I481V, V493L, E494N, and T519K. This attenuated feline calicivirus strain was named feline calicivirus strain 100.

[0058] Example 2: Study on the biological characteristics of 100 strains of feline calicivirus

[0059] 1. Pathogenicity test

[0060] Fifteen healthy, susceptible, antigen-antibody-negative cats aged 2 months were randomly divided into 3 groups of 5 each. The grouping and challenge details are shown in Table 1.

[0061] Table 1 Grouping of Pathogenicity Test Animals Group Inoculation strain Inoculation dosage 1 100 strains <![CDATA[Nasal drops 1ml (10 7.0 TCID 50 / ml)]]> 2 60 plants <![CDATA[Nasal drip 1ml (10 7.0 TCID 50 / ml)]]> 3 RPMI 1640 medium 1ml nasal drops

[0062] The cats were observed for 14 days after vaccination. Their body temperature, appetite, eye and nasal secretions, oral cavity, paw pads and other clinical manifestations were observed and recorded daily. The specific results are shown in Table 2.

[0063] Table 2. Pathogenicity of 60 strains to cats

[0064] The results showed that strain 60 of feline calicivirus could cause 100% (5 / 5) disease in cats, while strain 100 of feline calicivirus had normal body temperature, no clinical symptoms, and no changes in tissues and organs upon necropsy.

[0065] Pathogenicity tests showed that strain 100 of feline calicivirus had significantly reduced pathogenicity compared to strain 60 of its parental virulent strains, making it an attenuated virus strain.

[0066] Meanwhile, to verify the stability of the pathogenicity of feline calicivirus strain 100 at different passages, cultures of feline calicivirus strain 100 from passages 1, 20, 50, 80, and 110 were used to inoculate one group of five feline calicivirus antigen-antibody negative cats, with 1 ml (10 ml) administered intranasally. 7.0 TCID 50 / ml) / head, 5 cats served as the control group. Clinical changes in the cats were observed and recorded daily until 14 days after vaccination.

[0067] The results showed that 100 cultures of feline calicivirus from generations 1, 20, 50, 80, and 110, after inoculation and observation for 14 days, had normal body temperature, no clinical symptoms, and no changes in tissues and organs upon necropsy.

[0068] Pathogenicity tests at different passages showed that the pathogenicity of 100 feline calicivirus strains at different passages was significantly reduced, indicating that they were attenuated virus strains.

[0069] 2. Immunogenicity test

[0070] Ten healthy, susceptible, antigen-antibody-negative cats aged 2 months were randomly divided into two groups of 5 each. The grouping and immunization details are shown in Table 3.

[0071] Table 3 Grouping of animals for immunogenicity testing Group Inoculation strain Inoculation dosage 4 100 strains <![CDATA[subcutaneous 1 ml (10 7.0 TCID 50 / ml)]]> 5 RPMI 1640 medium 1ml subcutaneous

[0072] A second immunization was administered 21 days after the initial immunization. Fourteen days after the second immunization, five cats inoculated with feline calicivirus strain 100 and five control cats were given 1 ml of feline calicivirus strain 60 via nasal drops (10... 7.0 TCID 50 Cats were challenged with a dose of virus ( / ml) per head. After challenge, daily observations and records were made of clinical manifestations such as body temperature, appetite, eye and nasal secretions, oral cavity, and paw pads. Clinical symptoms were scored daily according to the scoring criteria in Table 4 based on their duration. The total clinical score for each cat within 14 days after challenge was calculated, and the differences in clinical scores between the immunized group and the control group were compared. Specific results are shown in Table 5.

[0073] Table 4. Clinical symptom scoring criteria after feline calicivirus challenge.

[0074] Table 5. Immunogenicity of 100 strains to cats

[0075] Note: Different superscript letters indicate significant differences (P < 0.05).

[0076] The results showed that the clinical symptom scores of cats inoculated with the feline calicivirus strain 100 were significantly lower than those of the challenge control group.

[0077] Immunogenicity tests showed that feline calicivirus strain 100 had good immunogenicity and could significantly reduce the clinical symptoms caused by challenge with feline calicivirus strain 60.

[0078] Meanwhile, to verify the stability of the immunogenicity of feline calicivirus strain 100 at different passages, on day 14 after secondary immunization with feline calicivirus strain 100 cultures at passages 1, 20, 50, 80, and 110, 21 days after the initial immunization, all immunization groups, including the control group, received 1 ml (10 ml) of feline calicivirus strain 60 via nasal drops. 7.0 TCID 50 / ml) / head dose of virus was used to challenge the cats. After the challenge, the cats' body temperature, appetite, eye and nasal secretions, oral cavity, paw pads and other clinical manifestations were observed and recorded daily. The clinical symptoms were scored daily according to the scoring criteria in Table 4. The total clinical score of each cat within 14 days after the challenge was calculated and the difference in clinical scores between the immune group and the control group was compared.

[0079] The results showed that the clinical symptom scores of cats inoculated with 100 strains of feline calicivirus from generations 1, 20, 50, 80, and 110 were significantly lower than those of the challenge control group, and all cats in the control group developed the disease.

[0080] Immunogenicity assays at different passages showed that all 100 strains of feline calicivirus at different passages had good immunogenicity and could significantly reduce the clinical symptoms caused by challenge with strain 60 of feline calicivirus.

[0081] 3. Virus reversion safety test

[0082] 100 strains of feline calicivirus, first generation virus fluid (10 9.5 TCID 50 Three healthy, susceptible cats were inoculated with 1 ml of the solution via intranasal drip. Temperature and clinical manifestations were monitored daily in the morning after inoculation. The cats were euthanized on day 5 post-inoculation for lesion observation. Histopathological examination of the tonsils, larynx, and trachea was performed using HE staining and immunohistochemical staining. Feline calicivirus nucleic acid was determined from the homologous fluids of the tonsils, larynx, and trachea. Fluids with high viral loads were selected, appropriately processed, and used as inoculum for the next generation of virulence reversion, thus allowing the virus to be transmitted to the fourth generation in the cats.

[0083] The tonsil, larynx, and trachea homologous fluids obtained from the fourth-generation virulence reversion test were then inoculated into three healthy, susceptible cats via intranasal drops (1 ml each). Temperature and clinical manifestations were monitored daily in the morning after inoculation. Cats were euthanized on day 21 post-inoculation for lesion observation, and samples from the tonsils, larynx, and trachea were collected for HE and immunohistochemical analysis, as well as viral nucleic acid determination.

[0084] If, during the virulence reversion subculture tests from the 1st to the 4th generation, feline calicivirus nucleic acid is undetectable in the tonsils, larynx, or trachea in a certain generation, the inoculation dose should be appropriately increased, and 6 cats should be inoculated in the same manner to improve the viral nucleic acid detection rate. If further tests confirm that feline calicivirus nucleic acid cannot be detected again in the tonsils, larynx, or trachea after subculture, it indicates that the virulence reversion test result is valid, and the strain has no possibility of virulence reversion.

[0085] The results showed that no abnormalities were observed in the body temperature, clinical observation, necropsy, or histopathological examination of cats vaccinated in generations 1 through 5. On the 5th day post-vaccination, FCV was isolated from the tonsils, larynx, and trachea of ​​cats in generations 1 through 3 during virulence reversion tests, but not from the tonsils, larynx, or trachea of ​​cats in generations 4 and 5. The viral load in the tonsils, larynx, and trachea of ​​cats in generations 1 through 5 also showed a gradual decreasing trend. No significant pathological changes were observed by HE staining. Only one cat in generation 1 showed FCV-positive immunohistochemical staining of the tonsils; all other organ tests were FCV-negative. In the cohabiting experimental cats during the virulence reversion test, no abnormalities were observed in the body temperature, clinical observation, necropsy, or histopathological examination, and immunohistochemical staining of the tonsils, larynx, and trachea was negative. FCV nucleic acid was detected in the ocular and nasal swabs of three cohabiting cats in the first-generation horizontal transmission experiment, but the virus was not isolated. Neutralizing antibodies against FCV were also detected in the serum of the first-generation cohabiting cats. This indicates that the feline calicivirus strain 100 does not exhibit virulence reversion ability, but it does demonstrate horizontal transmission and is a well-tolerated, attenuated strain with good safety.

[0086] FCV was tested at passages 20, 50, 80, and 110 of 100 strains of feline calicivirus (FCV) using the same viral reversion test procedure described above. The results showed no abnormalities in body temperature, clinical observation, necropsy, or histopathological examination of cats inoculated from passages 1 to 5. Quantitative real-time RT-PCR also showed a gradual decrease in viral load in organs during passage. FCV was isolated from the tonsils, larynx, and trachea in passages 1 to 3, but not from the tonsils, larynx, or trachea in passages 4 to 5.

[0087] Therefore, strain 100 of feline calicivirus will not re-evolve into a virulent strain and cause illness, and its safety is guaranteed.

[0088] 4. Gene sequence analysis

[0089] Genome amplification was performed on 100 feline calicivirus cultures from passage 1 to passage 110 using RT-PCR (different passage cultures were amplified separately). The amplified gene products were recovered, purified, and ligated into sequencing plasmid vectors. The nucleotide sequence of the viral gene was determined, and the amino acid sequence was converted using computer software. Sequence analysis software was used to compare the obtained amino acid sequences with those of 60 virulent parental strains to describe the viral amino acid sequence characteristics.

[0090] The results showed that the common amino acid sequence of each gene in the 1st to 110th generations of 100 feline calicivirus cultures led to mutations in the VP1 protein at the E428K, S443N, S450G, S465G, I481V, V493L, E494N, and T519K amino acid sites.

[0091] This suggests that the common characteristic changes in the amino acid encoding of the viral genes of 100 different generations of feline calicivirus cultures are likely the reason for the reduced virulence of their parent virulent strains.

[0092] Example 3: Preparation of a live attenuated feline calicivirus vaccine using 100 strains

[0093] 1. Viral replication

[0094] One hundred strains of feline calicivirus prepared in Example 1 were adsorbed and inoculated into F81 monolayer cells. Cell growth medium containing 90%–98% (v / v) RPMI 1640 medium and 2%–10% (v / v) newborn calf serum (pH adjusted to 6.8–7.2) was added, and the cells were incubated at 37°C with 5% CO2. After 80% cytopathic effect was observed, the virus was harvested, the virus titer was determined, and the cells were stored at low temperature.

[0095] 2. Preparation of the protective agent

[0096] Add 20g of sucrose and 2g of gelatin to every 100ml of deionized water, dissolve them completely, and then autoclave (121℃, 30min).

[0097] 3. Vaccine preparation

[0098] The prepared and preserved virus solution was mixed with the protective agent at a 1:1 (volume ratio) and then freeze-dried. The specific proportions of the vaccine content are shown in Table 6.

[0099] Table 6. Content ratio of 100 strains of attenuated live feline calicivirus vaccine Components Vaccine 1 Vaccine 2 Vaccine 3 <![CDATA[100 antigen units (TCID 50 ) / dose]]> <![CDATA[10 6.5 ]]> <![CDATA[10 7.5 ]]> <![CDATA[10 8.5 ]]> Protectant (V / V) 50％ 50％ 50％

[0100] Example 4: Immunogenicity test of 100 strains of feline calicivirus live attenuated vaccine

[0101] Twenty feline calicivirus antigen-antibody-negative cats aged 2 months or older were randomly divided into 4 groups of 5 each. Each group was immunized with a live attenuated feline calicivirus vaccine of strain 100 prepared in Example 3. Group 6 was immunized with vaccine 1, Group 7 with vaccine 2, Group 8 with vaccine 3, and Group 9 served as the control group. The immunizations were administered subcutaneously twice, 21 days apart. Fourteen days after the second immunization, the cats were challenged with feline calicivirus strain 60 at a dose of 1 ml (10... 7.0 TCID 50 Cats were challenged with a dose of virus ( / ml) per head. After challenge, daily observations and records were made of the cats' body temperature, appetite, eye and nasal discharge, oral cavity, and paw pads. Clinical symptoms were scored daily according to the scoring criteria in Table 4. The total clinical score for each cat within 14 days after challenge was calculated, and the differences in clinical scores between the immunized group and the control group were compared. The clinical symptom scores of the immunized group were determined to be significantly lower than those of the challenged control group (P<0.05). Specific results are shown in Table 7.

[0102] Table 7. Immunogenicity test results of 100 strains of feline calicivirus live attenuated vaccine.

[0103] Note: Different superscript letters indicate significant differences (P < 0.05).

[0104] The results showed that immunization of cats with the live attenuated feline calicivirus vaccine prepared in Example 3 significantly reduced the clinical symptoms caused by the virulent strain.

[0105] The study demonstrated that the live attenuated feline calicivirus vaccine strain 100 in the three experimental groups had good protective efficacy, showing excellent immunoprotection and safety.

[0106] Example 5: Broad-spectrum test of feline calicivirus live attenuated vaccine strain 100

[0107] Forty healthy, susceptible, antigen-antibody-negative cats aged 2 months were randomly divided into 8 groups of 5 cats each. Groups 10-13 received 1 ml / cat of live vaccine subcutaneously, while groups 14-17 received 1 ml of saline subcutaneously as a blank control. Two immunizations were administered 21 days apart. Fourteen days after the second immunization, the cats were challenged with one of four feline calicivirus strains (32, 33, 35, and 72) from Tianjin, Henan, Shanghai, and Sichuan, respectively, with 1 ml (10 ml) administered intranasally to each cat. 7.0 TCID 50 / ml), the clinical manifestations of the cats were observed and recorded daily after the challenge, including body temperature, appetite, eye and nasal secretions, oral cavity, and paw pads. The clinical symptoms were scored daily according to the scoring criteria in Table 4 based on the duration of the clinical symptoms. The total clinical score of each cat within 14 days after the challenge was calculated, and the differences in clinical scores between the immune group and the control group were compared. The specific results are shown in Tables 8, 9, 10, and 11.

[0108] Table 8. Results of broad-spectrum trial of 100 strains of feline calicivirus attenuated live vaccine (challenge against 32 strains of feline calicivirus).

[0109] Note: Different superscript letters indicate significant differences (P < 0.05).

[0110] Table 9. Results of broad-spectrum trial of feline calicivirus live attenuated vaccine strain 100 (challenge against 33 strains of feline calicivirus).

[0111] Note: Different superscript letters indicate significant differences (P < 0.05).

[0112] Table 10. Results of broad-spectrum trial of 100 strains of feline calicivirus attenuated live vaccine (challenge against 35 strains of feline calicivirus).

[0113] Note: Different superscript letters indicate significant differences (P < 0.05).

[0114] Table 11. Broad-spectrum test results of 100 strains of feline calicivirus attenuated live vaccine (challenge against 72 strains of feline calicivirus).

[0115] Note: Different superscript letters indicate significant differences (P < 0.05).

[0116] The results showed that all patients in the control group developed the disease, and the clinical scores of the patients immunized with the feline calicivirus strain 100 attenuated live vaccine were significantly lower than those of the control group after challenge.

[0117] This application demonstrates that the feline calicivirus strain 100 attenuated live vaccine provided can significantly reduce clinical symptoms after challenge with feline calicivirus from different geographical origins. The feline calicivirus strain 100 attenuated live vaccine of this application has broad-spectrum immunogenicity and can significantly reduce clinical symptoms after challenge with feline calicivirus from different geographical origins.

[0118] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

[0119] Note that the above are merely preferred embodiments and the technical principles employed in this application. Those skilled in the art will understand that this application is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the technical concept of this application, all of which fall within the scope of protection of this application.

Claims

1. One hundred strains of feline calicivirus, characterized in that, It was deposited at the China Center for Type Culture Collection on November 13, 2024, with accession number CCTCC NO:V2024109, and the deposit address is Wuhan University, Wuhan, China.

2. The use of the feline calicivirus strain 100 of claim 1 in any one of the following (a)-(c): (a) Preparation of products for detecting feline calicivirus or its antibodies; (b) To prepare products for the prevention and / or treatment of feline calicivirus infection; (c) Preparation of feline calicivirus antibodies.

3. The application according to claim 2, characterized in that, The product in (b) includes medicines, which may be vaccines.

4. A vaccine composition, characterized in that, The vaccine composition comprises an immunizing dose of feline calicivirus 100 antigen, wherein the feline calicivirus 100 antigen comprises live whole virus antigen of the aforementioned feline calicivirus 100 strain or its culture.

5. The vaccine composition according to claim 4, characterized in that, The antigen content of 100 strains of feline calicivirus is ≥10 6.5 TCID 50 / portion, can be set to 10 6.5 ~10 8.5 TCID 50 / portion, further optional 10 7.5 ~10 8.5 TCID 50 / Toufen.

6. The vaccine composition according to claim 4, characterized in that, The 100 strains of feline calicivirus were cultures from passage 1 to 110.

7. The vaccine composition according to any one of claims 4 to 6, characterized in that, It also includes freeze-drying protectants; Optionally, the lyophilization protectant includes at least one of sugars, proteins, buffer solutions, and protein-containing substances.

8. The vaccine composition according to claim 7, characterized in that, The sugars are selected from sorbitol, mannitol, starch, sucrose, glucose, or dextran; the proteins are selected from albumin or casein; the protein-containing substance is bovine serum or skim milk; and the buffer solution is phosphate buffer.

9. The vaccine composition according to claim 7, characterized in that, The freeze-drying protectant is an aqueous solution of 10 w / v%-30 w / v% sucrose and 1 w / v%-3 w / v% gelatin; more preferably, the freeze-drying protectant is an aqueous solution of 20 w / v% sucrose and 2 w / v% gelatin. Optionally, the ratio of the freeze-drying protectant to feline calicivirus strain 100 antigen is 1:(0.5-1.5) by volume.

10. The use of the vaccine composition according to any one of claims 4 to 9 in the preparation of a medicament for preventing feline calicivirus infection, wherein the medicament may be a vaccine.

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