Feline herpesvirus type 1 strain 110 and uses thereof

Abstract

The application belongs to the technical field of veterinary biological products, and particularly relates to a feline herpesvirus 1 type 110 strain and application thereof. The feline herpesvirus 1 type 110 strain is an artificially weakened virus strain, has stable biological characteristics and good immunogenicity, and a live attenuated vaccine prepared from the feline herpesvirus 1 type 110 strain can obviously alleviate clinical symptoms caused by wild feline herpesvirus 1 type, and can provide effective immunoprotection against epidemic strains in different regions.

Description

Technical Field

[0001] This application belongs to the field of veterinary biological products technology, specifically relating to a strain of feline herpesvirus type 1, strain 110 and its application. Background Technology

[0002] Feline infectious rhinotracheitis is a respiratory disease caused by feline herpesvirus type 1 (FHV-1), characterized by upper respiratory tract infection, keratitis, and conjunctivitis as the main symptoms. FHV-1 belongs to the Herpesviridae family, Alphaherpesvirinae subfamily, and Varicellavirus genus, and has only one serotype. FHV-1 is highly species-specific, primarily infecting cats. Cats of all breeds, sexes, and ages are susceptible, but it most commonly infects kittens aged 2–4 months. While it generally does not cause death in adult cats, the mortality rate in kittens can reach 50%. Besides cats, FHV-1 can also infect other felines such as Siberian tigers, South China tigers, and snow leopards, seriously endangering the health of rare wild felines and impacting species diversity.

[0003] Currently, vaccination is the primary means of preventing feline rhinotracheitis (FRT). There is no live FRT vaccine available in China, but the virulence of prevalent strains varies across different regions, leading to occasional outbreaks even after vaccination. Therefore, it is particularly important and urgent to screen for dominant strains in different regions of China and to develop and produce a safe and effective live FRT vaccine.

[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 a strain of feline herpesvirus type 1 (FHV-1) 110, providing an option for the prevention and detection of FHV-1.

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

[0007] A strain of feline herpesvirus type 1, strain 110, was deposited at the China Center for Type Culture Collection (CCTCC) on January 15, 2025, with accession number CCTCC NO:V202507 and deposit address: Wuhan University, Wuhan, China.

[0008] The use of feline herpesvirus type 1 strain 110 in any of the following (a)-(c):

[0009] (a) Prepare products for detecting feline herpesvirus type 1 or its antibodies;

[0010] (b) To prepare products for the prevention and / or treatment of feline herpesvirus type 1 infection;

[0011] (c) Preparation of feline herpesvirus type 1 antibody.

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

[0013] A vaccine composition comprising an immunizing dose of feline herpesvirus type 1 strain 110 antigen, wherein the feline herpesvirus type 1 strain 110 antigen comprises live whole virus antigen of the aforementioned feline herpesvirus type 1 strain 110 or its culture.

[0014] Furthermore, the antigen content of 110 strains of feline herpesvirus type 1 was ≥10. 5.5 TCID 50 / portion, can be set to 10 5.5 ~10 7.0 TCID 50 / portion, further optional 10 6.0 ~10 7.0 TCID 50 / Toufen.

[0015] Furthermore, the feline herpesvirus type 1 strain 110 culture is a culture that has undergone 1 to 120 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 herpesvirus type 1 strain 110 antigen is 1:(0.5-1.5) by volume.

[0021] The use of the above-mentioned vaccine composition in the preparation of a drug for preventing feline herpesvirus type 1 infection, wherein the drug may be a vaccine.

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

[0023] The feline herpesvirus type 1 strain 110 provided in this application exhibits significantly reduced pathogenicity compared to its parental virulent feline herpesvirus type 1 strain 64, and is an artificially attenuated virus strain. Furthermore, the feline herpesvirus type 1 strain 110 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 herpesvirus type 1 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 infected with the virus, effectively prevent outbreaks caused by feline herpesvirus type 1, and provide effective immune protection against feline herpesvirus type 1 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 herpesvirus type 1" (FHV-1) belongs to the Herpesviridae family, Alphaherpesvirinae subfamily. It is a typical enveloped, linear, double-stranded DNA virus that can cause infectious rhinotracheitis in cats and other felines. After infection with FHV-1, affected cats develop a fever, reaching 39.5–42°C, causing severe upper respiratory and ocular symptoms. It is characterized by latent infection, lifelong carrier status, and recurrent infections, seriously endangering the health and biodiversity of cats and other felines.

[0030] This application provides a strain of feline herpesvirus type 1 (Strain110), deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.V202507, located at Wuhan University, Wuhan, China, on January 15, 2025.

[0031] Pathogenicity tests showed that the pathogenicity of feline herpesvirus type 1 strain 110 was significantly reduced between passage 1 and passage 120 in cats. Cats inoculated at passage 120 and observed for 14 days showed no clinical symptoms, and no changes were observed in necropsy organs. Therefore, compared with the parental virulent feline herpesvirus type 1 strain 64, this virus has significantly reduced pathogenicity and is an artificially attenuated virus strain.

[0032] Immunogenicity tests showed that feline herpesvirus type 1 strain 110, cultured to passage 120, still possessed good immunogenicity. Fourteen days post-vaccination, compared to cats vaccinated with feline herpesvirus type 1 strain 64, the immunized group showed significantly reduced clinical symptoms induced by feline herpesvirus type 1 strain 64.

[0033] Virulence reversion tests showed that viruses cultured from generation 1 to generation 120, after inoculation and multiple 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, feline herpesvirus type 1 strain 64, the cultures of feline herpesvirus type 1 strain 110 from generation 1 to generation 120 showed mutations at three amino acid sites: V14F in gE protein, Q77R in TK protein, and L628S in ICP4 protein.

[0035] The gE and TK genes are associated with the virulence genes of herpesviruses, and the ICP4 protein is involved in the regulation of herpesvirus replication and latent infection. The common characteristic changes in the amino acid encoding of the genes of 110 different passages of feline herpesvirus type 1 strains are likely the reason for the reduced virulence of its parent virulent strain 64, while these changes did not lead to changes in immunogenicity.

[0036] The point mutations in the amino acid sequences of the gE, TK, and ICP4 proteins of the feline herpesvirus type 1 strain 110 in this application reduce the virulence compared to the parental virulent strain 64 and exhibit genetic stability.

[0037] This application also provides the use of the aforementioned feline herpesvirus type 1 strain 110 in the preparation of products for detecting feline herpesvirus type 1 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 herpesvirus type 1 strain 110 in the preparation of products for the prevention and / or treatment of feline herpesvirus type 1 infection. The products may be pharmaceuticals, such as vaccines.

[0039] In some implementations, the drug is a live attenuated feline herpesvirus type 1 vaccine.

[0040] This application also provides the use of the above-mentioned feline herpesvirus type 1 strain 110 in the preparation of feline herpesvirus type 1 antibodies.

[0041] This application provides a vaccine composition comprising an immunizing dose of feline herpesvirus type 1 strain 110 antigen, wherein the feline herpesvirus type 1 strain 110 antigen comprises the live whole virus antigen of the feline herpesvirus type 1 strain 110 or its culture thereof as described in this application.

[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 feline herpesvirus type 1 strain 110 is ≥10. 5.5 TCID 50 / portion, preferably 10 5.5 ~10 7.0 TCID 50 / portion, more preferably 10 6.0 ~10 7.0 TCID 50 / per dose. The antigen content of 110 strains of feline herpesvirus type 1 can be selected from 10... 5.5 TCID 50 / First serving, 10 5.6 TCID 50 / First serving, 10 5.7 TCID 50 / First serving, 10 5.8 TCID 50 / First serving, 10 5.9 TCID 50 / First serving, 106.0 TCID 50 / First serving, 10 6.1 TCID 50 / First serving, 10 6.2 TCID 50 / First serving, 10 6.3 TCID 50 / First serving, 10 6.4 TCID 50 / First serving, 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 / one serving or 10 7.5 TCID 50 / Toufen.

[0045] In a preferred embodiment, the feline herpesvirus type 1 strain 110 culture is a culture of passage 1 to 120. Specifically, the feline herpesvirus type 1 strain 110 culture can be selected from passages 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24. Cultures of 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 generations.

[0046] The term "culture of feline herpesvirus type 1 strain 110 from passage 1 to 120" refers to the culture obtained by continuously passaged naturally in F81 cells until the feline herpesvirus type 1 strain 110 is adapted to grow and replicate in F81 cells (i.e., cytopathic effects such as rounding, aggregation, and shedding appear on the cells), and then continuously passaged naturally in F81 cells from passage 1 to 120.

[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 herpesvirus type 1 strain 110 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 herpesvirus type 1 strain 110 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 herpesvirus type 1 infection. As used herein, the term "prevention" means the suppression of feline herpesvirus type 1 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 110 strains of feline herpesvirus type 1

[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 and inoculate 64 strains of feline herpesvirus type 1 into F81 monolayer cells, and continue to culture in cell growth medium containing 92%–98% (v / v) RPMI 1640 medium and 2%–8% (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 attenuated feline herpesvirus type 1 (FHV-1). Sequencing of the obtained FHV-1 attenuated strain revealed stable mutations at three amino acid sites: V14F in the gE protein, Q77R in the TK protein, and L628S in the ICP4 protein. This attenuated FHV-1 strain was named FHV-1 strain 110.

[0058] Example 2: Study on the biological characteristics of 110 strains of feline herpesvirus type 1

[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 1 110 strains <![CDATA[Nasal administration of 0.5 ml (10 4.5 TCID 50 / ml)]]> 2 64 strains <![CDATA[Nasal drip 0.5 ml (10 4.5 TCID 50 / ml)]]> 3 RPMI 1640 medium 0.5ml nasal drops

[0062] The cats were observed for 14 days after vaccination. Their body temperature, presence of conjunctivitis, eye and nasal discharge, sneezing and other clinical signs were observed and recorded daily. The specific results are shown in Table 2.

[0063] Table 2 Results of pathogenicity studies

[0064] The results showed that 64 strains of feline herpesvirus type 1 could cause 100% morbidity (5 / 5) in cats, while 110 strains of feline herpesvirus type 1 had normal body temperature, no clinical symptoms, and no changes in tissues and organs upon necropsy.

[0065] Pathogenicity tests showed that the pathogenicity of feline herpesvirus type 1 strain 110 was significantly reduced compared with that of the parental virulent strain 64, making it an attenuated virus strain.

[0066] Meanwhile, to verify the stability of the pathogenicity of feline herpesvirus 1 strain 110 at different passages, cultures of feline herpesvirus 1 strain 110 from passages 1, 30, 60, 90, and 120 were used to inoculate one group of five feline herpesvirus 1 antigen-antibody negative cats, with 0.5 ml (10 ml) administered intranasally to each cat. 4.5 TCID 50 ( / ml), with 5 cats serving as the control group. Clinical changes in the cats were observed and recorded daily until 14 days after vaccination.

[0067] The results showed that 110 cultures of feline herpesvirus type 1 from generations 1, 30, 60, 90 and 120, after 10 days of observation following inoculation, had normal body temperature, no clinical symptoms, and no changes in tissues and organs upon necropsy.

[0068] Different passage pathogenicity tests showed that the pathogenicity of 110 strains of feline herpesvirus type 1 was significantly reduced at different passages, making them 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 4 110 strains <![CDATA[1 ml subcutaneously (10 6.0 TCID 50 / ml)]]> 5 RPMI 1640 medium 1ml subcutaneously

[0072] A second immunization was administered 21 days after the initial immunization. Fourteen days after the second immunization, the test cats were challenged with feline herpesvirus type 1 strain 64, with 0.5 ml (10 ml) administered intranasally to each cat. 4.5 TCID 50 / ml), after challenge, the cats' body temperature, presence of conjunctivitis, eye and nasal discharge, sneezing, and other clinical manifestations were observed and recorded daily. 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. Specific results are shown in Table 5.

[0073] Table 4. Clinical symptom scoring criteria after feline herpesvirus type 1 challenge.

[0074] Table 5. Immunogenicity of 110 strains of feline herpesvirus type 1 to cats.

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

[0076] The results showed that all patients in the control group developed the disease, and the clinical scores of the patients immunized with the feline herpesvirus type 1 strain 110 vaccine combination were significantly lower than those of the control group after challenge.

[0077] Immunogenicity tests showed that strain 110 of feline herpesvirus type 1 had good immunogenicity, and challenge with virulent feline herpesvirus type 1 could significantly reduce clinical symptoms.

[0078] Meanwhile, to verify the stability of the immunogenicity of feline herpesvirus type 1 strain 110 at different passages, cultures of feline herpesvirus type 1 strain 110 were immunized at passages 1, 30, 60, 90, and 120, respectively. A second immunization was performed 21 days after the initial immunization. Fourteen days after the second immunization, the test cats were challenged with feline herpesvirus type 1 strain 64, with 0.5 ml (10 ml) administered intranasally to each cat. 4.5 TCID 50 / ml), after the challenge, the cats' body temperature, whether conjunctivitis, eye and nasal secretions, sneezing 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 110 strains of feline herpesvirus type 1 from generations 1, 30, 60, 90 and 120 were significantly lower than those of the challenge control group, and all cats in the control group developed the disease.

[0080] Immunogenicity tests at different passages showed that all 110 strains of feline herpesvirus 1 at different passages had good immunogenicity and could significantly reduce the clinical symptoms caused by challenge with 64 strains of feline herpesvirus 1.

[0081] 3. Virus reversion safety test

[0082] Administer 1 ml (10 ml) of feline herpesvirus type 1 strain 110 first-generation virus solution as nasal drops. 7.5 TCID 50Three healthy, susceptible cats were vaccinated with a dose of / ml. Clinical manifestations were observed daily after vaccination. The cats were euthanized on the fourth day post-vaccination to observe lesions. Samples from the trachea, tonsils, and submandibular lymph nodes were collected for HE staining and immunohistochemical analysis. Feline herpesvirus type 1 nucleic acid was determined from the homogenate of the trachea, tonsils, and submandibular lymph nodes. The homogenate samples with high viral loads were selected, appropriately processed, and used as inoculum for next-generation virulence reversion, thus allowing the virus to be transmitted to the fourth generation in the cats.

[0083] Three healthy, susceptible cats were inoculated with 1 ml of homogenate from the trachea, tonsils, and submandibular lymph nodes obtained from the fourth-generation virulence reversion test. Clinical manifestations were observed daily after inoculation. The cats were euthanized on day 21 post-inoculation, and lesions were observed. Samples from the trachea, tonsils, and submandibular lymph nodes were collected for HE staining and immunohistochemical analysis, and viral nucleic acid was determined.

[0084] If, during the virulence reversion subculture tests from generation 1 to 4, feline herpesvirus type 1 nucleic acid is undetectable in the trachea, tonsils, and submandibular lymph nodes 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 herpesvirus type 1 nucleic acid cannot be detected again in the trachea, tonsils, and submandibular lymph nodes 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 no abnormalities in body temperature, clinical observation, necropsy, or histopathological examination of cats vaccinated with generations 1 through 5. On the 4th day post-vaccination, FHV-1 was isolated from the trachea, tonsils, and submandibular lymph nodes in the first-generation virulence reversion test, but not from these organs in generations 2 through 5. The viral load in the trachea, tonsils, and submandibular lymph nodes gradually decreased from generation 1 to generation 5, and no significant pathological changes were observed after HE staining. Immunohistochemical staining showed that only the trachea of ​​two-thirds of the cats in the first-generation virulence reversion test was positive for FHV-1, while organs in generations 2 through 5 were negative for FHV-1. Therefore, feline herpesvirus type 1 strain 110 does not exhibit virulence reversion ability and is a safe, attenuated strain.

[0086] The viral loads of 110 strains of feline herpesvirus type 1 at passages 30, 60, 90, and 120 were verified according to the above-mentioned viral reversion test procedure. The results showed that no abnormalities were found in the body temperature, clinical observation, necropsy, and histopathological observation of the cats inoculated from passage 1 to passage 5. The results of quantitative real-time PCR also showed that the viral load in the trachea, tonsils, and submandibular lymph nodes gradually decreased during passage. FHV-1 could be isolated from the trachea, tonsils, and submandibular lymph nodes in the first-generation virulence reversion test, while FHV-1 could not be isolated from the trachea, tonsils, and submandibular lymph nodes of the cats in the second to fifth-generation virulence reversion tests.

[0087] Therefore, the feline herpesvirus type 1 strain 110 will not re-evolve into a virulent strain that causes disease, and its safety is guaranteed.

[0088] 4. Gene sequence analysis

[0089] Genome amplification was performed using PCR on 110 strains of feline herpesvirus type 1 from passage 1 to passage 120 (each passage was 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 64 highly virulent parental strains to describe the viral amino acid sequence characteristics.

[0090] The results showed that in the cultures of 110 strains of feline herpesvirus type 1 from generation 1 to generation 120, the common amino acid sequence of each gene encoded by the virus led to stable mutations at three amino acid sites: V14F in the gE protein, Q77R in the TK protein, and L628S in the ICP4 protein.

[0091] This indicates that the common characteristic changes in the amino acid encoding of the viral genes of 110 strains of feline herpesvirus type 1 from different generations of cultures are likely the reason for the reduced virulence of their parent virulent strains.

[0092] Example 3: Preparation of Feline Herpesvirus Type 1 Live Attenuated Vaccine (Strain 110)

[0093] 1. Viral replication

[0094] The feline herpesvirus type 1 strain 110, prepared in Example 1, was 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℃ for 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 feline herpesvirus type 1 110 strains attenuated live vaccine Components Vaccine 1 Vaccine 2 Vaccine 3 <![CDATA[110 strains of antigen (TCID 50 ) / dose]]> <![CDATA[10 5.5 ]]> <![CDATA[10 6.0 ]]> <![CDATA[10 7.0 ]]> Protectant (V / V) 50％ 50％ 50％

[0100] Example 4: Immunogenicity test of feline herpesvirus type 1 strain 110 attenuated live vaccine

[0101] Twenty feline herpesvirus type 1 antigen-antibody-negative cats aged 2 months or older were randomly divided into 4 groups of 5 cats each. All cats were immunized with the feline herpesvirus type 1 strain 110 attenuated live vaccine 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. All cats received subcutaneous injections, two times immunized, 21 days apart. Fourteen days after the second immunization, the cats were challenged with feline herpesvirus type 1 strain 64. Each cat received 0.5 ml (10 ml) intranasally. 4.5 TCID 50 / ml), after challenge, the cats' body temperature, presence of conjunctivitis, eye and nasal discharge, sneezing, and other clinical manifestations were observed and recorded daily. 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. Specific results are shown in Table 7.

[0102] Table 7. Immunogenicity test results of feline herpesvirus type 1 strain 110 attenuated live vaccine

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

[0104] The results showed that immunization of cats with the feline herpesvirus type 1 strain 110 attenuated live vaccine prepared in Example 3 significantly reduced the clinical symptoms caused by the virulent strain.

[0105] The results demonstrated that the live attenuated feline herpesvirus type 1 strain 110 vaccine in all three experimental groups had good protective efficacy, showing excellent immune protection and safety.

[0106] Example 5: Broad-spectrum test of feline herpesvirus type 1 strain 110 attenuated live vaccine

[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 control. A second immunization was administered 21 days after the initial immunization. Fourteen days after the second immunization, the cats were challenged with four strains of feline herpesvirus type 1 (FHV-1): 48 strains, 50 strains, 56 strains, and 62 strains, all from different provinces and cities. Each cat received 0.5 ml of FHV-1 intranasally (10... 4.5 TCID 50 / ml), after challenge, the cats' body temperature, presence of conjunctivitis, eye and nasal discharge, sneezing, and other clinical manifestations were observed and recorded daily. The clinical symptoms were scored daily according to the scoring criteria in Table 4, and the total clinical score of each cat within 14 days after challenge was calculated. The differences in clinical scores between the immunized 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 110 strains of feline herpesvirus 1 attenuated live vaccine (challenge against 48 strains of feline herpesvirus 1).

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

[0110] Table 9. Results of broad-spectrum trial of feline herpesvirus 1 strain 110 attenuated live vaccine (challenge against feline herpesvirus 1 strain 50)

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

[0112] Table 10. Broad-spectrum test results of 110 strains of feline herpesvirus 1 attenuated live vaccine (challenge against 56 strains of feline herpesvirus 1).

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

[0114] Table 11. Broad-spectrum test results of 110 strains of feline herpesvirus 1 attenuated live vaccine (challenge against 62 strains of feline herpesvirus 1).

[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 herpesvirus type 1 strain 110 attenuated live vaccine were significantly lower than those of the control group after challenge.

[0117] This application demonstrates that the feline herpesvirus 1 strain 110 attenuated live vaccine provided in this application can significantly reduce the clinical symptoms of feline herpesvirus 1 from different geographical locations after challenge. The feline herpesvirus 1 strain 110 attenuated live vaccine of this application has broad-spectrum immunogenicity and can significantly reduce the clinical symptoms of feline herpesvirus 1 from different geographical locations after challenge.

[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. A strain of feline herpesvirus type 1, comprising 110 strains, characterized in that, It was deposited on January 15, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:V202507, and the deposit address is Wuhan University, Wuhan, China.

2. The use of the feline herpesvirus type 1 strain 110 according to claim 1 in any one of the following (a)-(c): (a) Prepare products for detecting feline herpesvirus type 1 or its antibodies; (b) To prepare products for the prevention and / or treatment of feline herpesvirus type 1 infection; (c) Preparation of feline herpesvirus type 1 antibody.

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 herpesvirus type 1 strain 110 antigen, wherein the feline herpesvirus type 1 strain 110 antigen comprises the live whole virus antigen of the feline herpesvirus type 1 strain 110 or its culture as described in claim 1.

5. The vaccine composition according to claim 4, characterized in that, Feline herpesvirus type 1 strain 110 has an antigen content of ≥10 5.5 TCID 50 / portion, can be set to 10 5.5 ~10 7.0 TCID 50 / portion, further optional 10 6.0 ~10 7.0 TCID 50 / Toufen.

6. The vaccine composition according to claim 4, characterized in that, The 110 strains of feline herpesvirus type 1 were cultures from passage 1 to 120.

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 herpesvirus type 1 strain 110 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 herpesvirus type 1 infection, wherein the medicament may be a vaccine.

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