Fat calicivirus attenuated strain as well as preparation and application thereof

Abstract

The invention provides a feline calicivirus attenuated strain, which is characterized in that the amino acid sequence of the capsid protein (VP1) gene of the feline calicivirus attenuated strain is as shown in SEQ ID NO: 2, and the nucleotide sequence of the capsid protein (VP1) gene of the feline calicivirus attenuated strain is as shown in SEQ ID NO: 1. The cat calicivirus attenuated strain disclosed by the invention has good safety and immunogenicity, and can be used for preparing a monovalent live vaccine or a multi-combined live vaccine for cats. Compared with a traditional inactivated vaccine, the cat calicivirus attenuated strain can induce humoral immunity and cellular immunity, and provides a more reliable protection effect on prevention of FCV infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the preparation and application of an attenuated strain of feline calicivirus. Technical Background

[0002] Feline calicivirus (FCV) belongs to the family Caliciviridae and the genus Calicivirus. It is one of the viral pathogens widely spread among feline animals and can cause multiple oral inflammations and respiratory symptoms in cats. The clinical symptoms after FCV infection mainly include chronic stomatitis, oral ulcers, rhinitis, conjunctivitis, etc. Cats are the natural hosts of this virus, and almost all feline animals are susceptible to FCV. Since FCV was first isolated from diseased cats in 1957, FCV infections have been reported in feline animals such as cats, tigers, and leopards worldwide. FCV mainly infects kittens, among which kittens aged 56 - 84 days are the most susceptible. The incubation period after infection is 2 - 3 days, the body temperature rises to 39.5 - 40.5 °C, and typical clinical symptoms appear. Infected cats are the main sources of infection. FCV can be indirectly transmitted through the secretions of infected cats, or directly transmitted through contact or aerosols. FCV infection alone does not cause the death of infected animals. Usually, after mixed infection with other pathogens, the mortality rate of FCV infection will increase significantly.

[0003] Currently, there is no specific treatment method for FCV infection and disease. Clinically, vaccination is the main method to prevent FCV infection and disease. Different types of FCV vaccines have been developed successively at home and abroad, mainly including inactivated vaccines, attenuated live vaccines, virus-like particle vaccines, and vector vaccines. However, at present, only a multi - valent inactivated vaccine containing FCV in China has passed the emergency review by the competent department and obtained a temporary production approval number.

[0004] FCV belongs to RNA virus, and there is a large genetic variability among different strains. Research shows that there are 20% - 40% differences in the capsid variable regions of different FCV strains, and the homology of the whole - gene sequences of different strains varies greatly, with the homology range being 78.9% - 99.9%. The antigen diversity of FCV is the main reason for the current vaccine immune failure. The antigen diversity of the immunodominant sites in the variable region of the capsid protein gene explains the emergence of escape mutants. After immunization with the FCV inactivated vaccine, only humoral immune responses can be induced, generating neutralizing antibodies against the vaccine strain, and the cross - protection against wild strains with large homology variations is low. Compared with inactivated vaccines, attenuated live vaccines can not only induce humoral immunity but also stimulate cellular immunity, further improving the cross - protection effect against strains with lower homology after vaccination. Summary of the Invention

[0005] To solve the above problems, the present invention provides a feline calicivirus attenuated strain, which has good safety and immunogenicity, and can be used to prepare a monovalent live vaccine or a combined live vaccine for cats. Compared with traditional inactivated vaccines, this feline calicivirus attenuated strain can induce humoral immunity and cellular immunity, providing a more reliable protective effect against FCV infection.

[0006] The present invention discloses a feline calicivirus strain according to claim 1, wherein the amino acid sequence of the capsid protein (VP1) gene of the feline calicivirus attenuated strain is as shown in SEQ ID NO: 2.

[0007] The present invention discloses a feline calicivirus strain, wherein the nucleotide sequence of the capsid protein (VP1) gene of the feline calicivirus attenuated strain is as shown in SEQ ID NO: 1.

[0008] The present invention also discloses a vaccine composition, which contains any one of the above feline calicivirus attenuated strains or its derivative viruses.

[0009] Furthermore, the feline calicivirus attenuated strain selected in the vaccine composition is a live virus or an inactivated virus.

[0010] Furthermore, the vaccine composition contains the capsid protein (VP1) of the feline calicivirus attenuated strain or the nucleic acid encoding the capsid protein (VP1) of the feline calicivirus attenuated strain.

[0011] Furthermore, the preparation method of the vaccine composition includes the following steps:

[0012] Isolate the low-virulence strain of feline calicivirus and culture it to obtain a virus solution of the feline calicivirus attenuated strain;

[0013] Treat the virus solution of the attenuated strain, and the treatment includes cloning and purification;

[0014] Dilute the treated virus solution of the attenuated strain to a predetermined virus titer.

[0015] The preparation method of the feline calicivirus strain disclosed in the present invention includes the following steps: Isolate a low-virulence strain of feline calicivirus, inoculate it on CRFK cells and perform continuous subculture at temperature T; When the cytopathic effect rate is not less than 80%, freeze-thaw and harvest at a temperature below T1, and store the harvested culture at a temperature below T2; During the subculture process, perform plaque cloning and purification every N generations, N is 8 - 12, and a total of M times of cloning and purification are performed, M is 1 - 4. Each time of cloning and purification, pick the larger plaque for expansion culture; After cloning and purifying the feline calicivirus on CRFK cells to the Kth generation, where K is 36 - 42, it is okay.

[0016] Further, T is 39.5°C to 40.5°C, and can also be 38°C, 38.5°C, 39°C, 40°C; T1 is -10°C to -15°C, and can also be -11°C, -12°C, -13°C, -14°C; T2 is -60°C to -70°C, and can also be -65°C, or -66°C, or -67°C, or -68°C, or -69°C; N is 9, or 10, or 11; M is 2, or 3, or 4; K is 38 to 40, and can also be 39.

[0017] Further, the vaccine composition further comprises a combined live vaccine for cats or a combined inactivated vaccine containing an attenuated strain, and the vaccine composition further comprises a pharmaceutically acceptable carrier or adjuvant.

[0018] Further, the application of the vaccine composition in the preparation of a medicament for preventing feline systemic diseases caused by feline calicivirus.

[0019] Advantages of the present invention:

[0020] (1) By using a low-virulence feline calicivirus, continuous high-temperature passage culture was used for attenuation and cloning purification to obtain an almost non-pathogenic attenuated strain of feline calicivirus. To ensure the uniqueness of the strain, the VP1 gene sequence of the attenuated strain of feline calicivirus was protected.

[0021] (2) Using the attenuated strain of feline calicivirus to research and develop a monovalent live vaccine or inactivated vaccine for cats.

[0022] (3) Using the attenuated strain of feline calicivirus to research and develop a combined live vaccine or combined inactivated vaccine for cats.

[0023] The attenuated live vaccine disclosed in the present invention is that the inventor creatively obtained a weakly virulent strain with weakened pathogenicity required for preparing the vaccine. Artificially attenuated strains are usually obtained by physical, chemical methods, genetic modification or gene deletion. Since the virus has changed under certain external pressures, there will be certain biosafety problems after being put on the market as a live vaccine. Naturally attenuated strains are obtained by isolation from nature or by continuous subculture of cells. Since the virus is naturally attenuated, the possibility of reversion to virulence is reduced. Therefore, the present invention obtained a naturally attenuated weakly virulent strain of feline calicivirus and applied it to the research of feline calicivirus attenuated live vaccine, which is of great significance for the prevention of feline calicivirus disease.

[0024] The attenuated strain of feline calicivirus disclosed in the present invention has good safety and immunogenicity, and can be used to prepare a monovalent live vaccine or combined live vaccine for cats. Compared with traditional inactivated vaccines, the attenuated strain of feline calicivirus can induce humoral immunity and cellular immunity, providing a more reliable protective effect against feline calicivirus infection.

[0025] The significant characteristics of feline calicivirus are the diversity of viral genes and high genetic variability. The gene similarity rates among different strains vary greatly, which results in the fact that vaccines cannot provide good immune prevention for all feline calicivirus infections. Nevertheless, since there is no specific treatment method for feline calicivirus infection, immunoprophylaxis by developing vaccines is still relied on to prevent and control this disease. At present, the research on vaccines for preventing this disease mainly focuses on inactivated vaccines and live vaccines, and there are already inactivated vaccines approved for marketing in China. However, the inactivated vaccines can only play a protective role after multiple booster immunizations, and they can only stimulate the body to produce humoral immunity, and cannot provide good cross-protection effects against feline calicivirus that may have undergone gene mutations. Therefore, the research direction of vaccines for preventing this disease should be to improve the cross-reactivity and cross-protection effects of vaccines. Attenuated live vaccines can not only stimulate the body to produce humoral immunity, but also induce the body to produce cellular immunity, and the body has more persistent immune memory. Therefore, obtaining an attenuated strain of feline calicivirus that is safe, effective, and can be used in combination with other common feline virus strains has become the key to developing monovalent live attenuated vaccines and combined live vaccines for feline calicivirus.

[0026] In this invention, an attenuated strain of feline calicivirus was successfully obtained. The test results showed that this strain had good safety and immunogenicity. The monovalent live vaccine and combined live vaccine developed using this strain could induce the body to produce a relatively high antibody titer with a long duration, and had good protective effects against the challenge of virulent strains used for testing. Description of the Drawings

[0027] Figure 1 : The ocular and nasal secretions did not increase significantly;

[0028] Figure 2 : Oral ulcers appeared in the cats in the control group; Detailed Embodiments

[0029] The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.

[0030] Example 1 Passage and attenuation of feline calicivirus strain

[0031] The low-virulence feline calicivirus solution isolated in the laboratory was inoculated onto confluent monolayer CRFK cells at a ratio of 1‰ for continuous passage culture. When the cytopathic effect rate was not less than 80%, it was frozen and thawed at -15°C or below for harvesting, and the harvested culture was stored at -70°C or below. During the passage process, plaque cloning purification was performed every 10 generations, and a total of 3 times of cloning purification were carried out. Each time of cloning purification, larger plaques were picked for expanded culture. After the feline calicivirus strain was cloned and purified to the 40th generation on CRFK cells, healthy susceptible cats were inoculated through the intranasal infection route to test the virulence of the strain. The results showed that no clinical symptoms caused by feline calicivirus appeared in all animals after inoculation with the F40-generation virus, indicating that the pathogenicity of the F40-generation of this strain was further weakened, and a feline calicivirus attenuated strain had been cloned and domesticated.

[0032] Example 2 Sequencing of the VP1 Gene of the Feline Calicivirus Attenuated Strain

[0033] To determine the gene changes of the feline calicivirus attenuated strain after continuous passage on cells, primers were designed according to the literature to amplify, clone, and sequence its VP1 genome, and the changes in the VP1 genome sequence before and after passage were compared at the nucleotide and amino acid levels respectively. The alignment results showed that the similarity rate of the VP1 gene nucleotide sequence of the F40-generation of the feline calicivirus attenuated strain to that of the F2-generation before attenuation was 99.55%; the similarity rate of the VP1 gene amino acid sequence of the F40-generation of the attenuated strain to that of the F2-generation before attenuation was 98.95%

[0034] The nucleotide sequence of the VP1 gene of the F40-generation of the feline calicivirus attenuated strain:

[0035]

[0036] Amino acid sequence of VP1 gene of the attenuated strain of feline calicivirus at passage F40:

[0037] MCSTCANVLKYYNWDPHFKLTINPNDFLSIGFCDNPLMCCYPELLPEFGTVWDCNDSPLQIYLESILGDDEWSSTYEAIDPVVPPMHWVEMGKIFQPHPGVLMHHLIGEVAKGWDPNLPTFRLEADDGSITSPEQGTSVGGVIAEPSAQMATAADTATGKTVDSEWEAFFSFHTSVNWSTSETQGKILFKQNLGPLLNPYLEHISQLYVAWSGSVDVRFSISGSGVFGGKLAAIVVPPGVQPVQSTSMLQYPHVLFDARQVEPVIFSLPDLRNSLYHLMSDTDTTSLVIMVYNDLINPYANDSNSSGCIITVETKPGPDFRFHLLKPPGSMLVHGSIPSNLIPKSSSLWIGNRHWSDITDFVIRPSVFQANRHFDFKQETAGWSTPRFRPMTITISQKESEKLGIGIAHEAIVPGIPDGWPDTTIPGVLIPAGDYAITNERNSDITTDEAYIAATEIRNNTNFKSMYICGALQRAWGDKKISNTAFITTADLVGNKIKPNNVINQSRIVVFQDNHVNQQIQTSDVTLAMLGYTGIGEEVIGANRDRVVRINILPEVSARGGNHPIYYKNSLKLGYVIKTIDVFNSQILHTSRQLALNNYLLDPDSFAVYRITDSNGSWFDIGIDYSGFSFVGVSSIGNLEFPLTASYMGIQLAKIRLASNIRSSMTKL.

[0038] Example 3 Passage stability of the attenuated strain of feline calicivirus

[0039] The virus solution of the attenuated strain of feline calicivirus at passage F40 was inoculated onto confluent monolayer CRFK cells at a ratio of 1‰ and continuously passaged until passage F45. Samples from passage F41, F42, F43, F44, and F45 were taken for virus content determination. The results showed that the virus titers of each passage of the virus ranged from 10 9.5 to 10 9.9 TCID 50 / ml, with no obvious changes between different passages, indicating stable virus reproduction. The VP1 gene of the virus at passage F45 was amplified and sequenced. The results showed that the nucleotide sequence and amino acid sequence of the VP1 gene of the virus at passage F45 were both 100% similar to those of the VP1 gene of the virus at passage F40, indicating that within 5 consecutive passages, the virus gene did not mutate and had good stability.

[0040] Example 4 Pathogenicity test of the attenuated strain of feline calicivirus

[0041] Six healthy cats aged 10 - 14 weeks and negative for FCV antigen and antibody were selected to test the pathogenicity of the attenuated strains F41 and F45 of feline calicivirus. The cats were randomly divided into 2 groups, with 3 cats in each group. The first group was instilled with the attenuated strain F41 of feline calicivirus through nasal drip, and the second group was instilled with the attenuated strain F45 of feline calicivirus through nasal drip, 1 ml per cat (0.5 ml per nostril). Within 14 days after nasal drip, the rectal temperature was measured regularly every day and the clinical symptoms (such as spirit, appetite, oral cavity, eye and nasal secretions, sneezing, etc.) were observed. After 14 days, the test ended. According to the changes in body temperature and clinical symptoms, it was determined whether the attenuated strains F41 and F45 of feline calicivirus were pathogenic to cats.

[0042] The results showed that within 14 days after the cats were inoculated with the attenuated strains F41 and F45 of feline calicivirus through nasal drip, the body temperatures of the two groups of cats did not increase significantly and did not exceed the basal body temperature by 1°C; compared with before inoculation, there were no abnormalities in the spirit, appetite, respiratory symptoms and behavioral activities of the cats, and no phenomena of oral ulcer and a large increase in eye and nasal secretions were observed ( Figure 1 ). The results indicated that the attenuated strains F41 and F45 of feline calicivirus were almost non - pathogenic to cats.

[0043] Example 5 Immunogenicity test of the attenuated strain of feline calicivirus

[0044] Nine healthy cats aged 8 - 10 weeks and negative for FCV antigen and antibody were selected to test the immunogenicity of the attenuated strains F41 and F45 of feline calicivirus. The virus content of the attenuated strains F41 and F45 of feline calicivirus was diluted to 10 6.0 TCID 50 / ml, and 3 cats were subcutaneously injected respectively. After 21 days, a second immunization was carried out by the same route and at the same dose, 1.0 ml per cat per time; the remaining 3 cats were used as controls. Blood was collected 14 days and 21 days after the second immunization to detect antibodies. And 21 days after the second immunization, all cats were challenged with the virulent FCV JL strain for the nasal drip test, 1.0 ml per cat (0.5 ml per nostril). After challenge, the clinical symptoms (such as spirit, appetite, oral cavity, eye and nasal secretions, sneezing, etc.) were observed regularly every day and the body temperature was measured until 14 days after challenge. By comparing the differences in body temperature and clinical symptoms between the immunized group and the control group, the immunogenicity of the attenuated strains F41 and F45 of feline calicivirus was evaluated.

[0045] The antibody detection results showed that on the 14th day after the second immunization, the FCV neutralizing antibodies in the cats immunized with the F41 and F45 generations of the attenuated FCV strains could reach 1:23 to 1:64; on the 21st day after the second immunization, the antibodies in the two groups of immunized cats could reach 1:45 to 1:90. The results indicated that the F41 and F45 generations of the attenuated FCV strains had good immunogenicity, and there was no significant difference in the antibody levels produced by the immunized animals.

[0046] The results of the challenge protection showed that there was no obvious change in the body temperature of the immunized cats, and it did not exceed 1 °C compared with the basal body temperature; a small amount of ocular or nasal secretions appeared in the cats in the two immunized groups. According to the FCV symptom scoring system in the European Pharmacopoeia, the sum of the scores of each symptom in the immunized cats was between 3 and 10 points. All the cats in the control group showed typical FCV infection symptoms, such as increased nasal secretions, increased ocular secretions, oral ulcers, etc. ( Figure 2 ), and the sum of the scores of each symptom was between 17 and 27 points. The score of the immunized animals was significantly lower than that of the control group animals (P < 0.05). The results indicated that after immunizing animals with the F41 and F45 generations of the attenuated FCV strains, it could provide good protection against the infection of virulent FCV.

[0047] Example 6: Monovalent live vaccine for cats:

[0048] Preparation and potency test of a monovalent live vaccine containing an attenuated FCV strain

[0049] 1. Preparation of the virus liquid for vaccine production Take out the cryopreserved CRFK cells from the working cell bank, thaw them in a 37 °C water bath, transfer the cells into a cell culture flask containing DMEM culture medium with 4% - 6% bovine serum, and culture them in a 37 °C environment for 48 - 72 hours until the cells grow into a confluent monolayer. Discard the culture medium, digest with trypsin, and passage at a ratio of 1:3 - 1:4 to expand the culture. Inoculate the expanded CRFK cells into a culture flask and culture them at 37 °C. Inoculate the attenuated FCV strain seed virus into the well-grown monolayer CRFK cells at an MOI of 0.001 - 0.01, and culture them in a 37 °C environment. Harvest when the CPE reaches about 80%, and store at -15 °C or below. After the harvested virus liquid passes the purity test of the virus content, it is used as the virus liquid for vaccine production.

[0050] 2. Preparation and test of the monovalent live vaccine According to the virus content of the attenuated FCV strain in each dose of the vaccine during vaccine formulation, it should not be less than 10 5.5 TCID 50, calculate the dosage of the virus solution required for vaccine preparation. After supplementing the volume to 4 / 5 of the total vaccine preparation volume with serum-free DMEM diluent, mix it evenly with the lyophilization protectant in a ratio of 4:1 to obtain the vaccine stock solution. After aseptic quantitative sub-packaging, perform lyophilization. Lyophilization process: (1) Pre-freezing stage: Enter the chamber at room temperature, lower the chamber temperature to -45°C within 1 hour and maintain it for 2 hours; (2) Sublimation drying stage: Raise the chamber temperature from -45°C to -20°C within 1 hour and maintain it for 12 hours; (3) Desorption drying stage: Raise the chamber temperature from -20°C to -5°C within 3 hours and maintain it for 0.5 hour, then raise the product from -5°C to 30°C within 1 hour and maintain it for 5 hours. The lyophilization is completed. After the vaccine is taken out of the chamber and crimped, store it at 2-8°C. After the vaccine purity test is qualified, it is used for potency test.

[0051] 3. Potency Test of Monovalent Live Vaccine

[0052] 3.1 Monitoring of Antibody Level after Vaccine Immunization Dilute the vaccine with diluent to 1 dose / ml. Use 8 healthy susceptible cats at 8-10 weeks of age, 5 of which are subcutaneously injected with the vaccine, 1 dose per cat, and boost immunized with the same dose 21 days later. On the 21st day, 2 months, 3 months, 4 months, 5 months, and 6 months after the boost immunization, together with 3 control cats, collect blood to separate serum and detect the neutralizing antibody titer of feline calicivirus in the serum. The results show that on the 21st day after the boost immunization of the vaccine, the feline calicivirus antibody titers of the immunized animals were 1:16 - 1:90, all higher than the critical protection value of 1:11. The antibody level reached the highest value 2 months after immunization, and the antibody titers of the immunized animals were in the range of 1:51 - 1:101; by 6 months after the boost immunization, the antibody titers of the immunized animals were in the range of 1:25 - 1:45, still all higher than the critical value. It shows that the immune persistence period is not less than 6 months.

[0053] 3.2 Viral Challenge Protection Effect after Vaccine Immunization Dilute the vaccine with diluent to 1 dose / ml. Use 10 healthy susceptible cats at 8-10 weeks of age, randomly divide them into 2 groups, 5 in each group. Among them, 5 are each subcutaneously injected with 1 dose of the vaccine and boost immunized with the same dose 21 days later; the other 5 are not treated as controls. On the 21st day after the boost immunization, each cat is challenged intranasally with 1 ml of highly virulent feline calicivirus for testing, 0.5 ml per nostril. The challenge results show that all the animals in the control group showed typical symptoms such as increased nasal secretions, increased eye secretions, and oral ulcers, and the symptom scores were not less than 12 points, and the incidence rate was 5 / 5; although the immunized group showed mild symptoms, the symptoms did not exceed 6 points, and the protection rate was 5 / 5. The viral challenge protection results show that the monovalent live vaccine prepared from this feline calicivirus attenuated strain has good protection effect.

[0054] Example 7: Preparation Method of Feline Multivalent Live Vaccine

[0055] Preparation and Potency Test of Multivalent Live Vaccine Containing Feline Calicivirus Attenuated Strain

[0056] 1. Preparation of virus liquid for vaccine production

[0057] 1.1 The preparation of the virus liquid of the attenuated strain of feline calicivirus is the same as that of the virus liquid in the monovalent vaccine.

[0058] 1.2 Preparation of the virus liquid of the attenuated strain of feline parvovirus: Take out the cryopreserved CRFK cells from the working cell bank, thaw them in a 37°C water bath, transfer the cells into a cell culture flask containing DMEM culture medium with 4% - 6% bovine serum, and culture them in a 37°C environment for 48 - 72 hours until the cells grow into a confluent monolayer. Discard the culture medium, digest with trypsin, and passage at a ratio of 1:3 - 1:4 for expansion culture. Inoculate the expanded CRFK cells into a culture flask, and synchronously inoculate the attenuated strain of feline parvovirus seed virus at an MOI of 0.01 - 0.1 into the CRFK cells, and culture them in a 37°C environment. Harvest when the CPE reaches about 80%, and store at -15°C or below. After the harvested virus liquid passes the purity test of virus content, it is used as the virus liquid for vaccine production.

[0059] 2. Preparation and inspection of the combined live vaccine: According to the virus content of the attenuated strain of feline calicivirus in each dose of vaccine being 10 5.5 TCID 50 and the virus content of the attenuated strain of feline parvovirus being 10 4.0 TCID 50 , calculate the dosage of the virus liquid of the two viruses required for vaccine formulation. After supplementing the volume to 4 / 5 of the total vaccine formulation volume with serum-free DMEM diluent, then mix it evenly with the freeze-drying protectant at a ratio of 4:1 to obtain the vaccine stock solution. After aseptic quantitative dispensing, it is freeze-dried. Freeze-drying process: (1) Pre-freezing stage: Put it into the box at room temperature, and reduce the temperature in the box to -45°C within 1 hour and maintain it for 2 hours; (2) Sublimation drying stage: Raise the temperature in the box from -45°C to -20°C within 1 hour and maintain it for 12 hours; (3) Analytical drying stage: Raise the temperature in the box from -20°C to -5°C within 3 hours and maintain it for 0.5 hour, then raise the product from -5°C to 30°C within 1 hour and maintain it for 5 hours, and the freeze-drying is completed. After the vaccine is taken out of the box, crimped, and stored at 2 - 8°C, it is used for potency test after passing the purity test of the vaccine.

[0060] 3. Potency test of the combined live vaccine

[0061] 3.1 Monitoring of antibody levels after vaccination with the combined live vaccine The combined live vaccine was diluted with diluent to 1 dose / ml. Eight healthy and susceptible cats at 8 - 10 weeks of age were used. Five of them were subcutaneously injected with the vaccine at a dose of 1 dose per cat. Twenty - one days later, a second vaccination was carried out at the same dose. On the 21st day, 2 months, 3 months, 4 months, 5 months, and 6 months after the second vaccination, together with 3 control cats, blood was collected to separate serum, and the neutralizing antibody titers of feline calicivirus and feline parvovirus in the serum were detected. The results showed that on the 21st day after the second vaccination, the antibody titers of feline calicivirus in the immunized animals were 1:20 - 1:81, all higher than the critical protection value of 1:11; the antibody titers of feline parvovirus were 1:256 - 1:1446, all higher than the critical protection value of 1:45. The antibody levels reached the highest value 2 months after vaccination. The antibody titers of feline calicivirus in the immunized animals were 1:45 - 1:90, and the antibody titers of feline parvovirus were 1:811 - 1:2048. By the 6th month after the second vaccination, the antibody titers of the immunized animals were 1:25 - 1:45, still all higher than the critical value. The antibody detection results after vaccination indicated that the immunization duration of the combined live vaccine containing the attenuated strain of feline calicivirus was not less than 6 months.

[0062] 3.2 Challenge protection effect after vaccination with the vaccine The vaccine was diluted with diluent to 1 dose / ml. Twenty healthy and susceptible cats at 8 - 10 weeks of age were randomly divided into 2 groups, with 10 cats in each group. Ten of them were each subcutaneously injected with 1 dose of the vaccine, and 21 days later, a second vaccination was carried out at the same dose; the other 10 cats were not treated as the control. On the 21st day after the second vaccination, 5 cats were randomly selected from each group, and each cat was challenged intranasally with 1 ml of the virulent strain for feline calicivirus test, 0.5 ml per nostril; for the remaining 5 cats in each group, each was challenged with 2 ml of the virulent strain for feline parvovirus test. The results of the challenge with the virulent strain for feline calicivirus test showed that all the animals in the control group showed typical symptoms such as increased nasal secretions, increased eye secretions, and oral ulcers, and the symptom score was not less than 13 points, and the morbidity rate was 5 / 5; although the immunized group showed mild symptoms, the symptoms did not exceed 8 points, and the protection rate was 5 / 5. The results of the challenge with the virulent strain for feline parvovirus test showed that the proportion of the decrease in white blood cell count in the control group of animals exceeded 60%, and the morbidity rate was 4 / 5; the proportion of the decrease in white blood cell count in the immunized group of animals did not exceed 40%, and the protection rate was 5 / 5. The challenge protection results indicated that the combined live vaccine prepared from the attenuated strain of feline calicivirus had good protection effects.

[0063] Example 8: A vaccine composition,

[0064] A vaccine composition contains the attenuated strain of feline calicivirus or its derivative virus as described in any one of claims 1 - 2.

[0065] Example 9: A vaccine composition,

[0066] A vaccine composition contains the attenuated feline calicivirus strain described in claim 2 or a derivative virus thereof.

[0067] Example 10: A vaccine composition,

[0068] A vaccine composition contains the attenuated feline calicivirus strain described in claims 1 and 2 or a derivative virus thereof.

[0069] Example 11: A vaccine composition contains a derivative virus of the attenuated feline calicivirus strain described in claims 1 and 2.

[0070] Example 12: A vaccine composition

[0071] The vaccine composition contains the capsid protein (VP1) of the attenuated feline calicivirus strain or a nucleic acid encoding the capsid protein (VP1) of the attenuated feline calicivirus strain.

[0072] Those of ordinary skill in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples; under the concept of the present disclosure, the above embodiments, or the technical features between different embodiments, can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of brevity.

[0073] The embodiments of the present disclosure are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure shall be included within the protection scope of the present disclosure.

Claims

1. The feline calicivirus strain according to claim 1, characterized in that: The amino acid sequence of the capsid protein (VP1) gene of the attenuated strain of feline calicivirus is shown in SEQ ID NO:

2.

2. The feline calicivirus strain according to claim 1, characterized in that: The nucleotide sequence of the capsid protein (VP1) gene of the attenuated strain of feline calicivirus is shown in SEQ ID NO:

1.

3. A vaccine composition, characterized in that: The vaccine composition comprises the attenuated feline calicivirus strain or a virus derived therefrom according to any one of claims 1 to 2.

4. The vaccine composition according to claim 3, characterized in that: The attenuated feline calicivirus strain selected in the vaccine composition is a live virus or an inactivated virus.

5. The vaccine composition according to claim 3, characterized in that: The vaccine composition comprises the capsid protein (VP1) of the attenuated feline calicivirus strain or a nucleic acid encoding the capsid protein (VP1) of the attenuated feline calicivirus strain.

6. The vaccine composition according to claim 3, characterized in that: The preparation method of the vaccine composition comprises the following steps: Isolating the low-virulence strain of feline calicivirus and culturing it to obtain a virus liquid of the attenuated strain of feline calicivirus; Treating the attenuated virus liquid, wherein the treatment includes cloning and purification; The treated attenuated virus solution is diluted to a predetermined virus titer.

7. The feline calicivirus strain according to claim 1, characterized in that The preparation method of the feline calicivirus strain comprises the following steps: isolating a low-virulence strain of feline calicivirus, inoculating it on CRFK cells for continuous temperature T subculture; when the cytopathic rate is not less than 80%, freezing and thawing at a temperature below T1 for harvesting, and storing the harvested culture at a temperature below T2; during the subculture process, plaque cloning and purification are performed once every N generations, N is 8-12, and a total of M cloning and purification are performed, M is 1-4, and larger plaques are picked for expansion culture each time the cloning and purification is performed; after the feline calicivirus is cloned and purified on the CRFK cells to the Kth generation, the K is 36-42.

8. The feline calicivirus strain according to claim 7, characterized in that The T is 39.5°C to 40.5°C; the T1 is -10°C to -15°C; the T2 is -60°C to -70°C; the N is 10, the M is 3, and the K is 38 to 40.

9. The vaccine composition according to claim 3, characterized in that: The vaccine composition also includes a multiple live vaccine for cats or a multiple inactivated vaccine containing attenuated strains, and the vaccine composition also includes a pharmaceutically acceptable carrier or adjuvant.

10. Use of the vaccine composition according to claims 3 to 9 in the preparation of a medicament for preventing a systemic disease in a cat caused by feline calicivirus.

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