Feline calicivirus strain LZ-2016 and vaccine compositions

Abstract

The application provides a feline calicivirus LZ-2016 strain and a vaccine composition. The feline calicivirus LZ-2016 strain has a high virus titer, the strain has strong pathogenicity to cats, and can cause typical symptoms such as oral ulcer and conjunctivitis. The inactivated product of the strain can produce a good immune response when cats are immunized, has good immunogenicity, can better stimulate the body to produce high-level neutralizing antibodies against FCV, can provide high immunoprotection rate to FCV attack, and has basic potential for developing a vaccine. The strain can be prepared into a vaccine and used for feline calicivirus prevention and treatment, provides an important vaccine virus source for feline calicivirus prevention and control in China, and realizes effective feline calicivirus prevention and control.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and immunology, and particularly relates to a feline calicivirus LZ-2016 strain and a vaccine composition. BACKGROUND

[0002] Feline calicivirus (FCV) is one of the main pathogens causing upper respiratory tract disease in cats, which seriously threatens the life and health of pet cats and all wild cats. FCV belongs to the family Caliciviridae, and is a single-stranded, non-segmented RNA virus without a capsid. The mature FCV virion is spherical, with a diameter of about 35-40 nm. FCV infection is usually manifested as upper respiratory tract and oral lesions in cats, with frequent water blisters and ulcers on the tongue as the typical clinical features of the disease, and further leading to pneumonia and other symptoms. High virulence FCV strains can even cause death. The disease has strong infectivity, and sick cats and virus-carrying cats are the main sources of infection. FCV is transmitted through contact between infected and healthy animals or exposure to droplets in the air through sneezing. FCV mainly proliferates in oral and respiratory tissues, and due to different strains, the tissue tropism and pathogenicity are different, and a small number of FCV also exists in internal organs, feces and urine. FCV contains a single-stranded positive-sense RNA genome with multiple adenine chains, with a size of about 7.7 kb, encoding three open reading frames (ORFs). ORF1 is about 5.3 kb long, encoding 1763 amino acids (aa) of non-structural proteins; ORF2 is about 2.1 kb long, encoding a capsid protein VP1 precursor, and the capsid protein VP1 is also the main immunogenic protein of FCV; ORF3 is located at the 3' end of the genome, about 320 bp long, encoding a minor capsid protein VP2, which can also affect the replication, maturation, protein synthesis and virus particle formation process of FCV, and natural virus particles need to be assembled with VP1 and VP2.

[0003] Like many RNA viruses, there is a great deal of heterogeneity within FCV viral populations. Several FCV strains have been identified and isolated, notably the F9 strain (deposited at the American Type Culture Collection or ATCC, Accession Number VR-782), the 2280 strain (ATCC VR-2057), the KCD strain (ATCC VR-651) and the CFI strain (ATCC VR-654). Attenuated FCV strains, primarily the F9 strain, have been used for FCV vaccination since the late 1970s and these attenuated vaccines form the majority of commercial FCV vaccines. These vaccines stimulate a good protective rabbit immune response but have the problem of virulence reversion. There is evidence that live vaccine viruses can cause clinical signs in some cases and even cause epidemics. Inactivated vaccines are also commercially available and all of these inactivated vaccines contain an adjuvant. These inactivated vaccines primarily use the 255 and 2280 strains and usually contain an adjuvant to improve the immune response and induce better protection against heterologous FCV strains that are emerging in cat populations. However, they do not induce effective mucosal and cellular immune responses and thus are not effective in suppressing FCV replication in the respiratory tract, and in addition, the incidence of local adverse reactions is higher for adjuvanted vaccines than for non-adjuvanted vaccines (Gobar, 2002), thus increasing the risk of fibrosarcoma at the injection site associated with the vaccine. Non-adjuvanted FCV vaccines are usually modified live vaccines and usually contain the F9 strain described above, vaccination with FCV vaccines only protects cats from disease but not from infection and usually immunized cats can develop a subclinical or persistent infection and can shed virus for a long time to become a source of infection (Zhao Yanli, Dong Hongwei, Chen Xiaoqing, et al. Research Progress of Feline Infectious Rhinotracheitis Vaccine [J]. China Animal Health Magazine. 2014(09): 56-58), thus the safety of modified live vaccines is questionable. Due to antigenic drift over time, antisera raised against vaccine strains isolated in the 1960-70s, including strains such as F9, 255 or 2280, only neutralize some isolates of the calicivirus strains that were prevalent in the 1990s and 2000s. For example, anti-F9 serum neutralized 43% of US isolates between 1990-1996, while 56% of isolates between 1980-89 and 86% of isolates between 1958-79, 10% of isolates between 1990-96 (Lauritzen, 1997). Since most of the neutralizing epitopes are present in the hypervariable regions of the FCV capsid protein, the use of monovalent FCV vaccines can not completely prevent infection with the virus. Therefore, to improve the cross-neutralization response, multivalent vaccines have become a new research direction for FCV vaccines. Attenuated and inactivated vaccines from earlier FCV strains no longer provide sufficient protection against recent FCV epidemic strains and current calicivirus vaccines must be replaced by vaccines that are more adapted to the current epidemiological situation and are able to provide greater cross-neutralization against the isolates currently identified in the feline population. SUMMARY

[0004] The purpose of the present application is to provide a cat calicivirus LZ-2016 strain and its application. The strain has strong pathogenicity to healthy susceptible cats, and the inactivated product of the strain can induce high levels of neutralizing antibodies, thereby preventing FCV infection and achieving effective prevention and control of FCV.

[0005] The cat calicivirus LZ-2016 strain described in the present application is named cat calicivirus FCV LZ-2016 strain, which was preserved in China Center for Type Culture Collection on September 2, 2022, with a preservation number of CCTCC NO: V202276 and a preservation address of Wuhan, China.

[0006] The present application provides a vaccine composition containing a vaccine-acceptable carrier and the cat calicivirus strain or its derivative virus according to claim 1.

[0007] Preferably, the vaccine composition comprises FCV LZ-2016 capsid protein and a pharmaceutically acceptable carrier, wherein the capsid protein comprises SEQ ID NO: 2, and the capsid protein is provided in an effective amount to produce an immune response.

[0008] Preferably, the vaccine composition comprises an adjuvant and a cat calicivirus FCV LZ-2016 strain with a CCTCC preservation number of V202276, and the FCV LZ-2016 strain is selected from inactivated viruses.

[0009] Preferably, the vaccine composition further comprises at least one other cat calicivirus strain selected from FCV-F9, FCV LLK, FCV-M8, FCV-255 and FCV-2280.

[0010] Preferably, the vaccine composition comprises a nucleic acid encoding FCV-LZ-2016 capsid protein, wherein the nucleic acid has the sequence of SEQ ID NO: 1 or encodes SEQ ID NO: 2.

[0011] The nucleic acid consists of DNA having a nucleic acid sequence encoding the FCV capsid protein or its specific immunogenic fragment disclosed herein. The nucleic acid encoding the capsid protein or its specific immunogenic fragment is operably linked to a transcription promoter or a position adjacent thereto. This enables transcription of the capsid protein or its specific immunogenic fragment from the nucleic acid when the nucleic acid is inoculated into cat cells.

[0012] Preferably, the DNA molecule is a plasmid. Promoters useful in DNA vaccines are well known in the art and include, but are not limited to, the RSV LTR promoter, the CMV immediate early promoter, and the SV40 T antigen promoter. The nucleic acid is operably linked to a nucleic acid segment comprising a transcription termination signal and a poly(A) recognition signal at or near the position of the stop codon of the sequence encoding the capsid protein or the immunogenic fragment specific thereof. The DNA vaccine can be provided to the cat in an acceptable pharmaceutical carrier or in a lipid or lipid replacement vehicle (similar to those disclosed in Felgner, U.S. Patent No. 5703055). The DNA vaccine can be provided to the cat by various methods, such as intramuscular injection, intrajet injection, or biolistic bombardment. Methods of preparing DNA vaccines and methods of using the same are provided in Felgner, U.S. Patent Nos. 5589466 and 5580859. Finally, methods of preparing pharmaceutical grade plasmid DNA are provided in Marquet et al., U.S. Patent No. 5561064.

[0013] The FCV LZ-2016 strain described in the present application has a high virus titer, and the strain has strong pathogenicity to healthy susceptible cats and can cause typical symptoms such as oral ulcer and conjunctivitis. The inactivated preparation of the strain can produce a good immune response when the cats are immunized, has good immunogenicity, can better stimulate the body to produce high-level neutralizing antibodies against FCV, can provide high immunoprotection rate against FCV attack, and has basic potential for developing vaccines. The strain can be prepared into a vaccine for preventing and treating FCV, and provides an important vaccine strain source for preventing and controlling FCV in China, so as to achieve effective prevention and control of FCV. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 RT-PCR identification results of suspected infection samples (lane M is DL2000 Marker, lane 1 is a positive control, lane 2 is a negative control, and lane 3 is an FCV LZ-2016 strain ORF3 amplification product)

[0015] Figure 2 CPE observation results of FCV LZ-2016 strain cultured on F81 cells (A is F81 cells inoculated with FCV LZ-2016 strain for 24 h, and B is normal F81 cells);

[0016] Figure 3 Sucrose ultracentrifugal concentration and electron microscope observation results of FCV LZ-2016 strain (A is the ultracentrifugal concentration results of FCV LZ-2016 strain in different sucrose layers, and B is the electron microscope observation results of FCV LZ-2016 strain);

[0017] Figure 4FCV LZ-2016 strain whole genome RT-PCR amplification results (lane M is DL5000 Marker, lane 1 is negative control, lane 2 is FCV LZ-2016 strain Q1 segment PCR product, lane 3 is FCV LZ-2016 strain Q2 segment PCR product, lane 4 is FCV LZ-2016 strain Q3 segment PCR product);

[0018] Figure 5 FCV LZ-2016 strain whole genome nucleotide homology analysis;

[0019] Figure 6 FCV LZ-2016 strain capsid protein amino acid homology analysis;

[0020] Figure 7 FCV LZ-2016 strain whole genome nucleotide phylogenetic tree analysis;

[0021] Figure 8 FCV LZ-2016 strain challenge results on test cats (A, E are LZ-2016 strain 10 7.0 TCID 50 / mL challenge group; B, F are LZ-2016 strain 10 8.0 TCID 50 / mL challenge group; C, G are LZ-2016 strain 10 9.0 TCID 50 / mL challenge group; D, H are control group).

[0022] Figure 9 FCV remaining epidemic strain RT-PCR identification (lane M is DL2000 Marker, lane 1 is positive control, lane 2 is negative control, lane 3 is FCV BJ-2015 strain ORF3 amplification product, lane 4 is FCV LY-2017 strain ORF3 amplification product, lane 5 is FCV QD-2017 strain ORF3 amplification product, lane 6 is FCV SH-2016 strain ORF3 amplification product, lane 7 is FCV HZ-2015 strain ORF3 amplification product, lane 8 is FCV JN-2014 strain ORF3 amplification product). DETAILED DESCRIPTION

[0023] Experimental materials: the results of the identification of other domestic FCV epidemic strains involved in the examples after using the identification method of steps 2-3 in Example 1 are shown in Table 1. Figure 9The ORF2 gene nucleotide sequence homology between the BJ-2015 strain, the LY-2017 strain, the QD-2017 strain, the SH-2016 strain, the HZ-2015 strain and the JN-2014 strain and the reference strains shown in Table 2 and the LZ-2016 strain is 71.2% to 85.1%. Among them, the BJ-2015 strain and the QD-2017 strain are relatively close to the GX2019 strain (GenBank NO: MK867378) in genetic distance, and the ORF2 gene nucleotide sequence homology of the two is 79.7% and 78.5%, respectively; the LY-2017 strain, the SH-2016 strain and the HZ-2015 strain are relatively close to the HB-S4 strain (GenBank NO: KT267162) in genetic distance, and the ORF2 gene nucleotide sequence homology of the three is 83.1%, 83.3% and 82.0%, respectively; the JN-2014 strain is relatively close to the GX01-13 strain (GenBank NO: KT970059) in genetic distance, and the ORF2 gene nucleotide sequence homology of the two is 82.5%.

[0024] Definitions and Abbreviations

[0025] A "therapeutically effective amount" in the context of the present disclosure refers to an amount of antigen or vaccine that induces an immune response in an individual (e.g., a cat) receiving the antigen or vaccine sufficient to prevent or ameliorate a sign or symptom of disease (including adverse health effects) resulting from infection with a pathogen (e.g., a virus, such as FCV, a bacterium, a parasite, or a fungus) or a complication thereof. Either humoral or cell-mediated immunity, or both, can be induced. An animal's immunogenic response to a vaccine can be evaluated, for example, indirectly by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms following a wild-type strain challenge. The protective immunity conferred by a vaccine can be evaluated by measuring, for example, a reduction in clinical signs (e.g., mortality, morbidity, individual temperature values and overall physical condition, and overall health and performance). A therapeutically effective amount of a vaccine can vary depending on the particular virus used or the individual's condition, and can be determined by a physician.

[0026] "Pharmaceutically acceptable" means a substance that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of an individual without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for its intended use. The vaccine can also be mixed with a pharmaceutically acceptable adjuvant. In certain formulations of the vaccine of the present application, the vaccine is combined with other feline vaccines to produce a multivalent vaccine product that can provide protection to a cat against a broad spectrum of diseases caused by other feline pathogens. Currently, commercial producers of feline vaccines, as well as end users, prefer multivalent vaccine products. Thus, in a preferred embodiment, the present application provides a multivalent vaccine that can immunize a cat against feline calicivirus and at least one other feline pathogen, wherein the other feline pathogen is preferably selected from the group consisting of feline herpesvirus, feline leukemia virus, feline immunodeficiency virus, Chlamydia felis, and feline panleukopenia virus.

[0027] The vaccine composition can optionally include a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that is compatible with the vaccine and can serve as a pharmaceutical vehicle, excipient, or medium. Diluents can include water, saline, dextrose, ethanol, glycerol, and the like. Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, lactose, and the like. Stabilizers include albumin and the like. Any adjuvant known in the art can be used in the vaccine composition, including oil-based adjuvants, such as Freund's complete and incomplete adjuvants, mycolic acid-based adjuvants (e.g., trehalose dimycolate), bacterial lipopolysaccharide (LPS), peptidoglycans (i.e., murein, mucopeptide, or glycoproteins such as N Opaca, muramyl dipeptide [MDP], or MDP analogs), proteoglycans (e.g., extracted from Klebsiella pneumoniae), streptococcal preparations (e.g., OK432), Biostim™ (e.g., 01K2), "Iscoms" of EP 109 942, EP 180 564, and EP 231 039, aluminum hydroxide, saponin, DEAE-dextran, neutral oil (e.g., miglyol), vegetable oil (e.g., peanut oil), liposomes, Polyols. Adjuvants include, but are not limited to, RIBI adjuvant system (Ribi Inc.), alum, aluminum hydroxide gel, cholesterol, oil-in-water emulsion, water-in-oil emulsion, such as Freund's complete and incomplete adjuvants, block copolymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), adjuvants and additives useful in the context of the present application can be readily determined by one skilled in the art, the present application contemplates compositions comprising from about 50 μg to about 2000 μg of an adjuvant and, preferably, about 500 μg per 2 ml dose of the vaccine composition. In another preferred embodiment, the present application contemplates vaccine compositions comprising from about 1 μg / ml to about 60 μg / ml of an antibiotic, more preferably less than about 30 μg / ml of an antibiotic.

[0028] The immunogenic compositions of the present application can be prepared in various forms depending on the route of administration. For example, the immunogenic compositions can be prepared as sterile aqueous solutions or dispersions suitable for injectable use, or freeze-dried formulations using lyophilization techniques. The lyophilized immunogenic compositions are typically maintained at about 4°C and can be reconstituted in a stabilizing solution with or without an adjuvant, e.g., saline or / and HEPES.

[0029] In addition, the immunogenic compositions and vaccine compositions of the present application can include one or more pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial agents and antifungal agents, isotonic agents, absorption delaying agents, and the like. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the individual being immunized. Typically, the carrier will be sterile and pyrogen-free.

[0030] Example 1 Isolation and identification of FCV LZ-2016 strain

[0031] 1, Sample collection and processing: In May 2016, the upper respiratory tract swabs of cats suspected to be infected in Liuzhou, Guangxi were collected with sterile cotton swabs. The swabs were dissolved in 1 mL PBS buffer for 5 min, centrifuged at 3000 r / min for 10 min, the cotton swab was discarded, and then mixed and stored at -80°C for standby;

[0032] 2. RT-PCR identification: The virus RNA in the above preparation liquid was extracted according to the conventional Trizol RNA extraction method, and reverse transcription operation was performed according to the reverse transcription kit instruction of Novizen Biotechnology Co., Ltd.

[0033] 3. The cDNA was subjected to PCR amplification of FCV ORF3 gene.

[0034] According to the FCV FB-NJ-13 strain genome sequence (GenBank ID: KM111557) registered in GenBank, a pair of specific primers was designed to amplify the FCV ORF3 gene, and the primer sequences were as follows:

[0035] The upstream primer ORF3-F was 5'-GTTGACCCTTACTCATACAC-3'

[0036] The downstream primer ORF3-R was 5'-CCCTGGGGTTAGGCGC-3'

[0037] The amplification fragment size was 136 bp, and the reaction condition was as follows: after pre-denaturation at 94℃ for 5 min, the cycle parameters were as follows: 94℃ for 30 s, 54℃ for 30 s, and 72℃ for 30 s; after 35 cycles, 72℃ was extended for 10 min. 5 μl of the product was subjected to 1% agarose gel electrophoresis identification. The amplified PCR fragment size was 136 bp, which was consistent with the expected size (see Figure 1 );

[0038] 4. Virus isolation and culture: The sample was centrifuged at 3000 r / min for 10 min, and the supernatant was added with an equal amount of DMEM nutrient solution, filtered with a 0.22 um filter to remove bacteria, inoculated into F81 cells with a full monolayer, and cultured in a 37℃ 5% CO2 incubator. Obvious lesions appeared in the first generation of inoculation, and the cell lesions were stable in the third generation, showing cell rounding, aggregation, grape string-like, and finally complete necrosis and shedding (see Figure 2 );

[0039] 5. Plaque purification and virus content determination: The isolated FCV was diluted by 10 times successively (from 10 -1 to 10 -10), each dilution of virus liquid was inoculated into 6-hole cell culture plates of F81 cells, 2 holes for each dilution, adsorbed for 1 h at 37℃ in a 5% CO2 incubator, the virus liquid was aspirated, and 2% serum, 0.8% low-melting-point agarose-containing phenol red-free DMEM was overlaid, and continued to be cultured at 37℃ in a 5% CO2 incubator for 3-5 days. After obvious plaques were observed under a microscope, 0.1‰ neutral red was used for staining at 37℃ for 1 h, the staining liquid was aspirated, and the plaques were picked up in 200 μL of maintenance liquid, repeated freeze-thawing for 3 times, and inoculated into 24-hole cell culture plates of F81 cells, 0.8 mL of cell maintenance liquid was added after adsorption for 1 h at 37℃ in a 5% CO2 incubator, and cultured at 37℃ in a 5% CO2 incubator until complete lesion occurred. After that, the lesioned cell culture was collected, freeze-thawed for 3 times, and the TCID of each clone virus was determined 50 . The high-titer clone was selected, purified again as above for 2 times, and the high-titer clone virus was marked as F1 generation, and the virus content was determined, and the TCID was calculated according to the Reed-Muench method 50 . The virus content of the finally harvested FCV LZ-2016 strain virus liquid can reach 10 9.5 TCID 50 / mL;

[0040] 6. Virus sucrose ultracentrifugation concentration and purification and electron microscope observation: (1) The supernatant was taken after 12000 rpm centrifugation of a large amount of amplified FCV virus liquid for 1 h, and the supernatant was filtered with a 0.45 μm filter to remove cell debris; (2) 0.5 mol / L NaCl and 5% PEG6000 were added to the virus liquid, fully mixed, and then placed at 4℃ for 24 h. Subsequently, the virus liquid was centrifuged at 12000 rpm for 1 h, and the supernatant was discarded. A small amount of STE solution was added to the precipitate to dissolve overnight at 4℃; (3) the dissolved virus in the STE solution was added to the upper part of the centrifuge tube containing 20%, 35%, 50% and 65% 4 gradient sucrose solution, and centrifuged at 30000 rpm for 3 h. Samples were taken at 20%-35%, 35%-50% and 50%-65% layers, respectively; (4) the samples at different layers were placed in new centrifuge tubes, and the centrifuge tubes were filled with STE solution. After centrifugation at 30000 rpm for 4 h, the supernatant was discarded to remove sucrose, and a small amount of STE was added to the precipitate to dissolve overnight at 4℃; (5) the obtained virus suspension samples were dropped onto a copper mesh and adsorbed for 5 min. The virus suspension was gently absorbed with filter paper. After drying, 20 g / L phosphotungstic acid was added for negative staining for 1 min. After negative staining, the phosphotungstic acid was absorbed with filter paper. After drying, it was observed under an electron microscope. The results showed that typical cup-shaped structure virus particles with a diameter of 37-40 nm were observed (see Figure 3 );

[0041] Example 2 Whole genome sequencing analysis of FCV LZ-2016 strain

[0042] 1. Gene amplification: According to the known FCV genome sequence in GenBank (GenBank ID: KM111557), the primers in Table 1 were designed, the extracted viral RNA was reverse transcribed into cDNA, and the full-length gene was divided into three segments for segmented PCR amplification using the primers in Table 1. The PCR reaction used a 50 μl reaction system: Prime STAR Max DNA Polymerase 25 μl, template 3 μl, upstream and downstream primers (10 μmol / L) 2 μl each, and ddH2O 18 μl. The above reagents were mixed thoroughly, and amplification was performed under the following conditions: 98°C pre-denaturation for 1 min; 98°C denaturation for 10 s, 58°C annealing for 15 s, 72°C extension for 1 min, 35 cycles; and finally 72°C extension for 7 min. The electrophoresis results of the amplification products are shown in Figure 1. Figure 4 ;

[0043] Table 1 FCV full genome amplification primers

[0044]

[0045] Note: R is a commonly used degenerate base (R = A, G);

[0046] 2. Ligation transformation: Each segment of the gene was ligated with the vector, and the specific steps were as follows: 4 μl of gel recovery product was mixed with 1 μl of pEASY-T1 cloning vector, and after 16°C reaction for 30 min, it was placed on ice. Trans DH5a competent cells were taken out from the -70°C refrigerator, and the ligation product was added during melting. After mixing gently, it was ice-bathed for 30 min, 42°C heat shock for 1 min, and ice-bathing for 3 min. 500 μl of antibiotic-free LB liquid medium was added aseptically, and the bacteria were shaken at 37°C and 200 rpm for 1 h. After centrifugation at 3000 rpm for 3 min, 100 μl of supernatant was reserved, the precipitated bacteria were suspended, and they were spread on LB-Amp+ agar plates and cultured in a 37°C incubator for 12-14 h. After visible colonies appeared on the agar plates, each plate was aseptically picked and placed in 2 mL of LB-Amp+ liquid medium, and cultured at 37°C and 200 rpm for 10-12 h;

[0047] 3. PCR identification and sequencing: 1 μl of bacterial liquid was used as a template, and the universal primers M13F / R of the pEASY-T1 vector were used for PCR amplification. The PCR products were identified by agarose gel electrophoresis, and the positive plasmids were sequenced. The sequencing results were spliced using DNAStar-Seqman, and the FCV LZ-2016 strain genome sequence with a full length of 7683 bp was obtained. (The specific sequence is shown in SEQ ID NO: 1);

[0048] 4、Sequence analysis: The full genome sequence of FCV LZ-2016 strain was compared with the full genome nucleotide sequences of 25 strains (Table 2) registered in GenBank, including FCV-255 strain, FCV-F9 strain, FCV-F4 strain, FCV-GX2019 strain, FCV-SH strain, FCV-GD strain, FCV-FB-NJ-13 strain and FCV-HB-S4 strain, etc. The results showed that the nucleotide homology of the full genome sequence of LZ-2016 strain with the full genome sequences of the domestic and foreign reference strains was between 74.9% and 82.8%, and the genetic difference with the foreign epidemic strains was large. Phylogenetic tree analysis of the full-length genome nucleotide sequence of FCV showed that the CH-JL1 strain formed a small branch with strains FB-NJ-13, GD and 12Q087-1, and these strains were all isolated from China and East Asia (Fig. 1). Figure 5 、 7 );

[0049] Since the capsid protein encoded by the ORF2 gene fragment is the main structural protein of FCV and is the most important immunogenic protein for stimulating the body to produce neutralizing antibodies, the amino acid sequence of the capsid protein of LZ-2016 strain (the specific sequence is shown as SEQ ID NO: 2) was analyzed by alignment. The homology between the amino acid sequences of the domestic and foreign reference strains was between 80.6% and 89.5%, and the genetic difference with the foreign epidemic strains was large, further indicating that the antigenic difference between the current epidemic strains was large (Fig. 2). Figure 6 The cat calicivirus strain isolated in the application is named cat calicivirus FCV LZ-2016 strain, which was preserved in China Center for Type Culture Collection on September 2, 2022, and the preservation number is CCTCC NO: V202276.

[0050] Table 2 FCV reference strain information and homology analysis with LZ-2016 strain

[0051]

[0052] Example 3 Virulence study of FCV LZ-2016 strain

[0053] The virus liquid was diluted to 10 7.0 TCID 50 / mL, 10 8.0 TCID 50 / mL and 10 9.0 TCID 50 / mL with DMEM, respectively, 20 cats of 4-8 weeks old with negative cat calicivirus neutralizing antibodies and antigens were selected and divided into 4 groups, and the cats were infected by nasal instillation. The first group was the 10 7.0 TCID 50 / mL infection group, the second group was the 10 8.0TCID 50 / mL challenge group, the third group was 10 9.0 TCID 50 / mL challenge group, the fourth group was the control group, the challenge dose was 1 mL, the challenge group was isolated and fed, and the clinical observation was carried out for 14 days after challenge. The results showed that 10 7.0 TCID 50 / mL challenge group 3 / 5 test cats showed clinical symptoms such as depression, eyelid swelling, oral ulcer, serous or purulent eye and nose secretion; 10 8.0 TCID 50 / mL and 10 9.0 TCID 50 / mL challenge groups all 5 / 5 test cats showed clinical symptoms such as depression, eyelid swelling, oral ulcer, serous or purulent eye and nose secretion, and 2 / 5 eventually died; the control group had no obvious symptoms Figure 8 ).

[0054] Example 4 Inactivation, inactivation test and safety test of FCV LZ-2016 strain

[0055] 1. Inactivation and inactivation test of LZ-2016 strain: add virus liquid to 0.06% volume fraction of β-propiolactone and continuously stir, inactivate at 4°C for 24h, then hydrolyze β-propiolactone at 37°C for 2h, and then store at 4°C after completion. Take the inactivated virus and inoculate the logarithmic growth phase F81 cells at a ratio of 1:100, and blind passage for 2 generations. The result is no virus growth, which is complete inactivation of LZ-2016 strain; inactivated calicivirus can be treated by using formaldehyde, β-propiolactone (betapropriolactone, BPL) or binary ethyleneimine (BEI), or using other methods known in the art.

[0056] 2. Preparation of inactivated vaccine of LZ-2016 strain: determine the virus content of LZ-2016 strain virus liquid before inactivation, and dilute the completely inactivated LZ-2016 strain virus liquid to 10 8.5 TCID 50 / mL with sterile PBS;

[0057] 3. Safety test: select 10 healthy susceptible cats of 4-8 weeks old with negative cat calicivirus neutralizing antibody and antigen, and divide them into 2 groups, the first group of each test cat is injected subcutaneously with 2 doses of FCV LZ-2016 strain inactivated vaccine in the neck, and the second group is inoculated with the same amount of saline, and the clinical observation is carried out for 14 days, all 5 / 5 are healthy and alive, and no adverse reactions are found.

[0058] Example 5 Immunogenicity and efficacy of FCV LZ-2016 strain

[0059] Fifteen healthy, susceptible cats aged 4–8 weeks were randomly divided into three groups of five each. Group 1 was immunized with the FCV LZ-2016 strain inactivated vaccine; Group 2 was immunized with a commercially available vaccine (the only similar product currently available in China, Boehringer Ingelheim Animal Health's "Miamito" vaccine (Pfizer, USA) – a trivalent inactivated vaccine for feline rhinotracheitis, calicivirus, and panleukopenia)); and Group 3 served as the control group, immunized with an equal volume of physiological saline. Immunization was administered subcutaneously in the neck, 1.0 mL per cat. Blood was collected 21 days after the second immunization, and serum was separated. The FCV neutralizing antibodies in the serum were determined using the fixed-virus dilution serum method, as well as the serum's resistance to other prevalent FCV strains isolated and identified in our laboratory (the results of identification of other prevalent FCV strains in China using the methods described in steps 2–3 of Example 1 are attached). Figure 9 The neutralizing antibodies provided cross-protection. The virus was challenged 28 days after the second immunization via nasal drops, with a challenge dose of 10... 8.0 TCID 50 / cat, challenged with the LZ-2016 strain. Within 14 days post-challenge, experimental cats were observed for typical feline calicivirus (FCV) symptoms such as serous or purulent ocular and nasal discharge, oral ulcers, and death. Upper respiratory tract swabs were collected on day 7 post-challenge (if any cat died, samples were taken and tested on the same day), and RT-PCR was used for FCV pathogen identification.

[0060] The results showed that after immunization with the FCV LZ-2016 inactivated vaccine, the neutralizing antibody level in experimental cats ranged from 1:631 to 1:1024. The cats achieved 5 / 5 protection after challenge with the FCV LZ-2016 strain, and all FCV pathogen identification results from upper respiratory tract swab samples taken on day 7 post-challenge were negative. In contrast, after immunization with the commercial vaccine, the neutralizing antibody level in experimental cats ranged from 1:13 to 1:37. The cats achieved only 1 / 5 protection after challenge with the FCV LZ-2016 strain, and most of the FCV pathogen identification results from upper respiratory tract swab samples taken on day 7 post-challenge (or the day of death) were positive. All members of the control group developed the disease, and all FCV pathogen identification results from upper respiratory tract swab samples taken on day 7 post-challenge (or the day of death) were positive. The results of cross-reactivity testing of neutralizing antibodies against other domestically prevalent isolates in serum collected 21 days after the second immunization showed that the neutralizing antibody levels of serum against 7 representative domestically prevalent strains after immunization with the FCV LZ-2016 inactivated vaccine ranged from 1:128 to 1:1024. In contrast, after immunization with commercial vaccines, the neutralizing antibody levels against only a few strains were higher than 1:16. Commercial vaccines may not provide good immune protection against the homologous strains GX2019, GX01-13, and HB-S4. The FCV LZ-2016 inactivated vaccine of this invention provides better immune protection against the homologous strains GX2019, GX01-13, and HB-S4 than existing commercial vaccines.

[0061] Table 3 FCV neutralizing antibody detection results of sera from cats immunized with the vaccine

[0062]

[0063] Note: "-" represents negative for FCV pathogen identified by RT-PCR, "+" represents positive for FCV pathogen identified by RT-PCR, "-" represents no abnormality, "E" represents the appearance of serous eye and nose secretion, "F" represents the appearance of purulent eye and nose secretion, "I" represents the appearance of oral ulcer, "S" represents death;

[0064] Table 4 Neutralizing effect of sera from cats immunized with the vaccine on other FCV isolated strains

[0065]

[0066] In summary, the FCV LZ-2016 strain described in the present application has a high virus titer, and the strain has strong virulence to healthy susceptible cats and can cause typical symptoms such as oral ulcer and conjunctivitis. The inactivated product of the strain can produce good immune response when immunizing cats, has good immunogenicity, and can better stimulate the body to produce high-level neutralizing antibodies against FCV as an inactivated vaccine, can provide high immunoprotection rate against FCV attack, and has basic potential for developing vaccines.

[0067] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A feline calicivirus strain LZ-2016, characterized in that, The feline calicivirus strain is named feline calicivirus FCV LZ-2016 strain, which was preserved in China Center for Type Culture Collection on September 2, 2022, and the preservation number is CCTCC NO: V202276.

2. A vaccine composition, characterized in that, The composition contains a vaccine-acceptable carrier and the feline calicivirus strain of claim 1.

3. The vaccine composition of claim 2, wherein It comprises FCV LZ-2016 capsid protein and a pharmaceutically acceptable carrier, wherein the capsid protein comprises SEQ ID NO: 2, and the capsid protein is provided in an effective amount to produce an immune response.

4. The vaccine composition of claim 2, wherein It comprises an adjuvant and a feline calicivirus FCV LZ-2016 strain with a CCTCC preservation number of V202276, and the FCV LZ-2016 strain is selected from inactivated viruses.

5. The vaccine composition according to claim 2, characterized in that, It further comprises at least one other feline calicivirus strain selected from FCV-F9, FCV LLK, FCV-M8, FCV-255 and FCV-2280.

6. The vaccine composition of claim 2, wherein It comprises a nucleic acid encoding FCV LZ-2016 capsid protein, wherein the nucleic acid has the sequence of SEQ ID NO: 1 or encodes SEQ ID NO: 2.

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