Isolation and application of feline rhinotracheitis virus FHV-1 WH-2017 strain

Abstract

This invention relates to the fields of biological products and preventive veterinary medicine, specifically to the isolation and application of feline rhinotracheitis virus strain FHV-1WH-2017. A feline rhinotracheitis virus strain FHV-1WH-2017, with accession number CCTCC NO:V202296, was disclosed. Immunoprotection tests were conducted on 8-12 week old kittens after inactivation of the feline rhinotracheitis virus strain FHV-1WH-2017. The results showed that the strain not only had good safety but also high protective efficacy in the immunogenicity test. This demonstrates that the virus strain possesses good immunogenicity and can be used for vaccine research and development for this disease.

Description

Technical Field

[0001] This invention relates to the fields of biological products and preventive veterinary medicine, specifically to the feline rhinotracheitis virus strain FHV-1WH-2017 and its applications, vaccine compositions and their preparation methods. Background Technology

[0002] Feline herpesvirus 1 (FHV-1) belongs to the Herpesviridae family, Alphaherpesvirinae subfamily. It is an enveloped, double-stranded linear DNA virus with a full-length genome of approximately 136 kb. FHV-1 is a highly contagious and easily transmitted pathogen causing upper respiratory tract diseases in felines. Feline rhinotracheitis caused by FHV-1 is one of the leading causes of death in cats. Common respiratory symptoms after FHV-1 infection include sneezing, nasal congestion, squinting, and ulcerative rhinitis. Initially, it can cause serous nasal discharge (clear), which may become mucopurulent (pale yellow) after secondary bacterial infection, and even interstitial pneumonia. Severe disease progression can lead to anorexia, dehydration, and death due to secondary bacterial infections. The feline herpesvirus particle is oval or round, with icosahedral symmetry, and consists of four parts: nucleic acid, capsid, capsid, and envelope. Viral particle diameters vary depending on their location and maturity stage, with an average diameter of approximately 148 nm within the cell nucleus. The gD protein of feline rhinotracheitis virus (FRIV) is a characteristic protein of herpesviruses, composed of 374 amino acids encoded by the US6 gene. The gD gene is relatively conserved and suitable as a recognition gene for virus isolation and identification. The gI protein of FRIV, encoded by the US7 region of the FHV-1 genome, consists of 365 amino acids and is one of the main virulence proteins of FRIV. The extracellular portions of the gI and gE proteins form a complex in a non-covalent manner to jointly exert virulence. gI is a key gene distinguishing the transmissibility and virulence of FRIV, and the gI gene of FRIV has been found to be a variable region after sequence alignment; therefore, its encoding gene is often used in genetic evolutionary characterization and is a focus of vaccine development.

[0003] The main transmission routes of FHV-1 virus are respiratory droplet transmission, close contact transmission, and aerosol transmission in special environments. Transmission via bodily fluids and mother-to-child transmission is also possible. Most recovered cats are either lifelong or reinfected due to the latent presence of FHV-1 virus in the trigeminal nerve. Reactivation can occur during stress, illness, or weakened immunity. Currently, there are no vaccines for feline rhinotracheitis virus (FHV) in China, nor are there any vaccines specifically targeting the currently prevalent FHV infection. Therefore, it is necessary to search for circulating FHV strains in China that possess good immunogenicity and can serve as potential vaccine development strains. Summary of the Invention

[0004] This invention provides a domestically prevalent feline rhinotracheitis virus strain FHV-1WH-2017. This strain is highly pathogenic and has a high mortality rate in cats, causing typical upper respiratory symptoms (sneezing, nasal congestion, squinting, ulcerative rhinitis, initially causing serous nasal discharge, etc.). After inactivation, this strain exhibits good immunogenicity and, as an inactivated vaccine, can effectively stimulate the body to produce high levels of neutralizing antibodies against the domestically prevalent strain WH-2017, providing a high rate of immune protection against FHV challenge, thus possessing the basic potential for vaccine development. Furthermore, this strain has a high and stable titer, facilitating industrial-scale production.

[0005] This invention solves the above-mentioned technical problems by providing:

[0006] A strain of feline rhinotracheitis virus, FHV-1WH-2017, with accession number CCTCC NO:V202296.

[0007] Preferably, a vaccine strain is a combination of feline calicivirus strain and the aforementioned feline rhinotracheitis virus, wherein the feline calicivirus strain is named feline calicivirus FCV LZ-2016 strain, and was deposited at the China Center for Type Culture Collection on September 2, 2022, with accession number: CCTCC NO: V202276.

[0008] Preferably, a vaccine composition uses a combination vaccine strain of feline calicivirus and feline rhinotracheitis virus as an immunogen.

[0009] Furthermore, the raw materials of the vaccine composition include an inactivated virus of the viral strain and an adjuvant.

[0010] Furthermore, the method for preparing the vaccine composition includes the following steps:

[0011] Step 1: The feline rhinotracheitis virus strain FHV-1WH-2017 and the feline calicivirus strain FCV LZ-2016 were propagated and cultured separately to obtain the virus stock solution.

[0012] Step 2: Add the virus stock solution or its dilution obtained in Step 1 to β-propiolactone for inactivation;

[0013] Step 3: Dilute the inactivated virus solution with sterile PBS to obtain the final product.

[0014] In step 2, the viral solutions of feline rhinotracheitis virus strain FHV-1WH-2017 and feline calicivirus strain FCV LZ-2016 were added to β-propiolactone at volumes of 0.12% and 0.06%, respectively. After mixing evenly, the mixture was inactivated at 4°C for 24 hours and then hydrolyzed at 37°C for 2 hours.

[0015] In step 3, the inactivated feline rhinotracheitis virus strain FHV-1WH-2017 and feline calicivirus strain FCV LZ-2016 were diluted to 10 μL with sterile PBS. 8.5 TCID 50 / mL, 10 9.0 TCID 50 / mL, and aliquoted and stored at 4℃; the prepared antigen solutions of the two viruses and sterile PBS were mixed in a volume ratio of 1:1:1, so that the final concentration of FHV-1WH-2017 strain antigen in 1mL / head dose of antigen was 10 8.0 TCID 50 / mL, the final concentration of FCV LZ-2016 strain antigen was 10 8.5 TCID 50 / mL.

[0016] Furthermore, the vaccine composition is intended for use in the preparation of a formulation for use alone or in combination with other immunizing agents or other drugs to treat, prevent, mitigate and / or control feline rhinotracheitis virus and / or feline calicivirus infection.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1. The feline rhinotracheitis virus strain FHV-1WH-2017 provided by this invention, after simultaneous inoculation of F81 adherent cells with the virus and subsequent culture in DMEM with 2% new bovine serum at 37°C and 5% CO2, achieved a viral titer of up to 10. 8.5 TCID 50 / mL. The purified FHV-1WH-2017 strain was cultured under the standard conditions described above. The resulting viral fluid was used to challenge healthy, susceptible cats aged 14-18 weeks and adult cats (serum FHV-1 neutralizing antibody <1:2; negative FHV-1 test by pharyngeal swab PCR; unvaccinated against FHV-1) via nasal drops. All 14-18 week old cats showed varying degrees of FHV-1 infection, and 10 5.0 TCID 50 In the / mL challenge group, 4 / 5 of the experimental cats also developed feline rhinotracheitis-related symptoms; while adult cats, although exhibiting some resistance to FHV-1WH-2017 strain challenge, showed resistance when the viral titer increased to 10. 8.0 TCID 50 At a concentration of 100 mL, 4 / 5 of the test cats showed symptoms related to feline rhinotracheitis (serous or purulent ocular and nasal discharge, respiratory rales or paroxysmal coughing, sneezing, etc.).

[0019] 2. The safety test results of the inactivated feline rhinotracheitis virus strain WH-2017 provided by this invention showed that: when the inactivated virus content was 10... 8.0TCID 50 At a dose of / mL, cats that received single-dose, repeated single-dose, and overdose treatments all showed normal food and water intake, and their body temperature fluctuated within the normal range (37.5℃-39.5℃). No adverse reactions were observed at the injection site, and 5 out of 5 cats survived. This indicates good safety.

[0020] 3. The efficacy test results of the inactivated feline rhinotracheitis virus strain WH-2017 provided by this invention showed that: 10 7.0 TCID 50 / mL of inactivated feline rhinotracheitis virus vaccine was administered to cats. 21 days after challenge, 3 / 5 of the cats showed no abnormalities in body temperature, mental state, or appetite; 2 / 5 of the cats developed clinical symptoms such as sneezing and serous ocular and nasal discharge, of which 1 / 5 died; 10 7.5 TCID 50 / mL of feline rhinotracheitis virus inactivated vaccine was administered to cats. 21 days after challenge, 4 / 5 of the cats showed no abnormalities in body temperature, mental state, or appetite; 1 / 5 of the cats exhibited mild sneezing, serous ocular and nasal discharge, and other clinical symptoms. 10 8.0 TCID 50 / mL and 10 8.5 TCID 50 Cats tested with a / mL inactivated feline rhinotracheitis virus vaccine showed no abnormalities in body temperature, mental state, or appetite after 21 days of challenge, and 5 / 5 survived. In the non-immunized challenge control group injected with PBS buffer, 5 / 5 presented with serous or purulent ocular and nasal discharge, respiratory rales, or paroxysmal sneezing after challenge, and 3 / 5 died. Before death, these cats exhibited decreased body temperature and lethargy. The blank control group showed no abnormalities. This indicates that when the antigen content is not less than 10... 7.5 TCID 50 At a concentration of [value missing] / mL, it provided good protection for the experimental cats, and the level of activated antibodies was not lower than 1:32. This indicates that the WH-2017 strain of virus has good immunogenicity.

[0021] 4. The vaccine composition of the present invention can effectively immunize against infection by two viruses with a single dose, including feline rhinotracheitis virus strain FHV-1WH-2017 and feline calicivirus strain FCV LZ-2016. Attached Figure Description

[0022] Figure 1 These are normal adherent F81 cells;

[0023] Figure 2 These are PCR tests performed on F81 cells from pharyngeal swab samples of strain WH-2017 at passages 1, 5, 10, and 20 in a 20-passage blind passage.

[0024] Figure 3 This is an electron microscope image of WH-2017 strain after isolation and purification with sucrose gradient dilution.

[0025] Figure 4 This is a nucleotide homology analysis of FHV-1WH-2017 strain with 23 strains from the NCBI database using gI.

[0026] Figure 5 This is a nucleotide homology analysis of FHV-1WH-2017 strain with 23 strains of gE from the NCBI database;

[0027] Figure 6 This is a nucleotide homology analysis between strain FHV-1WH-2017 and strain TK from 23 NCBI databases;

[0028] Figure 7 This is a nucleotide homology analysis between strain FHV-1WH-2017 and 23 strains of the virus in the NCBI database;

[0029] Figure 8 This is a nucleotide homology analysis of FHV-1WH-2017 strain with 23 strains from the NCBI database;

[0030] Figure 9 This is a genetic evolution diagram of the gI gene of strain FHV-1WH-2017 and 23 strains from the NCBI database;

[0031] Figure 10 This is a genetic evolution diagram of the gE gene of strain FHV-1WH-2017 and 23 strains from the NCBI database;

[0032] Figure 11 This is a genetic evolution diagram of the FHV-1WH-2017 strain and 23 strains of the TK gene from the NCBI database;

[0033] Figure 12 This is a genetic evolution diagram of the gB gene of strain FHV-1WH-2017 and 23 strains from the NCBI database;

[0034] Figure 13 This is a genetic evolution diagram of the gC gene of strain FHV-1WH-2017 and 23 strains from the NCBI database;

[0035] Figure 14 These are the results of the cat's eye observations in each of the virus challenge test groups;

[0036] Figure 15 The observation of the eyelids and lungs after dissection of cats with clinical symptoms in each experimental group;

[0037] Figure 16 These are HE-stained sections of ocular lesions observed in each experimental group.

[0038] Figure 17The results are from a toxicity test of the FHV-1WH-2017 strain on adult cats.

[0039] Figure 18 The tissue analysis results are from the FHV-1WH-2017 strain after challenge to adult cats.

[0040] Figure 19 This is a histopathological observation of adult cats challenged with FHV-1WH-2017 strain;

[0041] Figure 20 The results of CPE observation of FCV LZ-2016 strain cultured on F81 cells (A is F81 cells 24h after inoculation with FCV LZ-2016 strain, and B is normal F81 cells).

[0042] Figure 21 The results are RT-PCR identification results of suspected infected samples (lane M is DL2000 Marker, lane 1 is positive control, lane 2 is negative control, and lane 3 is the ORF3 amplification product of FCV LZ-2016 strain).

[0043] Figure 22 The results of ultracentrifugation concentration and purification of sucrose in FCV LZ-2016 strain and electron microscopy observation are shown in Figure A (ultracentrifugation concentration results of different sucrose layers of FCV LZ-2016 strain, and electron microscopy observation results of FCV LZ-2016 strain).

[0044] Figure 23 The results are RT-PCR amplification of the whole genome of FCV LZ-2016 strain (lane M is DL5000 Marker, lane 1 is negative control, lane 2 is PCR product of FCV LZ-2016 strain Q1, lane 3 is PCR product of FCV LZ-2016 strain Q2, and lane 4 is PCR product of FCV LZ-2016 strain Q3).

[0045] Figure 24 This is a homology analysis of the whole genome nucleotides of FCV LZ-2016 strain;

[0046] Figure 25 This is a homology analysis of the amino acid composition of the capsid protein of FCV LZ-2016 strain;

[0047] Figure 26 Phylogenetic analysis of the whole genome nucleotides of FCV LZ-2016 strain;

[0048] Figure 27 The results of challenging experimental cats with FCV strain LZ-2016 (A and E represent LZ-2016 strain 10). 7.0 TCID 50 / mL challenge group; B and F were LZ-2016 strain 10 8.0 TCID 50 / mL challenge group; C and G are LZ-2016 strain 10 9.0 TCID 50 / mL challenge group; D and H are control groups);

[0049] Figure 28 The following are RT-PCR identifications of other circulating FCV isolates (lane M is DL2000 Marker, lane 1 is positive control, lane 2 is negative control, lane 3 is the ORF3 amplification product of FCV BJ-2015 strain, lane 4 is the ORF3 amplification product of FCV LY-2017 strain, lane 5 is the ORF3 amplification product of FCV QD-2017 strain, lane 6 is the ORF3 amplification product of FCV SH-2016 strain, lane 7 is the ORF3 amplification product of FCV HZ-2015 strain, and lane 8 is the ORF3 amplification product of FCV JN-2014 strain).

[0050] Figure 29 This is a graph showing the changes in body temperature after immunization in the bivalent inactivated vaccine overdose group, the immunization control group, and the blank control group. Detailed Implementation

[0051] In this invention, "per dose" or " / dose" refers to the vaccine dose used per cat per administration. Unless otherwise specified, in the embodiments of this invention, "per dose" or " / dose" refers to 1 mL.

[0052] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0053] Example 1: Acquisition, Identification and Testing of Feline Rhinotracheitis Virus

[0054] I. Isolation of the virus strain

[0055] In April 2017, pharyngeal swabs were collected from clinically suspected infected cats in Wuhan, Hubei Province, using sterile cotton swabs. PBS solution was added, and the samples were centrifuged at 12,000 rpm for 10 min. The supernatant was then aspirated and filtered through a 0.65 μm filter. The filtered supernatant was seeded into F81 cells at a volume ratio of 1% and cultured continuously. Cellular pathogenesis was observed daily, and the cells were harvested promptly upon the appearance of cytopathic effects. After sample processing, DNA was extracted according to the OMEGA Viral DNA Kit manual and stored below -15°C for later use. A pair of specific primers was designed based on the FHV-1 genome sequence registered in GenBank to amplify the conserved region of the FHV-1 gD gene (Table 1).

[0056] Table 1 PCR reaction system

[0057]

[0058] The primer sequences are:

[0059] Upstream primer F: 5'-GACGTGGTGAATTATCAGC-3'

[0060] Downstream primer R: 5'-CAACTAGATTTCCACCAGGA-3'

[0061] The amplified fragment was 288 bp in size, and the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0062] The reaction conditions were as follows: pre-denaturation at 94℃ for 5 min, followed by cycling with the following parameters: 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 30 s; after 35 cycles, extension at 72℃ for 10 min. The product was stored at 4℃ for testing.

[0063] The isolated FHV-1 was serially diluted 10-fold using DMEM containing 2% serum (from 10... -1 Up to 10 -8 Each dilution of virus solution was simultaneously inoculated into two wells of a 6-well F81 cell culture plate. The plates were incubated at 37°C with 5% CO2 for 1 hour for adsorption. The virus solution was then discarded, and the plates were covered with DMEM containing 2% serum and 0.8% low-melting-point agarose without phenol red. The plates were incubated at 37°C with 5% CO2 for 3-5 days. Once obvious plaques were observed under a microscope, the plates were stained with 0.1‰ neutral red at 37°C for 1 hour. The staining solution was discarded, and the plaques were picked and placed in 200 μL of maintenance medium. This mixture was then subjected to three freeze-thaw cycles and simultaneously inoculated into a 24-well F81 cell culture plate. After adsorption at 37°C with 5% CO2 for 1 hour, 0.8 mL of cell maintenance medium was added. The plates were incubated at 37°C with 5% CO2 until complete cytopathic effects were observed. The cytopathic cell cultures were then collected, and after three freeze-thaw cycles, the TCID values ​​of each clone were measured. 50 Select clones with high toxin titers and purify them twice more as described above to obtain pure FHV-1 virus, which is named FHV-1WH-2017 strain.

[0064] II. Routine Culture of Virus Strains

[0065] F81 cells were cultured adherently in cell culture flasks until a confluent monolayer was formed. The cells were then digested with 0.25% trypsin and collected for later use. The F81 cells were diluted with 2% new bovine serum cell maintenance medium to a concentration of (4.5-5.0) × 10⁻⁶ cells / flask. 6After adding the virus at a concentration of 100 cells / mL to a cell culture flask, inoculate with feline rhinotracheitis virus strain FHV-1WH-2017 at a MOI of 0.01. Observe for cytopathic effect (CPE) every 24 hours after inoculation. Harvest the virus solution 72 hours after the appearance of obvious visual cytopathic effect.

[0066] F81 cells frozen in liquid nitrogen were rapidly thawed at 37°C for 1 min, centrifuged at 1000 rpm, and the supernatant was discarded. The cells were resuspended in 1 mL of 10% new bovine serum DMEM cell culture medium and slowly added to a T25 flask, with an additional 4 mL of 10% new bovine serum DMEM cell culture medium added. Cells were cultured at 37°C and 5% CO2 for 24-48 h. Once a confluent monolayer was formed, the cells were digested with 0.25% trypsin and collected. Cells were then cultured at a rate of (5-6) × 10⁻⁶. 5 Cells were cultured at a density of 10 cells / mL (the density after adding 10% new bovine serum to DMEM cell culture medium) and allowed to adhere to the culture medium for 24-48 h at 37°C and 5% CO2.

[0067] The viral solution was serially diluted 10-fold in serum-free DMEM in EP tubes, starting from 10... -1 Up to 10 -8 Discard the culture medium containing a confluent monolayer of F81 cells from the 96-well plate, and add 100 μL of DMEM cell maintenance medium containing 2% serum to each well. Add the diluted virus solution (10... -3 Up to 10 -8 The cells were seeded into 8 wells of the above-mentioned 96-well culture plate, with each dilution seeded in one vertical row, 100 μL per well; 8 wells were set up as normal cell controls, with 100 μL of DMEM cell maintenance medium containing 2% serum added to each well. The 96-well culture plate was incubated at 37°C and 5% CO2 for 72 h. Cytopathic effects were observed and recorded. Normal cell controls should not show cytopathic effects. TCID was calculated according to the Reed-Muench method. 50 The results showed that the viral load should be ≥10 mmol / L. 8.5 TCID 50 .

[0068] III. Preparation of Feline Rhinotracheitis Virus FHV-1WH-2017 Strain Virus Fluid and Vaccine Preparation

[0069] 1. Virus propagation

[0070] Adherent F81 cells were cultured in DMEM medium containing 10% new bovine serum at a density of 0.5 × 10⁻⁶ cells / mL. 6.0 -1.0×10 6.0 Density per mL ( Figure 1The final volume of 5 mL was diluted and inoculated into T25 cell culture flasks. The cells were cultured at 37°C and 5% CO2 for 24-48 h. After the cells reached a confluent monolayer, 0.25% trypsin solution was added to digest the cells and the mixture was expanded into T75 cell culture flasks. The cells were then resuspended in 15 mL of 2% new bovine serum DMEM cell maintenance medium and inoculated with feline rhinotracheitis virus strain FHV-1WH-2017 at 0.01 MOI. The cells were cultured at 37°C and 5% CO2 for 72 h. The viral fluid was harvested, and the viral content was determined by observing cytopathic effects and PCR testing to detect purity. After meeting the requirements, the cells were quantitatively aliquoted and stored below -70°C.

[0071] 2. Preparation of viral fluid

[0072] Discard the culture medium from the F81 cells that had grown into a monolayer in the T75 cell culture flask, digest them with 0.25% trypsin solution, collect the cells, and expand them into T175 cell culture flasks. Then, resuspend the cells in 50 mL of 2% new bovine serum DMEM cell maintenance medium and inoculate them with feline rhinotracheitis virus strain FHV-1WH-2017 at 0.01 MOI. Continue culturing at 37℃ and 5% CO2 for 72 h, harvest the virus solution, observe cytopathic effects, and calculate the virus content.

[0073] 3. Vaccine preparation

[0074] ① Inactivation: Add β-propiolactone to the harvested virus stock solution or dilution at a volume of 0.12%, mix well, inactivate at 4℃ for 24h, and then hydrolyze at 37℃ for 2h.

[0075] ② Sterility test: The test shall be conducted in accordance with the appendix of the current Chinese Veterinary Pharmacopoeia, and no sterile growth should be observed.

[0076] ③ Inactivation test: The inactivated antigen was subjected to sterility test, and the inactivation solution was seeded into adherent F81 cells at 0.01 MOI. The cells were cultured at 37°C with 5% CO2 for 6 days and observed. The cells were then blindly passaged for 2 more times. The inactivation was determined by CPE.

[0077] ④ Vaccine preparation: Dilute the antigen solution with the known antigen content mentioned above to 10 using sterile PBS. 7.0 TCID 50 / mL, 10 7.5 TCID 50 / mL, 10 8.0 TCID 50 / mL and 10 8.5 TCID 50 / mL, dispensed into 1mL portions and stored at 4℃ for later use.

[0078] IV. Isolation and Identification of Feline Rhinotracheitis Virus FHV-1WH-2017 Strain

[0079] 1. Identification of feline rhinotracheitis virus

[0080] After treating the PCR-positive WH-2017 strain, the cells were inoculated into F81 cells and blindly passaged for 20 generations. PCR was then performed at generations 1, 5, 10, and 20. The results showed that a specific fragment of approximately 288 bp was detected in all cells after 20 generations of blind passage. Figure 2 This indicates that the isolated virus is infectious and can be continuously passaged.

[0081] 2. Isolation of feline rhinotracheitis virus

[0082] The isolated strain WH-2017 was purified using plaque purification. To minimize the number of passages, only the third generation of low-passage viruses was used for plaque purification. In the first round of plaque purification, ten clones were selected, inoculated into 24-well plates, and the viral load of clones exhibiting typical lesions was determined. Clones with high viral loads were then used for the next round of plaque purification, a process repeated three times (Table 2). The purified feline rhinotracheitis virus (FHV) strain FH-1WH-2017 was finally obtained through these three purification processes.

[0083] Table 2. Virus titers of 10 clone wells selected from the three purification processes of strain WH-2017.

[0084]

[0085] 3. Microscopic morphological observation of the purified clone of feline rhinotracheitis virus FHV-1WH-2017

[0086] F6 cell cultures of the WH-2017 clone were expanded and purified by ultrafiltration. The purified samples were then observed using transmission electron microscopy (TEM). Samples taken from the stratified areas between 20% and 35% sucrose concentrations showed isometric icosahedral viral particles with a diameter of approximately 128-168 nm under TEM, consistent with the morphology and size reported for FHV-1 in the literature. Figure 3 ).

[0087] 4. Molecular genetic evolutionary analysis of the cloned and purified strain FHV-1WH-2017

[0088] Primers were designed based on the FHV-1 genome sequence registered in GenBank to amplify different segments of the viral genome. The primer sequences are shown in Table 3. Five gene fragments (gI, gE, TK, gB, and gC) were sequenced and assembled into a whole genome sequence using Seqman software. The homology and phylogenetic relationships of multiple FHV-1 isolates from different regions both domestically and internationally were analyzed using DNASTAR and MEGA 7.0. The isolates included: FHV-1 strains 117-68 (2016), 124-68b (2016), 135-68a (2018), 384-75 (2016), 3224-04 (2016), 3232-05 (2016), 3236-06 (2016), C-27 (2008), CH-B (2020), Companion (2018), isolate Feligen (2016), FLOR_05 (2018), and GD. The nucleotide sequences of the five genomes (gI, gE, TK, gB, and gC) of 24 strains, including strains 2018 (2022), KANS_02 (2018), MILW_12 (2018), MM-3 (2018), NEWY_03 (2018), PEEBLES_1 (2018), PHIL_03 (2018), S5727 (2018), SANJ_01 (2018), WASH_03 (2018), 85-68 (2016), and our isolated strain WH-2017, were compared.

[0089] Table 3. Primers for FHV-1 gI, gE, TK, gB, and gC genome amplification.

[0090]

[0091] The nucleotide sequence of the gB genome of feline rhinotracheitis virus strain FHV-1WH-2017 is shown in SEQ ID NO.1, and the amino acid sequence of its expressed gB protein is shown in SEQ ID NO.2. The nucleotide sequence of the gC genome of feline rhinotracheitis virus strain FHV-1WH-2017 is shown in SEQ ID NO.3, and the amino acid sequence of its expressed gC protein is shown in SEQ ID NO.4. The nucleotide sequence of the gI genome of feline rhinotracheitis virus strain FHV-1WH-2017 is shown in SEQ ID NO.5, and the amino acid sequence of its expressed gI protein is shown in SEQ ID NO.6. The nucleotide sequence of the gE genome of feline rhinotracheitis virus strain FHV-1WH-2017 is shown in SEQ ID NO.7, and the amino acid sequence of its expressed gE protein is shown in SEQ ID NO.8. The nucleotide sequence of the TK genome of feline rhinotracheitis virus strain FHV-1WH-2017 is shown in SEQ ID NO.9, and the amino acid sequence of its expressed TK protein is shown in SEQ ID NO.9. As shown in NO:10, the results indicate that the homology of the five genomic sequences (gI, gE, TK, gB, gC) of strain WH-2017 with the five genomic sequences of these 23 viruses is between 99.7% and 100%. Figure 4-8 The results indicate that the strains isolated in recent years have a high degree of homology, but they are not completely identical to the five genome sequences of gI, gE, TK, gB, and gC of the 23 viruses.

[0092] The gI and gE genes are the main virulence genes of herpesviruses. They can mediate host cell fusion and affect tissue tropism, chemotactically attracting the virus to the nervous system. The thymidine kinase expressed by the TK gene is a major gene regulating the nucleic acid metabolism of FHV-1 virus. gB is a major immunogenic protein of herpesviruses, stimulating the production of neutralizing antibodies. The gC protein mainly plays a role in the adsorption of viral particles to host cells and can induce cellular immune responses. We designed this study to analyze the genetic differences among different FHV-1 strains using three virulence genes and two immunogenic genes.

[0093] To further understand the phylogenetic relationships among isolates from different parts of the world, strains isolated from different countries were selected from sequences published in GenBank, including FHV-1 strains 117-68 (2016), 124-68b (2016), 135-68a (2018), 384-75 (2016), 3224-04 (2016), 3232-05 (2016), 3236-06 (2016), C-27 (2008), CH-B (2020), Companion (2018), isolate Feligen (2016), FLOR_05 (2018), and GD. Phylogenetic trees of the gI, gE, TK, gB, and gC genes were constructed using MEGA 7.0 software for 24 strains: strain 2018 (2022), strain KANS_02 (2018), strain MILW_12 (2018), strain MM-3 (2018), strain NEWY_03 (2018), strain PEEBLES_1 (2018), strain PHIL_03 (2018), strain S5727 (2018), strain SANJ_01 (2018), strain WASH_03 (2018), strain 85-68 (2016), and strain WH-2017 (2017). Figures 9-13 ).

[0094] The results showed that the gI, gE, TK, gB, and gC genes of the isolated WH-2017 strain were located in the same major branch as the American strain and the Feligen vaccine strain. The gC gene of the WH-2017 strain and the FHV-1 standard strain C-27 were located in different branches, but the gI, gE, TK, and gB genes were still located in the same major branch, indicating that they were closely related to the domestic and international circulating strains, but their nucleotide sequences were not completely identical.

[0095] Microbial Preservation

[0096] The feline rhinotracheitis virus strain FHV-1WH-2017 isolated in this invention was deposited with a patent-approved depository, with the microbial accession number CCTCC NO:V202296; the classification name: feline rhinotracheitis virus strain FHV-1WH-2017; the deposit date: November 10, 2022; and the depository institution: China Center for Type Culture Collection (Wuhan University).

[0097] V. Virulence test of feline rhinotracheitis virus FHV-1WH-2017 strain

[0098] 1.14-18 week old healthy susceptible cat virulence test

[0099] Twenty healthy, susceptible kittens aged 14-18 weeks were randomly divided into four groups of five. Groups 1-3 were further divided into groups of 10... 5.0 TCID 50 / mL, 10 6.0 TCID 50 / mL and 10 7.0 TCID 50 Three different doses of FHV-1WH-2017 strain virus fluid were administered via nasal drops to each cat (1.0 mL / cat); group 4 served as the control group. Clinical manifestations of the experimental cats were observed post-infection for a total of 14 days.

[0100] The virus content was 10. 5.0 TCID 50 / mL, 10 6.0 TCID 50 / mL and 10 7.0 TCID 50 FHV-1WH-2017 strain virus was administered via nasal drops to healthy, susceptible cats aged 14-18 weeks, with 1 mL administered to each cat. Results showed that in 10 5.0 TCID 50 In the / mL challenge group, 3 / 5 of the cats developed symptoms. Among them, 1 (1 / 5) of the experimental cats did not show obvious clinical symptoms; 1 (1 / 5) of the experimental cats showed symptoms of depression, paroxysmal coughing or sneezing; and 3 (3 / 5) of the experimental cats showed symptoms of depression, paroxysmal coughing, paroxysmal sneezing, and eyelid redness and swelling. Figure 14 Symptoms included serous ocular and nasal discharge (lasting >2 days), and no deaths occurred during the entire monitoring period. 10 6.0 TCID 50 In the / mL challenge group, 5 out of 5 cats developed symptoms. Two (2 / 5) of the experimental cats exhibited paroxysmal coughing, paroxysmal sneezing, respiratory rales, and purulent ocular and nasal discharge, subsequently dying. The other three (3 / 5) cats all exhibited paroxysmal coughing, paroxysmal sneezing or respiratory rales, eyelid redness and swelling, serous ocular and nasal discharge (lasting >2 days), and purulent ocular and nasal discharge (lasting >2 days). Throughout the monitoring period, all experimental cats showed signs of depression, and some also exhibited hair loss around the eyes and nose or arched back and prone lying posture. 10 7.0 TCID 50 In the / mL challenge group, all experimental cats (5 / 5) exhibited symptoms including lethargy, paroxysmal coughing, paroxysmal sneezing or respiratory rales, and eyelid redness and swelling, with serous or purulent ocular and nasal discharge (lasting >2 days). In addition to these symptoms, two cats (2 / 5) showed signs of hair loss around the eyes and nose or arched back and prone position. Three experimental cats (3 / 5) eventually died during the monitoring period. The control group cats remained normal throughout the observation period. Figure 14 ), Observation of lesions in cat eyes as follows Figure 14 As shown.

[0101] Cats that died after viral infection were immediately autopsied. Surviving cats were euthanized on day 15 after a 14-day observation period, and their lesions in target organs were examined. Figure 15 As shown, the results of the FHV-1WH-2017 strain challenge group were: 10 5.0 TCID 50 Two (2 / 5) experimental cats in the / mL challenge group showed signs of disease, including mild eyelid redness and swelling, and a small number of white lesions and necrotic foci in the lungs; 10 6.0 TCID 50 / mL and 10 7.0 TCID 50 In the / mL challenge group, all experimental cats (5 / 5) exhibited varying degrees of eyelid congestion and swelling, as well as lung tissue congestion, partial atrophy, and white necrotic foci on the surface, indicating significant lesions. In the control group, the eyelids were pinkish-white, and the lungs were intact without lesions or necrotic foci. Pathological observations are as follows: Figure 16 As shown.

[0102] 2. Toxicity test in adult cats

[0103] With a viral load of 10 7.0 TCID 50 / mL, 10 8.0 TCID 50 Adult cats were infected with FHV-1WH-2017 strain 1.0 mL / cat via nasal instillation. Fifteen healthy adult cats were randomly divided into three groups of five. Group 1 was infected with 10 cats via nasal instillation. 7.0 TCID 50 / mL WH-2017 strain virus fluid, 1.0mL / animal; Group 2 was infected by nasal drops in 10 animals. 8.0 TCID 50 / mL of WH-2017 strain virus solution, 1.0mL / cat; Group 3 served as the control group. Clinical manifestations of the experimental cats were observed after infection for a total of 14 days.

[0104] The results showed that strain FHV-1WH-2017 had a growth rate of 10%. 7.0 TCID 50 After infecting healthy adult cats with a dose of / mL, two (2 / 5) of the test cats developed persistent lethargy starting on days 9-10, and paroxysmal sneezing at the end of the monitoring period; the remaining three (3 / 5) showed no other symptoms; the FHV-1WH-2017 strain at 10 8.0 TCID 50After infecting healthy adult cats with a dose of / mL, 4 cats (4 / 5) of the experimental cats developed symptoms including lethargy, respiratory rales, paroxysmal coughing or sneezing, eyelid swelling, and serous or purulent ocular and nasal discharge (lasting >2 days); 1 cat (1 / 5) of the experimental cat only showed lethargy. No adult cats died. The control group cats remained normal throughout the observation period. Figures 17-19 ).

[0105] VI. Inactivation, inactivation test and safety test of FHV-1WH-2017 strain

[0106] 1. Inactivation and inactivation test of WH-2017 strain: The virus solution was added to 0.12% β-propiolactone and stirred continuously. It was then inactivated at 4°C for 24 hours, followed by hydrolysis of β-propiolactone at 37°C for 2 hours. After the hydrolysis, it was stored at 4°C. The inactivated virus was inoculated into F81 cells in logarithmic growth phase at a ratio of 1:100 and passaged blindly for two generations. No virus growth was observed, indicating complete inactivation of the WH-2017 strain. Feline rhinotracheitis virus can be inactivated by treatment with formalin, β-propiolactone (BPL), or binary ethyleneimine (BEI), or other methods known in the art.

[0107] 2. Preparation of WH-2017 strain inactivated vaccine: The viral load of the WH-2017 strain virus solution before inactivation was measured, and the thoroughly inactivated WH-2017 strain virus solution was diluted to 10 μL using sterile PBS. 8.5 TCID 50 / mL;

[0108] 3. Safety test: Ten healthy susceptible cats aged 8-12 weeks with negative feline rhinotracheitis virus neutralizing antibodies and antigens were selected and divided into two groups. In the first group, each test cat was injected subcutaneously into the neck with two doses of FHV-1WH-2017 strain inactivated vaccine. In the second group, an equal amount of physiological saline was administered. After 14 days of clinical observation, all cats were 5 / 5 healthy and alive, and no adverse reactions were observed.

[0109] VII. Immunogenicity test of feline rhinotracheitis virus strain FHV-1WH-2017

[0110] 1. Test Plan

[0111] (1) Twenty-five kittens aged 8-12 weeks with neutralizing antibody titers not exceeding 1:2 were randomly divided into 5 groups (group 1, group 2, group 3, group 4, and group 5), with 5 kittens in each group. Groups 1, 2, 3, and 4 were immunized with antigens prepared in Example 2 with contents of 10, respectively. 7.0 TCID 50 / mL, 10 7.5 TCID50 / mL, 10 8.0 TCID 50 / mL and 10 8.5 TCID 50 One dose of inactivated vaccine per animal was administered in group 5 (mL). Group 5 served as the control group, with the same amount of sterile PBS buffer injected subcutaneously into the neck. Blood samples were collected on days 7, 14, 21, 28, and 35 after the first immunization to measure the neutralizing antibody levels.

[0112] (2) Thirty kittens aged 8-12 weeks with neutralizing antibody titers not exceeding 1:2 were randomly divided into 6 groups (group 6, group 7, group 8, group 9, group 10, and group 11), with 5 kittens in each group. Groups 6 to 9 were immunized with antigens prepared in Example 2 with contents of 10, respectively. 7.0 TCID 50 / mL, 10 7.5 TCID 50 / mL, 10 8.0 TCID 50 / mL and 10 8.5 TCID 50 One dose of inactivated vaccine per animal (1 mL) was administered as the immune challenge group. Group 10 was injected subcutaneously into the neck with the same amount of sterile PBS buffer as the non-immune challenge control group. Group 11 received no treatment as the blank control group. Twenty-one days after the first immunization, 1 mL of feline rhinotracheitis virus per animal was administered intranasally to groups 6, 7, 8, 9, and 10, with a viral load of 10. 7.0 TCID 50 / mL, body temperature and clinical manifestations were monitored daily after challenge. All experimental cats were euthanized and dissected 14 days after challenge.

[0113] 2. Test Results

[0114] Table 4. Neutralizing antibody levels against feline rhinotracheitis virus (FHV) strain FHV-1WH-2017 in kittens aged 8-12 weeks after immunization with inactivated feline rhinotracheitis virus vaccine.

[0115]

[0116] Table 5. Protection rates of kittens aged 8-12 weeks immunized with inactivated feline rhinotracheitis virus vaccine and those not immunized after challenge.

[0117]

[0118] The results showed that the neutralizing antibody levels in the test cats immunized with the FHV-1WH-2017 inactivated vaccine ranged from 1:4 to 1:128. 7.0 TCID 5021 days after immunization with the inactivated feline rhinotracheitis virus vaccine, 3 / 5 of the cats challenged with the vaccine showed no abnormalities in body temperature, mental state, or appetite; 2 / 5 of the cats exhibited mild clinical symptoms such as sneezing and serous ocular and nasal discharge, of which 1 / 5 died; 10 7.5 TCID 50 / mL of feline rhinotracheitis virus inactivated vaccine was administered to cats. 21 days after challenge, 4 / 5 of the cats showed no abnormalities in body temperature, mental state, or appetite; 1 / 5 of the cats exhibited mild sneezing, serous ocular and nasal discharge, and other clinical symptoms. 10 8.0 TCID 50 / mL and 10 8.5 TCID 50 Cats immunized with an inactivated feline rhinotracheitis virus vaccine (FRV) at a concentration of / mL showed no abnormalities in body temperature, mental state, or appetite after 21 days of challenge, and 5 / 5 survived. In the non-immunized control group injected with PBS buffer, 5 / 5 of the cats exhibited serous or purulent ocular and nasal discharge, respiratory rales, or paroxysmal sneezing after challenge, and 3 / 5 died. Before death, the cats in this control group showed decreased body temperature and lethargy. No abnormalities were observed in the blank control group. Experimental data are shown in Tables 4 and 5.

[0119] The experimental results showed that when the antigen content of the WH-2017 strain inactivated vaccine was higher than 10... 7.5 TCID 50 At a concentration of 1 / mL, immunizing one dose to 8-12 week old kittens can produce neutralizing antibodies against the WH-2017 strain of feline rhinotracheitis virus prevalent in my country. At least 4 / 5 of the test cats can be protected from feline rhinotracheitis virus infection 21 days after immunization.

[0120] Example 2: Acquisition, identification, and testing of feline calicivirus strain LZ-2016

[0121] I. Isolation of the virus strain

[0122] In May 2016, upper respiratory tract swabs were collected from clinically suspected infected cats in Liuzhou City, Guangxi Province using sterile cotton swabs. The swabs were dissolved in 1 mL of PBS buffer for 5 min, centrifuged at 3000 rpm for 10 min, and the cotton swabs were discarded. The samples were centrifuged at 3000 rpm for 10 min, and the supernatant was collected. An equal volume of DMEM nutrient solution was added, and the mixture was filtered through a 0.22 μm filter for sterilization. The resulting supernatant was inoculated into a confluent monolayer of F81 cells and cultured in a 37℃, 5% CO2 incubator. Cells were observed daily, and the virus was harvested promptly upon the appearance of cytopathic effects. Obvious cytopathic effects were observed in the first passage, and by the third passage, the cytopathic effects stabilized, manifesting as cell shrinkage, clumping, and a grape-like appearance, eventually leading to complete necrosis and detachment. (See attached image) Figure 20 After sample processing, viral RNA was extracted from the prepared solution using the standard Trizol RNA extraction method, and then reverse transcription was performed according to the instructions of the Novozymes Reverse Transcription Kit.

[0123] Based on the FCV FB-NJ-13 genome sequence (GenBank ID: KM111557) registered in GenBank, a pair of specific primers were designed to amplify the FCV ORF3 gene. The primer sequences are as follows:

[0124] Upstream primer ORF3-F: 5'-GTTGACCCTTACTCATACAC-3'

[0125] Downstream primer ORF3-R: 5'-CCCTGGGGTTAGGCGC-3'

[0126] The amplified fragment size was 136 bp. The reaction conditions were: pre-denaturation at 94℃ for 5 min, followed by cycling with the following parameters: 94℃ for 30 s, 54℃ for 30 s, and 72℃ for 30 s; followed by extension at 72℃ for 10 min after 35 cycles. 5 μl of the product was analyzed by 1% agarose gel electrophoresis. The amplified PCR fragment size was 136 bp, consistent with the expected size (see attached image). Figure 21 );

[0127] II. Cultivating Virus Strains

[0128] The isolated FCV was serially diluted 10-fold using DMEM containing 2% serum (from 10... -1 Up to 10 -10 The virus solution of each dilution was simultaneously inoculated into 6-well cell culture plates of F81 cells, with 2 wells inoculated for each dilution. The plates were incubated at 37°C with 5% CO2 for 1 hour for adsorption. The virus solution was then discarded, and the plates were covered with DMEM containing 2% serum and 0.8% low-melting-point agarose without phenol red. The plates were then incubated at 37°C with 5% CO2 for 3–5 days. After observing obvious plaques under a microscope, the plates were stained with 0.1‰ neutral red at 37°C for 1 hour. The staining solution was discarded, and the plaques were picked and placed in 200 μL of maintenance medium. The plates were then subjected to three freeze-thaw cycles and simultaneously inoculated into 24-well cell culture plates of F81 cells. After adsorption at 37°C with 5% CO2 for 1 hour, 0.8 mL of cell maintenance medium was added. The plates were then incubated at 37°C with 5% CO2 until complete cytopathic effects were observed. The cytopathic cell cultures were collected, and after three freeze-thaw cycles, the TCID of each clone was measured. 50 Select clones with high viral titers, purify them twice more as described above, and label the high-viral-titer clones as F1 generation. Perform virus content determination and calculate TCID using the Reed-Muench method. 50 The final harvested FCV LZ-2016 strain virus fluid contained up to 10 [units unspecified]. 9.5 TCID 50 / mL;

[0129] F1 generation cell cultures of FCV LZ-2016 strain were expanded and purified by ultrafiltration. The obtained viral suspension samples were individually adsorbed onto copper grids for 5 min. The viral suspension was then gently aspirated with filter paper and allowed to dry. After drying, 20 g / L phosphotungstic acid was added for negative staining for 1 min. The phosphotungstic acid was then removed with filter paper, and the samples were observed under an electron microscope after drying. The results showed typical goblet-shaped viral particles with a diameter of 37–40 nm (see attached image). Figure 22 ).

[0130] III. Molecular genetic evolutionary analysis of purified FCV strain LZ-2016

[0131] Primers listed in Table 6 were designed based on the known FCV genome sequence (GenBank ID: KM111557) in GenBank. The extracted viral RNA was reverse transcribed into cDNA. Using the cDNA as a template, the full-length gene was divided into three segments using the primers in Table 6 for segmental PCR amplification. The PCR reaction used a 50 μl reaction system: 25 μl Prime STAR Max DNA Polymerase, 3 μl template, 2 μl each of forward and reverse primers (10 μmol / L), and 18 μl ddH2O. The reagents were thoroughly mixed, and amplification was performed under the following conditions: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; and a final extension at 72℃ for 7 min. Electrophoresis results of the amplified products are shown in the appendix. Figure 23 ;

[0132] Table 6 Primers for FCV whole genome amplification

[0133]

[0134] Note: R is a commonly used degenerate base (R = A, G);

[0135] 2. Ligation and Transformation: Ligate each gene segment to the vector separately. The specific steps are as follows: Take 4 μl of the gel recovery product and gently mix it with 1 μl of pEASY-T1 cloning vector. Incubate at 16℃ for 30 min and then place on ice. Take Trans DH5α competent cells from a -70℃ freezer, add the ligation product while thawing, gently tap to mix, and incubate on ice for 30 min. Heat shock at 42℃ for 1 min, then incubate on ice for 3 min. Aseptically add 500 μl of antibiotic-free LB liquid medium and incubate at 37℃ and 200 rpm for 1 h. Centrifuge at 3000 rpm for 3 min, retain 100 μl of supernatant, resuspend the precipitated cells, and spread them on LB-Amp+ agar plates. Incubate upside down at 37℃ for 12–14 h. After visible colonies appear on the agar plates, aseptically pick colonies from each plate and incubate in 2 mL of LB-Amp+ liquid medium at 37℃ and 200 rpm for 10–12 h.

[0136] 3. PCR Identification and Sequencing: Using 1 μl of bacterial culture as a template, PCR amplification was performed using the universal primers M13F / R for the pEASY-T1 vector. The PCR products were identified by agarose gel electrophoresis, and positive plasmids were sequenced. The sequencing results were assembled using DNAStar-Seqman to obtain the genome sequence of FCV LZ-2016 strain, which is 7683 bp in length. (The specific sequence is shown in SEQ ID NO: 11), and the amino acid sequence of the expressed capsid protein is shown in SEQ ID NO: 12.

[0137] 4. Sequence Analysis: The whole genome sequence of FCV strain LZ-2016 was compared with the whole genome nucleotide sequences of 25 strains (Table 7) registered in GenBank, including FCV-255, FCV-F9, FCV-F4, FCV-GX2019, FCV-SH, FCV-GD, FCV-FB-NJ-13, and FCV-HB-S4. The results showed that the nucleotide homology of the whole genome sequence of strain LZ-2016 with the whole genome sequences of reference strains at home and abroad was between 74.9% and 82.8%, while the genetic differences from the prevalent strains abroad were significant. Phylogenetic analysis of the full-length FCV genome nucleotide sequence showed that strain CH-JL1 formed a small branch with strains FB-NJ-13, GD, and 12Q087-1. These strains were all isolated from China and East Asia (see attached table). Figure 24 , 26 );

[0138] Table 7. Information on FCV reference strains and homology analysis with LZ-2016 strain.

[0139]

[0140] Since the capsid protein encoded by the ORF2 gene fragment is the main structural protein of FCV and the most important immunogenic protein stimulating the body to produce neutralizing antibodies, comparative analysis of its encoded amino acids revealed the amino acid sequence of the capsid protein of strain LZ-2016 (specific sequence shown in SEQ ID NO: 2). The homology with domestic and international reference strains was between 80.6% and 89.5%, while the genetic differences with prevalent strains abroad were significant, further indicating that there are large antigenic differences among currently circulating strains (see appendix). Figure 25 The feline calicivirus strain isolated in this invention is named feline calicivirus FCV LZ-2016 strain and was deposited at the China Center for Type Culture Collection on September 2, 2022, with accession number CCTCC NO: V202276.

[0141] IV. Virulence test of feline calicivirus (FCV) strain LZ-2016

[0142] The virus solution was diluted to 10 with DMEM. 7.0 TCID 50 / mL, 10 8.0 TCID 50 / mL and 10 9.0 TCID 50 Twenty 4-8 week old feline cats that were negative for feline calicivirus neutralizing antibodies and antigens were selected and divided into four groups. The cats were challenged with intranasal drops. Group 1 received 10 mL of the drug. 7.0 TCID 50 Group 10 / mL challenged, Group 2 was 10 8.0 TCID 50 / mL challenge group, group 3 was 10 9.0 TCID 50 The challenge group was 1 mL, and the control group was 4 mL. The challenge dose was 1 mL. The animals were isolated and observed clinically for 14 days after challenge. Results showed that 10 7.0 TCID 50 / mL challenge group: 3 / 5 of the experimental cats showed clinical symptoms such as lethargy, eyelid swelling, oral ulcers, and serous or purulent ocular and nasal discharge; 10 8.0 TCID 50 / mL and 10 9.0 TCID 50 In the / mL challenge group, 5 out of 5 cats exhibited clinical symptoms such as lethargy, eyelid swelling, oral ulcers, and serous or purulent ocular and nasal discharge, and 2 out of 5 eventually died; the control group showed no obvious symptoms. Figure 27 ).

[0143] V. Inactivation, inactivation test and safety test of FCV LZ-2016 strain

[0144] 1. Inactivation and inactivation test of LZ-2016 strain: The virus solution was added to 0.06% β-propiolactone by volume and stirred continuously. The mixture was inactivated at 4°C for 24 hours, followed by hydrolysis of β-propiolactone at 37°C for 2 hours. After hydrolysis, the mixture was stored at 4°C. The inactivated virus was then inoculated into F81 cells in logarithmic growth phase at a ratio of 1:100 and passaged blindly for two generations. No virus growth was observed, indicating complete inactivation of the LZ-2016 strain. Caliciviruses can be inactivated by treatment with formalin, β-propiolactone (BPL), or binary ethyleneimine (BEI), or other methods known in the art.

[0145] 2. Preparation of LZ-2016 strain inactivated vaccine: The viral load of the LZ-2016 strain virus solution before inactivation was measured, and the thoroughly inactivated LZ-2016 strain virus solution was diluted to 10 μL using sterile PBS. 9.0 TCID50 / mL;

[0146] 3. Safety test: Ten healthy susceptible cats aged 8-12 weeks with negative feline calicivirus neutralizing antibodies and antigens were selected and divided into two groups. In the first group, each test cat was injected subcutaneously into the neck with two doses of FCV LZ-2016 strain inactivated vaccine. The second group was injected with an equal amount of physiological saline. After 14 days of clinical observation, all cats were 5 / 5 healthy and alive, and no adverse reactions were observed.

[0147] VI. Immunogenicity and efficacy study of FCV strain LZ-2016

[0148] 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 "Miamituron" 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. 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 (results of identification of other prevalent FCV strains in China using the identification method described in Example 2 are attached). Figure 28 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.

[0149] Table 8 Results of FCV neutralizing antibody detection in serum of vaccinated cats

[0150] Note: "-" indicates negative RT-PCR identification of FCV pathogen, "+" indicates positive RT-PCR identification of FCV pathogen, "—" indicates no abnormality, "E" indicates serous ocular and nasal discharge, "F" indicates purulent ocular and nasal discharge, "I" indicates oral ulceration, and "S" indicates death.

[0151]

[0152] Table 9. Neutralizing effect of vaccine-immunized cat serum on other FCV isolates.

[0153]

[0154] 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.

[0155] In summary, the FCV LZ-2016 strain described in this embodiment has a high viral titer. This strain is highly virulent in healthy, susceptible cats and can cause typical symptoms such as oral ulcers and conjunctivitis. Immunizing cats with the inactivated product of this strain can produce a good immune response and has good immunogenicity. As an inactivated vaccine, it can effectively stimulate the body to produce high levels of neutralizing antibodies against FCV and can provide a high rate of immune protection against FCV challenge, thus possessing the basic potential for vaccine development.

[0156] Example 3: Preparation of Combined Vaccines

[0157] (1) Preparation of viral fluid for vaccine production of feline rhinotracheitis virus FHV-1WH-2017 strain

[0158] Discard the culture medium from the confluent F81 cells in the T75 cell culture flask, digest them with 0.25% trypsin solution, collect the cells, and expand them into T175 cell culture flasks. Resuspend the cells in 50 mL of 2% new bovine serum DMEM cell maintenance medium and inoculate with feline rhinotracheitis virus strain FHV-1WH-2017 at 0.01 MOI. Continue culturing at 37°C and 5% CO2 for 72 hours. Harvest the viral fluid, observe cytopathic effects, and calculate the viral load. Then, inactivate the virus according to step three of Example 1. Dilute the inactivated antigen solution with known antigen content to 10 g / L using sterile PBS. 8.5 TCID 50 / mL, store at 4℃ for later use.

[0159] (2) Preparation of viral solution for vaccine production of feline calicivirus FCV LZ-2016 strain

[0160] Discard the culture medium from the confluent F81 cells in the T75 cell culture flask, digest them with 0.25% trypsin solution, collect the cells, and expand them into T175 cell culture flasks. Resuspend the cells in 50 mL of 2% new bovine serum DMEM cell maintenance medium and inoculate with feline calicivirus LZ-2016 strain at 0.01 MOI. Continue culturing at 37°C and 5% CO2 for 36 hours. Harvest the viral fluid, observe cytopathic effects, and calculate the viral load. Then, inactivate the virus according to step five of Example 2. Dilute the inactivated antigen solution with known antigen content to 10 g / L using sterile PBS. 9.0 TCID 50 / mL, store at 4℃ for later use.

[0161] (3) Preparation of a bivalent vaccine of feline rhinotracheitis virus FHV-1WH-2017 strain and feline calicivirus FCV LZ-2016 strain

[0162] The antigen solution prepared in steps (1) and (2) of Example 3 was mixed with sterile PBS in a ratio of 1:1:1 to make a final volume of 1 mL, and the final concentration of FHV-1WH-2017 strain antigen was 10. 8.0 TCID 50 / mL, the final concentration of FCV LZ-2016 strain antigen was 10 8.5 TCID 50 / mL. Store at 4℃ for later use, according to the specification of 1mL / dose.

[0163] (4) Effectiveness test

[0164] Twenty kittens aged 8-12 weeks were selected, all of whom tested negative for feline rhinotracheitis virus (FHV) and feline calicivirus (FCV) by PCR in mixed upper respiratory tract swabs and whose neutralizing antibody ratios for FHV and FCV were no higher than 1:2. The kittens were randomly divided into four groups of five each: Group 1 (animal numbers 41-45), Group 3 (animal numbers 51-55), Group 5 (animal numbers 61-65), and Group 7 (animal numbers 71-75). Each group received a subcutaneous injection of FHV-1 antigen prepared with a content of 10... 8.0 TCID 50 / mL and FCV antigen content 10 8.5 TCID 50 One dose / animal of the bivalent inactivated vaccine ( / mL) was administered; a second immunization was given 21 days later using the same dose and method. Groups 2 (animal numbers 46-50) and 6 (animal numbers 66-70) served as FHV-1 control groups, and groups 4 (animal numbers 56-60) and 8 (animal numbers 76-80) served as FCV-1 control groups, all without any treatment (blank control group). Blood samples were collected from groups 1, 2, 3, and 4 at 7, 14, 21, and 28 days after the second immunization for serum neutralizing antibody testing. 21 days after the second immunization, experimental cats in groups 5, 6, 7, and 8 were challenged with FHV-1 (challenge dose of 10 mL / mL). 7.0 TCID 50 Groups 7 and 8 were challenged with FCV (at a dose of 10 mL). 8.0 TCID 50 / mL). The antibody data after combined immunization with FHV-1WH-2017 strain + FCV LZ-2016 strain are as follows (immunization begins at 8 weeks of age, with a second immunization 21 days after the first immunization, and serum neutralizing antibody tests are performed every 7 days after the second immunization):

[0165] Table 10. Results of FHV-1 neutralizing antibody detection in healthy susceptible cats aged 8-12 weeks after secondary immunization.

[0166]

[0167] Table 11 Results of FCV neutralizing antibody detection in healthy susceptible cats aged 8-12 weeks after secondary immunization

[0168]

[0169] Table 12. Results of FHV-1 challenge 21 days after secondary immunization in healthy susceptible cats aged 8-12 weeks.

[0170]

[0171] Note: "—" indicates no abnormalities, "F" indicates purulent ocular and nasal discharge, "H" indicates respiratory rales or paroxysmal cough, and "S" indicates death.

[0172] Table 13 Results of FCV challenge 21 days after secondary immunization in healthy susceptible cats aged 8-12 weeks

[0173]

[0174] Note: "—" indicates no abnormalities, "F" indicates purulent eye and nasal discharge, "I" indicates oral ulcers, and "S" indicates death.

[0175] In summary, the results indicate that when FHV-1 and FCV are prepared and used in combination as a bivalent inactivated vaccine, high levels of antibodies (FHV-1: 1:50-1:178; FCV: 1:796-1:1175) can be induced in experimental cats 21 days after the completion of the established immunization schedule (first immunization at 8-12 weeks of age, followed by a second immunization 21 days later). This protects the experimental cats from infection by virulent strains.

[0176] (5) Safety test

[0177] Safety trial of the bivalent inactivated vaccine of FHV-1WH-2017 strain and FCV LZ-2016 strain

[0178] Fifteen 8-12 week old kittens were selected for the experiment. These kittens tested negative for feline rhinotracheitis virus and feline calicivirus in mixed upper respiratory tract swabs by PCR and had neutralizing antibodies against feline rhinotracheitis virus and feline calicivirus at a ratio not higher than 1:2.

[0179] The experimental cats were randomly divided into 3 groups of 5 cats each. Group 1 received a subcutaneous injection of FHV-1 antigen prepared with a content of 10 in the neck. 8.0 TCID 50 / mL and FCV antigen content 10 8.5 TCID 50 Group 1 received 2 doses / bird of the dual inactivated vaccine at a dose of / mL (overdose group); Group 2 received subcutaneous injection of physiological saline in the neck (immunization control group); Group 3 received no treatment (blank control group). All three groups were observed for 2 consecutive weeks. Figure 29 Basal body temperature was monitored 2 days before immunization, and body temperature was monitored daily after immunization for the same number of days as clinical observation.

[0180] Results: Compared with the control group, the overdose group and the immune control group showed normal food and water intake, body temperature fluctuated within the normal range (37.5℃-39.5℃), no adverse reactions were observed at the injection site, and 5 out of 5 survived.

[0181] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A feline rhinotracheitis virus strain FHV-1WH-2017, with accession number CCTCC NO:V202296, wherein the virus titer of the strain is ≥10^8.5 TCID50 / mL when cultured in F81 adherent cells for 72 hours.

2. A vaccine strain comprising a feline calicivirus strain and the feline rhinotracheitis virus of claim 1, wherein, The feline calicivirus strain was named feline calicivirus FCV LZ-2016 strain and was deposited at the China Center for Type Culture Collection on September 2, 2022, with accession number CCTCC NO: V202276.

3. A vaccine composition wherein the vaccine strain of claim 2 is used as an immunogen.

4. The vaccine composition according to claim 3, characterized in that, The raw materials of the vaccine composition include an inactivated virus of the viral strain and an adjuvant.

5. A method for preparing the vaccine composition of claim 3, comprising the following steps: Step 1: The feline rhinotracheitis virus strain FHV-1WH-2017 and the feline calicivirus strain FCV LZ-2016 were propagated and cultured separately to obtain the virus stock solution. Step 2: Add the virus stock solution or its dilution obtained in Step 1 to β-propiolactone for inactivation; Step 3: Dilute the inactivated virus solution with sterile PBS to obtain the final product. In step 2, β-propiolactone is added to the original virus solution or diluted solution at volumes of 0.12% and 0.06%, respectively. After mixing evenly, the mixture is inactivated at 4°C for 24 hours and then hydrolyzed at 37°C for 2 hours. In step 3, the inactivated feline rhinotracheitis virus strain FHV-1WH-2017 and feline calicivirus strain FCV LZ-2016 were diluted to 10 μL with sterile PBS. 8.5 TCID 50 / mL, 10 9.0 TCID 50 / mL, and aliquoted and stored at 4℃; the prepared antigen solutions of the two viruses and sterile PBS were mixed in a volume ratio of 1:1:1 to make a final volume of 1mL, and the final concentration of FHV-1WH-2017 strain antigen was 10. 8.0 TCID 50 / mL, the final concentration of FCV LZ-2016 strain antigen was 10 8.5 TCID 50 / mL.

6. The use of the vaccine strain of claim 2, the vaccine composition of any one of claims 3-4, in the preparation of an agent for use alone or in combination with other immunizing agents or other drugs, to treat, prevent, mitigate and / or control feline rhinotracheitis virus infection and / or feline calicivirus.

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