Celine-derived canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain and application thereof

Abstract

The invention discloses a cat canine parvovirus (CPV) / SH / CHN / 01 / 2022 / New CPV-2a strain and an application of the cat canine parvovirus (CPV) / SH / CHN / 01 / 2022 / New CPV-2a strain. The microbial preservation number of the cat canine parvovirus is CGMCC No.46465. The invention further discloses a vaccine combination taking the virus strain as an immunogen, the virus strain is good in immune efficacy, after the vaccine combination is immunized, high-titer neutralizing antibodies aiming at canine parvoviruses of different territorial sources can be generated, meanwhile, cat parvovirus neutralizing antibodies can also be generated, the vaccine combination can be used as an alternative vaccine for CPV, FPV can also be prevented, and the vaccine combination is safe and reliable. And the defects of current commercial CPV and FPV vaccines are overcome.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biological products, and particularly relates to a feline-derived canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain and its applications. Background Art

[0002] Parvovirus is a virus with a wide range of hosts and is currently one of the most frequently infecting animals. The host range of parvovirus is still constantly expanding and updating, and it has now become one of the main diseases of felines, canines, economic animals, and rare wild animals. Feline parvovirus (FPV), also known as feline panleukopenia virus, feline distemper virus, or feline infectious enteritis virus, belongs to the genus Parvovirus in the family Parvoviridae, and is a single-stranded non-enveloped linear DNA virus. It mainly infects cats and other feline animals, as well as various animals such as the Mustelidae and Procyonidae families. FPV infection can cause an acute, highly contagious disease characterized by high fever, vomiting, enteritis, and severe leukopenia, with a mortality rate generally ranging from 50% to 60%. Canine parvovirus (CPV) was isolated from the feces of diseased dogs by American scholars Eugster and Nairnl in 1977. Through research, it was found that it evolved from mutations at some amino acid sites of FPV. CPV has a variety of hosts, and the virus has been detected in wolves, foxes, cats, tigers, raccoons, bears, otters, jackals, leopards, and masked palm civets, etc. Both FPV and CPV belong to the order Carnivora, the family Parvoviridae, and the genus Parvovirus. Members of this genus include FPV, CPV, raccoon parvovirus (RaPV), and mink enteritis virus (MEV).

[0003] FPV and CPV are closely related and can cause diseases in their respective hosts. As early as around 1900, FPV was discovered. After decades of environmental changes and virus evolution (specific changes in the VP2 protein), CPV-2 that can infect dogs evolved around 1978. Subsequently, new genotypes such as CPV-2a, CPV-2b, CPV-2c, New CPV-2a, and New CPV-2b evolved successively. Among them, CPV-2a, CPV-2b, and CPV-2c can infect both dogs and cats. Compared with the original CPV-2, the antigen mutants CPV-2a, CPV-2b, and CPV-2c have higher pathogenicity to dogs, and the host range of the virus is continuously expanding. The VP2 protein is a key protein that determines the antigenic characteristics, host range, and receptor binding of FPV and CPV. It can stimulate the body to produce a large number of specific antibodies and also plays a key role in receptor recognition and tissue tropism. In addition, the VP2 protein also determines the pathogenicity, hemagglutination, etc. of the virus. Some studies have shown that the similarity of the VP2 gene and its expressed amino acids between FPV and CPV is greater than 99%, but mutations at several specific positions make FPV and CPV have huge differences in hemagglutination, host range, and antigenic characteristics.

[0004] Multiple mutations have occurred in the VP2 gene of each subtype strain of CPV-2, and continuous passage also adapts to the culture of feline cell lines. Vaccination is the main measure for preventing and controlling this disease, but not all vaccines can provide complete protection. The death of dogs caused by immune failure will cause a heavy blow to the dog breeding industry and pet owners. Therefore, isolating the CPV strains prevalent in China and preparing them into vaccines with good titers is of great significance for eliminating this disease. Summary of the Invention

[0005] For this reason, the present invention provides a feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain and its application.

[0006] In order to achieve the above object, the embodiments of the present invention provide the following technical solutions: In the first aspect, the present invention provides a feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain, and the feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain is a virus strain with a microorganism preservation number of CGMCC No. 46465, or a passaged virus strain or mutant virus strain whose clinical pathogenicity and immunogenicity have not changed; the full gene sequence of the virus strain is as shown in SEQ ID NO: 5.

[0007] In the second aspect, the present invention provides a vaccine composition, characterized in that: the vaccine composition uses the virus strain described in the first aspect as an immunogen.

[0008] Preferably, the raw materials of the vaccine composition include the immunogen and adjuvant.

[0009] In a third aspect, there is provided an application of the virus strain according to the first aspect and the vaccine composition according to the second aspect in the preparation of a medicament for preventing diseases caused by canine parvovirus.

[0010] The present invention has the following advantages: The virus strain of the present invention is a feline-derived canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain newly isolated and identified from the clinic. The neutralizing antibodies produced by the inactivated parvovirus vaccine can completely neutralize FPV and CPV, and have the characteristics of strong specificity and high sensitivity. Therefore, it can be used as a vaccine strain for preventing FPV and CPV. The object of the present invention is to provide a feline-derived canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain and its application, which makes up for the deficiencies of current commercial CPV and FPV vaccines and effectively prevents the epidemic caused by canine parvovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained according to the provided drawings.

[0012] Figure 1 It shows the cytopathic effect results of inoculating feline anal swab samples into F81 cells and blindly passing them for three generations (200×); Figure 2 It shows the agarose gel electrophoresis results of PCR amplification of FPV from cell supernatant; Figure 3 It shows the indirect immunofluorescence results of 4 isolates (200×); Figure 4 It shows the electron micrograph of virus particles of CPV isolates; Figure 5 It shows the phylogenetic tree of the VP2 gene of the isolates; Figure 6 It shows the one-step growth curve results of the CPV / SH / CHN / 01 / 2022 / New CPV-2a strain Figure 7 It shows the comparison results of the similarity of nucleotide sequences of the VP2 gene with reference strains; Figure 8 It shows the comparison results of the similarity of amino acid sequences of the VP2 protein with reference strains; Figure 9It is the immune antibody titer reduction diagram of the CPV / SH / CHN / 01 / 2022 / New CPV-2a inactivated vaccine. Detailed implementation manners

[0013] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0014] Example 1 Isolation of the original strain of feline parvovirus 1 F81 cell culture F81 cells (feline kidney cells) were purchased from Shanghai Xinyu Biotechnology Co., Ltd. The F81 cells were resuscitated and cultured in a culture medium containing 10% fetal bovine serum in a 37 °C, 5% CO2 incubator. After growing into a monolayer, they were digested with 0.1% (M / V) trypsin. Among them, fetal bovine serum was purchased from sigma company; DMEM medium and trypsin were purchased from GIBCO company; the F81 cell suspension was diluted with a cell culture medium with a serum concentration of 10% until the cell density reached 1×10 6 cells / mL and then reserved for use.

[0015] 2 Treatment, virus isolation, purification and titer determination of clinical samples 14 fecal swab samples of cats suspected of parvovirus infection were collected from pet hospitals in the Shanghai area. The fecal swab samples were vortex-mixed with DMEM at a ratio of 1:2 (M:V), repeatedly frozen and thawed 3 times, centrifuged at 5000 g for 10 min at 4 °C, and the supernatant was filtered through a 0.22 μm filter to remove bacteria. The filtrate was synchronously inoculated into the F81 cell suspension that had just been passaged and not adhered to the wall. After pipetting and mixing evenly, it was placed in a 37 °C, 5% CO2 constant temperature incubator for static culture, and the cytopathic effect (CPE) was observed every 12 h. When the CPE reached 80%, the virus was harvested. After repeatedly freezing and thawing 3 times, the virus was passaged.

[0016] After the CPE stably appeared for 5 generations, plaque purification of the isolated virus was carried out. The virus solution was diluted 10-fold with DMEM (containing 10% FBS), from 10 -1 dilution to 10 -5; 2 mL of diluted solutions with different concentrations were successively pipetted into a 6-well plate. DMEM (containing 10% FBS) culture medium was used as a negative control. It was statically cultured in an incubator at 37 °C and 5% CO₂ for 24 h. The supernatant was discarded. 2 mL of 1% agarose gel (already diluted) was added to each well. After cooling, it solidified into a covering layer. The 6-well plate was inverted and placed in an incubator at 37 °C and 5% CO₂ concentration for continued culture; CPE was observed daily. When obvious CPE appeared, 2 mL of 1% agarose gel containing 0.002% neutral red was added to each well, and it was allowed to cool and solidify to form a second covering layer. It was continued to be inverted and cultured, and plaques were observed. Single plaques were picked for proliferative culture, and the purified virus solution was stored at -80 °C.

[0017] The purified virus solution was diluted 10-fold with DMEM (containing 10% FBS), from 10 -1 dilution to 10 -12 ; 100 μL of the diluted solution was successively pipetted into a 96-well plate, with 8 wells for each dilution. It was cultured in an incubator at 37 °C and 5% CO₂ concentration. CPE was observed and recorded daily. At the same time, normal cells without virus inoculation were set as a control. According to the Reed-Muech method, the 50% Tissue Culture Infectious Dose (TCID 50 ) of the virus was calculated.

[0018] 14 samples of diseased materials were respectively inoculated into F81 cells. After three blind passages, obvious cell lesions (cytopathic effect, CPE) typical of FPV such as shedding, deformation, aggregation, and filamentous network formation appeared in the F81 cells inoculated with samples No. 4, No. 9, No. 11, and No. 12. The cells in the control group were closely arranged and grew vigorously without any lesions, as Figure 1 shown. After five blind passages, the isolates grew well on F81 cells. The virus titers after plaque purification could reach 10 7.29 TCID 50 •mL -1 ⁻¹, 10 7.57 TCID 50 •mL -1 ⁻¹, 10 7.33 TCID 50 •mL -1 ⁻¹, and 10 7.41 TCID 50 •mL -1 .

[0019] 3 Identification of the virus (1) PCR identification Extract the nucleic acid of the virus solution by the magnetic bead method according to the instruction manual of the nucleic acid extraction kit of Magen Company. Synthesize FPV identification primers according to the reference (Li Shaohan, You Xinyue, Fan Junwen, et al. Sequence analysis of VP2 and NS1 genes of 14 canine parvovirus isolates in Beijing area [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(01): 262-267.). The upstream primer FPV-P1 (SEQ ID NO: 1): 5'-TGATGGAGCAGTTCAACCAGA-3', the downstream primer FPV-P2 (SEQ ID NO: 2): 5'-TCAGATCTCATAGCTGCTGGA-3', and the size of the amplified target fragment is 574 bp. The primers were synthesized by Shanghai Personal Biotechnology Co., Ltd. PCR reaction system: 2×Taq PCR MasterMix 12.5 μL, upstream and downstream primers FPV-P1 and FPV-P2 (10 µmol / L) each 1 μL, DNA template 2 μL, supplemented with ddH2O to 25 μL. Reaction conditions: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 1 min, 35 cycles; extension at 72 °C for 10 min; store at 4 °C. Take 5 μL of the PCR amplification product and identify it by 1% agarose gel electrophoresis. The positive PCR product was sent to Shanghai Personal Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared and analyzed using the BLAST tool in NCBI to determine the virus species.

[0020] After PCR amplification of the isolates, agarose gel electrophoresis was performed, and the results were as Figure 2 shown. Target bands of 574 bp appeared in No. 4, No. 9, No. 11 and No. 12. After sequencing, the BLAST results showed that the amplified sequences had the highest homology of 100% with the partial VP2 gene sequences of FPV cat-2 strain, CPV CH-AH-D5 strain, FPV HN1806 strain and FPV BFPV-1 strain respectively.

[0021] (2) Indirect immunofluorescence assay (IFA) Synchronously inoculate the virus solution and F81 cells into a 24-well plate, and set F81 cells as a negative control at the same time. Culture at 37 °C and 5% CO2 concentration for 48 h.

[0022] Indirect immunofluorescence assay: I: Fix the cells, discard the supernatant, wash the cells twice with PBS, then fix the cells with 4% paraformaldehyde for 20 min, and wash three times with PBST; II: Permeabilize the cells, treat with pre-cooled (-20 °C) 100% methanol for 10 min, and wash three times with PBS; III: Block the cells, add the blocking agent (5% BSA + 0.3% Triton X-100) and block at room temperature for 1.5 h, then wash three times with PBS; IV: Incubate with the primary antibody, dilute the monoclonal antibody against FPV antigen at a ratio of 1:200, add 500 uL to each well, incubate at room temperature for 1 h, and wash twice each with PBS and PBST alternately; V: Incubate with the secondary antibody, dilute the FITC-labeled goat anti-mouse antibody at a ratio of 1:1000, add 500 µL to each well, incubate in the dark at room temperature for 1 h, and wash twice each with PBS and PBST alternately; VI: Take pictures, select the areas with obvious CPE, observe the fluorescence under a fluorescence microscope and take pictures.

[0023] The results of the indirect immunofluorescence assay are as Figure 3 shown. The F81 cells inoculated with the virus solution showed specific green fluorescence, while no specific fluorescence reaction was observed in the control group cells.

[0024] (3) Electron microscopy observation of the virus The collected cell cytotoxicity was repeatedly frozen and thawed three times at -80 °C and centrifuged at 10,000 r / min for 1 h to remove cell debris. Subsequently, the supernatant was mixed with polyethylene glycol 8000 (PEG8000) at a final concentration of 10% overnight. After centrifugation at 12,000 r / min for 2 h at 4 °C, it was resuspended with Tris-buffered saline (TBS). 10 μL of the virus solution was dropped onto a copper mesh carbon support film, and 10 μL of 2% phosphotungstic acid negative staining solution was added for negative staining. The morphology of the virus was observed by transmission electron microscopy.

[0025] The electron microscopy observation is as Figure 4 shown. Clear virus particles can be seen. The virus particles are round in shape, without an envelope on the surface, and about 23 nm in diameter, which conforms to the structural characteristics of parvovirus, indicating that the parvovirus strain was successfully isolated in this experiment.

[0026] Example 2 Gene sequencing and genetic evolution analysis of the isolated strain 1. FPV VP2 gene sequencing and genetic evolution analysis Extract the nucleic acid of the virus solution isolated and identified according to Example 1. Synthesize the VP2 gene amplification primers common to FPV and CPV according to the reference (Li Shaohan, You Xinyue, Fan Junwen, et al. Sequence analysis of VP2 and NS1 genes of 14 canine parvovirus isolates in Beijing area [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(01): 262-267.). The amplified fragment size is 1755 bp. The upstream primer VP2-F (SEQ ID NO: 3): 5'-CGGGATCCAT-GAGTGATGGAGCAGTTCAA-3'; the downstream primer VP2-R (SEQ ID NO: 4): 5'-GGAATTCTTAGTATAATTTTCTAGGTGCTAGTT-3'. The primers were synthesized by Shanghai Personal Biotechnology Co., Ltd. The total volume of the system is 50 μL: PCR reaction system: 2×Taq PCR MasterMix 12.5 μL, upstream and downstream primers VP2-F, VP2-R (10 µmol / L) 2 μL each, DNA template 4 μL, and ddH2O is added to make up to 50 μL. Reaction conditions: Pre-denaturation at 94 °C for 3 min; denaturation at 94 °C for 50 s, annealing at 55 °C for 50 s, extension at 72 °C for 1 min 30 s, 30 cycles; extension at 72 °C for 10 min; store at 4 °C. Take 5 μL of the PCR amplification product and identify it by 1% agarose gel electrophoresis. The positive PCR product was sent to Shanghai Personal Biotechnology Co., Ltd. for sequencing, and the VP2 gene fragment was spliced using the SeqMan software in Lasergene 7.0.

[0027] Use the Lasergene 7.0 software to align the VP2 gene sequence of the isolate with the reference sequences in GenBank. The reference sequences are shown in Table 1, and analyze the nucleotide homology differences; use the Neighbor-joining method (Bootstrap value is 1000) in the MEGA 6.0 software to construct a phylogenetic tree.

[0028] Table 1 Summary of VP2 gene reference sequences

[0029] Note: " / " indicates untyped.

[0030] Referring to 14 key amino acid sites of the VP2 protein of the reference strain, the genotypes of parvovirus isolates were identified. The results showed (Table 2) that the 9th isolate was consistent with the Pome strain of the New CPV-2a type at the key amino acid sites, so it belonged to CPV-2a of feline origin; it was named CPV / SH / CHN / 01 / 2022 / New CPV-2a. The remaining three isolates were consistent with the FPV-3.us_67 strain and belonged to the FPV strain, which were named FPV / SH / CHN / 01 / 2022, FPV / SH / CHN / 02 / 2022, and FPV / SH / CHN / 03 / 2022, respectively. Compared with their respective reference strains, no changes occurred in the 14 key amino acid sites of the 4 isolates.

[0031] Table 2 Genotype analysis of virus strains

[0032] The genetic evolutionary tree of the VP2 gene of the isolates was drawn. The results were as Figure 5 shown. The 4 isolates belonged to two major branches. Among them, the CPV / SH / CHN / 01 / 2022 / New CPV-2a strain belonged to the CPV branch, was the closest to the Chinese strains 06 / 09 and CPV-G15, and belonged to the same sub-branch. It had a relatively distant genetic relationship with the 4 CPV vaccine strains and was not in the same sub-branch. The 3 FPV isolates were all in the FPV branch, all had the closest genetic relationship with the Chinese strains, but had a relatively distant genetic relationship with each other and were in different sub-branches respectively, suggesting that the isolates might have different origins and had a relatively distant genetic relationship with the 2 FPV vaccine strains and were not in the same sub-branch.

[0033] 2. Plotting the one-step growth curve of virus isolates and whole-genome sequencing The CPV / SH / CHN / 01 / 2022 / New CPV-2a strain was further analyzed. 1 mL of virus solution with a titer of 1×10 5 TCID 50 •mL -1 was co-inoculated with F81 cells into a 25 cm 2 cell culture flask. Every 24 h, 100 μL of cell culture supernatant was taken for TCID 50 determination to plot the one-step growth curve of virus culture. The results were as Figure 6 shown. The DNA of the isolate (with a concentration of more than 2 ng / μL and a total amount of more than 200 ng) was sent to Shanghai Saiheng Biotechnology Co., Ltd. for library construction and sequencing by the second-generation high-throughput sequencing method to obtain the whole-genome sequence of the isolate (SEQ ID NO:5). Among them, the sequence information of SEQ ID NO: 5 was provided in the sequence listing submitted with this application.

[0034] The VP2 gene of the CPV / SH / CHN / 01 / 2022 / New CPV-2a isolate was amplified by PCR using specific primers, and the amplification product was sequenced. The sequence was assembled using the SeqMan software in Lasergene 7.0, and the full-length VP2 gene sequence of the isolate was successfully obtained. The results showed that the full length of the VP2 gene of the CPV / SH / CHN / 01 / 2022 / New CPV-2a isolate was 1755 bp, encoding 584 amino acids.

[0035] The similarity comparison with the reference sequence showed that the nucleotide and amino acid sequence similarities between CPV / SH / CHN / 01 / 2022 / New CPV-2a and CPV epidemic strains were 99.1% - 99.7% ( Figure 7 ), and 99.0% - 99.5% ( Figure 8 ), respectively. Among them, the nucleotide similarity was the highest with the Chinese strain CPV / BJ018 / 07 and the lowest with the Japanese strain V154; the nucleotide similarities with the FPV vaccine strains CU-4 and Purevax were 98.2% and 98.3%, respectively, and the nucleotide similarities with the CPV vaccine strains 790312, VAC_P vanguard, CPV-5.us.79, and CPVint(vaccine) were 99.1%, 98.7%, 99.1%, and 98.9%, respectively.

[0036] 3. Microbial Preservation The isolated feline parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain of the present invention was submitted to a depository institution recognized by the patent procedure for preservation. The microbial deposit number is CGMCC No. 46465. The taxonomic name is: Canine parvovirus, New CPV-2a type. The preservation time is: May 19, 2025; the preservation unit is: General Microbiology Center, China Microbial Culture Collection Center, preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. This virus strain is called Canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain, CPV CPV / SH / CHN / 01 / 2022 / New CPV-2a strain, CPV / SH / CHN / 01 / 2022 / New CPV-2a strain.

[0037] Example 3. Preparation of Canine Parvovirus Vaccine 1. Virus Amplification The feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / NewCPV-2a strain isolated in Example 1 and identified in Example 2 was amplified and cultured. It was examined according to the appendix of the current "Chinese Veterinary Pharmacopoeia". After the result showed no bacterial growth, the virus content was determined according to Example 2; it was filtered through a 0.22 μm filter membrane to obtain the virus solution of the feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain with cells and debris removed, and stored at low temperature.

[0038] 2. Preparation of Vaccine The virus solution was diluted with DMEM to 1×10 7 TCID 50 •mL -1 , formaldehyde with a final mass-volume percentage concentration of 0.2% was added to the diluted virus solution, and the virus was inactivated at 37 °C for 24 h; the inactivated virus solution was mixed with an equal volume of F81 cell suspension (1×10 6 cells / mL), added to a 6-well plate, 2 mL / well, cultured at 37 °C in a 5% (V / V) CO2 concentration incubator for 3 - 7 days, blindly passaged three generations, and the cytopathic effect was recorded simultaneously; the results showed that no CPE appeared in the cells after three generations of blind passage, indicating complete inactivation; then the virus solution with qualified inactivation verification was slowly injected into the aluminum adjuvant. The aluminum adjuvant was purchased from Chuangzhi Biotechnology Co., Ltd., and mixed and emulsified at a ratio of antigen:adjuvant volume ratio of 1:1 to prepare the inactivated vaccine.

[0039] Example 4. Potency Test of the Inactivated Vaccine of the Feline-Origin Canine Parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a Strain 1. Grouping and Immunization of Experimental Rabbits The experimental rabbits were randomly divided into two groups, three in each group. One group was used as the experimental group and immunized with the inactivated vaccine; the second group was used as the control group and immunized with DMEM. Among them, the experimental rabbits were clean-grade New Zealand rabbits, female, 3 months old, 6 rabbits, weighing 2 - 2.5 kg, purchased from Shanghai Puluoteng Biotechnology Co., Ltd.; the immunogen was the inactivated vaccine prepared in Example 3.

[0040] The immunogen was subcutaneously injected into the clean-grade New Zealand rabbits at multiple points. Each rabbit was injected 2 times, with an interval of 14 d each time. Each time, 4 points were injected, and 0.5 mL of the immunogen was injected at each point.

[0041] 2. Determination of Neutralizing Antibody Serum samples were collected on days 0, 7, 14, 21, 28, 35, 42, and 49 after the first immunization, and the neutralizing antibody titer was detected using the virus strain. The TCID of the virus strain was determined according to the method of Example 1 respectively 50Values. The serum neutralizing antibody was detected by the fixed virus-diluted serum method: The CPV / SH / CHN / 01 / 2022 / New CPV-2a strain, CPV / SH01 / 11 strain and FPV / SH / CHN / 01 / 2022 strain were propagated. Among them, the canine parvovirus CPV / SH01 / 11 strain was preserved in the veterinary laboratory of the Shanghai Animal Disease Prevention and Control Center; the virus titers were measured to be 10 7.57 TCID 50 •mL -1 、10 7.41 TCID 50 •mL -1 and 10 7.29 TCID 50 •mL -1 , respectively. The virus was diluted to 200 TCID 50 . The serum was diluted 20-fold and treated in a water bath at 56 °C for 30 min, and then serially diluted 2-fold (2 -1 ~2 -11 ). 100 μL of serum and 100 μL of virus were mixed and incubated at 37 °C for 1.5 h. At the same time, virus control, serum toxicity control, cell blank control and negative serum control were set. 50 μL of the incubated mixture and an equal volume of cell suspension were added to a 96-well plate simultaneously and cultured in an incubator at 37 °C with 5% CO2. The cytopathic effect was observed daily, and the observation was stopped after 4 - 5 d. The serum dilution at which the cytopathic effect finally occurred was recorded, and the maximum serum dilution that could neutralize the virus strain was determined. The immune antibody titer reduction diagram of the inactivated vaccine was drawn.

[0042] The results are as Figure 9 shown. Among them, the antibody titer against the feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain could reach up to 2133.4 at most, the antibody titer against the canine parvovirus CPV / SH01 / 11 strain could reach up to 853.4 at most, and the antibody titer against the feline parvovirus FPV / SH / CHN / 01 / 2022 strain could reach up to 1280 at most; the neutralizing antibody titer reached the peak 14 - 21 d after the second immunization and then gradually decreased. The antibody titer was still above 400 on the 35th day after the second immunization. Therefore, it can be used as a vaccine candidate strain for simultaneously preventing CPV and FPV infections.

[0043] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain, wherein the feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain is a virus strain with a microorganism deposit number of CGMCC No. 46465, or a passaged virus strain or mutant virus strain with unchanged clinical pathogenicity and immunogenicity.

2. The feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain according to claim 1, wherein, The complete gene sequence of the feline-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain is as shown in SEQ ID NO:

5.

3. A vaccine composition, characterized in that: The vaccine composition uses the virus strain described in claim 1 as an immunogen.

4. The vaccine composition according to claim 3, wherein, The raw materials of the vaccine composition include the immunogen and an adjuvant.

5. Use of the virus strain described in claim 1 and the vaccine composition described in any one of claims 3-4 in the preparation of a drug for preventing diseases caused by canine parvovirus.

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