A cat-derived canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain and its application

Abstract

The present invention discloses a feline-derived canine parvovirus (CPV / SH / CHN / 01 / 2022 / New CPV-2a) strain and its applications. The microbial deposit number is CGMCC No. 46465. The present invention also discloses a vaccine combination using the virus strain as an immunogen. The virus strain has good immune efficacy. After immunization, the vaccine combination of the present invention can not only produce high-titer neutralizing antibodies against canine parvoviruses of different geographical origins, but also produce neutralizing antibodies against feline parvovirus. This vaccine combination can serve as an alternative vaccine for CPV and can also prevent FPV, thus addressing the shortcomings of currently commercialized CPV and FPV vaccines.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biological products, and in particular to a cat-derived canine parvovirus (CPV) / SH / CHN / 01 / 2022 / New CPV-2a strain and applications thereof. Background Art

[0002] Parvovirus is a virus with a wide range of hosts and is currently one of the most common viruses infecting animals. Its host range continues to expand and renew, and it has become a major epidemic disease affecting cats, dogs, commercial animals, and rare wildlife. 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. It is a single-stranded, non-enveloped, linear DNA virus that primarily infects cats and other felids, as well as a variety of other animals, including mustelids and raccoons. 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 sick dogs by American researchers Eugster and Nairnl in 1977. Research has revealed that it arises from mutations in some amino acid sites of FPV. CPV has a diverse reservoir, including wolves, foxes, cats, tigers, raccoons, bears, otters, jackals, leopards, and civets. Both FPV and CPV belong to the order Carnivora, family Parvoviridae, and 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 disease in their respective hosts. FPV was discovered as early as 1900. After decades of environmental changes and viral evolution (specific changes in the VP2 protein), CPV-2, which can infect dogs, evolved around 1978. Subsequently, new genotypes, including CPV-2a, CPV-2b, CPV-2c, New CPV-2a, and New CPV-2b, have evolved. CPV-2a, CPV-2b, and CPV-2c can infect both dogs and cats. Compared to the original CPV-2, the antigenic variants CPV-2a, CPV-2b, and CPV-2c are more pathogenic to dogs, and the host range of the viruses continues to expand. The VP2 protein is a key protein that determines the antigenic properties, host range, and receptor binding of FPV and CPV. It stimulates the production of large amounts of specific antibodies and plays a key role in receptor recognition and tissue tropism. The VP2 protein also determines the pathogenicity and hemagglutination properties of the viruses. Studies have shown that the VP2 gene and the amino acid similarity expressed by FPV and CPV are greater than 99%, but variations in several specific positions lead to huge differences in hemagglutination, host range, and antigenic characteristics between FPV and CPV.

[0004] The VP2 gene of each CPV-2 subtype undergoes multiple mutations and, after serial passage, has also adapted to feline cell culture. Vaccination is the primary measure for preventing and controlling the disease, but not all vaccines provide complete protection. Dog deaths due to immunization failure are a significant blow to the dog industry and pet owners. Therefore, isolating the prevalent CPV strains in China and developing them into effective vaccines is crucial for eliminating the disease. Summary of the Invention

[0005] To this end, the present invention provides a cat-derived canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain and applications thereof.

[0006] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] In the first aspect, the present invention provides a cat-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain, wherein the cat-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain is a virus strain with a microbial preservation number of CGMCC No. 46465, or a passaged virus strain or mutant virus strain thereof whose clinical pathogenicity and immunogenicity have not changed; the full gene sequence of the virus strain is shown in SEQ ID NO: 5.

[0008] In a 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.

[0009] Preferably, the raw materials of the vaccine composition include the immunogen and adjuvant.

[0010] In the third aspect, the virus strain described in the first aspect and the vaccine composition described in the second aspect are used to prepare drugs for preventing diseases caused by canine parvovirus.

[0011] The present invention has the following advantages:

[0012] The virus strain described in the present invention is a newly isolated and identified feline canine parvovirus (CPV / SH / CHN / 01 / 2022 / New CPV-2a strain from clinical sources. The neutralizing antibodies produced by the prepared inactivated parvovirus vaccine can completely neutralize FPV and CPV, exhibiting strong specificity and high sensitivity. Therefore, it can be used as a vaccine strain for the prevention of FPV and CPV. The present invention aims to provide a feline canine parvovirus (CPV / SH / CHN / 01 / 2022 / New CPV-2a strain and its use, which address the shortcomings of currently commercialized CPV and FPV vaccines and effectively prevent epidemics caused by canine parvovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0014] Figure 1 The results of the third-generation blind cytopathic effect of F81 cells inoculated with cat anal swab samples (200×);

[0015] Figure 2 The results of FPV agarose gel electrophoresis were obtained by PCR amplification of cell supernatant;

[0016] Figure 3 Indirect immunofluorescence results of the four isolates (200×);

[0017] Figure 4 This is an electron micrograph of virus particles of CPV isolates;

[0018] Figure 5 This is the genetic evolutionary tree of the VP2 gene of the isolates;

[0019] Figure 6 One-step growth curve results for CPV / SH / CHN / 01 / 2022 / New CPV-2a strain

[0020] Figure 7The VP2 gene sequence nucleotide similarity comparison results with the reference strain;

[0021] Figure 8 The amino acid sequence similarity comparison results of VP2 protein and reference strains;

[0022] Figure 9 This is a graph showing the reduction in antibody titer of CPV / SH / CHN / 01 / 2022 / New CPV-2a inactivated vaccine. DETAILED DESCRIPTION

[0023] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0024] Example 1 Isolation of the original strain of cat-derived canine parvovirus

[0025] 1F81 cell culture

[0026] F81 cells (cat kidney cells) were purchased from Shanghai Xinyu Biotechnology Co., Ltd. F81 cells were revived and cultured in a 37°C, 5% CO2 incubator with culture medium containing 10% fetal bovine serum. After growing into a monolayer, they were digested with 0.1% (M / V) trypsin. Fetal bovine serum was purchased from Sigma; DMEM medium and trypsin were purchased from GIBCO. F81 cell suspension was diluted with cell culture medium with 10% serum concentration to a cell density of 1×10 6 / mL when ready for use.

[0027] 2. Clinical sample processing, virus isolation, purification and titer determination

[0028] Fourteen fecal swab samples from 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 1:2 (M:V) ratio, repeatedly frozen and thawed three times, and centrifuged at 5000g for 10 minutes at 4°C. The supernatant was sterilized by filtration through a 0.22μm filter. The filtrate was simultaneously inoculated into a suspension of freshly passaged, non-adherent F81 cells, mixed by pipetting, and incubated in a 37°C, 5% CO2 incubator. Cytopathic effect (CPE) was observed every 12 hours. Virus was harvested when CPE reached 80%, and after three cycles of freezing and thawing, the virus was passaged.

[0029] After CPE appeared stably for 5 generations, the virus was purified by plaque. The virus solution was diluted 10 times with DMEM (containing 10% FBS).-1 Dilute to 10 -5 ; 2 mL of dilutions of different concentrations were sequentially drawn into 6-well plates, DMEM (containing 10% FBS) culture medium was used as a negative control, and the plates were incubated in a 37°C, 5% CO2 constant temperature incubator for 24 h. The supernatant was discarded, 2 mL of 1% agarose gel (diluted) was added to each well, and the plates were cooled and solidified into a covering layer. The 6-well plates were inverted and placed in a 37°C, 5% CO2 concentration incubator for continued incubation; CPE was observed daily. When obvious CPE appeared, 2 mL of 1% agarose gel containing 0.002% neutral red was added to each well, cooled and solidified to form a second covering layer, and the plates were continued to be inverted for incubation. Plaques were observed, and single plaques were picked for proliferation culture. The purified virus solution was stored at -80°C.

[0030] The purified virus solution was diluted 10-fold with DMEM (containing 10% FBS). -1 Dilute to 10 -12 100 μL of the dilution solution was pipetted into 96-well plates, with 8 wells added for each dilution. The plates were then incubated at 37°C in a 5% CO2 incubator. CPE was observed and recorded daily. Uninfected normal cells were also used as controls. The 50% tissue culture infectious dose (TCID) of the virus was calculated according to the Reed-Muech method. 50 ).

[0031] Fourteen diseased samples were inoculated with F81 cells. After three blind passages, the F81 cells inoculated with samples No. 4, No. 9, No. 11, and No. 12 showed obvious FPV typical cytopathic effects (CPE), such as shedding, deformation, aggregation, and web formation. The cells in the control group were tightly arranged and grew vigorously, without any pathological changes. Figure 1 After five blind passages, the isolates grew well on F81 cells, and the virus titers after plaque purification could reach 10 7.29 TCID 50 mL -1 , 10 7.57 TCID 50 mL -1 , 10 7.33 TCID 50 mL -1 and 10 7.41 TCID 50 mL -1 .

[0032] 3 Virus identification

[0033] (1) PCR identification

[0034] Virus liquid nucleic acid was extracted using the magnetic bead method according to the instructions of the Magen nucleic acid extraction kit. FPV identification primers were synthesized 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 [J]. Journal of Animal Husbandry and Veterinary Medicine, 2021, 52(01): 262-267.), upstream primer FPV-P1 (SEQ ID NO: 1): 5'-TGATGGAGCAGTTCAACCAGA-3', downstream primer FPV-P2 (SEQ ID NO: 2): 5'-TCAGATCTCATAGCTGCTGGA-3', and the amplified target fragment size was 574 bp. Primers were synthesized by Shanghai Paisonno Biotechnology Co., Ltd. PCR reaction system: 12.5 μL of 2× Taq PCR Master Mix, 1 μL each of upstream and downstream primers FPV-P1 and FPV-P2 (10 μmol / L), 2 μL of DNA template, and ddH2O to 25 μL. Reaction conditions: 94°C initial denaturation for 2 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; extension at 72°C for 10 min; and storage at 4°C. 5 μL of PCR amplification product was identified by 1% agarose gel electrophoresis. Positive PCR products were sent to Shanghai Paisonno Biotechnology Co., Ltd. for sequencing. Sequencing results were compared using the NCBI BLAST tool to confirm viral species.

[0035] The isolates were amplified by PCR and then subjected to agarose gel electrophoresis. Figure 2 As shown, target bands of 574 bp appeared in strains 4, 9, 11, and 12. After sequencing, BLAST results showed that the amplified sequences had the highest homology of 100% with the partial sequences of the VP2 genes of FPV cat-2, CPV CH-AH-D5, FPV HN1806, and FPV BFPV-1, respectively.

[0036] (2) Indirect immunofluorescence assay (IFA)

[0037] The virus solution and F81 cells were inoculated simultaneously in a 24-well plate, and F81 cells were set as a negative control. The cells were cultured at 37°C with 5% CO2 for 48 hours.

[0038] Indirect immunofluorescence detection: I: Fix the cells, discard the supernatant, wash twice with PBS, fix the cells with 4% paraformaldehyde for 20 minutes, and wash three times with PBST; II: Permeabilization, treat with -20℃ pre-cooled 100% methanol for 10 minutes, wash three times with PBS; III: Block, add blocking agent (5% BSA + 0.3% Triton X-100) for 1.5 hours at room temperature, and wash three times with PBS; IV: Incubate with primary antibody, dilute anti-FPV antigen monoclonal antibody at 1:200, add 500uL to each well, incubate at room temperature for 1 hour, and wash twice alternately with PBS and PBST; V: Incubate with secondary antibody, dilute FITC-labeled goat anti-mouse antibody at 1:1000, add 500μL to each well, incubate at room temperature for 1 hour in the dark, and wash twice alternately with PBS and PBST; VI: Take pictures, select the obvious CPE area, observe the fluorescence under a fluorescence microscope and take pictures.

[0039] Indirect immunofluorescence test results Figure 3 As shown, F81 cells inoculated with the virus solution exhibited specific green fluorescence, while no specific fluorescence reaction was observed in the control group cells.

[0040] (3) Electron microscopic observation of viruses

[0041] The harvested cytotoxic cells were frozen and thawed three times at -80°C and centrifuged at 10,000 rpm for 1 hour to remove cell debris. The supernatant was then mixed with polyethylene glycol 8,000 (PEG8,000) at a final concentration of 10% overnight. After centrifugation at 12,000 rpm for 2 hours at 4°C, the cells were resuspended in Tris-buffered saline (TBS). 10 μL of the virus solution was dropped onto a copper-grid carbon support membrane and negatively stained with 10 μL of 2% phosphotungstic acid negative staining solution. Viral morphology was observed using transmission electron microscopy.

[0042] Electron microscopy observations Figure 4 As shown, clear virus particles can be seen. The virus particles are round in shape, have no envelope on the surface, and are about 23 nm in diameter, which is consistent with the structural characteristics of parvovirus, indicating that the parvovirus strain was successfully isolated in this experiment.

[0043] Example 2 Gene sequencing and genetic evolution analysis of isolated strains

[0044] 1. FPV VP2 gene sequencing and genetic evolution analysis

[0045] The isolated and identified viral liquid nucleic acid was extracted according to Example 1, and the VP2 gene amplification primers common to FPV and CPV were synthesized according to the reference (Li Shaohan, You Xinyue, Fan Junwen, et al. Sequence analysis of VP2 and NS1 genes of 14 canine parvovirus isolated strains in Beijing [J]. Journal of Animal Husbandry and Veterinary Medicine, 2021, 52(01): 262-267.). The amplified fragment size was 1755 bp, upstream primer VP2-F (SEQ ID NO: 3): 5'-CGGGATCCAT-GAGTGATGGAGCAGTTCAA-3'; downstream primer VP2-R (SEQ ID NO: 4): 5'-GGAATTCTTAGTATAATTTTCTAGGTGCTAGTT-3'. The primers were synthesized by Shanghai Paisonno Biotechnology Co., Ltd. The total volume of the system was 50 μL: PCR reaction system: 12.5 μL of 2× Taq PCR MasterMix, 2 μL each of the upstream and downstream primers VP2-F and VP2-R (10 μmol / L), 4 μL of DNA template, and ddH2O to 50 μL. Reaction conditions: 94°C initial denaturation for 3 min; 30 cycles of denaturation at 94°C for 50 s, annealing at 55°C for 50 s, and extension at 72°C for 1 min 30 s; extension at 72°C for 10 min; storage at 4°C. 5 μL of PCR amplification product was identified by 1% agarose gel electrophoresis. Positive PCR products were sent to Shanghai Paisonno Biotechnology Co., Ltd. for sequencing. VP2 gene fragments were assembled using SeqMan software in Lasergene 7.0.

[0046] Lasergene 7.0 software was used to align the VP2 gene sequences of the isolates with the reference sequences in GenBank (Table 1), and the nucleotide homology differences were analyzed. The neighbor-joining method (bootstrap value of 1000) in MEGA 6.0 software was used to construct a genetic evolutionary tree.

[0047] Table 1 Summary of VP2 gene reference sequences

[0048]

[0049] Note: “ / ” indicates unclassified.

[0050] The isolates were identified for parvovirus genotyping by referring to the 14 key amino acid sites of the VP2 protein of the standard strain. The results showed (Table 2) that isolate No. 9 was consistent with the Pome strain of the New CPV-2a type at the key amino acid sites and therefore belonged to the feline CPV-2a; it was named CPV / SH / CHN / 01 / 2022 / New CPV-2a. The remaining three strains 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 standard strains, none of the 14 key amino acid sites of the four isolates changed.

[0051] Table 2 Strain genotype analysis

[0052]

[0053] Draw the genetic evolutionary tree of the VP2 gene of the isolates. Figure 5 As shown, the four isolates belong to two major branches, among which the CPV / SH / CHN / 01 / 2022 / New CPV-2a strain belongs to the CPV branch, and is most closely related to the Chinese strains 06 / 09 and CPV-G15, belonging to the same sub-branch, and is distantly related to the four CPV vaccine strains, and is not in the same sub-branch; the three FPV isolates are all in the FPV branch, and are most closely related to the Chinese strain, but are distantly related to each other and are in different sub-branches, suggesting that the isolates may have different sources, and are distantly related to the two FPV vaccine strains, and are not in the same sub-branch.

[0054] 2. One-step growth curve drawing and whole-genome sequencing of virus isolates

[0055] The CPV / SH / CHN / 01 / 2022 / New CPV-2a strain was further analyzed. 5 TCID 50 mL -1 The virus solution was inoculated synchronously with F81 cells in 25 cm 2 Every 24 h, 100 μL of cell culture supernatant was aspirated and the TCID 50 The one-step growth curve of virus culture was drawn. The results were as follows Figure 6 As shown. The DNA of the isolate (concentration of 2 ng / μL or more, total amount of 200 ng or more) was sent to Shanghai Saiheng Biological Co., Ltd. for library construction and sequencing using second-generation high-throughput sequencing to obtain the complete gene sequence of the isolate (SEQ ID NO: 5). The sequence information of SEQ ID NO: 5 is provided in the sequence listing submitted with this application.

[0056] The VP2 gene of the CPV / SH / CHN / 01 / 2022 / New CPV-2a isolate was amplified by PCR using specific primers, and the amplified products were sequenced. The sequences were spliced ​​using SeqMan software in Lasergene 7.0 to successfully obtain the complete gene sequence of the VP2 isolate. 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.

[0057] The results of similarity comparison with the reference sequence showed that the nucleotide and amino acid sequence similarities of CPV / SH / CHN / 01 / 2022 / New CPV-2a and the CPV epidemic strains were 99.1% to 99.7%, respectively. Figure 7 ) and 99.0% to 99.5% ( Figure 8 ), among which the nucleotide similarity was highest with the Chinese strain CPV / BJ018 / 07 and 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.

[0058] 3. Microbial preservation

[0059] The present invention submits the isolated cat-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain to a patent procedure recognized depository institution for deposit, and its microbial deposit number is CGMCC No.46465. The classification name is: New CPV-2a canine parvovirus. The deposit time is: May 19, 2025: the depository unit is: General Microbiology Center of China Culture Collection Administration, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The 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, and CPV / SH / CHN / 01 / 2022 / New CPV-2a strain.

[0060] Example 3: Preparation of Canine Parvovirus Vaccine

[0061] 1. Viral amplification

[0062] The feline canine parvovirus (CPV) / SH / CHN / 01 / 2022 / NewCPV-2a strain isolated in Example 1 and identified in Example 2 was amplified and cultured. After sterile growth was confirmed according to the appendix of the current Chinese Pharmacopoeia, the viral content was determined as described in Example 2. The virus was filtered through a 0.22 μm filter membrane to obtain a virus solution of the feline canine parvovirus (CPV) / SH / CHN / 01 / 2022 / NewCPV-2a strain, free of cells and debris, which was then stored at low temperatures.

[0063] 2. Vaccine Preparation

[0064] Dilute the virus solution to 1 × 10 7 TCID 50 mL -1 The diluted virus solution was added with formaldehyde at a final concentration of 0.2% by volume, 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 The virus was mixed with 200 μg / mL of the virus and added to a 6-well plate at 2 mL / well. The culture was incubated at 37°C in a 5% (v / v) CO2 incubator for 3-7 days and blindly passaged for three generations. The cytopathic effect was recorded at the same time. The results showed that no CPE appeared in the cells after three blind passages, indicating complete inactivation. The virus liquid that passed the inactivation verification was then slowly injected into the aluminum gel adjuvant purchased from Chuangzhi Biotechnology Co., Ltd. and emulsified at a volume ratio of 1:1 antigen:adjuvant to prepare an inactivated vaccine.

[0065] Example 4: Efficacy test of inactivated vaccine of canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain from cats

[0066] 1. Grouping and immunization of experimental rabbits

[0067] The experimental rabbits were randomly divided into two groups, each with three rabbits. One group was used as the experimental group and immunized with the inactivated vaccine; the other group was used as the control group and immunized with DMEM (. The experimental rabbits were clean-grade female New Zealand rabbits, 3 months old, weighing 2-2.5 kg, purchased from Shanghai Proton Biotechnology Co., Ltd.; the immunogen was the inactivated vaccine prepared as described in Example 3.

[0068] The immunogen was injected subcutaneously at multiple points into clean New Zealand rabbits. Each rabbit was injected twice, with an interval of 14 days between each injection. Each injection was conducted at four points, with 0.5 mL of immunogen injected at each point.

[0069] 2. Determination of neutralizing antibodies

[0070] Serum samples were collected at 0, 7, 14, 21, 28, 35, 42 and 49 days after the first vaccination, and neutralizing antibody titers were tested using the virus strains. The TCID of the virus strains were determined according to the method in Example 1.50 The fixed virus-dilution serum method was used to detect serum neutralizing antibodies: CPV / SH / CHN / 01 / 2022 / New CPV-2a strain, CPV / SH01 / 11 strain and FPV / SH / CHN / 01 / 2022 strain were propagated, of which the canine parvovirus CPV / SH01 / 11 strain was preserved by the veterinary laboratory of Shanghai Animal Disease Prevention and Control Center; the virus titers were determined to be 10 7.57 TCID 50 mL -1 , 10 7.41 TCID 50 mL -1 and 10 7.29 TCID 50 mL -1 , dilute the virus to 200 TCID 50 After the serum was diluted 20 times, it was treated in a 56°C water bath for 30 minutes, and then serially diluted 2 times (2 -1 ~2 -11 Mix 100 μL of serum and 100 μL of virus and incubate at 37°C for 1.5 hours. Simultaneously, establish a virus control, a serum toxicity control, a cell blank control, and a negative serum control. Add 50 μL of the incubated mixture and an equal amount of cell suspension to a 96-well plate and incubate in a 37°C, 5% CO2 incubator. Observe cytopathic effects daily. Observe after 4-5 days. Record the serum dilution that ultimately produces cytopathic effects. Also record the maximum serum dilution that can neutralize the virus strain. Plot a graph of the reduction in immune antibody titer for the inactivated vaccine.

[0071] The results are as follows Figure 9 As shown, the antibody titer against cat-origin canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain can reach a maximum of 2133.4, the antibody titer against canine parvovirus CPV / SH01 / 11 strain can reach a maximum of 853.4, and the antibody titer against feline parvovirus FPV / SH / CHN / 01 / 2022 strain can reach a maximum of 1280; the neutralizing antibody titer reached a peak 14 to 21 days after the second vaccination, and then gradually decreased. On the 35th day after the second vaccination, the antibody titer still remained above 400, so it can be used as a vaccine candidate strain for the simultaneous prevention of CPV and FPV infections.

[0072] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A cat-derived canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain, wherein the cat-derived canine parvovirus CPV / SH / CHN / 01 / 2022 / New CPV-2a strain is a virus strain with a microbial preservation number of CGMCC No.46465.

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

5.

3. A vaccine composition, characterized in that: The vaccine composition uses the virus strain according to 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 adjuvant.

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

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