CN114921422B

CN114921422B - Canine parvovirus isolate and application thereof

Info

Publication number: CN114921422B
Application number: CN202210579322.0A
Authority: CN
Inventors: 汤承; 岳华; 王佳丽; 陈曦; 王远微
Original assignee: Southwest Minzu University
Current assignee: Southwest Minzu University
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2023-04-25
Anticipated expiration: 2042-05-25
Also published as: CN114921422A

Abstract

The invention discloses a canine cat parvovirus isolate, which belongs to the technical fields of biotechnology and virology, wherein the isolate is preserved in China center for type culture collection, the preservation address is China Wuhan, the preservation number is CCTCC NO: V202237, the preservation date is 2022, 5 months and 11 days, and the canine cat parvovirus isolate is classified and named as canine cat parvovirus S/CD/2021. The invention also relates to application of the canine feline parvovirus isolate. The canine and feline parvovirus can spread across hosts to cause systemic infection of dogs, can generate a higher level of neutralizing antibodies after being prepared into an inactivated vaccine, has the basic potential of the vaccine, and can protect dogs from being attacked by feline parvovirus.

Description

Canine parvovirus isolate and application thereof

Technical Field

The invention relates to the field of biotechnology and virology, in particular to a canine-source feline parvovirus isolate and application thereof.

Background

In recent years, with the continuous improvement of the living standard of people, pets become an indispensable part of life, and the rapidly expanding pet industry brings vigor to the social development and simultaneously has a plurality of problems to be solved urgently, for example, infectious diseases such as canine distemper, canine parvovirus diseases, canine coronavirus diseases and the like are widely prevalent worldwide, spread rapidly, and cause great harm to the canine industry. Among them, canine parvovirus disease is serious, the mortality rate is up to 50% -90%, and the development of the canine industry is seriously jeopardized.

Canine parvovirus (Canine parvovirus, CPV), which belongs to the genus parvoviridae, is a single-stranded negative-strand DNA virus that is free of a capsule, icosahedral structure, and is composed of 32 capsids, causes acute infectious diseases characterized by vomiting, diarrhea, leukopenia, and hemorrhagic enteritis, and has been found to be one of the most serious infections that endanger canine health.

CPV has more than 98% genomic homology to Feline Parvovirus (FPV) and only differs by a few amino acid sites, and is therefore considered to originate from FPV or evolved from FPV by some intermediate medium, CPV particle surface consists of 60 VP1 and VP2 alternating motifs, VP2 accounting for 90% and VP1 accounting for only 10%, so VP2 is the main component constituting capsid protein, and the difference of several amino acid sites on VP2 surface allows CPV, FPV to have respective specific host range, antigen type and hemagglutination properties. CPV is capable of agglutinating red blood cells of cats, pigs, rhesus monkeys, horses, and hamsters at a temperature of 4to 25℃and a pH of 6.0 to 7.2, and is incapable of agglutinating O-type red blood cells of cattle, rabbits, goats, guinea pigs, rats, mice, rats, geese, chickens, and humans. Host ranges for FPV include domestic and wild felines (felimia, subgrade) and some wild canines (cannifemia, subgrade), such as raccoons and foxes, but not dogs. Although FPV replicates in canine lymphoid tissues (thymus and bone marrow) after experimental inoculation, it does not bind to canine transferrin receptor (tfr), which is critical for infection, so FPV does not further infect dogs; in contrast, it is common for cats to be infected with CPV.

However, in recent years, there have been many reports of detection of feline parvovirus from canine feces, including china, vietnam, pakistan, thailand, etc.; however, VP2 protein characteristics were analyzed only in Vietnam (Hoang M, wu C N, lin C F, et al genetics characterization of feline panleukopenia virus from dogs in Vietnam reveals a unique Thr mutation in VP2[ J ]. PeerJ,2020,8 (160): e 9752.), china (Chen B, zhang X, zhu J, et al molecular Epidemiological Survey of Canine Parvovirus Circulating in China from 2014to 2019.2021.), and no strain was isolated, and therefore, the biological properties of canine feline parvovirus and the pathogenicity to canine were not clear.

After CPV infection, the incidence is quick and the death rate is higher, the current prevention and control measures for CPV are mainly vaccine immunity, CPV has only one antigen type, namely CPV-2, and commercially available CPV vaccines in the market are more, including imported and domestic vaccines, but belong to attenuated vaccines. However, CPV has antigenic drift, and then different subtypes CPV-2a, CPV-2b, CPV-2c (a) and CPV-2c (b) which vary in host range, coagulability and antigenicity appear, and as viruses vary continuously, new challenges are presented to the treatment and prevention of this disease. It is not clear whether the existing vaccine can protect the emerging canine feline parvovirus.

Thus, there is an urgent need to isolate the newly emerging canine feline parvovirus and develop a vaccine to prevent the occurrence of the disease and reduce the damage of the virus to dogs.

Disclosure of Invention

In order to solve the problems existing in the conventional CPV prevention, the invention aims to provide a canine-source feline parvovirus isolate and application thereof, wherein the canine-source feline parvovirus isolate (namely an S/CD/2021 isolate) is subjected to isolated culture, identification and pathogenicity research, and a vaccine composition prepared from the canine-source feline parvovirus isolate can generate high-level antibodies after immunization of puppies.

The technical scheme of the invention is as follows:

the first object of the invention is to provide a canine and feline parvovirus isolate, the microorganism of which has a collection number of CCTCC NO: V202237.

CPV is an autonomously replicating parvovirus whose genome is single-stranded, negative-stranded, linear DNA. The genome contains 5323 nucleotides, which is the smallest in DNA viruses. The genome contains two Open Reading Frames (ORFs), the first ORF consisting of the left half of the genome, encoding the early transcribed regulatory proteins-nonstructural proteins NSI and NS2; the second ORF is located in the right half of the genome and encodes the viral capsid proteins VP1 and VP2, which are mainly responsible for the capsid constituting the viral particle and play a very important role in the viral infection process. Studies have shown that VP2 protein on the surface of canine parvovirus can specifically bind to transferrin receptor (Transferrin receptor, tfR) on the surface of host cells, and the viruses enter cells through an clathrin-mediated endocytosis pathway; mutants lacking either the VP2 protein or the VP1 protein lose infectivity to host cells.

The VP2 protein key amino acid locus of the S/CD/2021 strain isolated by the invention is completely consistent with that of the traditional feline parvovirus, but the NS1 protein also has amino acid locus mutation, which proves that the feline parvovirus has a new evolution mode, is evolved by the NS1 protein, and is different from the prior research that the VP2 protein is evolved.

A second object of the present invention is to provide the use of the feline parvovirus isolate described above for the preparation of a product for preventing and/or treating diseases caused by canine feline parvovirus; the disease comprises acute gastroenteritis or acute myocarditis of dogs.

Further, the product is a vaccine.

Further, the vaccine is selected from one or more of inactivated vaccine, attenuated live vaccine, genetic engineering vaccine and polypeptide vaccine.

The term "inactivated vaccine", also referred to as an inactivated vaccine, as used herein refers to a suspension of an inactivated virus that is used as an antigen to generate immunity. Inactivated vaccines include, but are not limited to, whole virus vaccines and split vaccines. Inactivated vaccines can be readily produced using known methods. For example, whole virus inactivated vaccines can be obtained by treating the virus with formaldehyde solution, and split vaccines can be prepared from the virus envelope after treatment with ether. For example, the canine feline parvovirus S/CD/2021 isolate of the invention can be prepared into an inactivated vaccine by an inactivation method.

The third object of the present invention is to provide a vaccine composition comprising an effective amount of the canine feline parvovirus isolate and/or a pharmaceutically acceptable carrier or adjuvant.

By effective amount is meant that amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The exact amounts of ingredients used and the compositions to be administered will vary depending upon factors such as the type of disease being treated, the type and age of the animal to be treated, the manner of administration, and other ingredients in the composition.

The term "pharmaceutically acceptable carrier or adjuvant" as used herein means a carrier or adjuvant that when administered does not cause allergic reactions or other undesirable effects in the individual to whom it is administered. According to the present invention, the pharmaceutically acceptable carriers or excipients include, but are not limited to, the following agents: solvents, emulsifiers, suspending agents, disintegrants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants and the like.

Further, the use of the vaccine composition for the prevention and/or treatment of diseases caused by canine feline parvovirus;

the disease comprises acute gastroenteritis or acute myocarditis of dogs.

In the present invention, the vaccine composition includes, but is not limited to, an oil-in-water emulsion, a water-in-oil emulsion, or a double emulsion; the double emulsion is represented by an oil-in-water-in-oil emulsion.

The fourth object of the present invention is to provide a method for preparing the vaccine composition, wherein the vaccine is prepared by culturing and proliferating cat parvovirus isolate and inactivating the isolate.

A fifth object of the invention is to provide an article of manufacture comprising the aforementioned viral isolate or the aforementioned vaccine composition in a sterile vial, ampoule or syringe.

The sixth object of the present invention is to provide the use of the feline parvovirus isolate described above for the preparation of a diagnostic agent for the prevention and/or treatment of feline parvovirus.

Further, the diagnostic reagent comprises an antigen detection kit, an antibody detection kit and a nucleic acid detection kit.

The antigen in the antigen detection kit is selected from virus isolates, genetically engineered proteins of virus isolates or polypeptides of virus isolates; the antibody in the antibody detection kit is selected from monoclonal antibodies or polyclonal antibodies prepared by the virus isolate, genetically engineered proteins of the virus isolate or polypeptides of the virus isolate.

The term "prevention" according to the present invention refers to all the actions of inhibiting the onset of a disease caused by parvovirus by administering a vaccine composition according to the present invention.

The term "treatment" refers to all actions that lead to a alleviation or improvement of symptoms caused by a parvoviral infection by administration of a vaccine composition according to the invention.

The beneficial effects are that:

(1) The S/CD/2021 strain separated by the invention is obtained by separating the strain from the canine feces for the first time, and the genome characteristics and biological characteristics of the strain are studied for the first time, and the strain is aggregated with the feline parvovirus on a genome analysis evolutionary tree, but the hemagglutination, the cell tropism and the pathogenicity to dogs of the strain are different from those of the traditional feline parvovirus.

(2) The S/CD/2021 strain is obtained by separation, so that the epidemic of the canine and feline parvovirus is proved, the prophylaxis of the canine and feline parvovirus is more targeted and effective, and the vaccine prepared by the method has good immunogenicity, can stimulate organisms to generate high-titer specific serum neutralizing antibodies, can be used as candidate strains for developing the canine and feline parvovirus vaccine, and has important value in application research of the canine and feline parvovirus inactivated vaccine and the attenuated vaccine.

(3) The VP2 protein key amino acid locus of the S/CD/2021 strain isolated by the invention is completely consistent with that of the traditional feline parvovirus, and the NS1 protein has amino acid locus mutation, which proves that the feline parvovirus has a new evolution mode, is evolved by the NS1 protein, and is different from the prior research that the VP2 protein is evolved.

Drawings

The above and other objects and features of the present invention will become more apparent by referring to the following description, appended claims and accompanying drawings in which:

FIG. 1 is a diagram of F81 cytopathic effect of canine feline parvovirus S/CD/2021 isolate of the invention wherein A: normal F81 cells, B: f81 cells producing lesions; c: indirect immunofluorescence identification; d: a transmission electron microscope image;

FIG. 2 shows PCR detection of canine/feline parvovirus S/CD/2021 isolate of the present invention, wherein the gel electrophoresis loading sequence is in the order: lane 1: a negative control; lane 2: a positive control; lane 3: S/CD/2021; m: marker II is 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom;

FIG. 3 is a graph of genomic profile of canine feline parvovirus S/CD/2021 isolate of the invention versus all CPVs, FPVs;

FIG. 4 is a graph showing the measurement of hemagglutination titers of canine feline parvovirus S/CD/2021 isolates of the invention at different pH values;

FIG. 5 is a diagram showing MDCK cytopathy caused by canine feline parvovirus S/CD/2021 isolate of the present invention, wherein E: normal MDCK cells, F: diseased MDCK cells are produced.

FIG. 6 shows the results of detection of the feces and tissues of puppy infection test of canine cat parvovirus S/CD/2021 isolate of the present invention, wherein A is the result of detection of canine cat parvovirus pathogen in the feces every day after challenge; b is the detection result of the tissue organ canine and feline parvovirus pathogen after the puppy is dissected;

FIG. 7 is a graph showing the determination of neutralizing antibody levels after immunization of puppies with an inactivated vaccine prepared from canine/feline parvovirus S/CD/2021 isolate of the invention.

Detailed Description

The invention is further described below by means of the description of specific embodiments and with reference to the accompanying drawings, which are not intended to be limiting, but a person skilled in the art can make various modifications or improvements according to the basic idea of the invention, all without departing from the scope of the invention.

The method used in the present invention may be a method commonly used in the field of virus research, and is not limited to the specific description of the embodiments of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified.

The reagents, materials, etc. used in the examples described below are commercially available unless otherwise specified.

The reagents and materials used in the examples are as follows:

the incubator and the incubator were purchased from thermo forma company; high-speed centrifuge 5804 is available from Eppendorf corporation; the gel imaging system VersaDoc2000, a common PCR instrument, and a nucleic acid protein electrophoresis instrument powerpacuniversal (TM) are available from Bio-Rad company; quick Taq HS DyeMix available from TOYOBO Toyo Biotechnology Co., ltd; DNA Marker II was purchased from Dalianbao bioengineering Co., ltd; the MONTANIDE ISA 201VG adjuvant was purchased from specialty chemicals limited of sai (Shanghai). The incubator, the oven was purchased from thermo forma company, usa; single super clean bench from Bio X technologies, america; f81 cells, 1640 medium, fetal bovine serum were purchased from wunpro, inc; 0.25% EDTA (1×) trypsin was purchased from Gibco corporation.

Example 1 isolation and identification of canine feline parvovirus

1.1 isolation of canine feline parvovirus

Screening positive samples for canine feline parvovirus infection fecal samples were treated with PBS (100U/mL penicillin, 100g/mL streptomycin) 1:5 dilution, and centrifugation at 7000r/min for 5min to obtain supernatant, and filtering and sterilizing with a 0.22 μm filter membrane to obtain the disease material treatment liquid.

Mixing the disease treatment solution with F81 cell suspension according to the following ratio of 1:9 on 6-hole culture plate, and setting negative control hole containing 5% CO at 37deg.C ₂ Culturing in an incubator, observing cytopathy every 12h, sterilizing after culturing for 72h, repeatedly freezing and thawing the cytotoxin for 3 times, and inoculating F81 cells again, and carrying out passage until typical cytopathy appears.

After the disease treatment fluid is inoculated with the F81 cells, cytopathy appears in the diarrhea stool sample S/CD/2021 at the generation 2, cell cultures are harvested, typical cytopathy phenomena are generated after the F81 cells are inoculated again for 48 hours, the typical cytopathy phenomena are mainly represented by wiredrawing-like deformation of infected cells, cell aggregation and stacking, cell shrinkage and the like (see figure 1), most cells fall off after the disease is completely treated, other cells do not appear cytopathy, and cells in a control group grow well.

1.2 PCR identification of S/CD/2021 strains

1.2.1 viral PCR detection

CPV detection primers were sent to the Shanghai Biotechnology Co., ltd and synthesized with the following primer sequences:

TABLE 1 CPV detection primers

Taking 300 mu L of F81 cell culture of the isolated strain as an extraction sample of virus DNA, and extracting the DNA of feces by referring to the DNA extraction kit instruction of Beijing Aidelai biotechnology Co., ltd; the DNA is used as a template, and the PCR amplification reaction system is as follows: template DNA 2. Mu.L, 2 XrTaq PCR Premix 12.5. Mu.L, 1.0. Mu.L each of the upstream primer and the downstream primer, 8.5. Mu.LddH ₂ O is added up to 25 mu L.

The reaction procedure was as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 15s, annealing at 52℃for 15s, elongation at 72℃for 1min for a total of 35 cycles; extending at 72℃for 8min. The amplified product is detected by agarose gel electrophoresis, the result shows that the isolate obtains a specific band with the size of about 567bp (see figure 2), the PCR product is sent to the PRC for sequencing analysis, the nucleotide sequence of the PCR product is shown as SEQ ID NO.1, the determination result and CPV and FPV sequences published by NCBI are subjected to genotyping analysis by a genetic evolution tree, and the result of figure 3 shows that the isolate S/CD/2021 belongs to FPV.

1.2 Virus purification

Serial 10-fold dilution of virus liquid to 10-8, synchronous inoculation of the virus liquid into well-grown single-layer F81 cell 96-well culture plate, inoculation of 100 μl of virus diluent in each well, inoculation of 8 wells in each dilution, setting blank control wells, and placing at 37deg.C with 5% CO ₂ Is continuously cultured in the cell incubator. Observing cytopathy every day, continuously inoculating all virus solutions with maximum dilution and no dilution to 12-well plate filled with F81 cells, culturing, repeatedly freezing and thawing the virus solution with minimum dilution in 12-well plate for three times after 72H, collecting, and repeating the above steps for 3 times. And 3 times of purification, and then mass propagation of virus liquid is used as seed virus.

1.3 determination of viral titres

Spreading F81 cell suspension with good growth state on 96-well plate, serial diluting cell virus culture solution 10 times continuously, synchronously inoculating into 96-well cell plate, diluting 8 wells each with 100 μl, placing at 37deg.C and containing 5% CO ₂ Culturing in a cell incubator, observing pathological conditions of cells, and performing TCID on the isolated strain according to a Reed-Muench calculation method ₅₀ Measurement results showed that S/CD/2021=10 ^-3.8 /0.1mL。

EXAMPLE 2 exploration of genomic characteristics and biological Properties of canine feline parvovirus

2.1 genomic characterization of canine feline parvovirus

To obtain all the coding sequences of the isolated strains, 9 pairs of amplification primers (for details of the primer information, see Table 2) were designed using the Premier 5.0 software with reference to the complete gene sequence of Genbank accession number MT614366, in addition to the detection primers, which were synthesized by the Optimago Co., ltd. Amplifying all sequences by adopting a common PCR method, and obtaining the genome of the strain after sequence splicing, wherein the genome analysis result shows that the nucleotide and amino acid sequences of the strain gene are closest to FPV; compared with the traditional FPV and CPV, the NS1 sequence on the virus isolated strain sequence has special amino acid site mutation (I115T, L132V); the intron sequences of VP1 were mutated (g 2333g, a2369 g); the VP2 protein is identical to all specific amino acid positions of FPV.

TABLE 2

2.2 exploration of the biological Properties of canine feline parvovirus

2.2.1 hemagglutination

At 4 DEG CPerforming hemagglutination test on the separated strain by using 1% pig red blood cells by adopting PBS solutions with different pH values at 37 ℃; CPV is known to agglutinate porcine erythrocytes at pH6-pH8, whereas FPV is known to agglutinate porcine erythrocytes only below pH 6.8. The results show that the canine and feline parvovirus has different hemagglutination from the canine and feline parvovirus strain reported before, and the strain can agglutinate porcine erythrocytes at 4 ℃ but can not agglutinate porcine erythrocytes at 37 ℃; under different pH conditions, the strain has different agglutination titers, and the maximum value can reach 2 ⁸ . (as in figure 4).

2.2.2 cell infectivity assay

The conventional FPV cannot proliferate in MDCK cells, so that in order to explore the proliferation capacity of canine feline parvovirus in MDCK cells, the canine parvovirus is synchronously inoculated on the MDCK cells according to a 1.1 virus separation method, and the cell state is observed for multiple times. The results show that the strain can not only proliferate in MDCK cells, but also cause obvious cytopathy (as shown in figure 5).

2.2.3 pathogenicity test

To explore the pathogenicity of the virus on dogs, 10mL of the separated and purified virus liquid was fed to 3 test dogs, and the temperature was measured daily, the fecal detoxification condition was detected, and the clinical manifestation was observed. The results showed that canine parvovirus was detected in canine feces 8 days after challenge (see fig. 6A); serum before, on the 7 th day after and on the 11 th day after the challenge is adopted for the hemagglutination inhibition test, and the result shows that the antibody titer of 3 test dogs is obviously increased and the challenge is carried out on the 3 rd day, one test dog has diarrhea and bloody stool, and the test dogs return to normal on the fifth day; the other two dogs showed normal clinical manifestations. Test dogs were dissected and tested for S/CD/2021 strain in their thymus, heart, blood (FIG. 6B); the strain was shown to cause systemic infection in puppies.

Preservation information:

regarding canine cat parvovirus S/CD/2021 isolate with accession number CCTCC NO: V202237: the classification of the virus is named as canine cat parvovirus S/CD/2021Feline Parvovirus S/CD/2021, and is preserved in China center for type culture Collection at 5.11 of 2022: in the Wuhan university of No. 299 of Wuhan in Wuhan district of Wuhan, hubei province (first additional face of Wuhan university).

EXAMPLE 3 development of canine feline parvovirus inactivated vaccine

2.1 propagation of Virus

Taking F81 monolayer cells with good growth state, washing the cells with Hanks for 2 times, adding a proper amount of digestive pancreatin to digest the cells, discarding pancreatin, adding a proper amount of 1640 culture medium containing 10% fetal bovine serum, and blowing and uniformly mixing; the vaccine candidate strain S/CD/2021 cell culture solution with good immunogenicity is prepared according to the following ratio of 1:9 in proportion to F81 cells, and placing at 37deg.C with 5% CO ₂ When the cytopathy is about 80%, harvesting the virus culture solution, repeatedly freezing and thawing for 3 times, and centrifuging at 4 ℃ at high speed to obtain virus supernatant serving as the virus for the vaccine.

2.2 preparation of inactivated Virus vaccine

The vaccine candidate strain S/CD/2021 cell culture supernatant with good immunogenicity is fully and uniformly mixed with the beta-propiolactone with the final concentration of 0.5 percent, and the mixture is inactivated for 6 hours in a shaking table at 37 ℃. And respectively inoculating two inactivated virus solutions into F81 cells for inactivation and detection, carrying out blind transmission for two generations by the F81 cells, culturing for 48-72h for each generation, observing cytopathic effect, and simultaneously setting a positive virus control and a blank control.

The cells of the detection group and the negative control group inoculated with the inactivated virus liquid have no CPE, while the positive control group has typical cytopathy, and the detection group is subjected to 2-generation blind transmission, and CPE is not seen yet, so that the inactivation of the detected inactivated virus liquid is complete. Finally, mixing the inactivated virus liquid with the 201 adjuvant in equal volume, stirring the mixture, fully and uniformly mixing the mixture, emulsifying the mixture in an emulsifying machine for 5 minutes to prepare the inactivated vaccine of water-in-oil-in-water, subpackaging the inactivated vaccine in a transparent glass bottle, observing that the vaccine is milky uniform suspension, sucking a drop of the inactivated vaccine into a beaker containing water by using a syringe, uniformly dispersing the vaccine in the whole beaker, and centrifuging the inactivated vaccine for 5 minutes at 3000r/min, wherein layering phenomenon is not seen, and the vaccine is qualified in inspection and can be used for subsequent experiments.

TABLE 3 Table 3

6 healthy puppies with 1-3 month-old FPV antigen and antibody negative were selected. Three groups were divided, and all animals were bled for virus antibody level detection with daily observations of clinical abnormalities. First immunization was vaccinated subcutaneously through the neck (S/CD/2021), vaccinated at 2 mL/head; the blank group was inoculated with the same dose of virus-free cell fluid. The first vaccination was followed by a second immunization 21 days later, in the same manner as described above. The safety test result shows that the S/CD/2021 virus strain is safe to pups, has no side effect, and shows that the inactivated S/CD/2021 vaccine strain has good safety. Blood is collected at different times after vaccination, and the neutralizing antibody level of puppies after immunization is measured by an antibody neutralization test, wherein the highest antibody level reaches 1:1468 (FIG. 7).

The result of animal immune test shows that S/CD/2021 strain can induce high level antibody, can provide better immune protection effect for canine and feline parvovirus infection, and is ideal vaccine candidate strain.

While the foregoing description of the preferred embodiments of the invention has been presented and described, it is to be understood that the invention is not limited to the disclosed forms of the invention, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the scope of the inventive concept described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.

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ggtactttca ataatcagac ggaatttaaa tttttggaaa acggatgggt ggaaatcaca 2700

gcaaactcaa gcagacttgt acatttaaat atgccagaaa gtgaaaatta taaaagagta 2760

gttgtaaata atatggataa aactgcagtt aaaggaaaca tggctttaga tgatactcat 2820

gtacaaattg taacaccttg gtcattggtt gatgcaaatg cttggggagt ttggtttaat 2880

ccaggagatt ggcaactaat tgttaatact atgagtgagt tgcatttagt tagttttgaa 2940

caagaaattt ttaatgttgt tttaaagact gtttcagaat ctgctactca gccaccaact 3000

aaagtctata ataatgattt aactgcatca ttgatggttg cattagatag taataatact 3060

atgccattta ctccagcagc tatgagatct gagacattag gtttttatcc atggaaacca 3120

accataccaa ctccatggag atattatttt caatgggata gaacattaat accatctcat 3180

actggaacta gtggcacacc aacaaatgta tatcatggta cagatccaga tgatgttcaa 3240

ttttatacta ttgaaaattc tgtgccagtg cacttactaa gaacaggtga tgaatttgct 3300

acaggaacat ttttttttga ttgtaaacca tgtagactaa cacatacatg gcaaacaaat 3360

agagcattgg gcttaccacc atttttaaat tctttgcctc aatctgaagg agctactaac 3420

tttggtgata taggagttca acaagataaa agacgtggtg taactcaaat gggaaataca 3480

gactatatta ctgaagctac tattatgaga ccagctgagg ttggttatag tgcaccatat 3540

tattcttttg aagcatctac acaagggcca tttaaaacac ctattgcagc aggacggggg 3600

ggagcgcaaa cagatgaaaa tcaagcagca gatggtgatc caagatatgc atttggtaga 3660

caacatggtc aaaaaactac tacaacagga gaaacacccg agagatttac atatatagca 3720

catcaagata caggaagata tccagaagga gattggattc aaaatattaa ctttaacctt 3780

cctgtaacaa atgataatgt attgctacca acagatccaa ttggaggtaa aacaggaatt 3840

aactatacta atatatttaa tacttatggt cctttaactg cattaaataa tgtgccacca 3900

gtttatccaa atggtcaaat ttgggataaa gaatttgata ctgacttaaa accaagactt 3960

catgtaaatg caccatttgt ttgtcaaaat aattgtcctg gtcaattatt tgtaaaagtt 4020

gcgcctaatt taacaaatga atatgatcct gatgcatctg ctaatatgtc aagaattgta 4080

acttactcag atttttggtg gaaaggtaaa ttagtattta aagctaaact aagagcatct 4140

catacttgga atccaattca acaaatgagt attaatgtag ataaccaatt taactatgta 4200

ccaaataata ttggagctat gaaaattgta tatgaaaaat ctcaactagc acctagaaaa 4260

ttatattaa 4269

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence (2)

<400> 2

tgattgtaaa ccatgtagac taac 24

<210> 3

<211> 23

<212> DNA

<213> Artificial sequence (3)

<400> 3

taatgcagtt aaaggaccat aag 23

<210> 4

<211> 16

<212> DNA

<213> Artificial sequence (4)

<400> 4

aaaggtggcg ggctaa 16

<210> 5

<211> 18

<212> DNA

<213> Artificial sequence (5)

<400> 5

cgtagccatt taccagtt 18

<210> 6

<211> 22

<212> DNA

<213> Artificial sequence (6)

<400> 6

actgaggaag ttatggaggg ag 22

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence (7)

<400> 7

accaagtccc gcaaagtaca 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (8)

<400> 8

agcgcgggag aattcaaact 20

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence (9)

<400> 9

gcaacctggt gaatttatac ct 22

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (10)

<400> 10

tggaccagca agtacaggaa 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (11)

<400> 11

gattgcttgc cgcttgtgtc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (12)

<400> 12

aaattcacca ggttgcaaag 20

<210> 13

<211> 19

<212> DNA

<213> Artificial sequence (13)

<400> 13

caagactgtt cccaggccc 19

<210> 14

<211> 27

<212> DNA

<213> Artificial sequence (14)

<400> 14

gagacgactt ggattaaggt acgatgg 27

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence (15)

<400> 15

ttagttgcca atctcctg 18

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence (16)

<400> 16

aaacggatgg gtggaaat 18

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence (17)

<400> 17

tggtggtaag cccaatgctc tattt 25

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (18)

<400> 18

ttgaggcgtc tacacaaggg 20

<210> 19

<211> 25

<212> DNA

<213> Artificial sequence (19)

<400> 19

ttctaggtgc tagttgagat ttttc 25

<210> 20

<211> 17

<212> DNA

<213> Artificial sequence (20)

<400> 20

ttttggtgga aaggtaa 17

<210> 21

<211> 17

<212> DNA

<213> Artificial sequence (21)

<400> 21

aacatattct aagggca 17

Claims

1. Canine cat parvovirus @Feline Parvovirus) The isolated strain is characterized in that the microorganism has a collection number of CCTCC NO: V202237.

2. Use of the feline parvovirus isolate of claim 1 in the manufacture of a product for preventing and/or treating canine feline parvovirus-caused disease; the disease includes canine acute gastroenteritis or acute myocarditis.

3. The use according to claim 2, wherein the product is a vaccine.

4. The use according to claim 3, wherein the vaccine is selected from one or more of inactivated vaccine, attenuated live vaccine, genetically engineered vaccine, polypeptide vaccine.

5. A vaccine composition comprising an effective amount of the canine feline parvovirus isolate of claim 1 and/or a pharmaceutically acceptable carrier or adjuvant.

6. The method of preparing a vaccine composition according to claim 5, wherein the vaccine is prepared by culturing the propagated feline parvovirus isolate and inactivating the same.

7. An article of manufacture comprising the viral isolate of claim 1 or the vaccine composition of claim 5 in a sterile vial, ampoule or syringe.

8. Use of the feline parvovirus isolate of claim 1 in the preparation of a feline parvovirus diagnostic reagent.