CN114921422A

CN114921422A - Canine-derived feline parvovirus isolate and application thereof

Info

Publication number: CN114921422A
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: 2022-08-19
Anticipated expiration: 2042-05-25
Also published as: CN114921422B

Abstract

The invention discloses a canine parvovirus isolate, belonging to the technical field of biotechnology and virology, wherein the isolate is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address of Wuhan, China, the preservation number of CCTCC NO: V202237, the preservation date of 2022 years, 5 months and 11 days, and is named as canine parvovirus S/CD/2021 in classification. The invention also relates to application of the canine parvovirus isolate. The canine cat parvovirus can cause the whole body infection of dogs by cross-host transmission, can generate a high-level neutralizing antibody after being prepared into an inactivated vaccine, has the basic potential of the vaccine, and can protect dogs from being attacked by the cat parvovirus.

Description

Canine-derived feline parvovirus isolate and application thereof

Technical Field

The invention relates to the fields of biotechnology and virology, in particular to a canine parvovirus isolate and application thereof.

Background

In recent years, with the continuous improvement of living standard of people, pets become an indispensable part in the life of people, and the rapidly expanding pet industry brings vitality to social development and simultaneously has a plurality of problems to be solved urgently, for example, infectious diseases such as canine distemper, canine parvovirus disease, canine coronavirus disease and the like are widely prevalent and rapidly spread in the world, and cause great harm to the canine raising industry. Wherein, the canine parvovirus disease is particularly serious, the mortality rate is as high as 50 percent to 90 percent, and the development of the canine industry is seriously damaged.

Canine Parvovirus (CPV) belongs to the parvoviridae and parvovirus genera, is a single-stranded negative-strand DNA virus with an unencapsulated, icosahedral structure and a capsid consisting of 32 capsomeres, causes acute virulent infectious diseases with high contact degree and is characterized by vomiting, diarrhea, leukopenia and hemorrhagic enteritis, and is one of the most serious infectious diseases endangering the health of dogs since the discovery of the Canine Parvovirus (CPV) disease.

CPV has more than 98% genomic homology with Feline Parvovirus (FPV) and differs only at several amino acid sites, and is therefore considered to be derived from or evolved from FPV by certain intermediaries, the CPV particle surface is composed of alternating motifs of 60 VP1 and VP2, VP2 is 90% and VP1 is only 10%, so VP2 is the major component constituting the capsid protein, and the difference of several amino acid sites on the surface of VP2 allows CPV and FPV to have respective specific host range, antigenic type and hemagglutination properties. CPV agglutinates erythrocytes of cats, pigs, rhesus monkeys, horses and hamsters at a temperature of 4-25 ℃ and a pH of 6.0-7.2, but does not agglutinate O-type erythrocytes of cows, rabbits, goats, guinea pigs, rats, mice, hamsters, geese, chickens and humans. The host range for FPV includes domestic and wild felines (felimia suborder) and some wild canines (caniformia suborder), such as raccoons and foxes, but excludes domestic dogs. Although FPV replicates in canine lymphoid tissues (thymus and bone marrow) after experimental vaccination, it does not bind to canine transferrin receptor (tfr), which is critical for infection, so FPV does not further infect dogs; in contrast, CPV infection in cats is common.

In recent years, however, there have been many reports of the detection of feline parvovirus from canine feces, including china, vietnam, pakistan, thailand, and the like; but only Vietnam (Houng M, Wu C N, Lin C F, et al. genetic characterization of feline panleukapia viruses from dogs in vitamin derivatives a unique Thr101 mutation in VP2[ J ]. PeerJ,2020,8(160): e9752.) Chinese (Chen B, Zhang X, Zhu J, et al. molecular environmental Survey of Cansubviral circulation in China from 2014to 2019.2021.) analyzed the VP2 protein characteristics, and none isolated the strain, therefore, the Canine feline Parvovirus biological characteristics and pathogenicity to dogs are not clear.

After CPV infection, the morbidity is fast and the mortality is high, the prevention and control measures for CPV at present are mainly vaccine immunization, CPV only has one antigen type so far, namely CPV-2, and commercial CPV vaccines available in the market are more, including imported and domestic vaccines, but all belong to attenuated vaccines. However, CPV has antigenic drift, and then presents different subtypes CPV-2a, CPV-2b, CPV-2c (a) and CPV-2c (b) which are changed in host range, hemagglutination and antigenicity, and with the continuous variation of viruses, the CPV provides new challenges for the treatment and prevention of the disease. It is unclear whether the existing vaccines can protect the newly emerging canine feline parvovirus.

Therefore, the need of isolating new canine feline parvovirus and developing vaccines to prevent the disease and reduce the damage of the canine parvovirus is urgent.

Disclosure of Invention

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

The technical scheme of the invention is as follows:

the invention aims to provide a canine feline parvovirus isolate, the microbial preservation number of which is 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 among 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, the nonstructural proteins NSI and NS 2; the second ORF, located in the right half of the genome, encodes the viral capsid proteins, structural proteins VP1 and VP2, which are mainly responsible for the capsid constituting the virion and play a very important role in the viral infection process. Research shows that the VP2 protein on the surface of canine parvovirus can be specifically combined with a Transferrin receptor (TfR) on the surface of host cells, and the virus enters the cells through a clathrin-mediated endocytosis pathway; the mutants lacking either VP2 protein or VP1 protein lost infectivity of the host cells.

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

The second purpose of the invention is to provide an application of the feline parvovirus isolate in preparing products for preventing and/or treating diseases caused by canine feline parvovirus; the disease includes canine acute gastroenteritis or acute myocarditis.

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 called inactivated vaccine, as used herein, refers to a suspension of inactivated virus 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 viruses with formaldehyde solutions, and split vaccines can be prepared from viral envelopes after treatment with ether. For example, the canine feline parvovirus S/CD/2021 isolate of the present invention can be used to prepare an inactivated vaccine by inactivation.

The third invention aims to provide a vaccine composition, which comprises an effective amount of the canine feline parvovirus isolate and/or a pharmaceutically acceptable carrier or auxiliary material.

The effective amount refers to the amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The precise amounts of the ingredients used and the composition to be administered will vary depending on factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.

The term "pharmaceutically acceptable carrier or adjuvant" as used herein, means a carrier or adjuvant which, 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 carrier or adjuvant includes, but is not limited to, the following agents: solvents, emulsifiers, suspending agents, disintegrating agents, 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 includes canine acute gastroenteritis or acute myocarditis.

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 behaves as a water-in-oil-in-water emulsion.

The fourth object of the present invention is to provide a method for producing the vaccine composition, which comprises culturing and propagating a feline parvovirus isolate, inactivating the feline parvovirus isolate, and producing a vaccine.

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

The sixth invention aims to provide the application of the feline parvovirus isolate in preparing a diagnostic reagent for preventing and/or treating feline parvovirus.

Furthermore, 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 a virus isolate, a gene engineering protein of the virus isolate or a polypeptide of the virus isolate; the antibody in the antibody detection kit is selected from the virus isolate, a monoclonal antibody or a polyclonal antibody prepared from genetic engineering protein of the virus isolate or polypeptide of the virus isolate.

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

The term "treatment" refers to all actions leading to a reduction or amelioration of symptoms caused by a parvovirus infection by administration of a vaccine composition according to the invention.

Has the advantages that:

(1) the S/CD/2021 strain separated by the invention is obtained by separating dog feces for the first time, and the characteristics and biological characteristics of the genome are researched for the first time, the genome analysis evolutionary tree and the feline parvovirus are gathered into one strain, but the hemagglutination, cell tropism and pathogenicity to the dog of the strain are all 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 feline parvovirus is verified, the canine feline parvovirus is prevented with pertinence and effectiveness, and the vaccine has good immunogenicity after being prepared, can stimulate an organism to generate a high-titer specific serum neutralizing antibody, can be used as a candidate strain for developing the canine feline parvovirus vaccine, and has important value in the application research aspect of the canine feline parvovirus inactivated vaccine and the attenuated vaccine.

(3) The key amino acid site of the VP2 protein of the S/CD/2021 strain separated by the invention is completely consistent with that of the traditional feline parvovirus, and the NS1 protein has amino acid site mutation, so that the fact that the feline parvovirus has a new evolution mode is proved, wherein the evolution mode is from NS1 protein, and is different from the existing research that the evolution mode is from VP2 protein.

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, wherein:

FIG. 1 is a graph showing F81 cell pathology caused by S/CD/2021 isolate of canine feline parvovirus of the present invention, wherein A: normal F81 cells, B: producing diseased F81 cells; c: performing indirect immunofluorescence identification; d: a transmission electron microscope image;

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

FIG. 3 is a graph of the genomic characteristics of canine feline parvovirus S/CD/2021 isolates of the present invention compared to all CPV, FPV;

FIG. 4 is a graph showing the determination of the coagulation titer of canine feline parvovirus S/CD/2021 isolates of the present invention at different pH' S;

FIG. 5 is a picture of MDCK cell pathology caused by canine feline parvovirus S/CD/2021 isolate of the present invention, wherein E: normal MDCK cells, F: diseased MDCK cells were generated.

FIG. 6 shows the feces and tissue detection results of the infection test of puppies with canine parvovirus S/CD/2021 isolates of the present invention, wherein A is the detection result of canine parvovirus pathogens in feces every day after challenge; b is a result of detecting the canine parvovirus pathogen of the tissue organ of the puppy after the cesarean section;

FIG. 7 is a graph showing the measurement of the level of neutralizing antibodies after immunization of puppies with inactivated vaccines prepared from S/CD/2021 isolates of canine feline parvovirus according to the present invention.

Detailed Description

The present invention is further described in the following description of the embodiments with reference to the drawings, which are not intended to limit the invention, and those skilled in the art may make various modifications or improvements based on the basic idea of the invention, but within the scope of the invention, unless departing from the basic idea of the invention.

The method applied in the present invention can adopt 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 procedures used in the following examples are all conventional procedures unless otherwise specified.

Reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

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

the constant temperature incubator and the constant temperature shaking incubator were purchased from Thermoforma corporation; high speed centrifuge 5804 is available from Eppendorf; the gel imaging system VersaDoc2000, the general PCR instrument, and the nucleic acid protein electrophoresis instrument powerpaciniaversal TM were purchased from Bio-Rad company; quick Taq HS DyeMix was purchased from TOYOBO Toyobo Biotech Co., Ltd; DNA Marker II was purchased from Dalibao bioengineering, Inc.; the MONTANIDE ISA 201VG adjuvant was purchased from Sepetidae (Shanghai) specialty Chemicals, Inc. Constant temperature incubator, constant temperature shaking incubator purchased from Thermoforma, USA; single person clean bench was purchased from Bio X technologies, USA; f81 cells, 1640 medium, fetal bovine serum were purchased from Wuhan Proxel, Inc.; 0.25% EDTA (1X) trypsin was purchased from Gibco.

Example 1 isolation and identification of Canine-derived feline parvovirus

1.1 isolation of Canine-derived feline parvovirus

Positive samples infected with canine feline parvovirus were screened, and fecal samples were treated with PBS (100U/mL penicillin, 100g/mL streptomycin) 1: 5, diluting, centrifuging at 7000r/min for 5min, and filtering and sterilizing the obtained supernatant through a 0.22-micron filter membrane to obtain the disease material treatment fluid.

Mixing the disease material treatment liquid with the F81 cell suspension according to the ratio of 1: 9 is inoculated on a 6-well culture plate synchronously, and negative control wells containing 5% CO at 37 ℃ are arranged at the same time ₂ Culturing in an incubator, observing cytopathy every 12h, collecting toxin after culturing for 72h, repeatedly freezing and thawing the cell toxin for 3 times, then inoculating the F81 cells again, and carrying out passage until typical cytopathy occurs.

After the pathogen treatment fluid is inoculated with F81 cells, the diarrhea fecal sample S/CD/2021 has cytopathic effect in the 2 nd generation, cell culture is harvested, F81 cells are inoculated again for 48h to generate typical cytopathic effect which is mainly shown in the phenomena of filamentation deformation of infected cells, cell accumulation and cell shrinkage (see figure 1), most of cells fall off after complete pathological change, other cells have no cytopathic effect, and the cells of a control group grow well.

1.2 PCR identification of S/CD/2021 Strain

1.2.1 Virus PCR detection

The CPV detection primer is synthesized by Shanghai Biotechnology Limited, and the primer sequence is as follows:

TABLE 1 CPV detection primers

Taking 300 mu L of F81 cell culture of the separated strain as an extracted sample of virus DNA, and extracting DNA of excrement by referring to the specification of a DNA extraction kit of Beijing Edelay Biotechnology Limited company; taking DNA as a template, the reaction system for PCR amplification is as follows: template DNA 2. mu.L, 2 XrTaq PCR Premix 12.5. mu.L, upstream primer and downstream primer each 1.0. mu.L, 8.5. mu.LddH ₂ The total amount of O was 25. mu.L.

The reaction procedure was as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 15s, annealing at 52 ℃ for 15s, and extension at 72 ℃ for 1min for 35 cycles; extension at 72 ℃ for 8 min. The amplified product was detected by agarose gel electrophoresis, and the results showed that the isolate obtained a specific band of about 567bp (see FIG. 2), and the PCR product was sent to Oncology Limited for sequencing analysis, the nucleotide sequence of which is shown in SEQ ID NO.1, and the results of the determination and CPV and FPV sequences published by NCBI were analyzed by gene tree typing, and the results of FIG. 3 showed that the isolate S/CD/2021 belongs to FPV.

1.2 Virus purification

Serially diluting the virus solution 10 times to 10-8, synchronously inoculating the virus solution into a well-grown monolayer culture plate with 96 wells for F81 cells, inoculating 100 μ L of virus diluent into each well, inoculating 8 wells for each dilution, simultaneously setting blank control wells, placing at 37 deg.C and 5% CO ₂ The cell culture box of (2) continues culturing. Observing the cytopathic condition every day, independently and continuously inoculating all virus solutions with the maximum dilution times of the lesions and the next dilution times of the lesions after 6 days into a 12-pore plate which is fully paved with F81 cells for continuous culture, repeatedly freezing and thawing the virus solution with the lesions and the lowest dilution in the 12 pores for three times after 72 hours, collecting the virus solution, and then repeating the steps for 3 times. After 3 times of purification, virus liquid is propagated in large batch as seed virus.

1.3 determination of viral titre

Spreading F81 cell suspension with good growth state in 96-well plate, serial diluting cell virus culture solution by 10 times, synchronously inoculating to 96-well plate with 8 wells per dilution and 100 μ L per well, and placing at 37 deg.C with 5% CO ₂ Culturing in cell culture box, observing pathological change of cells, and performing TCID on isolate according to Reed-Muench calculation method ₅₀ The result of the measurement was S/CD/2021 ═ 10 ^-3.8 /0.1mL。

Example 2 exploration of genomic and biological characteristics of Canine feline parvovirus

2.1 genomic characterization of Canine-derived feline parvoVirus

To obtain all coding sequences of isolated strains, 9 additional pairs of amplification primers (detailed primer information in table 2) were designed using Premier 5.0 software with reference to the entire gene sequence of Genbank accession No. MT614366, in addition to the detection primers, and were synthesized by optisco bio ltd. Amplifying all sequences by adopting a common PCR method, obtaining the genome of the strain after splicing the sequences, wherein the genome analysis result shows that the gene nucleotide and the amino acid sequence of the strain are most approximate to FPV; compared with the traditional FPV and CPV, the NS1 sequence on the virus isolate sequence has special amino acid site mutation (I115T, L132V); the intron sequence of VP1 has mutations (g2333g, a2369 g); the VP2 protein is identical to FPV at all specific amino acid positions.

TABLE 2

2.2 exploration of the biological Properties of Canine-derived feline parvoVirus

2.2.1 hemagglutination

Performing hemagglutination test on the isolate by using 1% pig red blood cells in PBS (phosphate buffer solutions) with different pH values at the temperature of 4 ℃ and the temperature of 37 ℃; CPV is known to agglutinate pig red blood cells at pH 6-8, while FPV agglutinates pig red blood cells only at pH6.8 or lower. The result shows that the canine feline parvovirus has different hemagglutination properties from previously reported canine and feline parvovirus strains, and the strains can agglutinate porcine red blood cells at 4 ℃ and cannot agglutinate porcine red blood cells at 37 ℃; under different pH conditions, the strain has different agglutination titers, and the maximum agglutination titer can reach 2 ⁸ . (see fig. 4).

2.2.2 cell infectivity assay

Previous researches show that the traditional FPV cannot proliferate in MDCK cells, so in order to explore the proliferation capacity of the canine feline parvovirus in MDCK cells, the canine feline parvovirus is synchronously inoculated on MDCK cells according to a method for separating 1.1 viruses, and the cell state is observed for many times. The results show that the strain can not only proliferate in MDCK cells but also cause obvious cytopathic effect (as shown in figure 5).

2.2.3 pathogenicity test

In order to explore the pathogenicity of the virus to dogs, 10mL of separated and purified virus liquid is fed to 3 test dogs, the body temperature is measured every day, the excrement detoxification condition is detected, and the clinical performance is observed. The results showed that canine parvovirus was detected in canine feces 8 consecutive days after challenge (see figure 6A); blood coagulation inhibition tests are carried out by adopting the sera before challenge, on the 7 th day after challenge and on the 11 th day after challenge, and the results show that the antibody titer of 3 test dogs is obviously increased on the 3 rd day of challenge, one test dog has diarrhea and bloody stool, and the test dog is recovered to be normal on the fifth day; the clinical manifestations of the two remaining test dogs were normal. Test dogs were dissected and tested for S/CD/2021 strain in their thymus, heart, blood (see FIG. 6B); indicating that the strain can cause systemic infection of puppies.

Preservation information:

the canine feline parvovirus S/CD/2021 isolate with the preservation number of CCTCC NO: V202237: the virus is classified and named as canine Feline Parvovirus S/CD/2021Feline Parvovirus S/CD/2021, and is deposited at the China Center for Type Culture Collection (CCTCC) at 5/11 th 2022 with the addresses: eight-way 299 Wuhan university school in Wuchang district, Wuhan City, Hubei province (the first small facing of the first appendix of Wuhan university).

EXAMPLE 3 development of Canine-derived feline parvovirus inactivated vaccine

2.1 propagation of the Virus

Taking F81 monolayer cells with good growth state, cleaning for 2 times by Hanks, adding a proper amount of pancreatin to digest the cells, then discarding the pancreatin, adding a proper amount of 1640 culture medium containing 10% fetal calf serum, and blowing, beating and uniformly mixing; the vaccine candidate strain S/CD/2021 cell culture solution with good immunogenicity is prepared by mixing the following components in percentage by weight according to 1: 9 proportion was synchronously inoculated into F81 cells, placed at 37 ℃ with 5% CO ₂ The virus culture solution is obtained when the cell disease is about 80 percent, and then the virus culture solution is repeatedly frozen and thawed for 3 times and centrifuged at high speed at 4 ℃ to obtain virus supernatant which is used as the virus for the vaccine.

2.2 preparation of inactivated Virus vaccine

And (3) sufficiently and uniformly mixing the cell culture supernatant of the candidate vaccine strain S/CD/2021 with good immunogenicity with beta-propiolactone with the final concentration of 0.5%, and inactivating for 6 hours in a shaking table at 37 ℃. Respectively inoculating two inactivated virus solutions to F81 cells for inactivation test, performing blind passage for two generations through F81 cells, culturing for 48-72h in each generation, observing cytopathic condition, and simultaneously setting a positive virus control and a blank control.

Cells of the detection group inoculated with the inactivated virus solution and the negative control group have no CPE, while the positive control group has typical cytopathic effect, the detection group is subjected to 2-generation blind transmission, and no CPE is still seen, which indicates that the detected inactivated virus solution is completely inactivated. And finally, mixing the inactivated virus solution and the 201 adjuvant in the same volume, stirring, fully and uniformly mixing, emulsifying for 5min in an emulsifying machine to prepare the water-in-oil-in-water inactivated vaccine, subpackaging the inactivated vaccine in transparent glass bottles, observing that the vaccine is milky uniform suspension, sucking a drop of the inactivated vaccine into a beaker containing water by using an injector, uniformly dispersing the vaccine in the whole beaker, and centrifuging the inactivated vaccine at 3000r/min for 5min, wherein no layering phenomenon exists, and the vaccine is qualified in inspection and can be used for subsequent experiments.

TABLE 3

6 healthy puppies with 1-3 months old FPV antigen and negative antibody were selected. The test is divided into three groups, and the clinical abnormality is observed every day, and all animals are subjected to blood sampling for detecting the virus antibody level. The first immunization was performed by neck subcutaneous vaccination (S/CD/2021), at a vaccination dose of 2 mL/head; the blank control group was inoculated with the same dose of virus-free cell sap. The second immunization was carried out 21 days after the first vaccination, the mode of inoculation and the dose were as described above. The safety test result shows that the S/CD/2021 virus strain of the invention is safe to puppies without side effect, which indicates that the inactivated S/CD/2021 vaccine strain has good safety. Blood is collected at different time after vaccination, and the neutralizing antibody level after immunization of puppies is determined by using an antibody neutralizing test, and the highest antibody level is 1: 1468 (fig. 7).

The results of animal immunity tests show that the S/CD/2021 strain can induce and generate high-level antibodies, can provide better immune protection effect on canine feline parvovirus infection, and is an ideal vaccine candidate strain.

While several preferred embodiments of the invention have been illustrated and described, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not intended to be exhaustive of other embodiments, and that various other combinations, modifications, and environments may be used and modifications may be made within the scope of the inventive concept as described herein, either by the above teachings or by the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> university of southwest ethnic group

<120> canine feline parvovirus isolate and application thereof

<130> 5.25

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4269

<212> DNA

<213> Artificial sequence (1)

<400> 1

atgtctggca accagtatac tgaggaagtt atggagggag taaattggct aaaaaaacat 60

gcagaaaatg aagcattttc gtttgttttt aaatgtgaca acgtccaact aaatggaaag 120

gatgttcgct ggaacaacta taccaaacca attcaaaatg aagagctaac atctttaatt 180

agaggagcac aaacagcaat ggatcaaacc gaagaagaag aaatggactg ggaatcggaa 240

gttgatagtc tcgccaaaaa gcaagtacaa acttttgatg cattaattaa aaaatgtctt 300

tttgaagttt ttgtttctaa aaatatagaa ccaaatgaat gtatttggtt tattcaacat 360

gaatggggaa aagatcaagg ctggcattgt catcttttac ttcatagtaa gaacttacaa 420

caagcaactg gtaaatggct acgcagacaa atgaatatgt attggagtag atggttggtg 480

actctttgtt cggtaaactt aacaccaact gaaaagatta agctcagaga aattgcagaa 540

gatagtgaat gggtgactat attaacatac agacataagc aaacaaaaaa agactatgtt 600

aaaatggttc attttggaaa catgatagca tattactttt taacaaagaa aaaaattgtc 660

cacatgacaa aagaaagtgg ctatttttta agtactgatt ctggttggaa atttaacttt 720

atgaagtatc aagacagaca aactgtcagc acactttaca ctgaacaaat gaaaccagaa 780

accgttgaaa ccacagtgac gacagcacag gaaacaaagc gcgggagaat tcaaactaaa 840

aaggaagtgt caatcaaatg tactttgcgg gacttggtta gtaaaagagt aacatcacct 900

gaagattgga tgatgttaca accagatagt tatattgaaa tgatggcaca accaggaggt 960

gaaaatcttt taaaaaatac acttgaaatt tgtactttga ctttagcaag aacaaaaaca 1020

gcatttgaat taatacttga aaaagcagat aatactaaac taactaactt tgatcttgca 1080

aattctagaa catgtcaaat ttttagaatg cacggatgga attggattaa agtttgtcac 1140

gctatagcat gtgttttaaa tagacaaggt ggtaaaagaa atacagttct ttttcatgga 1200

ccagcaagta caggaaaatc tattattgct caagccatag cacaagctgt ggggaatgtt 1260

ggttgttata atgcagcaaa tgtaaatttt ccatttaatg actgtaccaa taaaaattta 1320

atttggattg aagaagctgg taactttggt caacaagtta atcaatttaa agcaatttgt 1380

tctggacaaa caattagaat tgatcaaaaa ggtaaaggaa gtaagcaaat tgaaccaact 1440

ccagtaatta tgacaactaa tgaaaatata acaattgtaa gaattggatg tgaagaaaga 1500

cctgaacata cacaaccaat aagagacaga atgttgaaca ttaagttagt atgtaagctt 1560

ccaggagact ttggtttggt tgataaagaa gaatggcctt taatatgtgc atggttagtt 1620

aaacatggtt atgaatcaac catggctaac tatacacatc attggggaaa agtaccagaa 1680

tgggatgaaa actgggcgga gcctaaaata caagaaggta taaattcacc aggttgcaaa 1740

gacttagaga cacaagcggc aagcaatcct cagagtcaag accaagttct aactcctctg 1800

actccggacg tagtggacct tgcactggaa ccgtggagta ctccagatac gcctattgca 1860

gaaactgcaa atcaacaatc aaaccaactt ggcgttactc acaaagacgt gcaagcgagt 1920

ccgacgtggt ccgaaataga ggcagacctg agagccatct ttacttctga acaattggaa 1980

gaagattttc gagacgactt ggattaaggt acgatggcac ctccggcaaa gagagccagg 2040

agaggtaagg gtgtgttagt aaggtggggg gaggggaaag atttaataac ttaactaaat 2100

atgtgttttc ttataggact tgtgcctcca ggttataaat atcttgggcc tgggaacagt 2160

cttgaccaag gagaaccaac taacccttct gacgccgctg caaaagaaca cgacgaagct 2220

tacgctgctt atcttcgctc tggtaaaaac ccatacttat atttctcgcc agcagatcaa 2280

cgctttatag atcaaactaa ggacgctaaa gattgggggg ggaaaatagg acattatttt 2340

tttagagcta aaaaggcaat tgctccagta ttaactgata caccagatca tccatcaaca 2400

tcaagaccaa caaaaccaac taaaagaagt aaaccaccac ctcatatttt catcaatctt 2460

gcaaaaaaaa aaaaagccgg tgcaggacaa gtaaaaagag acaatcttgc accaatgagt 2520

gatggagcag ttcaaccaga cggtggtcaa cctgctgtca gaaatgaaag agctacagga 2580

tctgggaacg ggtctggagg cgggggtggt ggtggttctg ggggtgtggg gatttctacg 2640

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. A canine feline parvovirus isolate is characterized in that the microbial preservation number is CCTCC NO: V202237.

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

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

4. The use according to claim 2, wherein the vaccine is selected from one or more of inactivated vaccine, attenuated live vaccine, genetic engineering vaccine, and 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 vaccine composition according to claim 5, wherein the vaccine composition is for use in the prevention and/or treatment of a disease caused by canine feline parvovirus.

7. The vaccine composition of claim 6, wherein the disease-causing condition comprises canine acute gastroenteritis or acute myocarditis.

8. The method for producing the vaccine composition according to claim 5, wherein the feline parvovirus isolate is cultured and propagated, and inactivated to produce a vaccine.

9. 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.

10. Use of the feline parvovirus isolate of claim 1 in the preparation of a diagnostic agent for the prophylaxis and/or treatment of feline parvovirus.