Canine coronavirus strain as well as inactivated vaccine and application thereof

Abstract

The invention provides a canine coronavirus strain as well as an inactivated vaccine and application thereof. The canine coronavirus strain is named as CCoV2015, and the genome full-length cDNA sequence of the canine coronavirus strain is as shown in SEQ ID NO. 1. The canine coronavirus strain is collected from intestinal tissues of naturally diseased dogs, sequence comparative analysis shows that the strain is a novel canine coronavirus strain, the infection ability of the strain is high, the strain is highly homologous with canine coronavirus strains separated in recent years, and the canine coronavirus strain is inactivated to prepare the canine coronavirus vaccine. Meanwhile, the canine coronavirus vaccine also fills the blank of a single canine coronavirus vaccine, and plays a very important role in prevention of canine coronavirus.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a canine coronavirus strain, an inactivated vaccine prepared using the same, and applications thereof. Background Art

[0002] Canine coronavirus disease (CCoV) is a viral infectious disease in dogs caused by canine coronavirus (CCoV). Canine coronavirus is an alphacoronavirus that infects dogs and causes gastroenteritis as its primary symptom. It comprises two distinct genotypes, CCoV-I and CCoV-II. CCoV-II is further subdivided into CCoV-IIa and CCoV-IIb. Recent studies have reported the possible existence of a new subgenotype, CCoV-IIc. A total of 378 fecal samples from diarrheal dogs were collected from six regions in Heilongjiang Province, including Daqing, Harbin, Jixi, Mudanjiang, Jiamusi, and Qiqihar. Of these, 74 samples were positive for CCoV, with an overall CCoV positivity rate of 19.58%. CCoV-II was the predominant strain.

[0003] Canine coronavirus can infect dogs of all breeds and ages (puppies are particularly affected). It is a common, acute gastrointestinal infectious disease in dogs. The main source of infection is sick dogs, and the main route of transmission is through contaminated feed and drinking water, which infects the digestive tract. The disease has an acute onset, rapid transmission, a short course, and a high mortality rate. It is often mixed with canine parvovirus or rotavirus, which aggravates the condition and leads to rapid death from dehydration due to acute diarrhea and vomiting. Clinically, it mainly manifests as frequent vomiting, diarrhea, depression, and anorexia. It is a major viral infectious disease that endangers the dog breeding industry. Canine coronavirus disease is distributed and prevalent worldwide, and coronavirus is also an important pathogen of canine infectious respiratory syndrome. Therefore, canine coronavirus vaccine is of great significance in the prevention of canine coronavirus disease.

[0004] However, at present, there are few studies on single canine coronavirus vaccines. For example, CN111097044A discloses a triple inactivated vaccine of canine leptospira, icterohaemorrhagiae, and canine coronavirus. Meanwhile, the only vaccines available for purchase on the market that can be used in mass production are Zoetis's canine distemper, adenovirus type 2, parainfluenza, and parvovirus quadruple live vaccine - canine leptospirosis (canine type, icterohaemorrhagiae) bivalent inactivated vaccine - canine coronavirus disease inactivated vaccine to obtain immune protection against canine coronavirus. Although multi-vaccines are more convenient to use, on the one hand, there is a problem of large side effects, which causes dogs to become ill after being vaccinated. On the other hand, there is also a problem of poor immune effect and the problem of being infected by canine coronavirus and becoming ill after immune failure.

[0005] Therefore, there is a need in the art to develop a new, single canine coronavirus vaccine with better immune effect. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a canine coronavirus strain and an inactivated vaccine and application thereof.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a canine coronavirus strain, wherein the full-length cDNA sequence of the genome of the canine coronavirus strain comprises any one of the base sequences shown in (I), (II) or (III):

[0009] (I) the base sequence shown in SEQ ID NO.1;

[0010] (II) a base sequence having ≥98% homology with the base sequence shown in SEQ ID NO. 1;

[0011] (III) A base sequence obtained by modifying, replacing, deleting or adding at least one base to the base sequence shown in SEQ ID NO. 1.

[0012] As a preferred technical solution of the present invention, the canine coronavirus is named CCoV2015, and the full-length cDNA sequence of its genome is shown in SEQ ID NO.1.

[0013] The present invention collects intestinal tissue from naturally ill dogs in Jilin Province, and determines that it is a new canine coronavirus strain with a different gene sequence from the existing canine coronavirus through sample preparation, PCR identification, sequencing, and NCBI sequence comparison. The canine coronavirus strain is then obtained by cell separation, and further subjected to viral titer determination, PCR identification, and sequence comparison.

[0014] Then, the viral liquid of canine coronavirus was inactivated by BEI, and after inactivation, it was verified for inactivation and the vaccine was prepared to obtain a canine coronavirus vaccine. The effectiveness and safety of the vaccine were determined through safety tests, immunogenicity tests and virus protection tests.

[0015] As a preferred technical solution of the present invention, the virus content of the third generation virus liquid of canine coronavirus is (10 5.0 ~10 8.0 )TCID 50 For example, the virus content of the third generation virus solution of canine coronavirus in the present invention can be 10 6.0 TCID 50 / ml, 10 6.5 TCID 50 / ml, 10 7.0 TCID 50 / ml or 10 7.5 TCID 50 / ml, etc.

[0016] In a second aspect, the present invention provides use of the canine coronavirus strain as described in the first aspect in preparing vaccines, antibodies, hybridoma cells or detection kits.

[0017] Wherein, the vaccine includes an inactivated vaccine and / or a recombinant protein vaccine; the antibody includes an egg yolk antibody prepared using the canine coronavirus strain.

[0018] In a third aspect, the present invention provides a canine coronavirus vaccine, which is prepared using the canine coronavirus strain described in the first aspect or the recombinant protein expressed thereby.

[0019] As a preferred technical solution of the present invention, the canine coronavirus vaccine is an inactivated vaccine; wherein the content of canine coronavirus is (10 5.0 ~10 8.0 )TCID 50 / ml, for example, 10 6.0 TCID 50 / ml, 10 6.5 TCID 50 / ml, 10 7.0 TCID 50 / ml or 10 7.5 TCID 50 / ml, etc.

[0020] Preferably, the method for preparing the inactivated vaccine comprises treating canine coronavirus with an inactivating agent.

[0021] Preferably, the inactivation agent comprises any one of formaldehyde, β-propiolactone, ethyleneimine, and BEI, or a combination of at least two thereof, wherein the concentration of formaldehyde may be 0.01%-0.2% w / v, the concentration of β-propiolactone may be 0.03%-0.2% w / v, and the concentration of ethyleneimine may be 0.5-20 mM.

[0022] As a preferred technical solution of the present invention, the canine coronavirus vaccine further includes a pharmaceutical carrier and / or a vaccine adjuvant.

[0023] As a preferred technical solution of the present invention, the dosage form of the vaccine adjuvant includes oil-in-water type, water-in-oil-in-water type, water-in-oil type or hydrophilic adjuvant, for example, it can be any one of: aluminum hydroxide, aluminum phosphate, carbomer or Montanide Gel 02PR or a combination of at least two.

[0024] The inactivated virus solution and vaccine adjuvant are mixed in a certain ratio depending on the type of adjuvant used. For example, when Montanide Gel 02PR is used as the adjuvant, the volume ratio of the inactivated virus solution to the vaccine adjuvant in the canine coronavirus vaccine is (5-10):1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0025] Preferably, the canine coronavirus vaccine of the present invention is a BEI inactivated vaccine. The greatest feature of the BEI inactivated vaccine is that BEI can destroy the viral nucleic acid, completely losing the virus's infectivity, without damaging its protein capsid, thereby preserving its protective antigens and hardly affecting the immunogenicity of the virus.

[0026] In a fourth aspect, the present invention protects an egg yolk antibody prepared using the canine coronavirus strain as described in the first aspect.

[0027] In a fifth aspect, the present invention protects a reagent for detecting canine coronavirus vaccines, comprising an antibody obtained using the canine coronavirus strain described in the first aspect as an immunogen; or, a specific primer based on the whole genome of the canine coronavirus strain.

[0028] The numerical range described in the present invention includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention isolates and screens a new canine coronavirus strain and names it CCoV2015. Sequence comparison and analysis show that the strain is a new canine coronavirus strain, typed as CCoV-Ⅱa. The strain can be extracted from sick dogs and has a strong infectivity. Sequence analysis and comparison show that the sequence homology between the strain and multiple prevalent strains isolated in recent years is 92% or more. The present invention inactivates the strain to prepare a canine coronavirus vaccine, and determines the effectiveness and safety of the vaccine through safety tests, immunogenicity tests, and challenge protection tests.

[0031] The inactivated canine coronavirus vaccine provided by the present invention fills the gap in single canine coronavirus vaccines and plays a very important role in the prevention of canine coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the result of CCoV2015 nucleic acid identification, where lane M represents Marker (100-2000bp); lane 1 represents CCoV2015 virus culture fluid; lane 2 represents the negative control (normal cell culture fluid).

[0033] Figure 2 This is the evolutionary tree of the CCoV2015 genome sequence. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further illustrated below with reference to the accompanying drawings and through specific implementation methods. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0035] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0036] Example 1

[0037] 1. Sample preparation

[0038] Intestinal tissue was collected from naturally ill dogs in Jilin Province. A portion of the intestinal tissue was minced with sterilized scissors. The tissue was then added to 10 ml of PBS and slowly ground in a sterilized mortar until no visible tissue fragments remained. The mixture was then shaken and centrifuged at 10,000 rpm for 20 minutes at 4°C. The supernatant was aliquoted in small, 1 ml tubes in a sterile laminar flow hood and stored at -70°C until ready for use.

[0039] 2. PCR identification

[0040] (1) RNA extraction

[0041] The prepared intestinal tissue grinding solution was used to extract sample RNA using an RNA kit. The RNA was stored at -70°C or immediately reverse transcribed into cDNA.

[0042] (2) cDNA preparation

[0043] Use the PrimeScript RT reagent Kit with gDNAEraser kit. The reaction system is shown in Tables 1 and 2. The total volume is 20 μL. Reagents 1 and 3 must be operated on ice.

[0044] Table 1 Reverse transcription system 1

[0045]

[0046] Table 2 Reverse transcription system 2

[0047]

[0048] A Reverse Transcriptase XL (AMV) kit, Randoma primer, Oligo primer, RRI, and dNTPs (10 mM) were used. The reaction system is shown in Table 3.

[0049] The total volume is 50 μL, in which AMV and RRI reagents need to be operated on ice. The above system is reverse transcribed in a 42°C water bath for 1 hour to obtain cDNA.

[0050] Table 3 Reverse transcription system 3

[0051]

[0052]

[0053] 3. PCR amplification

[0054] (1) PCR reaction system

[0055] The PCR reaction system is shown in Table 4. The total volume is 30 μL. Mix with a vortex mixer, centrifuge briefly, and set aside.

[0056] Upstream primer (CCoV-nsp14-F, SEQ ID NO.2):

[0057] 5′-GTGATGCTATCATGACTAG-3′;

[0058] Downstream primer (CCoV-nsp14-R, SEQ ID NO.3):

[0059] 5′-CACCATTACAACCTTCTAA-3′.

[0060] Table 4 PCR reaction system

[0061] Reagents Dosage (μL) DreamTaqGreenPCRMasterMix(2X) 15 10 mM upstream primer 2 10 mM downstream primer 2 Double distilled water 7 cDNA 4

[0062] (2) Amplification procedure and reaction conditions

[0063] Place the PCR reaction tube in a PCR amplifier. The reaction parameters are: 95°C denaturation for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 48°C for 30 seconds, and extension at 72°C for 45 seconds; then extension at 72°C for 10 minutes, followed by storage at 4°C. PCR products were analyzed by 1% agarose gel electrophoresis.

[0064] 4. Electrophoresis detection of PCR products

[0065] Prepare a 1% agarose plate with TAE electrophoresis buffer. Place the plate in a horizontal electrophoresis tank and add 1×TAE electrophoresis buffer just above the gel surface. Add 3 μL of the PCR amplification product to each sample well and 3 μL of DNAMarkerDL 2000 to the standard molecular weight control well for electrophoresis.

[0066] 5. Gel imaging system analysis

[0067] The PCR amplification product showed a band around 400 bp, which was consistent with the expected size, indicating that the canine coronavirus nucleic acid was positive. Figure 1 .

[0068] Example 2

[0069] 1. Virus isolation and culture

[0070] Take part of the canine coronavirus intestinal tissue and grind it thoroughly in a dry heat sterilized mortar. Then put the ground tissue fluid into a sterile centrifuge tube and centrifuge it at 10,000 rpm for 20 minutes. Take the supernatant, add 5% double antibody and refrigerate overnight for later use.

[0071] Passage Vero cells to T25 cell flasks. When the cells grow into a monolayer, pour out the culture medium and add the sample to the cell culture flask at 1 ml / flask. Shake gently and incubate in a 37°C, 5% CO2 incubator. Shake gently every 15 minutes. Discard the sample after incubation for 1 hour. Then add 10 ml of culture medium to the cell flask, mix gently and culture in a 35°C, 5% CO2 incubator.

[0072] The cells were observed for cytopathic effects every day, and the virus was harvested by repeated freezing and thawing three times to obtain the virus solution (P1 generation). The P1 generation virus solution was then inoculated into Vero cell monolayers to the third generation. Cell controls were set up at each generation.

[0073] No macroscopic cytopathic effects were found in the first generation.

[0074] In the second generation, a small amount of cells showed stringiness and rounding.

[0075] The third generation cells showed large-scale cell stringing, rounding and shedding, while the control cells did not show cytopathic effects.

[0076] 2. Determination of viral content

[0077] One day in advance, cells were plated into 96-well cell culture plates, 100 μl / well, and the cell concentration was 1×10 5 / ml, and then incubate in a 37°C, 5% CO2 incubator for one day to grow into a good cell monolayer.

[0078] On the second day, the third generation virus solution was diluted 10-fold in MEM culture medium containing 2% serum, starting from 10 -1 Dilute to 10 -12 Then, the samples of each dilution were inoculated into 96-well plates, and 4 replicates were made for each dilution, 100 μl / well, and cultured in a 35°C, 5% CO2 incubator for 5 days. The cytopathic effect of each dilution was recorded, and the TCID was calculated according to the Reed-Muench method. 50 .

[0079] The virus content of the third generation virus solution is 10 7.0 TCID 50 / ml.

[0080] 3. Indirect immunofluorescence detection

[0081] The harvested third-generation virus solution was inoculated into a 96-well Vero cell monolayer. After 48 hours of culture, the supernatant was discarded and fixed with 80% cold acetone at room temperature for 30 minutes. After washing three times with PBST, a 200-fold diluted canine coronavirus monoclonal antibody (mouse source) was added at 50 μl / well and incubated at 37°C for 1 hour. After washing three times with PBST, a FITC-labeled goat anti-mouse enzyme-labeled secondary antibody was added at 50 μl / well and incubated at 37°C for 1 hour. After washing three times with PBST, the results were observed. The results showed that the Vero cells inoculated with the virus showed specific green fluorescence, while the control cells had no specific fluorescence. The results showed that the isolated virus was canine coronavirus and was named CCoV2015.

[0082] Sequencing, the obtained full genome sequence of CCoV2015 is shown in SEQ ID NO.1, combined with Figure 2 It can be seen that sequence analysis and comparison show that its high homology with the sequences of multiple epidemic strains isolated in recent years is 92% or more.

[0083] Example 3

[0084] 1. Preparation of inactivated CCoV vaccine

[0085] Canine coronavirus antigen was diluted to 10% with MEM culture medium. 6.0 TCID 50 / ml, add BEI to make the final concentration of 0.002mol / L, then put it in a water bath at 30℃ for inactivation for 24 hours, and then add the inactivation terminator sodium thiosulfate to make the final concentration of 0.002mol / L.

[0086] 2. Inactivation Verification

[0087] Take the inactivated antigen and inoculate it into a T25 cell flask that has grown into a well-grown Vero monolayer. Each inactivated sample is inoculated into two T25 cell culture flasks, 1 ml / flask. After gently shaking, place the flask in a 35°C, 5% CO2 incubator for incubation. Gently shake once every 15 minutes during the incubation period. Incubate for 1 hour, then add culture medium to the cell flask to 10 ml, continue to culture in a 35°C, 5% CO2 incubator, and subculture once after 3 days.

[0088] Set up virus and cell controls. If the virus control shows cytopathic effects, while the inactivated sample and cell control do not, the inactivation is considered qualified.

[0089] 3. Vaccine preparation

[0090] The antigens that have passed the inactivation verification can be mixed with the adjuvant according to the ratio of 7:1 = CCoV2015:Montanide Gel 02PR to prepare the inactivated vaccine and stored at 2-6°C.

[0091] Example 4

[0092] 1. Safety evaluation

[0093] Five healthy beagle dogs aged 2 to 3 months (CCoV neutralizing antibody titer no higher than 1:4) were selected and each dog was injected subcutaneously with 2 ml of vaccine. The dogs were observed for 14 consecutive days, and their mental state, appetite, body temperature and stool characteristics were recorded daily.

[0094] After a one-time double dose of the vaccine was administered subcutaneously to dogs, the dogs were observed for 14 consecutive days, and no local or systemic adverse reactions occurred in the dogs. Anatomical and histological examinations were performed on some of the immunized dogs, and no visible pathological changes were found.

[0095] 2. Immunogenicity evaluation

[0096] (1) Vaccination

[0097] Five 2- to 3-month-old Beagle dogs (CCoV neutralizing antibody titers no higher than 1:4) were subcutaneously injected with 0.5 ml of the inactivated vaccine. Five other dogs were subcutaneously inoculated with 0.5 ml of MEM medium in the neck to serve as controls. Immunizations were repeated twice, 21 days apart. Twenty-one days after the booster immunization, blood was collected, serum was isolated, and CCoV neutralizing antibody titers were determined.

[0098] (2) Determination of CCoV neutralizing antibody titer

[0099] The serum to be tested was diluted 2-fold in series, starting from 2 1 Dilute to 2 times 12 times, with an equal volume of 100 TCID 50The serum-virus mixture was mixed with 50 μl of CCoV and incubated at 37°C for 1 hour. The serum-virus mixture was transferred to a monolayer of Vero cells, with 100 μl per well, and incubated at 35°C for 5 days. The pathological changes in each well were assessed daily, and the serum neutralizing antibody titer was calculated.

[0100] After the prepared vaccine was immunized in dogs, the CCoV neutralizing antibody titer was shown in Table 5. The antibody titer of the blank control group after immunization was no higher than 1:4. The vaccine immunization group could stimulate the body to produce CCoV neutralizing antibody titer one week after the first immunization; one week after the booster immunization, the antibody titer increased. Three weeks after the booster immunization, the CCoV neutralizing antibody titer was no less than 1:32, indicating that the vaccine prepared using CCoV2015 has good immunogenicity.

[0101] Table 5 CCoV neutralizing antibody titer determination results

[0102]

[0103]

[0104] 3. Attack and protection test

[0105] Ten healthy beagle dogs aged 2 to 3 months (CCoV neutralizing antibody titer no higher than 1:4) were selected and randomly divided into two groups: the immunization group and the control group, with 5 dogs in each group.

[0106] The immunization group received a subcutaneous injection of the prepared inactivated vaccine, 0.5 ml per dog, and the control group received a subcutaneous injection of MEM culture medium, 0.5 ml per dog. A booster immunization was performed every 21 days. 21 days after the booster immunization, each dog was inoculated with CCoV2015 strain (diluted to a virus titer of 10 6.0 TCID 50 After 14 days of observation, all dogs were scored according to the scoring criteria in Table 6. The data up to 14 days after the challenge were counted, and the total score for the 14 days was calculated.

[0107] If the scores between the vaccine group and the control group were significantly different (P < 0.05), the vaccine group was determined to be protected; if the difference was not significant (P > 0.05), the vaccine group was determined to be unprotected.

[0108] Table 6 Health status and clinical symptom scoring system

[0109]

[0110]

[0111] The scoring results of the animals in each group after immunization and challenge are shown in Table 7. There is a significant difference in the scores between the vaccine group and the control group (P < 0.0001), that is, the immunized group is protected, which shows that the canine coronavirus inactivated vaccine can protect the body against viral attacks after immunization, effectively alleviate the symptoms after challenge, reduce the score after challenge, and shorten the course of the disease.

[0112] Table 7 Scoring details after challenge

[0113]

[0114]

[0115] In summary, the canine coronavirus vaccine provided by the present invention was administered subcutaneously to dogs at a double dose, and no local or systemic adverse reactions occurred in the dogs, indicating good safety. The vaccine-immunized group was able to stimulate the body to produce CCoV neutralizing antibody titers one week after the first immunization, and the antibody titer increased one week after the booster immunization. Three weeks after the booster immunization, the CCoV neutralizing antibody titer was not less than 1:32, so the immunogenicity was also good. In addition, in the challenge protection test, the vaccine group was well protected, effectively alleviating the symptoms after the challenge, reducing the score after the challenge, and shortening the course of the disease.

[0116] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A canine coronavirus strain, characterized in that The full-length cDNA sequence of the canine coronavirus genome comprises any one of the base sequences shown in (I), (II) or (III): (I) the base sequence shown in SEQ ID NO.1; (II) a base sequence having ≥98% homology with the base sequence shown in SEQ ID NO. 1; (III) A base sequence obtained by modifying, replacing, deleting or adding at least one base to the base sequence shown in SEQ ID NO.

1.

2. The canine coronavirus strain according to claim 1, characterized in that The canine coronavirus is named CCoV2015, and the full-length cDNA sequence of its genome is shown in SEQ ID NO.

1.

3. The canine coronavirus strain according to claim 1, characterized in that The virus content of the third generation virus liquid of canine coronavirus is (10 5.0 ~10 8.0 )TCID 50 / ml.

4. Use of the canine coronavirus strain according to any one of claims 1 to 3 in the preparation of vaccines, antibodies, hybridoma cells or detection kits; The vaccines include inactivated vaccines and / or recombinant protein vaccines; The antibodies include egg yolk antibodies prepared using the canine coronavirus strain.

5. A canine coronavirus vaccine, characterized in that The canine coronavirus vaccine is prepared using the canine coronavirus strain according to any one of claims 1 to 3 or the recombinant protein expressed therein.

6. The canine coronavirus vaccine according to claim 5, characterized in that The canine coronavirus vaccine is an inactivated vaccine; The content of canine coronavirus in the canine coronavirus vaccine is (10 5.0 ~10 8.0 )TCID 50 / ml.

7. The canine coronavirus vaccine according to claim 6, characterized in that The canine coronavirus vaccine is an inactivated vaccine, and the preparation method includes treating the canine coronavirus with an inactivating agent; The inactivation reagent includes any one of formaldehyde, β-propiolactone, ethyleneimine, and BEI, or a combination of at least two of them.

8. The canine coronavirus vaccine according to any one of claims 5 to 7, characterized in that The canine coronavirus vaccine further comprises a pharmaceutical carrier and / or a vaccine adjuvant; The dosage form of the vaccine adjuvant includes oil-in-water type, water-in-oil-in-water type, water-in-oil type or hydrophilic adjuvant; The vaccine adjuvant includes any one of aluminum hydroxide, aluminum phosphate, carbomer or Montanide Gel 02PR, or a combination of at least two of them.

9. Egg yolk antibody prepared using the canine coronavirus strain according to any one of claims 1 to 3.

10. A reagent for detecting canine coronavirus vaccine, characterized in that It comprises an antibody obtained using the canine coronavirus strain according to any one of claims 1 to 3 as an immunogen, or a specific primer based on the whole genome of the canine coronavirus strain.

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