Canine coronavirus attenuated strain CCoV AS22 and application thereof

Abstract

The invention discloses a canine coronavirus attenuated strain CCoVAS22 and application thereof, and relates to the technical field of biological medicines. The canine coronavirus attenuated strain CCoVAS22 is preserved in the China Center for Type Culture Collection on July 12, 2024, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: V202413. According to the invention, the technical means of cross passage in MDCK and CRFK cell lines is utilized to obtain the autonomously separated CCoVAS22 strain, and the strain has the advantages of high virus titer and stable passage on cells, has the characteristics of high safety and good immunogenicity after being inactivated by beta-propiolactone, can be used as a CCoV inactivated vaccine candidate strain, and can be used as a CCoV inactivated vaccine candidate strain. The problem that domestic canine coronavirus isolates are few can be relieved to a certain extent, and technical support is provided for preventing diseases caused by canine coronavirus.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a canine coronavirus attenuated strain CCoV AS22 and an application thereof. Background Art

[0002] Canine coronavirus disease is a disease caused by canine coronavirus (CCoV) that mainly infects canines such as dogs, raccoon dogs and foxes. The highest incidence rate is in puppies aged 2-4 months. When CCoV infects alone, it usually causes self-limited enteritis and mild diarrhea in dogs, but when mixed with other pathogens, it can cause severe clinical symptoms and even death.

[0003] The research on CCoV in China started later than that in foreign countries. In terms of pathogen isolation, the method of effectively isolating CCoV is still immature, resulting in fewer isolates obtained in my country, which restricts the research on CCoV. At present, there is no specific drug for treating CCoV viral enteritis, and the prevention of the disease mainly relies on vaccination. With the reports of multiple lethal CCoV highly pathogenic variant strains in Europe in recent years, more and more researchers have also increased their attention to CCoV vaccines. The present invention intends to develop a canine coronavirus attenuated strain, thereby providing technical support for the prevention of diseases caused by canine coronavirus. Summary of the invention

[0004] The purpose of the present invention is to provide a canine coronavirus attenuated strain CCoV AS22 and its application to solve the problems existing in the above-mentioned prior art. The strain has the characteristics of high safety and good immunogenicity after inactivation, and the vaccine prepared by the strain can resist infection by some existing CCoV epidemic strains in China, providing technical support for preventing diseases caused by canine coronavirus.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a canine coronavirus, which was deposited in the China Center for Type Culture Collection on July 12, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCCNO: V202413.

[0007] The present invention also provides the use of the canine coronavirus attenuated strain CCoV AS22 in preparing a medicine for preventing diseases caused by canine coronavirus.

[0008] Furthermore, the medicine is a vaccine.

[0009] Furthermore, the vaccine is an inactivated vaccine.

[0010] The present invention also provides a medicine for preventing diseases caused by canine coronavirus, wherein the active ingredient comprises the canine coronavirus attenuated strain CCoV AS22.

[0011] Furthermore, the medicine is a vaccine.

[0012] Furthermore, the vaccine is an inactivated vaccine.

[0013] Furthermore, the vaccine also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the auxiliary material includes aluminum hydroxide adjuvant.

[0015] The present invention also provides a method for preparing a canine coronavirus inactivated vaccine, comprising the following steps: inactivating the canine coronavirus attenuated strain CCoV AS22 to obtain an inactivated virus antigen;

[0016] The inactivated virus antigen and the vaccine adjuvant are evenly mixed to obtain the canine coronavirus inactivated vaccine.

[0017] The present invention discloses the following technical effects:

[0018] The invention utilizes the technical means of cross-passaging in MDCK and CRFK cell lines to obtain an independently isolated CCoVAS22 strain, which has the advantages of high toxicity and stable passage on cells, has the characteristics of high safety and good immunogenicity after being inactivated by beta-propiolactone, can be used as a candidate strain for CCoV inactivated vaccine, can alleviate the problem of less canine coronavirus isolates in China to a certain extent, and provides technical support for preventing diseases caused by canine coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the growth curve of CRFK cells infected with CCoV AS22;

[0021] Figure 2The figure is an electrophoresis diagram of RT-PCR detection of exogenous virus of CCoV AS22 virus, wherein, from left to right, lane 1: 2000bp DNA marker; lane 2: CAV positive control (1030bp); lane 3: CCoV positive control (409bp); lane 4: CDV positive control (455bp); lane 5: CPV positive control (559bp); lane 6: CPIV positive control (182bp); lane 7: CAV negative control (no band); lane 8: CCoV negative control (no band); lane 9: CDV negative control (no band); lane 10: CPV negative control (no band); lane 11: CPIV negative control (no band); lane 12: CCoV AS22 virus sample (CAV negative no band); lane 13: CCoV AS22 virus sample (CCoV positive 409bp); Lane 14: CCoV AS22 virus sample (CDV negative no band); Lane 15: CCoV AS22 virus sample (CPV negative no band); Lane 16: CCoV AS22 virus sample (CPIV negative no band);

[0022] Figure 3 The results of the exogenous virus test of CCoV AS22 10th generation strain using IFA;

[0023] Figure 4 TCID for long-term storage of CCoV AS22 strains at different temperatures 50 The results of the drug price test;

[0024] Figure 5 These are microscopic pathological changes of some tissues after dogs were inoculated with CCoV AS22 strain, where A to D are HE staining images of tissue sections of spleen (A), jejunum (B), ileum (C) and cecum (D) of dogs inoculated with the strain, respectively;

[0025] Figure 6 This is the result of testing the neutralizing antibody levels in dogs after immunization with CCoV AS22 inactivated vaccine. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1

[0032] 1. Isolation and cultivation of CCoV AS22 strain

[0033] 1.1 Cells and viruses

[0034] CRFK and MDCK cells were provided by Wuhan Keqian Biotechnology Co., Ltd. The above cells were cultured in 10% FBS-DMEM medium in a 37°C, 5% CO2 incubator. The CCoV AS22 strain was isolated from canine anal swabs by the Rabies Virus Laboratory of Veterinary Public Health, Huazhong Agricultural University, and was identified and preserved.

[0035] 1.2 Virus culture and passaging

[0036] 1.2.1 Virus culture

[0037] The CCoV AS22 strain was inoculated into the above-mentioned well-growing cell monolayer, adsorbed for 1 hour, the inoculum was discarded, and 1% FBS-DMEM medium was added to continue culturing in a 37°C, 5% CO2 incubator. After 72 hours, when more than 80% of the cells became pathological, freeze-thawed once at -20°C to -80°C, and the cell culture virus fluid was harvested.

[0038] 1.2.2 Cell passaging method:

[0039] The MDCK / CRFK cell cross-passaging method was used. CCoV AS22 virus was first cultured in MDCK cells (inoculated into new MDCK cells at a ratio of 1 / 10), and then continuously passaged to the fifth generation, and then passaged to the 60th generation on CRFK cells in the same way.

[0040] 1.3 Virus growth curve and TCID 50 Determination

[0041] CCoV AS22 was passaged to the sixth generation, and the virus solution was appropriately diluted and inoculated into CRFK cells cultured in 12-well plates at MOIs of 0.01, 0.05, 0.1, and 1, respectively. Three replicate wells were used, and one 12-well plate was taken out every 12 hours and temporarily stored at -80°C until 72 hpi. After freezing and thawing once, TCID 50 Determination.

[0042] CCoV virus culture was serially diluted 10-fold with maintenance solution, 10 -1 ~10 -10 , inoculate CRFK cells cultured in 96-well plates, 100 μL / well, 8 replicate wells, culture at 37°C, 5% CO2 incubator until 72 hpi, observe cytopathic effects under a microscope, and calculate TCID according to the Reed-Muench method 50 The results are shown in Figure 1 , the highest titer of CCoV AS22 with an MOI of 0.05 was 10 5.5 TCID 50 / 0.1mL.

[0043] 1.4 Mycoplasma testing

[0044] Tested according to the current appendix of "Chinese Veterinary Pharmacopoeia", no mycoplasma growth was found.

[0045] 1.5 Exogenous virus detection

[0046] 1.5.1 Inspected according to the current appendix of the Chinese Pharmacopoeia of Veterinary Medicine, free from exogenous virus contamination.

[0047] 1.5.2 PCR test for exogenous viruses

[0048] Using CCoV AS22 10th generation virus as template, PCR amplification was performed with specific primers for canine adenovirus (CAV1 / CAV2) E3 protein gene and canine parvovirus (CPV) VP2 gene; RT-PCR amplification was performed with specific primers for CCoV M protein gene, canine distemper virus (CDV) H gene, and canine parainfluenza virus (CPIV) N gene. The primer sequences are shown in Table 1. The results showed that the CCoV AS22 10th generation virus sample was positive only for the CCoV M protein gene, and the amplification results of other exogenous virus primers were negative, see Table 1. Figure 2 At the same time, IFA was used to test the exogenous virus of CCoV AS22 10th generation virus, and the results are shown in Figure 3 , further proving that the CCoV AS22 virus strain was not contaminated by related exogenous viruses.

[0049] Table 1 Primers used in PCR testing of exogenous viruses

[0050]

[0051] 1.6 Microbial Deposit

[0052] The present invention submits the CCoV AS22 strain to a preservation institution for preservation, and its microbial preservation number is CCTCC NO: V202413; the preservation time is: July 12, 2024; the preservation unit is: China Center for Type Culture Collection; the preservation address is: Wuhan University, Wuhan, China.

[0053] 2. CCoV AS22 virus storage period test

[0054] CRFK cells were used to culture CCoV AS22 virus, and the virus was divided into 1 mL / vial and stored in -80℃, -20℃, and 4℃ refrigerators. After a certain period of time, one vial was taken out from each of the vials to determine the TCID of the virus. 50 , evaluate the shelf life of CCoV virus strains. Figure 4 The virus was stored in a -80℃ refrigerator for 12 months. 50 No significant change, stored in -20℃ refrigerator for 12 months TCID 50 1 titer drop; stored in 4℃ refrigerator for 3 months TCID 50 The results showed that the virus strain should be stored at -80℃ for at least 12 months.

[0055] 3.CCoV AS22 virulence test

[0056] 3.1 Experimental dogs

[0057] Eight dogs aged 7-8 weeks without maternal antibodies (serum CCoV neutralizing antibody titer ≤ 1:2) were provided by the experimental animal base (breeding dogs were not vaccinated). The experimental dogs were isolated and raised in the animal room of Huazhong Agricultural University, fed with commercial feed and free drinking water, and used in this experiment after 1 week of adaptation. Starting 3 days (-3 days) before the experiment, fecal samples were tested with CCoV antigen rapid detection kit (Anjie) (IC) to prove that there was no CCoV infection.

[0058] 3.2 Grouping and virus inoculation

[0059] Six dogs aged 7-8 weeks were selected, and four of them were inoculated with 10 4 TCID 50 / 0.1mL of CCoV AS22 virus, 1mL nasal drops, 4mL oral administration. The other two dogs were inoculated with the same amount of cell maintenance fluid as the control group.

[0060] After virus inoculation, all dogs except dog 4# were observed for a total of 14 days. The clinical symptoms of the experimental dogs were observed every day, the rectal temperature was monitored, fecal detoxification was detected using immunochromatographic test strips (IC) and virus isolation (VI) methods, and anticoagulated blood was collected every 3 days for white blood cell classification and counting.

[0061] On the 7th day after virus inoculation, dog No. 4 was selected from the experimental group and dog No. 6 was selected from the control group. The dogs were anesthetized, killed, and dissected. Related tissue samples were taken and fixed with formaldehyde, sliced, and stained with HE for pathological histological observation under a microscope.

[0062] 3.3 Virulence test results

[0063] The results of the virulence test of CCoV AS22 are shown in Table 2. During the entire observation period, the body temperature of dogs inoculated with CCoV AS22 (1#, 2#, 3#, and 4#) occasionally increased, and obvious clinical symptoms appeared after vaccination, mainly manifested as depression, decreased food intake, and diarrhea. The lymphocyte count increased acutely 3 days after vaccination, and in severe cases it increased to 43.7%. The dogs showed obvious inflammatory reactions. The results of pathological histological observations of dog 4# showed that pathological changes mainly occurred in the spleen, jejunum, ileum, and cecum, with a decrease in splenic lymphocytes and bleeding. The villi of the jejunum partially fell off, the goblet cells decreased, and the lymphocytes and plasma cells were scattered. The intestinal epithelium of the ileum fell off, the goblet cells decreased, and the lymphocytes and plasma cells were scattered. The villi of the cecum partially fell off, the lymphocytes and plasma cells were scattered, the intestinal glands were dilated, and necrotic cell fragments were occasionally seen in the glandular cavity of the intestinal glands. There were no obvious pathological changes in other tissues. Figure 5 The healthy control dogs (5# and 6#) remained normal throughout the observation period, and no pathological changes were found in the histopathological observation of dog 6#.

[0064] Table 2 Virulence and safety characteristics of CCoV AS22 strain

[0065]

[0066] Dpi: days after virus inoculation; IC: colloidal gold test strip test; VI: virus isolation test; A: depression, loss of appetite; B: diarrhea; “none”: no clinical symptoms; “+” indicates positive, and “-” indicates negative.

[0067] 4. CCoV inactivated vaccine

[0068] The inactivated canine coronavirus vaccine was prepared using the CCoV AS22 strain.

[0069] 4.1 The brief process of virus culture and inactivation is as follows: CCoV AS22 was expanded and cultured with CRFK cells, centrifuged at 4000r / min to remove cell debris, and the supernatant was inactivated by adding 1‰ of β-propiolactone to obtain CCoV AS22 inactivated virus antigen, which was stored at 4°C for use.

[0070] 4.2 Preparation of CCoV aluminum hydroxide adjuvant inactivated vaccine

[0071] Take the above CCoV AS22 inactivated virus antigen, slowly add an equal amount (V:V) of commercial aluminum hydroxide gel adjuvant (adjuvant 2%, InvivoGen USA), mix well, and make a virus particle with a final content of 10 6 TCID 50 inactivated vaccine.

[0072] The amino acid sequence of the target antigen protein, Spike protein, designed by the inactivated vaccine is as follows (SEQ ID NO.1):

[0073]

[0074] 4.3 Immunization

[0075] Take 8 dogs aged 5-6 weeks (serum CCoV neutralizing antibody titer ≤ 1:2), randomly select 5 dogs as the experimental group, and vaccinate 1 mL / dog with the inactivated vaccine prepared in 4.2. The other 3 dogs were vaccinated with the same amount of cell maintenance fluid as the control group in the same way. Both the experimental group and the control group were vaccinated by subcutaneous injection, twice at week 0 and week 3. The second vaccination dose and route were the same as the first. Continue to observe for 2 weeks, and collect blood samples every week for the determination of neutralizing antibodies.

[0076] 4.4CCoV serum neutralizing antibody assay

[0077] Canine serum was inactivated at 56°C for 30 min before testing. Serum was diluted in 2% FBS-DMEM medium in a 96-well cell culture plate at a ratio of 1:2 (50 μL system), with 4 rows of replicate wells for each dilution, and 50 μL / well containing 200 TCID 50 The CCoVAS22 virus solution was incubated at 37°C for 1.5 h, and 100 μL / well (about 60% cells) of the 96-well CRFK cell plate prepared the day before was added. The cells were cultured in a 37°C CO2 incubator for 4 days. The CPE was observed under a microscope, and the neutralizing antibody titer was calculated based on the logarithm of the maximum dilution of serum that completely inhibited the cytopathic effect. The results are shown in Figure 6 The neutralizing antibody titer of the secondary immunization of CCoV AS22 inactivated vaccine reached 1:181, which has good immunogenicity.

[0078] In summary, the CCoV AS22 strain has potential application value in developing into an inactivated vaccine.

[0079] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A canine coronavirus attenuated strain CCoV AS22, characterized in that: The canine coronavirus attenuated strain CCoV AS22 was deposited in the China Center for Type Culture Collection on July 12, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: V202413.

2. Use of the canine coronavirus attenuated strain CCoVAS22 as claimed in claim 1 in the preparation of a medicament for preventing diseases caused by canine coronavirus.

3. The use according to claim 2, characterized in that: The medicine is a vaccine.

4. The use according to claim 3, characterized in that: The vaccine is an inactivated vaccine.

5. A drug for preventing diseases caused by canine coronavirus, characterized in that: The active ingredient comprises the canine coronavirus attenuated strain CCoVAS22 described in claim 1.

6. The drug according to claim 5, characterized in that The medicine is a vaccine.

7. The drug according to claim 6, characterized in that The vaccine is an inactivated vaccine.

8. The drug according to claim 6, characterized in that The vaccine also includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that The auxiliary materials include aluminum hydroxide adjuvant.

10. A method for preparing a canine coronavirus inactivated vaccine, characterized in that: The method comprises the following steps: inactivating the canine coronavirus attenuated strain CCoVAS22 according to claim 1 to obtain an inactivated virus antigen; The inactivated virus antigen and the vaccine adjuvant are evenly mixed to obtain the canine coronavirus inactivated vaccine.

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