Porcine D-type coronavirus SC-MS strain, vaccine prepared from porcine D-type coronavirus SC-MS strain and application of vaccine

Abstract

The invention discloses a swine D-type coronavirus SC-MS strain, a vaccine prepared from the swine D-type coronavirus SC-MS strain and application of the swine D-type coronavirus SC-MS strain, and belongs to the technical field of veterinary biological products. The porcine D-coronavirus SC-MS strain is preserved in China Center for Type Culture Collection in Wuhan University, Wuhan, China on November 20, 2024, and the preservation number of the porcine D-coronavirus SC-MS strain is CCTCC NO: V202428. According to the invention, the porcine D-type coronavirus SC-MS strain which can be stably proliferated on ST cells and has high virus content is obtained through separation, and an inactivated vaccine prepared from the strain has good safety and immunogenicity to piglets, can induce the piglets to generate high-level neutralizing antibodies and has long antibody maintenance time.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and specifically relates to a swine delta coronavirus SC-MS strain, a vaccine prepared therefrom, and uses thereof. Background Art

[0002] Porcine delta coronavirus (PDCoV) is an enveloped single-stranded positive-strand RNA virus with a genome length of about 25.4 kb. It belongs to the order Nidovirales, the family Coronaviridae, and the genus Deltacoronavirus. It is a newly discovered porcine enterovirus in recent years. PDCoV infects pigs of all ages, with newborn piglets being the most susceptible and having a high mortality rate. Clinical symptoms after infection include acute diarrhea, vomiting, and dehydration. In 2014, the first report of the prevalence of PDCoV in American pigs was published. Subsequently, cases of PDCoV appeared in China, Canada, Japan, and other countries. PDCoV quickly became a common pathogen in pigs around the world. PDCoV not only threatens the healthy development of the pig industry, but recent studies have found that the hosts of PDCoV can also be cattle, chickens, and turkeys. PDCoV has even been found in children's plasma, posing a potential threat to human health.

[0003] Due to the lack of effective drugs and vaccines for the treatment and prevention of PDCoV, in the face of the increasing trend of PDCoV infection rate in my country in recent years, the prevention and control pressure on pig farms is increasing, and a safe and effective PDCoV vaccine is urgently needed. We found that the patents related to PDCoV vaccines authorized by the State Intellectual Property Office have different technical defects. For example, a strain of swine D-coronavirus and its application (ZL202310493286.0) has problems such as low virus content, low antibody titer, and short immunity duration. A strain of swine D-coronavirus attenuated strain and its application (ZL202310493301.1) also has problems or risks such as low antibody titer, short immunity duration, strong virulence, and interference from maternal antibodies. Therefore, it is particularly urgent to isolate a strain with high virus content, good immunogenicity, and high safety. Summary of the invention In response to the above shortcomings in the prior art, the present invention provides a pig tin coronavirus SC-MS strain and its vaccine and uses. The pig tin coronavirus SC-MS strain provided by the present invention has a high virus content and good immunogenicity. After preparation into an inactivated vaccine, it can induce higher levels of neutralizing antibodies, and has a high protection rate for piglets and a long duration of immunity.

[0004] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is: The object of the present invention is to provide a SC-MS Porcine delta coronavirus strain, which was deposited at the China Model Culture Collection Center of Wuhan University, Wuhan, China on November 20, 2024, and its collection number is CCTCC NO: V202428.

[0005] Another object of the present invention is to provide a recombinant vector comprising the S gene of the above-mentioned swine delta coronavirus SC-MS strain.

[0006] Another object of the present invention is to provide the use of the above-mentioned swine delta coronavirus SC-MS strain or recombinant vector in the preparation of a preparation for treating or preventing swine delta coronavirus infection.

[0007] Another object of the present invention is to provide a vaccine comprising the above-mentioned swine delta coronavirus SC-MS strain and a pharmaceutically acceptable adjuvant thereof.

[0008] Furthermore, the vaccine is a live attenuated vaccine or an inactivated vaccine.

[0009] Furthermore, the virus content of the SC-MS strain of swine delta coronavirus in the vaccine is not less than 10 7.5 TCID 50 / mL.

[0010] Furthermore, the adjuvant is SEPPIC MONTANIDE TM ISA 15A VG.

[0011] Beneficial effects of the present invention: The present invention isolates a pig Dermocoronavirus SC-MS strain. The inactivated vaccine prepared using this strain has good safety and immunogenicity, and can induce the body to produce high levels of neutralizing antibodies and the antibodies last for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The cytopathic effect of ST cells inoculated with PDCoV SC-MS strain; A: ST cells infected with PDCoV SC-MS strain; B: normal ST cell control; Figure 2 The PCR amplification results of the S gene of the PDCoV SC-MS strain; M: D2000 DNA Marker; 1-4: S1-S4 gene fragment amplification products; 5: negative control; Figure 3 Phylogenetic tree analysis of the S gene of PDCoV SC-MS strain; Figure 4The indirect immunofluorescence results of PDCoV SC-MS strain; A: ST cells infected with PDCoV SC-MS strain; B: ST cell control without virus inoculation; Figure 5 TCID of each passage of PDCoV SC-MS strain 50 ; Figure 6 This is the antibody growth and decline pattern of piglets immunized with PDCoV SC-MS strain inactivated vaccine. DETAILED DESCRIPTION

[0013] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0014] Example 1 Isolation and Identification of Swine D-Coronavirus Strain 1. Pathological materials and cell lines Small intestinal tissue and intestinal contents of piglets with watery diarrhea were collected from a large-scale pig farm in Meishan, Sichuan in 2018. ST cells were preserved by Huapai Biotechnology (Group) Co., Ltd.

[0015] 2. Isolation and culture of viruses The small intestinal tissue identified as PDCoV positive by RT-PCR was taken and ground with PBS. After grinding, it was placed in a -80℃ refrigerator and repeatedly frozen and thawed 3 times. It was centrifuged at 2000 r / min for 10 min at 4℃, and the supernatant was separated and sterilized by filtration with a 0.22 µm pore filter. The filtrate was used for subsequent virus isolation. When the ST cells in the T75 cell culture flask were full of monolayers, the monolayer ST cells were washed 3 times with PBS, 2 mL of filtrate was added, and incubated in a 37℃, 5% CO2 incubator for 1 h, shaking once every 30 min. After the incubation, the liquid in the cell culture flask was discarded, 20 mL of serum-free MEM medium (containing 8 µg / mL trypsin) was added, and a normal cell control without virus was set up. It was placed in a 37℃, 5% CO2 incubator for culture, and CPE was observed every day. If CPE did not appear after 72-96 h of culture, the cell culture was harvested after repeated freezing and thawing 3 times, and the cell culture continued to be blindly cultured on ST cells.

[0016] According to this method, obvious CPE appeared at the fifth generation of blind propagation, which was manifested by ST cells becoming larger and rounder, and then developing into cell fusion and shedding. The cells in the negative control group grew well ( Figure 1 ).

[0017] 3. Identification of isolated strains (1) Sequencing and sequence analysis of the S gene Referring to the genome sequence of the PDCoV HB-BD strain (accessory number: MF948005) published in GenBank, four pairs of specific primers were designed using Primer Premier 5.0 software to identify the isolated strain and amplify the full-length S gene. The primers were synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd., and the information related to the primers is shown in Table 1.

[0018] Table 1 Primer sequences

[0019] According to the instructions of TRIzol™ LS Reagent and the instructions of the high-efficiency reverse transcription kit of Toyobo (Shanghai) Biotechnology Co., Ltd., the total cytotoxic RNA of the F5 generation of PDCoV SC-MS strain was extracted and reverse transcribed to synthesize cDNA. The obtained cDNA was used as a template and PCR amplified using the four pairs of primers in Table 1. PCR reaction system: PrimeSTAR Max Premix (2×) 25μL, 2μL of upstream and downstream primers, 2μL of cDNA, and 19μL of ddH2O. The reaction program was set as 95℃ pre-denaturation for 5 min, 95℃ denaturation for 45 s, 58℃ annealing for 30 s, and 72℃ extension for 90 s, for a total of 30 cycles; finally, 72℃ extension for 10 min.

[0020] The PCR product was detected by 1% agarose gel electrophoresis, and the results showed that four specific bands consistent with the expected fragment size were obtained ( Figure 2 ), the product was sent to Shenggong Bioengineering (Shanghai) Co., Ltd. for sequencing. The splicing sequencing results obtained the S gene of the PDCoV SC-MS strain 3480bp. The S gene of SC-MS strain was analyzed in phylogenetic analysis with representative strains from different countries published on GeneBank. The results showed that the isolate belongs to the G1b subgroup as well as most Chinese strains ( Figure 3 ).

[0021] (2) Indirect immunofluorescence test The cell growth medium was discarded from the 96-well cell culture plate covered with a monolayer of ST cells, and the PDCoV SC-MS strain virus solution was inoculated. After the virus infected the cells for 24 hours, the maintenance medium was discarded, and the cells were fixed with a methanol-acetone mixture for 40 minutes. The fixative was discarded and the 96-well cell culture plate was dried. 100 μL of PDCoV positive serum diluted 1:100 was added to each well, incubated at 37°C for 1.5 hours, and then washed 4 times with PBS. 100 μL of FITC-labeled goat anti-pig IgG diluted 1:200 was added to each well, incubated at 37°C for 1 hour, and then washed 3 times with PBS. The test results were observed under a fluorescence microscope. The results are shown in Figure 4 .

[0022] like Figure 4 It was shown that ST cells infected with PDCoV showed specific green fluorescence, further proving that the isolate strain was PDCoV. The isolated strain was named the SC-MS Porcine delta coronavirus strain, and was deposited at the China Model Culture Collection Center of Wuhan University, Wuhan, China on November 20, 2024. The collection number is CCTCC NO: V202428.

[0023] (3) Virus content determination Inoculate ST cell suspension at 100 µL / well in a 96-well cell culture plate, then place it in 37°C and 5% CO2 incubator for 48 to 72 h. Wait for the ST cells in the 96-well cell culture plate to grow a monolayer, discard the cell growth solution, and wash the cells with PBS 3 times. The virus was diluted 10 times continuously using serum-free MEM medium (containing 5 µg / mL of trypsin) and diluted it to 10. -1 -10 -8 Inoculate in a 96-well cell culture plate, seed 8 wells per dilution, with a seeding amount of 100 µL / well. At the same time, negative cell control was set up, and CPE was placed in a 37°C, 5% CO2 incubator for 72~96 hours for observation and recording, and calculated according to the Reed-Muench method.

[0024] The PDCoV SC-MS strain was continuously subcultured on ST cells, and the virus titer was measured every 5 generations. The titer of the cytotoxic F5 to F30 generations of the PDCoV SC-MS strain was 10 4.3 TCID 50 / mL to 10 8.2 TCID 50 / mL, with the increase of virus generation, the virus titer showed an upward trend and gradually stabilized ( Figure 5 ).

[0025] 4. Pathogenicity test Ten piglets that had just been born and had not eaten colostrum were selected and randomly divided into 5 piglets / group. Each piglet in the challenge group was orally administered with 4 mL of PDCoVSC-MS strain F15 cytotoxin, and each piglet in the control group was orally administered with 4 mL of MEM culture medium. During the experiment, they were fed by artificial feeding. The piglets in the challenge group became ill 5 / 5 days after the challenge, showing symptoms of depression, anorexia, and watery diarrhea. The diarrhea piglets were found to be bloated with gas in the small intestine, and a large amount of yellow liquid accumulated in the intestinal cavity. The intestinal wall was significantly thinned and translucent. The spirit and appetite of the piglets in the control group were normal, and no clinical symptoms were observed. After the autopsy, no visible pathological changes in the intestine were observed.

[0026] Example 2 Preparation and testing of PDCoV SC-MS strain inactivated vaccine 1. Preparation of inactivated vaccine of PDCoV SC-MS strain Take the well-grown ST monolayer cells, discard the original culture medium and wash the monolayer ST cells with PBS 3 times, add the PDCoV SC-MS strain F24 virus solution in a 1% ratio, and put it in a 37°C incubator. When the cell lesions are more than 80% freeze and thaw 3 times to harvest the virus solution as a virus for preparing vaccines. The virus content of the virus solution should not be less than 10. 7.5 TCID50 / mL. Use formaldehyde solution with a final concentration of 0.2% at 37°C to inactivate the virus solution for 36 hours, and perform an inactivation test on the inactivated virus solution. Inactivation test steps: The inactivated virus solution is placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in PBS for 24 hours; take 5 bottles of ST cells that have grown a monolayer, inoculate 1 mL of the dialyzed virus solution in each T25 cell bottle, and then add 5 mL of serum-free MEM culture medium (containing 5 µg / mL trypsin), and culture in a 37°C, 5% CO2 incubator. No CPE appears after blind passage for 3 generations, indicating that the virus is completely inactivated. MONTANIDE TM ISA 15A VG is used as the oil phase and the inactivated virus solution is used as the aqueous phase. The oil phase is added to the aqueous phase at a ratio of 1:9 at room temperature, and emulsified at 1000 r / min for 30 min to prepare a PDCoV inactivated vaccine. The sterile test is carried out according to the current Appendix of the "Chinese Veterinary Pharmacopoeia", and no bacterial growth is achieved.

[0027] 2. Safety testing of the PDCoV SC-MS strain inactivated vaccine 10 healthy piglets of 14-21 day-old who were negative for PDCoV antigen and whose serum neutralizing antibody titers were not higher than 1:4 were randomly divided into 5 heads / group. The first group of neck muscles was vaccinated with PDCoV SC-MS inactivated vaccine 4mL / head, and the second group of neck muscles was vaccinated with 4mL / head. After 10 consecutive days of observation, the piglets were in good spirits, with normal appetite and body temperature, and no local or systemic adverse reactions caused by the vaccine occurred.

[0028] 3. Efficacy testing of the inactivated vaccine of PDCoV SC-MS strain Ten healthy piglets aged 14 to 21 days with negative PDCoV antigen and serum neutralizing antibody titer not higher than 1:4 were screened and randomly divided into 5 piglets / group. The first group was inoculated with 2 mL / head of PDCoV SC-MS strain inactivated vaccine in the neck muscle, and the second group was inoculated with 2 mL / head of normal saline in the neck muscle. After 14 days, the same dose was used for booster immunization. Blood was collected from the anterior vena cava 14 days after the second immunization, and the serum neutralizing antibody titer was detected by the fixed virus-dilution serum method. At the same time, each piglet in the immunization group and the control group was orally administered with 4 mL of PDCoVSC-MS strain F15 cytotoxicity. The piglet status was observed every day and the diarrhea was recorded. The results are shown in Table 2.

[0029] Table 2 Results of the efficacy study of the inactivated vaccine of PDCoV SC-MS strain Piglet grouping Neutralizing antibody titer Number of cases Protection rate Inactivated vaccine immunization group 1:256、1:256、1:256、1:512、1:512 0 5 / 5 Normal saline control group <1:2、<1:2、1:2、1:2、1:2 5 0 / 5 4. Duration of immunity of PDCoV SC-MS strain inactivated vaccine Ten healthy piglets aged 14 to 21 days with negative PDCoV antigen and serum neutralizing antibody titer not higher than 1:4 were screened and randomly divided into 5 piglets / group. The first group was inoculated with 2mL / head of PDCoV SC-MS strain inactivated vaccine in the neck muscle, and the second group was inoculated with 2mL / head of normal saline in the neck muscle. After 14 days, the same dose was used for booster immunization. Blood was collected before immunization and 14 days, 28 days, 60 days, 90 days, 120 days, and 150 days after the first immunization, and the serum neutralizing antibody titer was detected by fixed virus-dilution serum method.

[0030] The results showed that the serum neutralizing antibodies of the vaccine immunized group turned positive in 14 days after immunization, but the antibodies were low. After strengthening the immunization, the serum neutralizing antibody titers increased rapidly, and the neutralizing antibody level reached the highest 28 days after immunization, and remained at a higher antibody level 120 days after immunization ( Figure 6 ).

[0031] Finally, it should be explained that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the invention has been described in detail with reference to examples, ordinary skill in the art should understand that the technical solutions of the present invention can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present invention, and they should be covered within the scope of the claims of the present invention.

Claims

1. A SC-MS Porcine delta coronavirus strain, characterized in that: The swine delta coronavirus SC-MS strain was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on November 20, 2024, and its deposit number is CCTCC NO: V202428.

2. A recombinant vector, characterized in that: Comprising the S gene of the swine delta coronavirus SC-MS strain described in claim 1.

3. Use of the SC-MS strain of swine delta coronavirus according to claim 1 or the recombinant vector according to claim 2 in the preparation of a preparation for treating or preventing swine delta coronavirus infection.

4. A vaccine, characterized in that It comprises the swine delta coronavirus SC-MS strain as described in claim 1, and a pharmaceutically acceptable adjuvant thereof.

5. The vaccine according to claim 4, characterized in that The vaccine is a live attenuated vaccine or an inactivated vaccine.

6. The vaccine according to claim 5, characterized in that The virus content of SC-MS strain of swine delta coronavirus in the vaccine is not less than 10 7.5 TCID 50 / mL.

7. The vaccine according to claim 6, characterized in that Adjuvant: SEPPIC MONTANIDE TM ISA 15A VG.

Images