Porcine reproductive and respiratory syndrome virus NADC30 strain SCqq, inactivated vaccine and application

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain, an inactivated vaccine and application, and belongs to the technical field of veterinary medicine. The SCqq strain is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC NO: V202486, and the preservation time is September 29, 2024. According to the invention, a porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain is screened, and an inactivated vaccine prepared from the virus strain is high in immunogenicity, safe and reliable, has a very good immune protection effect on porcine reproductive and respiratory syndrome virus NADC30 infection, can induce an organism to generate a high-level antibody, and achieves 100% virulent virus attack protection.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary medicine, and specifically relates to a porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain, an inactivated vaccine and uses thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a viral infectious disease that harms the pig industry. The World Organization for Animal Health (OIE) lists it as a reportable animal disease. my country lists highly pathogenic porcine reproductive and respiratory syndrome as a Category II animal infectious disease. The pathogen is the porcine reproductive and respiratory syndrome virus, which is a single-stranded positive-strand RNA virus. The virus particles are spherical with a diameter of 55 to 60 nanometers. There are currently two serotypes, the European type and the American type. The strains isolated in my country are mainly the American type.

[0003] In recent years, the PRRSV strain prevalent in my country's pig farms has a close genetic relationship with the NADC30 strain reported in the United States. This strain has recombined with other subgroup strains prevalent in China (classical PRRSV, HP-PRRSV or vaccine strains), and a new type of virus has been produced through genetic mutation. Domestic scholars collectively refer to this type of virus as NADC30-like strains, which have become the main prevalent strain of PRRSV. After sows are infected, they mainly suffer from abortion, and piglets develop severe respiratory symptoms after infection, causing huge economic losses to the global pig industry.

[0004] The most effective means of preventing PRRSV infection is vaccination. Currently, research on PRRSV NADC30 strains in my country is in its infancy, and no such vaccines or diagnostic products are available on the market. Therefore, there is an urgent need to develop a vaccine against porcine reproductive and respiratory syndrome virus NADC30 strains. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain, an inactivated vaccine and uses thereof, which can effectively prevent diseases caused by porcine reproductive and respiratory syndrome virus NADC30 infection.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is: The purpose of the present invention is to provide a porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain, which is preserved in the China Type Culture Collection, the preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the preservation number is: CCTCC NO: V202486, and the preservation time is September 29, 2024.

[0007] Furthermore, the SCqq strain has a nucleotide sequence encoding the GP5 protein as shown in SEQ ID NO.1 and a nucleotide sequence encoding the Nsp2 protein as shown in SEQ ID NO.2.

[0008] The purpose of the present invention is to provide the use of the porcine reproductive and respiratory syndrome virus NADC30-like strain SCqq strain in the preparation of a preparation for diagnosing, preventing or treating NADC30-like porcine reproductive and respiratory syndrome.

[0009] Furthermore, the preparation is a vaccine.

[0010] The object of the present invention is to provide an inactivated vaccine for preventing NADC30-like porcine reproductive and respiratory syndrome, which comprises the inactivated SCqq strain and a pharmaceutically acceptable adjuvant.

[0011] Furthermore, the adjuvant is a veterinary adjuvant.

[0012] Furthermore, the adjuvant is SEPPIC MONTANIDE TM ISA 15A VG.

[0013] The purpose of the present invention is to provide the use of the porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain in constructing a porcine reproductive and respiratory syndrome virus infection model.

[0014] Furthermore, the infection model is a cell infection model or an animal infection model.

[0015] The purpose of the present invention is to provide use of the porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain in preparing an antibody detection reagent.

[0016] Beneficial effects of the present invention: The present invention has screened a porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain. The inactivated vaccine prepared using the virus strain has high immunogenicity, is safe and reliable, has a good immune protection effect on porcine reproductive and respiratory syndrome virus NADC30 infection, can induce the body to produce high levels of antibodies, and achieve 100% protection against strong toxin attacks.

[0017] In addition, this virus strain can also be used to screen, prepare, detect and / or evaluate porcine reproductive and respiratory syndrome virus NADC30 antibodies, and to prepare porcine reproductive and respiratory syndrome virus NADC30 infected cell models or animal models, and then to study the pathogenic mechanism of porcine reproductive and respiratory syndrome virus NADC30 infection, screen antiviral drugs, develop new diagnostic strategies and / or treatment strategies, and evaluate the immune protection effect of vaccines, and has broad clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the pathological observation results of PAMs cells inoculated with the porcine reproductive and respiratory syndrome virus SCqq strain; A is the PAMs cells after inoculation, and B is the normal PAMs cell control; Figure 2 This is the electrophoresis diagram of PCR amplification of ORF5 and Nsp2 genes of porcine reproductive and respiratory syndrome virus SCqq strain; Figure 3 Genetic evolutionary tree drawn for the ORF5 gene sequence of porcine reproductive and respiratory syndrome virus SCqq strain; Figure 4 This is a map of the amino acid deletion positions (black boxes) after translation of the Nsp2 gene sequence of porcine reproductive and respiratory syndrome virus SCqq strain. DETAILED DESCRIPTION

[0019] 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.

[0020] Example 1 Isolation and Identification of Porcine Reproductive and Respiratory Syndrome Virus SCqq Strain 1. Isolation of porcine reproductive and respiratory syndrome virus (1) Treatment of diseased materials In 2023, lung tissue was collected from a suspected case of porcine reproductive and respiratory syndrome in a pig farm in Sichuan. The lung tissue was crushed with a grinder and made into a tissue suspension with PBS. 2000 IU / mL of penicillin and streptomycin were added, and the suspension was placed in a -80°C refrigerator and frozen and thawed three times. The suspension was then centrifuged at 10,000 rpm for 10 minutes. The supernatant was filtered and sterilized with a 0.22μm microporous filter membrane, and the filtrate was stored at -20°C for use as inoculation material.

[0021] (2) Virus isolation The PAMs cells that have grown into a monolayer were discarded from the culture medium, washed once with PBS buffer, inoculated with 1 mL of the melted filtrate, placed in an incubator for adsorption for 1-2 hours, then the culture flask was removed and the filtrate was discarded, 5 mL of DMEM maintenance solution containing 2% fetal bovine serum was added, and cultured in a cell culture incubator at 37°C and 5% CO2 for 3 days. The state of the cells was observed every day, and the diseased cells were collected in time and frozen and thawed three times at -20°C to obtain the virus solution. Another 2 mL was inoculated into the PAMs cells that had grown into a monolayer, and blind passage was continued for 3 generations, and the cell lesions were observed daily. The cell culture was harvested after three freeze-thaw cycles. 2. Virus identification (1) PCR identification of American type / European type PRRSV is divided into two genotypes: European type (represented by strain Lelystad virus, LV) and American type (represented by strain VR-2332), whose gene sequence homology is 55-80%. Based on the PRRSV ORF5 gene sequence published on Genbank, DNAStar compared the nucleotide sequences of all PRRSV ORF5 genes and designed two primers in the conserved region using Oligo 6.0. The amplified product is expected to be 851bp. The primer sequences are as follows: Table 1 ORF5 gene primer information

[0022] Extract viral RNA: RNA was extracted according to the Tiangen RNA extraction kit RNAprep Pure Cell / Bactterla Kit operating procedure.

[0023] RT-PCR reaction system: PrimeScript 1 Step Enzyme Mix 0.6μL; 2×1 Step Buffer (Dye Plus) 12.5μL; RNase free water 7.9μL; 1μL of upstream and downstream primers each; 2μL of RNA.

[0024] Reaction conditions: 50°C for 30 min; 94°C for 5 min; 94°C for 45 s, 55°C for 30 s, 72°C for 90 s, 30 cycles; 72°C for 10 min.

[0025] After the reaction, the PCR product was identified by electrophoresis using 1% agarose gel. Take 5 μL of the reaction product and electrophoresed it in 1% agarose gel at 120V for 40 min. After the electrophoresis, take out the gel and observe the results in a gel imager. The results showed that a specific band of about 851 bp was amplified ( Figure 2), and the PCR products were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing analysis. The sequencing results were compared with the sequences published by NCBI and the phylogenetic tree was analyzed. The results showed that the isolated PRRSV SCqq strain was an American type ( Figure 3 ).

[0026] (2) PCR identification of PRRSV classic strains / variant strains / NADC30-like strains Primers for amplifying the Nsp2 gene were designed based on the comparison results of PRRSV American strains published in GenBank, and the amplified product was expected to be 1941 bp.

[0027] Table 2 Nsp2 gene primer information

[0028] Extract viral RNA: RNA was extracted according to the Tiangen RNA extraction kit RNAprep Pure Cell / Bactterla Kit operating procedure.

[0029] RT-PCR reaction system: PrimeScript 1 Step Enzyme Mix 0.6μL; 2×1 Step Buffer (Dye Plus) 12.5μL; RNase free water 7.9μL; 1μL of upstream and downstream primers each; 2μL of RNA.

[0030] Reaction conditions: 50°C for 30 min; 94°C for 5 min; 94°C for 45 s, 55°C for 30 s, 72°C for 90 s, 30 cycles; 72°C for 10 min.

[0031] After the reaction, the PCR product was identified by electrophoresis using 1% agarose gel. Take 5 μL of the reaction product and electrophoresed it in 1% agarose gel at 120V for 40 min. After the electrophoresis, take out the gel and observe the results in a gel imager. The results showed that a specific band of about 1941 bp was amplified ( Figure 2 ), and the PCR products were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing analysis. The sequencing results were translated and compared, and the results showed that "111+1+19" amino acid deletions appeared at positions 149-259, 310, and 328-346, which were the same as the genomic characteristics of the NADC30 genotype strain, indicating that the SCqq strain belongs to the NADC30-like strain ( Figure 4 ). It was deposited in China Center for Type Culture Collection, the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is: CCTCC NO: V202486, and the deposit time is September 29, 2024.

[0032] (3) Specificity experiments The isolated PRRSV virus was added with an appropriate amount of PRRSV positive serum and allowed to act at 37°C for 1 hour. At the same time, a virus control and a cell control were set up. The cells were cultured in an incubator at 37°C with 5% carbon dioxide for 5 days. The virus control cells all showed cytopathic effects, while the normal cells and virus neutralized cells did not show cytopathic effects, indicating that the isolated virus can be neutralized by the characteristic positive serum of porcine reproductive and respiratory syndrome virus, with good specificity.

[0033] Example 2 Determination of the titer and virulence of porcine reproductive and respiratory syndrome virus SCqq strain 1. Toxicity determination PAMs cells were dispersed into 1×10 6 The cell suspension was inoculated into a 96-well plate at 100 μL / well. After the cells adhered to the wall, the culture medium in the well was discarded. 100 μL of the virus solution diluted 10 times with the cell maintenance medium (10 -1 ~10 -8 ) F4 generation, each dilution was repeated for 4 wells, normal PAMs cells were used as control, and cultured in a 37℃, 5% CO2 incubator for 72 h. The cytopathic effect was observed under an inverted biological microscope, and the number of wells with and without cytopathic effect was recorded. The virus TCID was calculated using the Reed- Muench method. 50 The virus content is 10 6 .0 TCID 50 / mL.

[0034] 2. Toxicity determination Dilute the PRRSV SCqq strain F4 virus solution to 10 5 .0 TCID 50 / mL, inoculated 5 3-4 week old piglets, 2mL of nasal drops for each pig. Another 5 3-4 week old piglets were set up as blank controls. After vaccination, 100% (5 / 5) of the piglets showed respiratory symptoms such as fever, depression, loss of appetite, coughing or wheezing. The lungs of the challenge test pigs showed patchy consolidation. Comprehensive temperature response, clinical manifestations and pathological autopsy observation results showed that all the challenge test pigs became ill (5 / 5 became ill). The blank control pigs had no clinical symptoms or fever, and no visible pathological changes in the lungs were observed after autopsy. This shows that the isolated PRRSV SCqq strain is a highly virulent epidemic strain. Example 3 Preparation and testing of inactivated vaccine of porcine reproductive and respiratory syndrome virus SCqq strain 1. Vaccine preparation (1) Virus reproduction The nutrient solution was discarded after the PAMs cells were fully grown in a monolayer, and the virus seeds of porcine reproductive and respiratory syndrome virus SCqq strain were inoculated into the cells at a ratio of 5% of the nutrient solution. The cells were adsorbed at 37°C for 30 to 60 minutes, and then the maintenance solution was added. The cells were cultured at 37°C and observed for 48 to 72 hours. The cells were harvested when more than 80% of the cells showed pathological changes. After freezing and thawing three times, the cells were harvested as antigen solution and sampled for identification. (2) Virus inactivation Use formaldehyde to inactivate the virus. Add the formaldehyde solution to the harvested antigen solution to a final concentration of 0.2%, and inactivate at 37°C for 48 hours. After inactivation, take samples for sterility and inactivation tests. The sterility test is carried out according to the method in the appendix of the third volume of the Chinese Pharmacopoeia of Veterinary Medicine. The inactivation test method is to inoculate the inactivated virus solution of the porcine reproductive and respiratory syndrome virus SCqq strain into PAMs monolayer cells, discard the virus solution after adsorption for 1 hour, add maintenance solution, culture at 37°C for 3 days, and harvest the cell culture solution after repeated freezing and thawing at -20°C for 3 times. Then blindly propagate for 2 generations according to the same method. If the inoculated PAMs cells do not show specific cytopathic effects, they are considered qualified.

[0035] (3) Vaccine preparation Antigen and SEPPIC MONTANIDE TM ISA 15 A VG is emulsified and mixed in a certain proportion to prepare an inactivated vaccine.

[0036] 2. Inspection (1) Property inspection Drop the vaccine into the water and spread it evenly.

[0037] (2) Sterility test Tested according to the appendix of the current "Chinese Veterinary Pharmacopoeia", no sterile growth was found. (3) Safety inspection Five healthy susceptible piglets aged 3 to 4 weeks were selected and 4 mL of the vaccine was injected intramuscularly into the neck of each pig. Five control pigs were also selected and observed for 14 days. No clinical symptoms caused by porcine reproductive and respiratory syndrome virus or local or systemic adverse reactions caused by the vaccine appeared in both the experimental group and the control group. The results are shown in Table 3.

[0038] Table 3 Safety test results

[0039] 3. Effectiveness test (1) Serological methods Five healthy susceptible piglets aged 3 to 4 weeks were used, and the vaccine was injected intramuscularly in the neck at 2.0 mL / pig. A second vaccination was performed 14 days after the first vaccination using the same route and dose, and five control piglets were set up. Blood was collected 14 days after the second vaccination to separate serum and detect neutralizing antibodies against porcine reproductive and respiratory syndrome virus. The neutralizing antibody detection method is as follows: 1) Serum preparation: Aseptically collect blood samples, place at room temperature for 2 hours, centrifuge at 6000-8000 rpm for 5 minutes, collect the supernatant and incubate in a 56°C water bath for 30 minutes as the serum to be tested.

[0040] 2) Serum dilution: In a 96-well plate, add 50 μL of serum-free DMEM to each well, add 50 μL of the serum to be tested to the first well of each column (i.e., row A of the 96-well plate), make a 2-fold serial dilution, dilute to row H, and discard 50 μL.

[0041] 3) Virus solution dilution: Take out a tube of virus solution with determined virus content from the -70℃ refrigerator and dilute it to 2000TCID with serum-free DMEM. 50 / mL, from 2000TCID 50 Take 100 μL of the solution and dilute to 200 TCID / mL 50 / mL, 20TCID 50 / mL and 2TCID 50 / mL.

[0042] 4) Add 50 μL of 2000 TCID to each well of the diluted serum. 50 / mL virus solution, set up four columns with only 50μL DMEM, add 50μL of 2000TCID 50 / mL virus solution; 50μL of 200TCID50 / mL virus solution was added to each well in the second column; 50μL of 20TCID50 / mL virus solution was added to each well in the third column 50 / mL virus solution; add 50μL of 2TCID to each well in the fourth column 50 / mL virus solution as 100TCID 50 / well, 10TCID 50 / well, 1TCID 50 / well, 0.1TCID 50 / well of virus control, at 36-37℃ for 1h, then add 100μL of PAMs cell suspension, at 36-37℃, 5% CO 2 Incubate incubator for 72-120 hours and observe the results.

[0043] 5) Reading: When reading at 72-120h, the entire well should be observed. If any part shows cytopathic effect, it will be counted as cytopathic in that well.

[0044] 6) Result establishment condition: 100TCID 50 / well, 10TCID 50 / well virus control wells should show cytopathic effect, 1TCID 50 / well virus control wells should show cytopathic effect in wells 1 to 7, 0.1TCID50 If the virus control in each well shows cytopathic effect in well 0, the result is valid.

[0045] 7) Result determination: The highest dilution without cytopathic effect is taken as the neutralizing antibody titer of the serum.

[0046] The results of neutralizing antibody detection showed that the vaccine prepared from the porcine reproductive and respiratory syndrome virus PRRSV SCqq strain can induce the production of high levels of neutralizing antibodies, see Table 4.

[0047] Table 4 Potency test results (serological method) Grouping Neutralizing antibody titer Experimental Group 1:128；1:64；1:128；1:128；1:64 Control group 1:2；1:2；＜1:2；1:2；1:2 (2) Immune attack method Five healthy susceptible piglets aged 3 to 4 days were used. The vaccine was injected intramuscularly in the neck at a dose of 2.0 mL / pig. A second vaccination was performed 14 days after the first vaccination using the same route and dose. Five control piglets were set up. Fourteen days after the second vaccination, the porcine reproductive and respiratory syndrome virus SCqq strain was challenged by intranasal inoculation, with 2.0 mL (10 6.0 TCID 50 / mL), body temperature was measured after vaccination and observation was conducted for 14 days. The comprehensive body temperature and clinical symptoms showed that the protection rate was 100%, see Table 5.

[0048] Table 5 Efficacy test results (immune challenge method) Grouping Temperature response Clinical symptoms Protection rate Experimental Group 0 / 5 0 / 5 5 / 5 Control group 3 / 5 5 / 5 0 / 5 In summary, the present invention provides a porcine reproductive and respiratory syndrome virus NADC30 strain, which has good immunogenicity, can induce the body to produce a high level of neutralizing antibody titer, and can resist strong toxin attacks, and is expected to become a safe and effective vaccine strain.

[0049] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain, characterized in that: It is deposited in the China Center for Type Culture Collection, the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit number is: CCTCC NO: V202486, and the deposit time is September 29, 2024.

2. Use of the porcine reproductive and respiratory syndrome virus NADC30-like strain SCqq strain according to claim 1 in the preparation of a preparation for diagnosing, preventing or treating NADC30-like porcine reproductive and respiratory syndrome.

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

4. An inactivated vaccine for preventing porcine reproductive and respiratory syndrome of the NADC30 class, characterized in that: It comprises the inactivated SCqq strain described in claim 1, and a pharmaceutically acceptable adjuvant thereof.

5. The inactivated vaccine according to claim 4, characterized in that The adjuvant is a veterinary adjuvant.

6. The inactivated vaccine according to claim 5, characterized in that Adjuvant: SEPPIC MONTANIDE TM ISA 15 AVG.

7. Use of the porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain according to claim 1 in constructing a porcine reproductive and respiratory syndrome virus infection model.

8. The use according to claim 7, characterized in that The infection model is a cell infection model or an animal infection model.

9. Use of the porcine reproductive and respiratory syndrome virus NADC30 strain SCqq strain according to claim 1 in the preparation of antibody detection reagents.

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