Porcine reproductive and respiratory syndrome virus NADC30 attenuated strain and live vaccine thereof

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus NADC30 attenuated strain and a live vaccine thereof. The attenuated strain is classified and named as a porcine reproductive and respiratory syndrome virus NADC30 attenuated strain, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on July 2, 2024, the preservation number is CGMCC No.45997, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No.1 yard, West Beichen Road, Chaoyang District, Beijing. According to the present invention, the PRRSV FJ1402 is successfully and continuously passed to F123, the pathogenicity of the obtained attenuated strain on piglets is significantly reduced, the PRRSV FJ1402F100 and the PRRSV F123 can induce the piglets to produce humoral immunity, and can protect the piglets against the attack of the PRRSV FJ1402, such that the good immune protection effect is provided, and the important foundation is provided for the development of the PRRSV live vaccine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a porcine reproductive and respiratory syndrome virus NADC30 attenuated strain and a live vaccine thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that currently threatens the world's pig industry. It can cause reproductive disorders in sows, such as abortion, premature birth, and mummified fetuses, as well as respiratory diseases in pigs of different ages, causing huge economic losses to the global pig industry (Wang, et al., 2015). PRRSV mutates rapidly, and the PRRSV NADC30-Like strain (lineage 1) has become a new epidemic strain, and the immune protection effect of commercial vaccines against it is still not ideal (Bai, et al., 2016). In this study, we passaged the PRRSV NADC30-Like strain FJ1402 from Marc-145 to F123. The F7, F100, F113, and F123 generations of FJ1402 virus piglet infection experiments showed that the pathogenicity of F100, F113, and F123 was significantly reduced. The results of the piglet immune protection test showed that FJ1402 F100 and F123 can effectively induce immune response and protect piglets against NADC30-LikeFJ1402 F7 generation strong virus attack, indicating that FJ1402 F100 and F123 can be used as candidate viruses for PRRSV NADC30-Like strain attenuated vaccine. Summary of the invention

[0003] The purpose of the present invention is to provide a porcine reproductive and respiratory syndrome virus NADC30 attenuated strain and a live vaccine thereof.

[0004] The purpose of the present invention can be achieved by the following technical solutions:

[0005] In the first aspect, the present invention requests protection for a weak strain of porcine reproductive and respiratory syndrome virus NADC30, which is classified and named as a weak strain of porcine reproductive and respiratory syndrome virus NADC30. It was deposited in the General Microbiology Center of China Microorganism Culture Collection on July 2, 2024, with the deposit number CGMCC No.45997, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] In the second aspect, the present invention seeks to protect the use of the above-mentioned porcine reproductive and respiratory syndrome virus NADC30 attenuated strain in the preparation of porcine reproductive and respiratory syndrome vaccine.

[0007] In the third aspect, the present invention seeks protection for a porcine reproductive and respiratory syndrome vaccine, which comprises the above-mentioned porcine reproductive and respiratory syndrome virus NADC30 attenuated strain.

[0008] Furthermore, the vaccine is a live vaccine. Furthermore, the vaccine is a nasal drop or an injection. Furthermore, the nasal drop is in the form of a solution, a suspension or an emulsion.

[0009] Beneficial effects of the present invention:

[0010] The present invention successfully continuously passaged PRRSV FJ1402 to F123, and the pathogenicity of the obtained attenuated strains to piglets was significantly reduced. PRRSV FJ1402 F100 and F123 can induce humoral immunity in piglets and protect piglets against the attack of PRRSV FJ1402, have good immune protection effects, and lay an important foundation for the development of PRRSV live vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is different generations of FJ1402 CPE;

[0012] (A) CPE of FJ1402 at different passages infected with Marc-145, (B) CPE of FJ1402 at different passages infected with PAM, (C) Plaque morphology of FJ1402 at different passages infected with Marc-145; the red arrows represent CPE.

[0013] Figure 2 Statistics of clinical symptoms of piglets after virus attack;

[0014] (A) Rectal temperature, (B) Body weight changes during the challenge, and (C) Clinical symptom statistics after the challenge.

[0015] Figure 3 PRRSV viremia and PRRSV viral load in organs (lungs, thymus, lymph nodes) of piglets after virus challenge;

[0016] (A) Viremia, (B) Lung viral load, (C) Lymph node viral load, (D) Thymus viral load.

[0017] Figure 4 The levels of serum inflammatory factors (IL-2, IL-4, IL-10, IFN-α and TNF-α) in piglets 2 weeks after infection.

[0018] Figure 5 To observe the thymus status of piglets 2 weeks after infection;

[0019] (A) F123, (B) F113, (C) F100, (D) F7, (E) PBS; Note: The red arrows indicate the thymus on both sides of the trachea and the chest, respectively.

[0020] Figure 6 The pathological changes of lung tissue in piglets after virus attack;

[0021] (A)F123, (B)F113, (C)F100, (D)F7, (E)PBS, HE.200×.

[0022] Figure 7 This is the immunohistochemistry of the lungs of piglets after virus challenge;

[0023] (A)F123, (B)F113, (C)F100, (D)F7, (E)PBS, HE.200×.

[0024] Figure 8 To test the levels of serum inflammatory factors in pigs after immunization.

[0025] Fig. 9 These are the observation results of clinical symptoms after the experimental pigs were challenged with the virus.

[0026] (A) Rectal temperature, (B) Body weight change within 2 weeks, (C) Clinical symptom score.

[0027] Fig.10 To test the viremia and organ viral load of pigs after virus challenge.

[0028] Fig.11 This is the macroscopic lesions of the thymus of immunized pigs after virus challenge;

[0029] (A) F123, (B) F100, (C) challenge control group; Note: The red arrows indicate the thymus on both sides of the trachea and the chest, respectively.

[0030] Fig.12 To show the pathological changes and immunohistochemistry of lung tissues in immunized pigs after virus challenge, 200×;

[0031] (A / D) FJ1402 F123 immunization group, (B / E) FJ1402 F100 immunization group, (C / F) challenge control group. DETAILED DESCRIPTION

[0032] The present invention is described in detail below in conjunction with specific embodiments. According to the following description and implementation, those skilled in the art can determine the basic characteristics of the present invention, and can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention to make it suitable for various uses and conditions.

[0033] Unless otherwise specified, the reagents and materials used in the following examples can be obtained through commercial channels or prepared using methods disclosed in the prior art.

[0034] Example 1

[0035] 1 Materials and Methods

[0036] 1.1 Cells, strains, sera, and experimental animals

[0037] Marc-145 cells were preserved by our laboratory; PRRSV FJ1402 F7 (GenBank: KX169191.1) virus fluid was isolated, identified and preserved by our laboratory; PRRSV positive and negative pig serum was prepared, identified and preserved by our laboratory; the antibody against PRRSV N protein (2H7) was prepared and preserved by a graduate student in our laboratory; 3-4 week old healthy piglets (both PRRSV antigen and antibody were negative) were provided by the Experimental Animal Center of Jiangsu Nannong High-tech Co., Ltd.

[0038] 1.2 Main Reagents

[0039] RNA extraction kit was purchased from Shanghai Suobao Biotechnology Co., Ltd.; 2×Phanta Max Master Mix, 2×Taq Mix, reverse transcriptase HiScript II 1st Strand cDNA Synthesis Kit and qPCR Green Master Mix was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; Gel Extraction Kit was purchased from BioFlux; DL2000 DNA Marker and pClone007 vector were purchased from Beijing Qingke Biotechnology Co., Ltd.; Tetramethylbenzidine (TMB) and Alex 488-labeled goat anti-mouse fluorescent secondary antibody were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; Porcine IL-2, IL-4, IL-10, IFN-α and porcine TNF-α Elisa detection kits were purchased from Beijing Solebao Technology Co., Ltd. All other conventional reagents were of analytical grade.

[0040] 1.3 Virus subculture

[0041] Marc-145 cells for virus culture were cultured with DMEM cell culture medium containing 10% (volume fraction, v / v) fetal bovine serum and 1% (volume fraction, v / v) penicillin / streptomycin. The Marc-145 cells were revived with the cell culture medium or passaged into a T25 cell culture bottle. After a good cell monolayer was grown, the growth medium was discarded and replaced with fresh serum-free DMEM culture medium. 1% (v / v) NADC30-Like FJ1402 virus solution was added and cultured at 37°C. Cell lesions were observed once in the morning and afternoon every day (cells were seen to aggregate and form clusters in the early stage). When more than 80% of the cells were lesioned, the cells were harvested, frozen and thawed twice at -70°C, and 5 ml was retained.

[0042] 1.4 Plaque assay

[0043] Marc-145 cells were plated on a six-well plate, and the experiment could be conducted only after the cells were aggregated into a monolayer. The virus was diluted 10 times with pure DMEM, the cell plate was removed, the original nutrient solution was aspirated, the cell surface was washed 2-3 times with pure DMEM, the diluted virus was added, and it was placed in a 37°C incubator for incubation for 1 hour. During this period, the cell plate should be removed and tilted and mixed every 15 minutes to ensure uniform distribution of the virus. After the incubated cell plate was removed, the incubation solution was discarded, the lid was covered for standby use, 5% (volume fraction, v / v) 2×DMEM and melted low-melting point agar were mixed evenly in a 1:1 volume ratio, added to the cell plate, placed in a 4°C refrigerator for 5 minutes, and the agar was completely solidified. After solidification, the cell plate was placed in a 37°C incubator for several days. The cell plate was removed and 1 ml of crystal violet staining solution was added to cover the cell wells, and the cell plate was placed in a 37°C incubator for 2-3 hours. Take out the cell plate, flush the agar in the cell plate wells with water, and clearly visible plaques can be seen. 1.5 Molecular characterization of different generations of NADC30-like PRRSV FJ1402

[0044] 1.5.1 Viral genome extraction

[0045] Take 200 μl of FJ1402 F7, F40, F60, F100, F113 and F123 virus liquid, extract total RNA according to the OMEGA Bio-Tek Virual RNA kit kit, dissolve it in DEPC water, and freeze it at -70°C for use.

[0046] 1.5.2 Whole-genome sequencing

[0047] The above RNA was reverse transcribed into cDNA, and the primers in Table 1 were used as templates to amplify the fragments of the whole genome of FJ1402 F7, F40, F60, F100, F113 and F123 viruses. PCR system (25μL system): 1μL of upstream and downstream primers, 12.5μL of 2×Phanta Max Master Mix, 1μL of cDNA, and 9.5μL of double distilled water. Reaction cycle parameters: 95℃5min; 95℃30s, 56℃30s, 72℃2min, a total of 35 cycles; finally 72℃5min. The reaction product was detected by 1% agarose gel electrophoresis. The gene amplification product was recovered and purified by the PCR product purification kit, cloned into the pClone007 vector, and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0048] 1.5.3 Genetic stability analysis

[0049] The whole genome sequencing results of FJ1402 F7, F40, F60, F100, F113 and F123 were analyzed, and the amino acid stability of each protein in each generation was compared.

[0050] Table 1 Primer sequences for PRRSV whole genome sequencing

[0051]

[0052] 1.6 Real-time fluorescence quantitative PCR

[0053] Absolute fluorescence quantitative detection of PRRSV RNA content in serum, lung, thymus and lymph nodes. Collect 200μl serum / lung / lymph node / thymus, weigh 1g at different lobe points, cut into pieces and add 5ml sterile PBS to prepare homogenate. After repeated freezing and thawing 3 times, RNA was extracted according to the instructions of the OMEGA kit, reverse transcribed into cDNA, and used as a template for qPCR to detect viral load. 20μL reaction system: SBYR Green Master Mix, double distilled water, standard template, upstream and downstream primers and ROX were 10μL, 7.8μL, 1μL, 0.4μL, and 0.4μL, respectively. Reaction conditions: 50℃2min, 95℃2min, 95 15s, 60℃1min, collect fluorescence signals, 40 cycles. Primer sequences are F: 5'-AATAACAACGGCAAGCAGCAG-3'; R: 5'-CCTCTGGACTGGTTTTGTTGG-3'. A standard curve was constructed using a recombinant plasmid containing the PRRSV ORF7 gene, and the PRRSV virus content was calculated based on the CT value according to the standard curve (Zhang Jie, et al., 2019).

[0054] 1.7 Neutralization test

[0055] Dilute the FJ1402 F7 virus solution to 200 TCID using 4% (v / v) DMEM. 50 , the serum was inactivated at 56°C for 45 minutes, filtered with a 0.22um filter in a clean bench, and the serum was diluted 2 times with pure DMEM. The diluted virus was added to the diluted serum at a volume ratio of 1:1, and after sufficient vortex mixing, it was placed in a 37°C incubator for 1 hour and inoculated onto the marc-145 cells that had grown a monolayer of a 96-well plate. Positive serum control, negative serum control, virus control, and normal cell control were set up. The original nutrient solution in the 96-well plate was discarded, and the incubated virus-serum mixture was added to the well. Four parallel wells can be set for each dilution according to the situation. The cell plate was cultured in a 37°C incubator for 4 days for indirect immunofluorescence (IFA).

[0056] 1.8 Indirect immunofluorescence (IFA)

[0057] Marc145 cells were plated in 96-well plates, and the experiment was performed when the cells grew to a monolayer state of 80-90%. The cells were gently washed with PBST, 200μl / well, washed 3 times; fixed with 100μl of anhydrous ethanol, 4℃, 30min; the fixative was discarded, washed 3 times with PBST; N antibody (1:200) was added, and allowed to act at room temperature for 1h; washed 3 times with PBST, 5min / time; the fluorescently labeled secondary antibody diluted in proportion was added and incubated in the dark, 37℃, 45min; washed 3 times with PBST, and observed with a fluorescence microscope.

[0058] 1.9 Virulence test of high-generation PRRSV FJ1402 in piglets

[0059] 25 healthy piglets aged 3-4 weeks were randomly divided into 5 groups and weighed. Groups 1-4 were injected intramuscularly with 2 ml and intranasally with 2 ml FJ1402 F7 (10 6 TCID 50 / ml)、F100(10 7 TCID 50 / ml)、F113(10 7.3 TCID 50 / ml) and F123(10 7.5 TCID 50 / ml) virus solution, and the last group was treated with PBS in the same way. All pigs were isolated and raised, and clinical symptoms (mental state, breathing, coughing, death) were observed for 15 days. Blood was collected on days 0, 3, 6, 9, 12 and 15 of the challenge to separate serum and measure viremia. All pigs were killed on day 15, their body weights were measured, and their lungs, thymus and lymph nodes were collected to measure viral load and histopathological examination.

[0060] 1.10 Piglet immune protection test

[0061] 1.10.1 Animal grouping and immunization

[0062] Twenty healthy piglets aged 3-4 weeks were randomly divided into 4 groups, namely A, B, C and D. Group A and Group B were injected intramuscularly with 1 ml FJ1402 F100 (F100 (10 7 TCID 50 / ml) and 1ml FJ1402 F123 (10 7.5 TCID 50 Group C was the challenge control group (PBS group in the immune protection experiment), and group D was the blank control group (MOCK group).

[0063] 1.10.2 Attack

[0064] 28 days after the piglets were immunized, 2 ml of FJ1402 F7 (106TCID50 / ml) virus solution was injected intramuscularly and 2 ml was dripped into the nose of the piglets in groups A, B and C. The piglets were observed for 15 days after the challenge.

[0065] 1.10.3 Clinical symptom observation and statistics

[0066] After the piglets were challenged with the virus, they were kept in isolation, and their clinical symptoms were observed and scored every day. The scoring items were mental state, breathing and coughing. The score of each item was an integer between 0 and 4, with 0 points for normal condition, 1 point for extremely mild symptoms, 2 points for mild symptoms, 3 points for severe symptoms, and 4 points for death. Each indicator was scored every day after the challenge and the sum of each indicator was calculated. Finally, the average value of each group was statistically analyzed.

[0067] 1.10.4 Antibody monitoring

[0068] PRRSVN Elisa antibodies were measured according to the IDEXX kit method 2 weeks and 4 weeks after immunization; PRRSV GP5 antibody and neutralizing antibody levels were measured in the serum 4 weeks after immunization.

[0069] 1.10.5 Viremia and viral load

[0070] Blood was collected from piglets on days 0, 3, 6, 9, 12, and 15 after infection, and serum was separated to determine the PRRSV viral load. All pigs were killed on day 15 after infection, and their body weights were measured. Their lungs, thymus, and inguinal lymph nodes were collected to determine the viral load and conduct histopathological examinations.

[0071] 1.11 Indirect Elisa for determination of PRRSV GP5 antibody levels

[0072] (1) Coating: The coating concentration of purified GP5 protein was adjusted to 2 μg / mL in carbonate buffer (pH 9.6), 100 μL / well was added to the ELISA plate, and the coating was carried out at 37°C for 2 h.

[0073] (2) Washing: Discard the liquid in the wells, add 200 μL PBST to each well, shake and wash three times, and then pat dry with absorbent paper;

[0074] (3) Blocking: Add 200 μL of 5% (mass fraction, m / m) skim milk to each well and block at 37°C for 2 h, then wash;

[0075] (4) Serum incubation: Dilute the separated serum at a 2-fold ratio of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, add 100 μL to each well, set up standard positive and negative serum controls, incubate at 37°C for 1 h, and then wash;

[0076] (5) Enzyme-labeled antibody incubation: HRP-labeled goat anti-mouse IgG antibody was diluted 1:1000 in PBST as the secondary antibody, 100 μL / well, incubated at 37°C for 30 min, and then washed;

[0077] (6) Substrate color development: Add 100 μL of TMB color development solution to each well in a light-proof environment and react at 37°C for 10 min;

[0078] (7) Stop: Add stop solution, 50 μL / well, then immediately read OD450nm and calculate the P / N value.

[0079] (8) Judgment criteria: P / N greater than or equal to 2.1 is considered positive; P / N less than 2.1 is considered negative.

[0080] 1.12 Pathological observation

[0081] All piglets were killed 14 days after the infection, and the pathological changes in the lungs of each piglet were observed. Edema, interstitial widening and meat changes were observed and counted. 2 The tissue blocks were fixed with 4% paraformaldehyde; dehydrated with alcohol gradient; transparent with xylene; embedded in paraffin; trimmed; sliced; stained with hematoxylin and eosin. Lung tissue sections were observed under a microscope, and the pathological scoring rules were as follows: 0-no lesions, 1-mild, focal multifocal interstitial pneumonia (<50%), 2-moderate, multifocal merger (50-75%), 3-severe, patchy and extensive (75-90%), 4-severe diffuse (>90%). GraphPad 7 software was used for statistical analysis of the data, and the differences among the groups were compared. P<0.05 indicated significant differences (* indicates P<0.05)

[0082] 1.13 Immunohistochemistry (IHC) detection of PRRSV antigens

[0083] The sectioning method is the same as above, including spreading, sticking, dewaxing, inactivation, antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, counterstaining, dehydration, transparency and sealing.

[0084] 2 Results

[0085] 2.1 PRRSV FJ402 cell passaging and plaque purification

[0086] FJ1402 was cultured and serially subcultured on Marc-145 cells. Viruses from the 7th, 40th, 60th, 100th and 113th generations were used for virus plaque assay. Plaques were selected for purification and serially subcultured to the 123rd generation. Cytopathic effects were observed. The results are shown in the figure. Figure 1 A and Table 2. As the virus generation increases, the time of CPE in inoculated cells gradually becomes faster and more obvious. F7 generation virus inoculated Marc-145 cells, vacuolization, shrinkage, and clustering appeared 4-5 days after culture (dpi), and foci flakes fell off around 7dpi. Cells inoculated with the 100th generation virus solution, vacuolization, shrinkage, and clustering appeared 24-36hpi, and foci flakes fell off at 48hpi.

[0087] The 7th, 40th, 60th, 100th, 113th and 123rd generations of PAM cells were inoculated. The results showed that the low-generation virus fluid caused PAM to swell and break, while the high-generation (F100, F113 and F123) viruses failed to cause obvious CPE in PAM. Figure 1 B, Table 2.

[0088] Virus plaque assay was performed by inoculating Marc-145 cells with the 7th, 40th, 60th, 100th, 113th and 123rd generation viruses. The results showed that CPE appeared at 7 dpi after inoculation of Marc-145 cells with FJ1402 F7, F40 and F60 generation virus liquids, and the CPE was in the form of dots with different sizes. FJ1402 F100, F113 and F123 showed obvious CPE at 5 dpi, with larger plaques and uniform size. Figure 1 C, Table 2.

[0089] Table 2 Biological characteristics, CPE and plaque morphology of FJ1402 at different generations

[0090]

[0091] 2.2 Sequencing analysis of viral genes of different generations of PRRSV FJ1402

[0092] Take the virus liquid of F7, F40, F60, F100, F113 and F123 of PRRSV FJ1402, extract the genomic RNA, and reverse transcribe to obtain cDNA. PCR amplification was performed according to the primers in Table 1 to amplify the whole genome of the virus, connect it to the intermediate vector pCLONE007 (commercially available vector, item number: TSV0101), and send it to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results are shown in Table 3. The results of whole genome sequencing show that the full length of the FJ1402 genome of each generation is 15051bp, and the homology of each generation is as high as 99.9%. There is no addition or deletion of ORF in each generation, but there are amino acid mutations, as shown in Table 3.

[0093] Only two stable amino acid mutations appeared in Nsp2 in the virus liquids of F100, F113 and F123, and one stable amino acid mutation appeared in Nsp1β, Nsp3 and ORF6. The amino acid sequences of Nsp2 in the virus liquids of F113 and F123 were completely consistent, indicating that the viral gene inheritance of FJ1402 was basically stable after the F100 generation, and the genetic characteristics of the viral genes of F113-F123 generations were stable.

[0094] Table 3 Comparison analysis results of amino acid sequences of viral genomes of different generations of PRRSV FJ1402

[0095]

[0096]

[0097] 2.3 Pathogenicity test of PRRSV FJ1402 virus fluids of different generations in piglets

[0098] 2.3.1 Clinical symptoms

[0099] During the 0-15 days after the piglets were challenged with the virus, the piglets' body temperature was measured daily. Figure 2 A) Body weight within 2 weeks ( Figure 2 B) and clinical symptoms ( Figure 2 C), the results are as follows: FJ1402 F7 group had elevated body temperature 3-7 days after infection, and showed clinical symptoms of loss of appetite, depression, lethargy, rapid breathing and mild diarrhea; 7 days after infection, body temperature returned to normal, and the clinical symptoms of PRRSV infection gradually disappeared. FJ1402 F100, F113 and F123 challenge groups did not develop high fever during the entire observation period, and no clinical symptoms of PRRSV infection were found.

[0100] 2.3.2 Viremia and organ viral load

[0101] No viremia was detected in the PBS group, and very low viremia levels (≤10 2The FJ1402 F7 challenge group showed a high level of viremia 3 days after the challenge, and the viremia level decreased slightly 9 days after the challenge, but was still significantly higher than that of the FJ1402 high-generation challenge group ( Figure 3 A). Compared with the PBS group, the lungs of the FJ1402 challenge group ( Figure 3 B) Inguinal lymph nodes ( Figure 3 C) and thymus ( Figure 3 D) The viral loads showed different degrees. The viral loads in the lungs, lymph nodes and thymus of the FJ1402 F7 challenge group were significantly higher than those of the FJ1402 high-generation challenge group (p < 0.0001).

[0102] 2.3.3 Levels of inflammatory factors

[0103] Two weeks after the piglets in the PRRSV FJ1402 F123, F113, F100, and F7 groups were challenged with PRRSV, blood was collected from all piglets to separate serum, and the ELISA method was used to detect the content of corresponding inflammatory factors (IL-2, IL-4, IL-10, IFN-α, TNF-α) in the serum. The results were as follows: There was no significant difference in IL-2, IL-4, IFN-α, and TNF-α among the F123, F113, F100, and F7 challenge groups; while the IL-10 in the PRRSV F7 challenge group was significantly higher than that in the other challenge groups, such as Figure 4 .

[0104] 2.3.4 Observation of thymus status

[0105] The piglets in each group were killed 2 weeks after infection and the thymus atrophy of each group was observed. Figure 5 PBS group ( Figure 5 E) The thymus of piglets is distributed in the neck and chest. The neck is located on both sides of the trachea, in a cord-like shape, reaching the throat forward; it becomes thinner in the neck and chest backward, located in the mediastinum in the front of the chest cavity, and reaches the bottom of the heart backward. High-generation PRRSV FJ1402 F123 ( Figure 5 A) F113( Figure 5 B) and F100( Figure 5 C) There was no significant difference between the challenge group and the PBS group. Two weeks after the challenge, the thymus of the FJ1402 F7 group piglets was significantly atrophied compared with the other groups, with no visible thymus on both sides of the trachea and only a part of the chest thymus remaining.

[0106] 2.3.5 Pathological changes in piglet lungs (HE)

[0107] The lungs were observed by histopathology, and the results showed Figure 6: Compared with the PBS group, the lung interstitium of piglets in the high-generation PRRSVFJ1402FJ1402 F123, FJ1402 F113 and FJ1402 F100 challenge groups was slightly widened, and the lung interstitium of the FJ1402F7 group was significantly widened with inflammatory cell infiltration.

[0108] 2.3.6 Immunohistochemical detection of PRRSV

[0109] The results of immunohistochemical test on lung pathological sections of piglets in each group showed that Figure 7 . The results of immunohistochemistry showed that there were a large number of brown PRRSV antigen-positive cells in the lungs of the FJ1402 F7 challenge group. PRRSV antigens were mainly distributed in bronchial epithelial cells and surrounding alveolar macrophages, and were distributed in the cytoplasm and nucleus. The lung tests in the PBS group were all negative. The PRRSV antigen in the lung macrophages of the FJ1402 F123, F113, and F100 challenge groups was positive, but significantly lower than that of the FJ1402 F7 challenge group.

[0110] 2.4 PRRSV high-generation virus immune protection test for piglets

[0111] 2.4.1 Humoral Immunoassay Results

[0112] ELISA antibody: Blood was collected from piglets at 14 and 28 days after immunization, and serum was separated and tested by IDEXX kit for PRRSV ELISA, as shown in Table 4. All immunized piglets were positive for PRRSV antibodies, and the S / P values ​​were basically the same. The serum antibody of the PBS control group was negative, indicating that humoral immunity can be induced 14 days after F100 and F123 virus immunization.

[0113] Table 4 PRRSV ELISA antibody in serum of experimental piglets after immunization

[0114]

[0115]

[0116] GP5 antibody: PRRSV GP5 ELISA antibody was measured with serum 28 days after immunization, and the results are shown in Table 5. FJ1402F100 immunization group: GP5 antibody titers of 3 pigs were above 1:3200, the highest was 1:12800, and two were lower. FJ1402F123 immunization group: GP5 antibody titers of 5 piglets were all above 1:1600, the highest was 1:6400. PBS group was negative for GP5 antibody.

[0117] Table 5 PRRSV GP5 ELISA antibody titer after immunization of experimental pigs

[0118]

[0119]

[0120] Neutralizing antibodies: The neutralizing antibody levels for FJ1402 were determined using sera from piglets immunized with FJ1402 F100 and FJ1402 F123 for 28 days, as shown in Table 5. The neutralizing antibody levels for PRRSV FJ1402 in all immunized piglets were between 1:3.2 and 1:5, indicating that pigs have the ability to neutralize FJ1402.

[0121] Table 5 PRRSV FJ1402 neutralizing antibody levels in serum of experimental pigs 28 days after immunization

[0122]

[0123] Cytokines: The serum 28 days after immunization was used to measure the levels of IL-2, IL-4, IL-10, IFN-α, and TNF-α. Figure 8 The IL-2 level in the immunized group was significantly higher than that in the challenged control group; the IL-10 and TNF-α in the immunized group were significantly lower than those in the challenged control group; while there were no significant differences in IL-4 and IFNα between the immunized group and the non-immunized group (P>0.05).

[0124] 2.4.2 Clinical symptoms of immunized pigs after challenge

[0125] During the 0-15 days after the piglets were infected with the virus, the piglets' body temperature was measured daily ( Fig. 9 A) Body weight within 2 weeks ( Fig. 9 B) and clinical symptoms ( Fig. 9 C), the results are as follows: the piglets in the challenge control group began to have high fever 4 days after the challenge, and the body temperature of the piglets returned to normal after the 8th day; none of the immunized piglets had fever; the weight gain of the immunized piglets in 2 weeks was significantly higher than that of the challenge control group; none of the immunized piglets showed clinical symptoms of PRRSV infection. The body temperature and clinical manifestations of the PBS group were normal.

[0126] 2.4.3 Viremia and organ viral load

[0127] The PRRSV load in the sera of the immunized group, challenge control and negative control on days 0, 3, 6, 9, 12 and 15 was measured respectively. On day 15 of the challenge, all piglets were killed and the lungs, inguinal lymph nodes and thymus were weighed to measure the PRRSV virus load. Fig.10: The immunized piglets had a low level of PRRSV load before the challenge, and the PRRSV load dropped to below 100 copies / ml after the challenge. The piglets in the challenge control group reached the highest level 6 days after the challenge, and the serum virus dropped and finally stabilized at a certain level on the 9th day, but it was higher than that of the immunized group. No PRRSV was detected in the serum of the negative control group. The viral loads in the lungs and thymus of the immunized group were significantly lower than those in the challenge control group (P>0.05), and the viral loads in the inguinal femoral lymph nodes of the immunized group were lower than those in the challenge control group (P>0.05). No PRRSV was detected in the organs of the negative control group.

[0128] 2.4.4 Observation of thymus status

[0129] The immune piglets were killed 2 weeks after infection and the thymus atrophy of each group was observed. Fig.11 ). Immunization group ( Fig.11 A, B) Piglets and challenge control group ( Fig.11 C), the thymus of the immunized piglets developed normally without atrophy; while the thymus of the chest and neck of the challenged control group was severely atrophied, with only thin flakes of tissue remaining.

[0130] 2.4.5 Pathological changes of lung tissue

[0131] HE staining results of lung tissue sections of piglets in each group are as follows Fig.12 AC: FJ1402 F123 immune group ( Fig.12 A) and FJ1402 F100 immunization group ( Fig.12 B) and the challenge control group ( Fig.12 C), the lungs of non-immunized piglets after challenge showed typical interstitial pneumonia characteristics such as alveolar wall thickening and increased inflammatory cells, while the immunized piglets also had alveolar wall thickening, but the degree of lesions was significantly reduced compared with the non-immunized piglets. Immunohistochemistry showed (12D-F) that PRRSV positive particles were detected in the immunized piglets (12D, E), but the virus particles were reduced compared with the challenge control group.

[0132] 3 Discussions

[0133] Since PRRS was introduced into my country in 1995 (Guo Baoqing, et al., 1996), it has seriously affected the development of my country's pig industry. More importantly, it has caused significant economic losses to the pig industry worldwide (VanderWaal and Deen, 2018). Lineage 1 PRRSV, especially NADC30-Like PRRSV, has become the most prevalent PRRSV lineage in North America and Asia (Fang, et al., 2022, Paploski, et al., 2021, Sun, et al., 2020, Yu, et al., 2020). In 2013, a new PRRSV strain was isolated from clinical materials in my country. Because of its high homology with the PRRSV NADC30 strain in the United States, it was named NADC30-Like PRRSV. It has a unique and untrained 131 aa deletion in the NSP2 protein (Zhao, et al., 2015, Zhou, et al., 2015). At present, NADC30-Like PRRSV has become the main epidemic strain in my country (Guo, et al., 2019). The pathogenicity of PRRSV NADC30-Like isolates to piglets ranges from moderate (Sun, et al., 2016, Wang, et al., 2018, Zhang, et al., 2019, Zhang, et al., 2022, Zhang, et al., 2016) to highly pathogenic (Chen, et al., 2021, Liu, et al., 2017, Zhao, et al., 2015), and most of them are moderately virulent strains. It is reported that, in addition to recombination with classical PRRSV strains, most of the current NADC30-Like PRRSVs have recombined with highly pathogenic PRRSV strains (Shi, et al., 2018, Zhang, et al., 2022, Zhang, et al., 2016), and the pathogenicity has been improved to a certain extent (Chen, et al., 2021). The recombination sites of NADC30-Like PRRSV are relatively random, but statistical analysis shows that the nucleotide positions of the recombination sites are approximately in 5'UTR, nsp1-9, nsp12, ORF3 and ORF5-3'UTR (Guo, et al., 2019, Yu, et al., 2021, Zhang, et al., 2019). Recombination at these sites may be related to PRRSV replication ability and cell tropism (Yu, et al., 2020). Our laboratory isolated and characterized the NADC30-like PRRSV FJ1402, which can be passaged in Marc-145 cells.In order to select PRRSV live vaccine seed, PRRSV FJ1402 was serially passaged to F123 in this study. Whole genome analysis found that the genome of FJ1402 F100 remained relatively stable, and the pathogenicity to piglets was significantly reduced. Piglet immune protection experiments showed that PRRSV FJ1402 F100 and F123 could induce humoral immunity in piglets and protect piglets against the attack of PRRSV FJ1402, showing good immune protection.

[0134] PRRS vaccines mainly include attenuated live vaccines, inactivated vaccines, subunit vaccines, live vector vaccines and DNA vaccines (Li, et al., 2022). Genetically engineered vaccines have the advantages of simple design and good safety. Vaccines expressing PRRSV structural proteins in viruses, bacteria, fungi, plants or DNA vectors have been evaluated in pigs or mice. A study constructed a recombinant TGEV expressing GP5 and M proteins of PRRSV, but it only provided partial protection against PRRSV (Cruz, et al., 2010). In addition, attenuated pseudorabies virus (PRV) is also a potential replication vector of PRRSV. Pigs immunized with PRV expressing GP5 and M proteins can reduce viremia, viral load and lung lesions (Jiang, et al., 2007). In addition, the baculovirus expression system is widely used as a gene delivery and vaccine development tool due to its efficient transgenic ability. The commercialized PRRSV vaccines in my country include 9 strains, namely PRRS MLV / RespPRRS MLV, CH-1R, HuN4-F112, JXA1-P80, R98, TJM-F92, GDr180, PC and CH-1a (Li, et al., 2021). PRRSV RespPRRS MLV and R98 belong to lineage 5, while the other strains belong to lineage 8. The currently popular NADC30-Like PRRSV belongs to lineage 1 (Li, et al., 2022, Wei, et al., 2019, Zhang, et al., 2016, Zhou, et al., 2017). The existing commercialized vaccines provide limited protection against it, and this limitation is also the reason why NADC30-Like PRRSV has become popular in China. In this study, we passaged the lineage 1PRRSV FJ1402 strain from Marc-145 to F123. The results of the piglet immune protection test showed that infection with FJ1402F100 and F123 did not cause obvious clinical symptoms and could protect piglets against attack by the NADC30-Like FJ1402 strain, laying an important foundation for the research and development of lineage 1PRRSV attenuated live vaccine.

[0135] Vertical transmission poses a challenge to the safety of PRRSV-MLVs, including a reduction in the number of live piglets and persistent infection in vaccinated hosts. Charerntantanakul and Wang et al. demonstrated that MLVs-vaccinated pigs had viremia lasting up to 4 weeks after vaccination, leading to transmission of the vaccine virus to unvaccinated pigs (Charerntantanakul, 2012, Wang, et al., 2013). Therefore, the possibility of PRRSV-MLV virus transmission should be considered. Since the PRRS MLV vaccine of Ingelvac was approved for marketing in 1994, it has been widely used in pig farms in China and the United States. Subsequently, it was found that the virus that caused PRRS outbreaks in these pig farms had almost the same nucleotide sequence as the vaccine strain ( et al., 1997). In addition, the recombination between MLV and wild-type strains exacerbates the outbreak of PRRS (Wenhui, et al., 2012). The PRRSV MLV FJ1402 selected in this study was genetically stable after being passed to the F100 generation. The pathogenicity test showed that the selected vaccine strain did not cause obvious clinical symptoms of PRRSV infection in piglets, but PRRSV could be detected in the lungs, lymph nodes and thymus of vaccinated piglets. The new PRRSV MLVs vaccine should make breakthroughs in effectiveness and safety. Therefore, in the later stage of strain selection, immunized piglets and healthy piglets should be mixed and raised to detect whether the vaccine strain has spread. In addition, PRRSV MLVs may have a strong virulence, and it is also particularly important to pass on the selected strains in pigs and detect their pathogenicity in piglets.

[0136] PRRSV MLVs show advantages over inactivated vaccines in terms of immune response, duration of immunity, and better mediated cellular immunity. Infected or vaccinated pigs responded to PRRSV proteins and produced virus-specific antibodies, but early antibodies did not show virus neutralizing activity (Labarque, et al., 2000). The FJ1402 strain selected in this study produced PRRSV antibodies in piglets 14 days after immunization, and antibodies with neutralizing PRRSV FJ1402 were produced 28 days after immunization. It is well known that PRRSV has immunosuppressive effects to evade host immune responses. It has been reported that the production of IFN-α in lung secretions of PRRSV-infected pigs and in PRRSV-infected PAMs and DCs is negligible (Albina, et al., 1998). The thymus is the main organ that supports T cell proliferation, differentiation, and repertoire selection ( -Pflücker, 2004). PRRSV infects thymocytes, and the viral load and whether the thymus is atrophic are related to the virulence of the virus (He, et al., 2012, Ogno, et al., 2019). Thymocyte apoptosis may lead to a decrease in the number of mature T lymphocytes (Butler, et al., 2019). The pathogenicity of FJ1402 F100, F113 and F123 piglets showed that high-passage FJ1402 did not cause thymic atrophy in piglets. This may explain why more severe clinical symptoms are observed in pigs infected with highly pathogenic PRRSV strains. Studies have shown that virulent PRRSV strains can trigger a characteristic inflammatory cytokine storm response, especially mediated by interleukin 1α (IL-1α), IL-1β, IL-6 and TNF-α, accompanied by obvious clinical symptoms, fever and severe lung tissue pathological changes (Amarilla, et al., 2015, Li, et al., 2017, Liu, et al., 2010, Renson, et al., 2017, Sánchez-Carvajal, et al., 2020). FJ1402 F100 and F123 immunized piglets induced lower levels of TNF-α. Studies have reported that IL-2 can be used as a vaccine adjuvant, significantly enhancing the protective efficacy of PRRSV vaccines against PRRSV infection models (Charerntantanakul, 2009). On the one hand, IL-10 can inhibit other inflammatory factors (IL-1β, IL-6, TNF and IL-12) (Enk, 2005), and on the other hand, it can also inhibit the function of effector T lymphocytes (Vicari and Trinchieri, 2004). The sera of piglets immunized with FJ1402 F123 and F100 contain high levels of IL-2 and low levels of IL-10, which supports their use as vaccine strains. Currently available vaccines can only provide protection against homologous strains, but cannot provide effective cross-protection against heterologous strains.

[0137] This study demonstrated through piglet immune protection experiments that FJ1402 F100 and F123 can protect against the attack of the homologous strain FJ1402 (Sublineage1.8).

[0138] The results showed that FJ1402 F100 and F123 can be selected as seed viruses for the attenuated vaccine of PRRSV NADC30-Like strain. FJ1402 F100 was deposited in the General Microbiology Center of China Microbiological Culture Collection on July 2, 2024, with the deposit number CGMCC No.45997, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Claims

1. A weak strain of porcine reproductive and respiratory syndrome virus NADC30, which is classified and named as a weak strain of porcine reproductive and respiratory syndrome virus NADC30. It was deposited in the General Microbiology Center of China Microorganism Culture Collection on July 2, 2024, with the deposit number CGMCC No.45997. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the porcine reproductive and respiratory syndrome virus NADC30 attenuated strain according to claim 1 in the preparation of porcine reproductive and respiratory syndrome vaccine.

3. A porcine reproductive and respiratory syndrome vaccine, characterized in that: The vaccine comprises the porcine reproductive and respiratory syndrome virus NADC30 attenuated strain as described in claim 1.

4. The porcine reproductive and respiratory syndrome vaccine according to claim 3, characterized in that The vaccine is a live vaccine.

5. The porcine reproductive and respiratory syndrome vaccine according to claim 3 or 4, characterized in that: The vaccine is in the form of nasal drops or injection.

6. The porcine reproductive and respiratory syndrome vaccine according to claim 5, characterized in that The dosage form of the nasal drops is solution type, suspension type or emulsion type.

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