A porcine reproductive and respiratory syndrome virus and its application

Abstract

This invention belongs to the field of animal vaccine technology, and specifically relates to a porcine reproductive and respiratory syndrome virus (PRRSV) strain NJ-1106R, with accession number CGMCC No. 12008. Strain NJ-1106R provides excellent protection against HP-PRRSV and NADC30-like strains, providing a certain material basis for further in-depth research on the pathogenesis of PRRSV and vaccine application.

Description

Technical Field

[0001] This invention belongs to the field of animal vaccine technology, and in particular relates to a porcine reproductive and respiratory syndrome virus strain NJ-1106R, as well as its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as "blue ear disease," is caused by porcine reproductive and respiratory syndrome virus (PRRSV). Infection with the virus can lead to abortion, return to estrus, stillbirths, weak piglets, piglet mortality, and varying degrees of respiratory symptoms in pigs of all ages. It can also damage the pig's immune system, causing mixed or secondary infections. Therefore, research into vaccines targeting PRRSV variants is imperative.

[0003] Due to the high pathogenicity of PRRSV variants, the current approach is to artificially attenuate these variants and then use the attenuated strains to prepare vaccines. Since artificially attenuated strains have similar immunogenicity to the original strains but significantly reduced pathogenicity, the resulting vaccines exhibit excellent immunogenicity and, most importantly, high safety. Furthermore, with the emergence of HP-PRRSV and NADC30-like strains, developing vaccines that protect against both types of strains is a hot research topic and a significant challenge. HP-PRRSV exhibits substantial mutations compared to previously prevalent PRRSV strains, with the deletion of 30 amino acids in the Nsp2 gene (based on the VR-2332 strain, positions 482 and 538–566) often used as a marker for this type of variant. The NADC 30 strain is characterized by the deletion of 131 amino acids in the Nsp2 region, namely 111 amino acids at positions 323–433, 1 amino acid at position 482, and 19 amino acids at positions 534–552, with a deletion pattern of "111+1+19". Summary of the Invention

[0004] In response to HP-PRRSV and NADC30-like strains, this invention, based on the isolation and identification of a moderately virulent PRRSV strain NJ-1106, obtained an artificially attenuated strain NJ-1106R through continuous passage.

[0005] In 2011, piglets at a large-scale pig farm in Nanjing exhibited mild respiratory problems. Lungs and lymph nodes from piglets showing mild high fever and cough were collected, and a moderately virulent PRRSV strain, NJ-1106, was isolated. Identification using reverse transcription-polymerase chain reaction (RT-PCR), indirect immunofluorescence assay (IFA), and whole-genome sequencing confirmed the isolate to be PRRSV. After 150 generations of continuous passage, an artificially attenuated strain, NJ-1106R, was obtained.

[0006] A porcine reproductive and respiratory syndrome virus (PRRSV), strain NJ-1106R, with accession number CGMCC No. 12008.

[0007] A porcine reproductive and respiratory syndrome virus, characterized by the following features at the following amino acid sites: (Based on the VR-2332 strain)

[0008] ①ORF1a: Amino acid at position 122 is His, amino acid at position 151 is Ser, amino acid at position 780 is Asn, amino acid at position 799 is Asn, amino acid at position 1152 is Pro, amino acid at position 1664 is Gly, and amino acid at position 2109 is Phe.

[0009] ②ORF1b: Amino acid at position 656 is Leu, and amino acid at position 991 is Ala;

[0010] ③GP2: The 10th amino acid is Phe, and the 51st amino acid is Val;

[0011] ④GP3: The amino acid at position 85 is Thr;

[0012] ⑤GP4: The amino acid at position 160 is Gly;

[0013] ⑥GP5: The amino acid at position 55 is Phe, and the amino acid at position 64 is Ala;

[0014] ⑦N: The amino acid at position 48 is Thr, and the amino acid at position 118 is Ile.

[0015] The application of the porcine reproductive and respiratory syndrome virus (PRRSV) in the preparation of drugs for the treatment or prevention of PRRSV infection.

[0016] The application of the porcine reproductive and respiratory syndrome virus in the preparation of drugs for preventing infection with HP-PRRSV strains or NADC30-like strains.

[0017] The drug in question is a vaccine.

[0018] The drug contains live porcine reproductive and respiratory syndrome virus.

[0019] The application of the porcine reproductive and respiratory syndrome virus (PRRSV) in the preparation of drugs for the treatment or prevention of fever, loss of appetite, cough, wheezing, or wild-type viremia caused by PRRSV infection.

[0020] The application of the porcine reproductive and respiratory syndrome virus in the preparation of a drug for preventing lung lesions caused by PRRSV infection.

[0021] The application of the porcine reproductive and respiratory syndrome virus in the preparation of drugs that produce PRRSV antibodies.

[0022] Beneficial effects:

[0023] 1. The attenuated PRRSV strain NJ-1106R did not cause disease in piglets after 10 reversion tests, indicating that the attenuated strain did not revert to virulence after 10 generations of inoculation in experimental animals and can be used for vaccine research.

[0024] 2. After single-dose, single-dose repeated, and 10-fold super-dose vaccination of piglets with the attenuated PRRSV strain NJ-1106R, the body temperature was normal and there were no clinical symptoms. Necropsy showed no granulation of the lungs, proving that the attenuated PRRSV strain NJ-1106R is safe for pigs.

[0025] 3. After immunizing piglets with the attenuated PRRSV strain NJ-1106R, their body temperature remained normal, and the vaccine viremia disappeared 9 days after immunization, indicating that the vaccine virus is highly safe. 60% of the pigs had ELISA antibodies that turned positive 14 days after immunization, and all of them turned positive 21 days after immunization, which can induce the body to produce PRRS-specific antibodies.

[0026] 4. The attenuated PRRSV strain NJ-1106R provides good protection against HP-PRRSV and NADC30-like strains, providing certain material and foundation for further in-depth research on the pathogenesis of PRRSV and vaccine application.

[0027] Information on strain preservation

[0028] Deposit date: January 8, 2016

[0029] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0030] Accession number: CGMCC No. 12008

[0031] Address of depositary institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Classification and nomenclature: Porcine reproductive and respiratory syndrome virus. Attached Figure Description

[0032] Figure 1The product is the RT-PCR amplification product of the PRRSV NJ-1106N protein gene of this invention, wherein M is DL2,000 DNA Marker, 1 is the RT-PCR amplification product of PRRSV NJ-1106 strain MARC-145 cell toxicity, 2 is the RT-PCR amplification product of normal MARC-145 cells, 3 is the negative control, and 4 is the PRRSV positive control.

[0033] Figure 2 The images show the IFA results of PRRSVNJ-1106 in this invention, where A represents the IFA results of normal MARC-145 cells and B represents the IFA results of MARC-145 cells infected with the PRRSVNJ-1106 strain.

[0034] Figure 3 The graph shows the changes in body temperature of piglets after immunization with the attenuated PRRSV strain NJ-1106R and challenge with HP-PRRSV.

[0035] Figure 4 This is a graph showing the changes in body temperature of piglets after immunization with the attenuated PRRSV strain NJ-1106R and challenge with the NADC 30-like strain of the present invention.

[0036] Figure 5 The changes in ELISA antibodies in piglets after immunization with the attenuated PRRSV strain NJ-1106R and challenge with HP-PRRSV are shown in the figure. V represents the number of days after immunization and C represents the number of days after challenge.

[0037] Figure 6 The changes in ELISA antibodies in piglets after immunization with the attenuated PRRSV strain NJ-1106R and challenge with the NADC 30 strain are shown in the figure. V represents the number of days after immunization and C represents the number of days after challenge. Detailed Implementation

[0038] The PRRSV attenuated strain NJ-1106R of the present invention will be further described below with reference to specific embodiments.

[0039] 1. Isolation, identification, and attenuation of PRRSVNJ-1106 strain

[0040] A moderately virulent strain of PRRSVNJ-1106 (GenBank accession number JX880029) was isolated from the lungs and lymph nodes of piglets suspected of having porcine reproductive and respiratory syndrome (PRRS) at a large-scale pig farm in Nanjing. Lung and lymph node tissues from piglets exhibiting mild fever and cough symptoms were aseptically collected, minced, ground, and diluted to a 1:10 suspension in DMEM medium. Penicillin and streptomycin were added to a concentration of 100 U / ml, and the suspension was subjected to three freeze-thaw cycles at -80°C. The suspension was centrifuged at 10,000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm microporous membrane for sterilization before being inoculated into a monolayer of well-growing MARC-145 cells. The cells were incubated at 37°C for 1 h for adsorption, supplemented with DMEM maintenance medium, and cultured in a 37°C, 5% CO2 incubator. Cytopathic effects (CPE) were observed daily. When the CPE reached approximately 80%, the virus culture was harvested. After three freeze-thaw cycles, it was centrifuged at 10,000 rpm for 5 minutes. The supernatant was then used to inoculate MARC-145 cells for passage as described above. The second-generation virus was purified by three plaque erosions. The purified cloned virus strain was inoculated onto a monolayer of well-growing MARC-145 cells. When the CPE reached 80%, the virus solution was harvested and named PRRSV NJ-1106. PRRSV NJ-1106 was identified by RT-PCR, IFA, and whole-genome sequencing, confirming that the isolate was PRRSV (see 1.1-1.3 below). PRRSV NJ-1106 was passaged for 150 generations to obtain the artificially attenuated strain NJ-1106R.

[0041] 1.1 Preliminary identification of PRRSVNJ-1106 strain by RT-PCR

[0042] The PRRSV NJ-1106 strain virus solution was repeatedly frozen and thawed three times, and total RNA was extracted using TRIzol reagent as a template for RT-PCR. Negative and positive controls were also set up.

[0043] Primers designed targeting the conserved N protein gene of PRRSV are as follows:

[0044] PRRSV-N1:5'-ATGCCAAATAACAACGG-3',

[0045] PRRSV-N2:5'-TGCTGAGGGTGATGCTGT-3'

[0046] RT-PCR reaction system: 25 μl 2×1Step Buffer, 2 μl PrimeScript 1Step Enzyme Mix, 2 μl PRRSV-N1 (10 μM), 2 μl PRRSV-N2 (10 μM), 3 μl template RNA, and 16 μl RNase Free H2O to make the total reaction volume 50 μl.

[0047] The PCR reaction program was as follows: 50℃ for 30 min; 94℃ for 2 min; 94℃ for 30 sec, 57℃ for 30 s, 72℃ for 30 s, 30 cycles; extension at 72℃ for 10 min.

[0048] The products were extracted and subjected to 1% agarose gel electrophoresis. The results showed bright, specific bands in both the isolated NJ-1106 strain and the PRRSV positive control, consistent with the theoretically designed 369bp band. However, normal MARC-145 cells and the negative control failed to amplify any bands. Figure 1 Preliminary results indicate that the isolated virus is PRRSV.

[0049] 1.2 IFA

[0050] After MARC-145 cells had grown into a confluent monolayer in a 24-well plate, they were inoculated with 1.0 MOI of PRRSVNJ-1106 virus strain, with normal cells serving as a negative control. After 24 hours of culture, the culture medium was discarded when mild CPE appeared. The cells were fixed with pre-cooled methanol (-20℃) at 4℃ for 10 min, and washed three times with PBS. Then, 1:500 diluted PRRSVN protein monoclonal antibody SDOW-17 was added, and the cells were incubated at 37℃ for 1.0 h, followed by three washes with PBS. Finally, 1:200 diluted FITC-labeled goat anti-mouse IgG was added, and the cells were incubated at 37℃ for 45 min, followed by three washes with PBS. The cells were observed under a fluorescence microscope. The results showed that virus-infected diseased cells exhibited specific green fluorescence, while normal MARC-145 cells showed no fluorescence. Figure 2 This result further confirms that the isolated virus strain NJ-1106 is PRRSV.

[0051] 1.3 Whole genome sequencing analysis

[0052] The whole genome of PRRSV NJ-1106 strain was sequenced using next-generation sequencing. The sequencing results were uploaded to GenBank (accession number JX880029). Further isolation confirmed that the virus strain NJ-1106 was PRRSV.

[0053] 2. Whole genome sequencing analysis and gene characteristics of the attenuated PRRSV strain NJ-1106R

[0054] PRRSV NJ-1106 strain was passaged for 150 generations to obtain the artificially attenuated strain NJ-1106R. Using next-generation sequencing, the whole genome of the attenuated PRRSV strain NJ-1106R from generations 100, 110, 120, 130, 140, and 150 was sequenced. The genomes of these generations were found to be identical, indicating good genetic stability of the attenuated strain. Compared with PRRSV NJ-1106 strain (GenBank accession number JX880029) and other known PRRSV gene coding sequences, the attenuated PRRSV strain NJ-1106R has the following characteristics: Based on the VR-2332 strain, ① ORF1a: amino acid position 122 is His, amino acid position 151 is Ser, amino acid position 780 is Asn, amino acid position 799 is Asn, amino acid position 1152 is Pro, 1 ① Amino acid at position 664 is Gly, and amino acid at position 2109 is Phe; ② ORF1b: amino acid at position 656 is Leu, and amino acid at position 991 is Ala; ③ GP2: amino acid at position 10 is Phe, and amino acid at position 51 is Val; ④ GP3: amino acid at position 85 is Thr; ⑤ GP4: amino acid at position 160 is Gly; ⑥ GP5: amino acid at position 55 is Phe, and amino acid at position 64 is Ala; ⑦ N: amino acid at position 48 is Thr, and amino acid at position 118 is Ile.

[0055] 3. Test for virulence reversion of attenuated PRRSV strain NJ-1106R

[0056] The first virulence reversion test used 120th generation PRRSV NJ-1106R to inoculate 4-6 week old healthy susceptible piglets that were negative for PRRSV antigen and antibodies. 2 ml / pig was injected intramuscularly into the neck. 6.0 TCID 50 / ml, with a negative control piglet not inoculated. For the 2nd to 10th virulence reversion tests, serum from piglets that were PRRSV positive in the previous test was mixed and used as the inoculum for the next animal inoculation. All inoculations were performed via intramuscular injection into the neck, with uninoculated piglets serving as a negative control each time. Rectal temperature of piglets was measured at fixed points for 14 consecutive days after each inoculation, and clinical observation was conducted for 14 days. Blood samples were collected every other day to detect the virus in the serum for use as the inoculum for the next virulence reversion passage. The 10th virulence reversion test was observed up to 21 days after inoculation. From the 2nd test onwards, the virus inoculum could not be amplified by in vitro cell culture. After 10 consecutive tests, piglets inoculated with the virus had normal body temperature and showed no clinical symptoms. The results indicate that PRRSV NJ-1106R reverted to susceptible animals, and did not cause disease in piglets after 10 reversion tests, indicating that this attenuated strain did not exhibit virulence reversion after 10 generations of inoculation and can be used for vaccine research.

[0057] 4. Safety study of the attenuated PRRSV strain NJ-1106R

[0058] Safety trials were conducted on piglets infected with the 120th generation PRRSV NJ-1106R strain. Twenty healthy, susceptible piglets aged 4-6 weeks, negative for both PRRSV antigen and antibody, were randomly divided into four groups of five each, housed individually. Group 1 received a single-dose vaccination via intramuscular injection into the neck (10... 6.0 TCID 50 Group 2 received a single-dose repeated vaccination, administered via intramuscular injection in the neck (10 ml) 1 ml / head; Group 2 received a single-dose repeated vaccination, administered via intramuscular injection in the neck (10 ml) 6.0 TCID 50 After administering 1 ml / head, repeat the same dose after 24 hours; Group 3 received a 10-fold super-dose injection, administered via intramuscular injection in the neck (10 ml / head). 7.0 TCID 50 (1 ml / pig); Group 4 consisted of 5 uninoculated piglets as a control. After inoculation with the attenuated strain, rectal temperature was measured daily at fixed points for 14 consecutive days, and clinical symptoms were observed. Piglets were necropsized 21 days after inoculation to observe for lung lesions. Changes in various indicators in piglets after single-dose, repeated single-dose, and 10-fold super-dose inoculation with the PRRSV attenuated strain NJ-1106R are shown in Table 1.

[0059] Table 1. Record of changes in various indicators in piglets after single-dose, repeated single-dose, and 10-fold super-dose vaccination with PRRSV attenuated strain NJ-1106R.

[0060]

[0061]

[0062] The results showed that after single-dose, repeated single-dose, and 10-fold super-dose vaccination of piglets, the piglets maintained good mental state and appetite, normal body temperature, and showed no clinical symptoms. Necropsy examination 21 days post-vaccination revealed no granulation of the lungs. This demonstrates that the PRRSV attenuated strain NJ-1106R is safe for pigs.

[0063] 5. Immunopotency test of attenuated PRRSV strain NJ-1106R

[0064] Twenty-five healthy, susceptible piglets aged 4-6 weeks that were negative for PRRSV antigen and antibodies were randomly divided into 5 groups of 5 piglets each, and housed in single-room isolation. Groups 1 and 2 were the immunization groups, and both groups received an intramuscular injection of 1 ml (10 ml) of the 120th generation PRRSV NJ-1106R strain in the neck. 6.0 TCID 50 Groups 3 and 4 served as challenge control groups, and all received 1 ml of DMEM maintenance solution via intramuscular injection in the neck. Group 5 served as the DMEM maintenance solution control group, and all received 1 ml of DMEM maintenance solution via intramuscular injection in the neck.

[0065] After immunization, the clinical manifestations of piglets were observed and rectal temperature was measured daily. Blood samples were collected on days 5, 7, 9, 11, 14, 21, and 28 to separate serum for the detection of vaccine viremia and PRRS antibodies.

[0066] 28 days after immunization, groups 1 and 3 were treated with the HP-PRRSV strain (10 5.0 TCID 50 Attack with 3ml / head via intramuscular injection; Groups 2 and 4 were treated with a NADC30-like strain (10 ml). 6.0 TCID 50 Group 5 was the DMEM maintenance solution control group, administered 3 ml / pig intramuscularly. Piglets were observed for clinical signs and rectal temperature daily after challenge, up to day 21. Serum was collected on days 3, 6, 9, 12, 15, 18, and 21 post-challenge for viremia and PRRS antibody detection using wild-type virus (HP-PRRSV or NADC30-like strain).

[0067] 5.1 Detection of clinical symptoms and changes in body temperature

[0068] After immunization with PRRSV NJ-1106R strain, pigs in groups 1 and 2 (immunization groups) had normal appetite and spirit, and did not show respiratory symptoms such as coughing or wheezing, and their body temperature remained normal; pigs in groups 3 and 4 (challenge control group) and group 5 (DMEM maintenance solution control group) all had normal appetite and spirit, and did not show respiratory symptoms such as coughing or wheezing, and their body temperature remained normal.

[0069] Following HP-PRRSV challenge, five pigs in Group 1 (immunization group) experienced a slight increase in body temperature two days post-challenge, which subsequently returned to normal. All five pigs exhibited normal appetite and alertness, and did not display respiratory symptoms such as coughing or wheezing; their body temperatures remained below 40.0℃. In Group 3 (challenge control group), five piglets experienced a rise in body temperature on day 2 post-challenge, with temperatures exceeding 41℃ from days 4 to 8. One piglet died seven days after inoculation, and another died eight days after inoculation. The remaining three pigs exhibited significant decreased appetite and alertness, conjunctivitis, and respiratory symptoms such as coughing and wheezing. Pigs in Group 5 (DMEM maintenance solution control group) maintained normal appetite and alertness, and did not display respiratory symptoms such as coughing or wheezing; their body temperatures remained normal. The changes in body temperature in Groups 1, 3, and 5 after challenge are shown in the figure. Figure 3 .

[0070] Following challenge with the NADC30-like strain, five pigs in Group 2 (immunized group) experienced a slight increase in body temperature three days post-challenge. Only one pig exhibited mild anorexia and respiratory symptoms such as coughing and wheezing, with its temperature ranging from 40.0-40.5℃ on days 8-10. The other four pigs maintained normal appetite and alertness, without coughing or wheezing, and their temperatures remained below 40.0℃. In Group 4 (challenge control group), five pigs experienced a rise in body temperature on days 2-3 post-challenge, exceeding 40℃ on days 7-14. All five pigs exhibited significant decreased appetite and alertness, conjunctivitis, and respiratory symptoms including dyspnea, rapid breathing, coughing, and wheezing. Pigs in Group 5 (DMEM maintenance solution control group) maintained normal appetite and alertness, without coughing or wheezing, and their temperatures remained normal. The changes in body temperature in Groups 2, 4, and 5 after challenge are shown in the figure. Figure 4 .

[0071] 5.2 Detection of viremia in vaccines and wild-type viruses

[0072] The vaccine virulence was detected using RT-PCR, as described in section 1.1 above. Five days after immunization with the PRRSV NJ-1106R strain, all 10 pigs in the immunized group showed vaccine viremia; seven days after immunization, only two pigs in the immunized group showed vaccine viremia; and from 9 to 28 days after immunization, vaccine viremia disappeared in all pigs in the immunized group. This indicates that the PRRSV NJ-1106R strain has high safety. The results are shown in Table 2.

[0073] Table 2. Viremia of swine vaccines in immunized groups (Groups 1 and 2)

[0074]

[0075] Viremia detection of wild-type virus (HP-PRRSV or NADC30-like strain) was performed using RT-PCR, following the method described in 1.1, except that the primers were targeted at the Nsp2 gene. The primer sequences are as follows:

[0076] PRRSV-Nsp2-Fwd:5'-TGATTGGRATGTTGTGCT-3'

[0077] PRRSV-Nsp2-Rev:5'-ATRATGGCTTGAGCTGAG-3' (Italics represent degenerate bases)

[0078] The HP-PRRSV amplification fragment is 981 bp, and the NADC30-like strain amplification fragment is 678 bp.

[0079] After challenge with the HP-PRRSV strain, in Group 1 (immunization group), 4 out of 5 pigs had wild-type virus on days 3, 6, 9, and 12, 2 pigs had wild-type virus on day 15, 1 pig had wild-type virus on day 18, and none had wild-type virus on day 21. In Group 3 (challenge control group), the 2 pigs that died after challenge had wild-type virus (pig numbers 61 and 62), and the remaining 3 pigs had wild-type virus throughout the observation period, lasting until day 21.

[0080] After challenge with the NADC30-like strain, all 5 pigs in group 2 (immunized group) had wild-type virus on days 3, 6, 9, and 12 post-challenge; 3 pigs had wild-type virus on day 15; 1 pig had wild-type virus on day 18; and none had wild-type virus on day 21. All 5 pigs in group 4 (challenge control group) had wild-type virus throughout the entire observation period after challenge, continuing until day 21. The results are shown in Table 3.

[0081] The pigs in Group 5 (DMEM maintenance fluid control group) did not contain either vaccine virus or wild virus.

[0082] Table 3. Viremia of wild-type virus detected by RT-PCR.

[0083]

[0084] 5.3 PRRS antibody detection

[0085] PRRS antibodies were detected using the Porcine Reproductive and Respiratory Syndrome Virus Antibody Detection Kit (HerdCheck*PRRSX3).

[0086] In Group 1 (immunization group), 3 out of 5 pigs tested positive for ELISA antibodies 14 days after immunization, and all 5 pigs tested positive on day 21. ELISA antibody levels further increased after day 28. ELISA antibody levels decreased on days 3, 6, and 9 after challenge, but increased and remained at a high level on days 12, 15, 18, and 21, indicating immune memory following immunization with the attenuated PRRSV strain NJ-1106R. In Group 3 (challenge control group), 5 pigs were negative for ELISA antibodies 3 days after challenge, and all were positive on day 6. Subsequently, ELISA antibody levels increased from days 9 to 21, with an S / P value reaching 2.6, indicating that infection with the wild-type HP-PRRSV strain caused a rapid increase in ELISA antibodies. This also demonstrates the high virulence and pathogenicity of the HP-PRRSV strain, enabling it to rapidly proliferate in pigs. No ELISA antibodies were detected in pigs in Group 5 (DMEM maintenance solution control group). See the results below. Figure 5 .

[0087] In Group 2 (immunization group), 3 out of 5 pigs tested positive for ELISA antibodies 14 days after immunization, and all 5 pigs tested positive on day 21. ELISA antibody levels further increased after day 28. ELISA antibody levels decreased on days 3, 6, and 9 after challenge, and increased on days 12, 15, 18, and 21, indicating that immune memory was generated after immunization with the attenuated PRRSV strain NJ-1106R. In Group 4 (challenge control group), 5 pigs tested negative for ELISA antibodies 3 days after challenge, 3 out of 5 pigs tested positive on day 6, and subsequently tested positive and increased on days 9-21, with an S / P value reaching 2.5. This indicates that infection with the wild-type NADC30 strain caused a rapid increase in ELISA antibodies, and also suggests that the NADC30-like strain is highly virulent and pathogenic, and can rapidly proliferate in pigs. No ELISA antibodies were detected in pigs in Group 5 (DMEM maintenance solution control group). See the results below. Figure 6 .

[0088] 5.4 Detection of pathological changes in the lungs

[0089] After challenge with HP-PRRSV strain, the lungs of the 5 pigs in Group 1 (immunization group) were normal upon dissection; 2 of the 5 pigs in Group 3 (challenge control group) died. The necropsy of the dead piglets and the remaining 3 piglets 21 days later showed obvious patchy consolidation in the lungs, hemorrhage in 60-90% of the lung area, hemorrhage in the hilar lymph nodes, and hemorrhage in the submandibular lymph nodes.

[0090] After challenge with the NADC30-like strain, 4 out of 5 pigs in Group 2 (immunization group) had normal lungs upon dissection, and only 1 pig showed mild consolidation in the diaphragmatic lobe region of its lungs; all 5 pigs in Group 4 (challenge control group) showed significant consolidation in their lungs upon dissection, with hemorrhage in 30-70% of the lung area, hemorrhage in the hilar lymph nodes, and hemorrhage and edema in the submandibular lymph nodes.

[0091] The lungs of pigs in Group 5 (DMEM maintenance fluid control group) were all normal upon dissection.

[0092] After challenge with HP-PRRSV and NADC30-like strains, all piglets immunized with the attenuated PRRSV strain NJ-1106R, except for one pig in the NADC30-like strain challenge group, had normal body temperature, mental state, and appetite. Viremia from wild-type virus (HP-PRRSV or NADC30-like strain) gradually disappeared, and ELISA antibodies initially decreased and then increased, remaining at a high level. This indicates that immunization with the attenuated PRRSV strain NJ-1106R produced immune memory, providing immune protection against HP-PRRSV and NADC30-like strains. Post-mortem examination revealed normal lungs. In contrast, all pigs in the HP-PRRSV and NADC30-like strain challenge control groups developed symptoms, exhibiting typical characteristics of PRRSV infection, including high fever, decreased mental state and appetite, coughing, wheezing, persistent wild-type virus viremia, rapidly increasing ELISA antibodies, and significant lung consolidation.

[0093] Based on the above experiments, it is demonstrated that the porcine reproductive and respiratory syndrome virus NJ-1106R can be used to prepare a drug for treating or preventing PRRSV infection. This drug can prevent infection by HP-PRRSV strains or NADC30-like strains. The drug is preferably a vaccine.

[0094] The drug may contain live porcine reproductive and respiratory syndrome virus (PRRSV) NJ-1106R, such as a live virus vaccine.

[0095] The drug prepared from the porcine reproductive and respiratory syndrome virus NJ-1106R can treat or prevent fever, loss of appetite, cough, wheezing or wild-type viremia caused by PRRSV infection, and can also prevent pulmonary sarcoma caused by PRRSV infection.

[0096] The aforementioned drugs can induce pigs to produce PRRSV immune antibodies.

Claims

1. A porcine reproductive and respiratory syndrome virus (PRRSV) NJ-1106R, characterized in that... The porcine reproductive and respiratory syndrome virus (PRRSV) NJ-1106R has the accession number CGMCC No. 12008.

2. The use of the porcine reproductive and respiratory syndrome virus NJ-1106R as described in claim 1 in the preparation of a medicament for preventing PRRSV infection.

3. The application according to claim 2, characterized in that... PRRSV is either the HP-PRRSV strain or a NADC30-like strain.

4. The application according to claim 2, characterized in that... The drug in question is a vaccine.

5. The application according to claim 2, characterized in that... The drug contains live porcine reproductive and respiratory syndrome virus (PRRSV) NJ-1106R.

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