CN116790515A - Porcine reproductive and respiratory syndrome virus and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of animal vaccines, and particularly relates to a porcine reproductive and respiratory syndrome virus NJ-1106R strain with the preservation number of: CGMCC No.12008. The strain NJ-1106R can provide good protection for the attack of HP-PRRSV and NADC 30-like strains, and provides a certain material and foundation for further developing the pathogenesis of PRRSV and vaccine application research.
Description
Technical Field
The invention belongs to the technical field of animal vaccines, and particularly relates to a porcine reproductive and respiratory syndrome virus NJ-1106R strain and application thereof.
Background
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS), commonly known as "porcine reproductive and respiratory syndrome", is caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV), and can cause abortion, oestrus return, stillbirth or weaning of sows, death of piglets, and respiratory tract symptoms of pigs of various ages to different degrees after virus infection, and can destroy the immune system of the pigs, causing mixed infection or secondary infection. Therefore, it is imperative to study vaccines against PRRSV variants.
Because of the high pathogenicity of PRRSV variants, the current thinking is to artificially attenuate PRRSV variants and prepare vaccines from the artificially attenuated strains. Because the artificially attenuated strain has similar immunogenicity compared with the original strain and the pathogenicity is obviously reduced, the prepared vaccine has excellent immunity performance, and most importantly, has high safety. In addition, due to the advent of HP-PRRSV and NADC 30-like strains, the development of vaccines that are protective against both types of strains is a hotspot and difficulty of research. HP-PRRSV has a large degree of variation from previously popular PRRSV strains, wherein the deletion of 30 amino acids (positions 482 and 538-566 based on VR-2332 strain) from the Nsp2 gene is often used as a marker for such variant strains. The NADC 30-like strain is characterized in that 131 amino acids, respectively 111 amino acids at 323-433 positions, 1 amino acid at 482 positions and 19 amino acids at 534-552 positions are deleted in the Nsp2 region, and the deletion mode is 111+1+19".
Disclosure of Invention
Aiming at HP-PRRSV and NADC 30-like strains, the invention obtains an artificially attenuated strain NJ-1106R through serial passage attenuation on the basis of separating and identifying a moderately virulent PRRSV NJ-1106 strain.
In 2011, piglets in a Nanjing scale pig farm show mild respiratory problems, lung and lymph nodes of piglets with mild high fever and cough symptoms are collected, a PRRSVNJ-1106 strain with medium toxicity is separated, and the separated strain is identified by methods of reverse transcription-polymerase chain reaction (RT-PCR), indirect Immunofluorescence (IFA), whole gene sequencing analysis and the like, so that the separated strain is proved to be PRRSV. The artificial attenuated strain NJ-1106R is obtained through continuous passage to 150 generations.
Porcine reproductive and respiratory syndrome virus, porcine reproductive and respiratory syndrome virus NJ-1106R strain accession number is: CGMCC No.12008.
A porcine reproductive and respiratory syndrome virus, characterized by the following amino acid positions: amino acid position is based on VR-2332 strain,
(1) ORF1a: amino acid 122 is His, amino acid 151 is Ser, amino acid 780 is Asn, amino acid 799 is Asn, amino acid 1152 is Pro, amino acid 1664 is Gly, and amino acid 2109 is Phe;
(2) ORF1b:656 amino acid Leu,991 amino acid Ala;
(3) GP2: amino acid 10 is Phe and amino acid 51 is Val;
(4) GP3: amino acid 85 is Thr;
(5) GP4: amino acid 160 is Gly;
(6) GP5: amino acid 55 is Phe and amino acid 64 is Ala;
(7) n: amino acid 48 is Thr and amino acid 118 is Ile.
The application of the porcine reproductive and respiratory syndrome virus in preparing a medicament for treating or preventing PRRSV infection.
The application of the porcine reproductive and respiratory syndrome virus in preparing a medicament for preventing HP-PRRSV strain or NADC 30-like strain infection.
The medicament is a vaccine.
The medicine contains live porcine reproductive and respiratory syndrome virus.
The application of the porcine reproductive and respiratory syndrome virus in preparing the medicine for treating or preventing the body temperature rise, the mental appetite drop, the cough, the asthma or the wild virus blood disease caused by PRRSV infection.
The application of the porcine reproductive and respiratory syndrome virus in preparing a medicine for preventing lung sarcoidosis caused by PRRSV infection.
The application of the porcine reproductive and respiratory syndrome virus in preparing a PRRSV antibody-producing medicament.
The beneficial effects are that:
1. the PRRSV attenuated strain NJ-1106R does not cause the disease of piglets after 10 times of back-strengthening tests, which indicates that 10 generations of animals inoculated with the attenuated strain do not have virulence back-strengthening, and the attenuated strain can be used for vaccine research;
2. after single-dose and single-dose repeated inoculation and 10-time overdose inoculation of the PRRSV attenuated strain NJ-1106R on piglets, the body temperature is normal, no clinical symptoms exist, and the lung is not changed by section inspection, so that the PRRSV attenuated strain NJ-1106R is proved to have good safety on pigs;
3. the body temperature of the PRRSV attenuated strain NJ-1106R after immunization of the piglet keeps normal, and the vaccine virus viremia disappears 9 days after immunization, which indicates that the vaccine virus safety is high; 60% of the ELISA antibodies of the pigs turn positive after 14 days of immunization, and all the ELISA antibodies turn positive after 21 days, so that the organisms can generate PRRS specific antibodies;
4. the PRRSV attenuated strain NJ-1106R can provide good protection force for the attack of HP-PRRSV and NADC 30-like strain, and provides a certain material and foundation for further developing the pathogenic mechanism of PRRSV and the research of vaccine application.
Information on preservation of strains
Preservation time: 2016 1 month 8 days
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Preservation number: CGMCC No.12008
Deposit unit address: the institute of microbiology, national institute of sciences, no. 3, north chen west way 1, region of korea, beijing city, postal code: 100101 class naming: porcine reproductive and respiratory syndrome virus.
Drawings
FIG. 1 shows the RT-PCR amplification product of PRRSV NJ-1106N protein gene of the present invention, wherein M is DL2,000DNA Marker,1 is RT-PCR amplification product of PRRSV NJ-1106 strain MARC-145 cell venom, 2 is RT-PCR amplification product of normal MARC-145 cell, 3 is negative control, 4 is PRRSV positive control;
FIG. 2 shows the IFA results of PRRSVNJ-1106 of the present invention, wherein A is the IFA results of normal MARC-145 cells and B is the IFA results of MARC-145 cells infected with PRRSVNJ-1106 strain;
FIG. 3 is a diagram showing the temperature change of piglets after immunization of PRRSV attenuated strain NJ-1106R and challenge of HP-PRRSV;
FIG. 4 is a diagram showing the temperature change of piglets after immunization by PRRSV attenuated strain NJ-1106R and challenge by NADC 30-like strain;
FIG. 5 shows the variation of ELISA antibody of piglets after immunization of PRRSV attenuated strain NJ-1106R and challenge of HP-PRRSV, wherein V represents days after immunization and C represents days after challenge;
FIG. 6 shows the changes of ELISA antibodies of piglets after immunization of PRRSV attenuated strain NJ-1106R and challenge of NADC 30-like strain, V represents days after immunization, and C represents days after challenge.
Detailed Description
The PRRSV attenuated strain NJ-1106R of the present invention is further described below in connection with the specific embodiments.
Isolation, identification and passaging of PRRSVNJ-1106 strain
A moderate virulent PRRSVNJ-1106 strain (GenBank accession number is JX 880029) is obtained by separating the lung and lymph nodes of a piglet suspected of porcine reproductive and respiratory syndrome in a pig farm in Nanjing scale. Lung and lymph node tissues of piglets showing mild high fever and cough symptoms are collected aseptically, cut and ground, diluted into 1:10 suspension with DMEM culture medium, added with penicillin and streptomycin to 100U/ml, and repeatedly frozen and thawed for 3 times at-80 ℃. Centrifuging at 10,000r/min for 30min, collecting supernatant, filtering with 0.22 μm microporous membrane, sterilizing, inoculating to MARC-145 cell monolayer with good growth state, adsorbing at 37deg.C for 1 hr, adding DMEM maintaining solution, standing at 37deg.C with 5% CO 2 Culturing in an incubator. Cytopathy (CPE) was observed daily. When CPE reaches about 80%, collecting virus culture, repeating freezing and thawing for 3 times, centrifuging at 10,000r/min for 5min, and inoculating MARC-145 cells with supernatant to carry out virus passage. And (3) carrying out plaque purification on the 2 nd generation virus for 3 times, inoculating the purified clone virus strain to a MARC-145 cell monolayer with good growth state, and harvesting the virus solution when the CPE reaches 80%, wherein the virus solution is named PRRSV NJ-1106 strain. For PRRSVNJ-1106 strainThe isolated strain is confirmed to be PRRSV by RT-PCR, IFA, whole gene sequencing analysis and other methods, and the isolated strain is 1.1-1.3. The PRRSVNJ-1106 strain was serially passaged to 150 passages to obtain an artificially attenuated strain NJ-1106R.
1.1 preliminary identification of PRRSVNJ-1106 Strain by RT-PCR
The PRRSV NJ-1106 strain virus liquid is repeatedly frozen and thawed for 3 times, and TRIzol reagent is used for extracting total RNA as a template of RT-PCR. Negative and positive controls were also established.
Primers were designed for PRRSV conserved N protein genes as follows:
PRRSV-N1:5’-ATGCCAAATAACAACGG-3’，
PRRSV-N2:5’-TGCTGAGGGTGATGCTGT-3’
RT-PCR reaction system: 25 μl2×1step 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 H were added 2 O makes the total reaction volume 50. Mu.l.
The PCR reaction procedure was: 30min at 50 ℃;94 ℃ for 2min;94 ℃ for 30sec,57 ℃ for 30s,72 ℃ for 30s,30 cycles; extending at 72℃for 10min.
Taking out the product to carry out 1% agarose gel electrophoresis, and separating the toxic NJ-1106 strain and PRRSV positive control to obtain bright specific bands which are consistent with the 369bp of theoretical design; while neither normal MARC-145 cells nor negative controls amplified any bands (FIG. 1). The results initially indicate that the isolated virus is PRRSV.
1.2IFA
After MARC-145 cells were confluent with a monolayer on a 24-well plate, PRRSVNJ-1106 strain virus solution at 1.0MOI was inoculated, while normal cells were used as negative control. Culturing for 24h, and discarding the culture solution when slight CPE appears; fixing with pre-cooled methanol at-20deg.C and 4deg.C for 10min, and washing with PBS for 3 times; dripping PRRSVN protein monoclonal antibody SDOW-17 diluted by 1:500 respectively, reacting at 37 ℃ for 1.0h, and washing with PBS for 3 times; FITC-labeled goat anti-mouse IgG was added at 1:200 dilution, allowed to act at 37℃for 45min, and washed 3 times with PBS; and observed under a fluorescence microscope. The results showed that virus-infected diseased cells showed specific green fluorescence and normal MARC-145 cells did not respond with fluorescence (FIG. 2). The results further demonstrate that the isolated virus NJ-1106 strain is PRRSV.
1.3 Whole gene sequencing analysis
The PRRSVNJ-1106 strain was subjected to whole gene sequencing using the second generation sequencing method. The sequencing results were uploaded to GenBank (accession number JX 880029). Further, the virus NJ-1106 strain was isolated with certainty to be PRRSV.
Whole gene sequencing analysis and Gene characterization of PRRSV attenuated strain NJ-1106R
The PRRSVNJ-1106 strain was serially passaged to 150 passages to obtain an artificially attenuated strain NJ-1106R. And the PRRSV attenuated strain NJ-1106R of the 100 th, 110 th, 120 th, 130 th, 140 th and 150 th generations is subjected to full-gene sequencing by using a second-generation sequencing method, and the generation genes are found to be completely identical, so that the genetic stability of the attenuated strain is good. Compared with PRRSV attenuated strain NJ-1106 strain (GenBank accession number JX 880029) and other PRRSV gene coding sequences currently known at home and abroad, the PRRSV attenuated strain NJ-1106R has the following characteristics: amino acid position is based on VR-2332 strain, (1) ORF1a: amino acid 122 is His, amino acid 151 is Ser, amino acid 780 is Asn, amino acid 799 is Asn, amino acid 1152 is Pro, amino acid 1664 is Gly, and amino acid 2109 is Phe; (2) ORF1b:656 amino acid Leu,991 amino acid Ala; (3) GP2: amino acid 10 is Phe and amino acid 51 is Val; (4) GP3: amino acid 85 is Thr; (5) GP4: amino acid 160 is Gly; (6) GP5: amino acid 55 is Phe and amino acid 64 is Ala; (7) n: amino acid 48 is Thr and amino acid 118 is Ile.
PRRSV attenuated strain NJ-1106R virulence return test
The 1st virulence return test uses the 120 th PRRSV NJ-1106R to inoculate 4-6 week old PRRSV antigen, antibody negative healthy susceptible piglet, neck intramuscular injection 2 ml/head, 10 6.0 TCID 50 Per ml, while no negative control piglets were vaccinated. The 2 nd to 10 th virulence return test the sera positive for PRRSV of the first 1st piglet were mixed and used as the next animal inoculum to inoculate the piglets. All the piglets are inoculated by neck intramuscular injection, and each time, the piglets which are not inoculated are taken as negative control. The rectal temperature of the piglet is measured at fixed points for 14 days after each inoculation of virus, clinical observation is carried out for 14 days, and blood sampling is carried out every other day to detect the serumThe virus was used for the next virulent return passage of the inoculum. The 10 th virulence return test was observed 21 days after virus inoculation. From the start of trial 2, the viral inoculum could not be passaged up by in vitro cell culture. The piglets were normothermic after 10 consecutive trials of virus vaccination and did not show any clinical onset. The result shows that PRRSV NJ-1106R returns to susceptible animals, and the piglets do not get ill after 10 times of back-strength tests, which indicates that 10 generations of the attenuated strain inoculated test animals do not generate virulence back-strength, and the attenuated strain can be used for vaccine research.
Safety study of PRRSV attenuated strain NJ-1106R
The safety test is carried out on the 120 th-generation PRRSV NJ-1106R strain infected piglets, 20 healthy and susceptible piglets which are negative to PRRSV antigens and antibodies of 4-6 weeks are selected, and are randomly divided into 4 groups, 5 piglets are fed in a single room. Group 1 was single dose vaccinated, neck intramuscular injection (10 6.0 TCID 50 /ml) 1 ml/head; group 2 was single dose repeat vaccinations, neck intramuscular injection (10 6.0 TCID 50 Per ml) 1 ml/head, the same dose was repeated at 24 hour intervals; group 3 was vaccinated with 10-fold overdose, neck intramuscular injection (10 7.0 TCID 50 /ml) 1 ml/head; group 4 was 5 non-vaccinated piglets as a control. After the inoculation of the attenuated strain, the rectal temperature of the piglets is measured at fixed points every day for 14 continuous days, clinical symptoms are observed, and the piglets are examined by dissecting and observing whether the lung is diseased or not 21 days after the inoculation of the attenuated strain. The PRRSV attenuated strain NJ-1106R is inoculated in single dose and repeated in single dose, and inoculated in 10 times of overdose, and the change of each index of the piglets is shown in table 1.
TABLE 1 Single dose, single dose repeat Vaccination, 10 times overdose Vaccination of PRRSV attenuated strain NJ-1106R, piglet index change recording Table
The results show that: after the single dose and the single dose of the attenuated strain are repeatedly inoculated to 10 times of the super dose of the attenuated strain to inoculate piglets, the mental state and appetite of the piglets are good, the body temperature is normal, and no clinical symptoms exist. The lungs were examined by dissecting 21 days after inoculation for no meat changes. The PRRSV attenuated strain NJ-1106R is proved to be safe for pigs.
PRRSV attenuated strain NJ-1106R immunopotency assay
25 healthy and susceptible piglets which are negative for PRRSV antigen and antibody at the age of 4-6 weeks are randomly divided into 5 groups, 5 piglets are fed in a single room in a separated mode. Groups 1 and 2 were immunized groups, and were neck intramuscular injected with 1ml of the 120 th generation PRRSV NJ-1106R strain (10) 6.0 TCID 50 Per ml), groups 3 and 4 are challenge control groups, and 1ml of DMEM maintenance solution is injected into neck muscle. Group 5 was DMEM maintenance solution control group, and 1ml DMEM maintenance solution was injected into the neck muscle.
The piglets were observed daily for clinical manifestations and rectal temperature was measured, and serum was collected and isolated on days 5, 7, 9, 11, 14, 21, 28 for detection of vaccinoma viremia and PRRS antibodies.
After 28 days of immunization, groups 1 and 3 were treated with HP-PRRSV strain (10 5.0 TCID 50 /ml) challenge, intramuscular injection 3 ml/head; NADC 30-like strain for group 2 and 4 (10 6.0 TCID 50 /ml) challenge, intramuscular injection 3 ml/head; group 5 is a DMEM maintenance fluid control group, without challenge. The clinical manifestations of piglets were observed daily after challenge and rectal temperature was measured for 21 days. Serum was collected and isolated on days 3, 6, 9, 12, 15, 18, 21 after challenge for viremia and PRRS antibody detection of wild virus (HP-PRRSV or NADC 30-like strain).
5.1 clinical symptom and body temperature Change detection
After PRRSV NJ-1106R strain is immunized, the spirits of pigs in the 1st and 2 nd groups (immunized groups) are normal in appetite, respiratory tract symptoms such as cough and asthma are not generated, and the body temperature is kept normal; pigs in the 3 rd group, the 4 th group (the challenge control group) and the 5 th group (the DMEM maintenance solution control group) have normal appetite, have no respiratory symptoms such as cough, asthma and the like, and have normal body temperature.
After the HP-PRRSV strain attacks the virus, the body temperature of the 5 pigs in the 1st group (immune group) is raised for 2 days after the virus attack, and then the pigs return to normal, the spirit appetite of the 5 pigs is normal, the respiratory tract symptoms such as cough, asthma and the like do not appear, and the body temperature is below 40.0 ℃; the temperature of 5 piglets in group 3 (the toxicity attack control group) rises on day 2 after toxicity attack, the temperature of 4-8 days is above 41 ℃,1 piglet dies on 7 days after inoculation, 1 piglet dies on 8 days after inoculation, and the other 3 pigs have obvious respiratory symptoms such as mental appetite reduction, conjunctivitis, cough, asthma and the like. The group 5 (DMEM maintenance solution control group) has normal appetite, no respiratory tract symptoms such as cough and asthma, and normal body temperature. The body temperature changes of pigs in groups 1, 3 and 5 after the toxin is attacked are shown in figure 3.
After the NADC 30-like strain attacks the toxin, the body temperature of 5 pigs in the group 2 (immune group) is raised in 3 days after the toxin attack, only 1 pig in the 5 pigs has the respiratory symptoms such as slight anorexia, cough, asthma and the like, the body temperature is between 40.0 and 40.5 ℃ in 8-10 days, the spirit appetite of the other 4 pigs is normal, the respiratory symptoms such as cough, asthma and the like do not appear, and the body temperature is below 40.0 ℃; the temperature of 5 pigs in group 4 (the toxicity attack control group) rises from day 2 to day 3 after toxicity attack, the temperature is above 40 ℃ from day 7 to day 14, and the 5 pigs all have obvious respiratory symptoms such as mental appetite reduction, conjunctivitis, dyspnea, shortness, cough, asthma and the like. The group 5 (DMEM maintenance solution control group) has normal appetite, no respiratory tract symptoms such as cough and asthma, and normal body temperature. The changes in pig body temperature after challenge in groups 2, 4, and 5 are shown in figure 4.
5.2 detection of viremia of vaccine and viremia of wild virus
The vaccine toxicity detection method adopts RT-PCR, see 1.1. Vaccine virus viremia exists in 10 pigs of an immune group 5 days after PRRSV NJ-1106R strain is immunized; 7 days after immunization, only 2 pigs in the immune group had vaccine virus toxemia; 9-28 days after immunization, all porcine vaccine virus blood symptoms of the immunized group disappear. The PRRSVNJ-1106R strain is high in safety. The results are shown in Table 2.
TABLE 2 immunization groups (groups 1, 2) piglet vaccine toxemia
The viremia detection of wild virus (HP-PRRSV or NADC 30-like strain) adopts RT-PCR, the method is referenced 1.1, the difference is that the primer is aimed at Nsp2 gene, the primer sequence is as follows:
PRRSV-Nsp2-Fwd:5’-TGATTGGRATGTTGTGCT-3’
PRRSV-Nsp2-Rev 5'-ATRATGGCTTGAGCTGAG-3' (italics for the degenerate base)
The amplified fragment of the HP-PRRSV is 981bp, and the amplified fragment of the NADC 30-like strain is 678bp.
After the HP-PRRSV strain attacks the virus, 4 pigs in group 1 (immune group) have wild virus in 3, 6, 9 and 12 days after the virus attack, 2 pigs in 15 days have wild virus, 1 pig in 18 days have wild virus, and no wild virus exists in 21 days; group 3 (challenge control) had wild toxicity in 2 pigs (pigs No. 61, 62) that died after challenge, and the remaining 3 pigs had wild toxicity throughout the entire observation for 21 days.
After the NADC 30-like strain is challenged, 5 pigs in group 2 (immune group) have wild toxicity on 3, 6, 9 and 12 days after the challenge, 3 pigs on 15 days have wild toxicity, 1 pig on 18 days have wild toxicity, and no wild toxicity is generated on 21 days; the 5 pigs in group 4 (the challenge control group) had wild toxicity during the whole observation process after challenge for 21 days. The results are shown in Table 3.
Pigs of group 5 (DMEM maintenance fluid control group) had no vaccine and wild type toxins.
TABLE 3RT-PCR detection of viremia with wild toxin
5.3PRRS antibody detection
PRRS antibody detection was performed using the porcine reproductive and respiratory syndrome virus antibody detection kit (HerdCheck. PRRS X3).
The ELISA antibodies were raised further after 14 days after immunization of the 5 pigs of group 1 (immunization group) with 3 pigs of ELISA antibodies turning positive, 21 days with all 5 pigs turning positive, and 28 days. ELISA antibody is decreased on days 3, 6 and 9 after virus challenge, and ELISA antibody is increased and maintained at a higher level on days 12, 15, 18 and 21, which indicates that PRRSV attenuated strain NJ-1106R is immunized to generate immune memory; ELISA antibodies are negative 3 days after the virus attack of the group 3 (virus attack control group) 5 pigs, ELISA antibodies are positive 6 days, then ELISA antibodies are raised 9-21 days, and the S/P value reaches 2.6, which indicates that the ELISA antibodies are rapidly raised due to the wild virus HP-PRRSV strain infection, and also indicates that the toxicity and pathogenicity of the HP-PRRSV strain are strong, and the wild virus HP-PRRSV strain can be rapidly proliferated in pigs. ELISA antibodies were not detected in pigs of group 5 (DMEM-maintenance fluid control group), and the results are shown in FIG. 5.
The ELISA antibodies were raised further after 14 days after immunization of the 5 pigs of group 2 (immunization group) with 3 pigs of ELISA antibodies turning positive, 21 days with all 5 pigs turning positive, and 28 days. ELISA antibody is decreased on days 3, 6 and 9 after virus challenge, ELISA antibody is increased on days 12, 15, 18 and 21, which shows that PRRSV attenuated strain NJ-1106R is immunized to generate immune memory; 3 days after the 5 pigs of the 4 th group (the toxicity attack control group) attack the toxicity, 3 pigs of the 5 pigs of the 6 days ELISA antibody are positive, and then the ELISA antibodies are positive and rise in 9-21 days, and the S/P value reaches 2.5, which indicates that the ELISA antibodies are quickly raised due to the wild-type NADC30 strain infection, and also indicates that the toxicity and pathogenicity of the NADC30 strain are strong, and the NADC30 strain can be quickly proliferated in pigs. ELISA antibodies were not detected in pigs of group 5 (DMEM-maintenance fluid control) and the results are shown in FIG. 6.
5.4 detection of pathological changes in the lung
After the HP-PRRSV strain attacks the virus, the dissected lung of 5 pigs in group 1 (immune group) is normal; 2 pigs in group 3 (the toxicity attack control group) die, and the 3 pigs left after the death of piglets and 21 days are examined by dissection, and obvious lamellar solid changes appear in the lung, 60-90% area of the lung bleeds, the pulmonary lymph node bleeds and the submandibular lymph node bleeds.
After the NADC 30-like strain attacks the virus, 4 pigs in group 2 (immune group) have normal dissected lung, and only 1 pig has slight actual change in the diaphragmatic lobe area; the 5 pig anatomic lungs of group 4 (the toxicity attack control group) all show obvious actual changes, 30-70% area bleeding of the lungs, bleeding of the pulmonary lymph nodes and bleeding edema of the submaxillary lymph nodes.
Porcine dissected lungs were normal in group 5 (DMEM maintenance fluid control).
After the HP-PRRSV and the NADC 30-like strains are challenged, except 1 piglet is challenged by the PRRSV attenuated strain NJ-1106R, the body temperature and the spirit appetite of all other piglets (9 piglets) are normal, the viremia of wild virus (HP-PRRSV or the NADC 30-like strains) gradually disappears, the ELISA antibody is firstly reduced and then increased and maintained at a higher level, which indicates that the PRRSV attenuated strain NJ-1106R generates immune memory after being immunized, and can generate immune protection on the HP-PRRSV and the NADC 30-like strains, and the lung is normal after the analysis. And pigs of the HP-PRRSV and NADC 30-like strain virus attack control group are ill, and typical characteristics of high fever, reduced mental appetite, cough, asthma, continuous presence of wild virus blood diseases, rapid rise of ELISA antibodies, obvious actual changes of lungs and the like of PRRSV infection appear.
Based on the above experiments, it is proved that the porcine reproductive and respiratory syndrome virus NJ-1106R can be used for preparing a medicament for treating or preventing PRRSV infection. The medicine can prevent HP-PRRSV strain or NADC 30-like strain infection. The medicament is preferably a vaccine.
The medicine can contain live viruses of porcine reproductive and respiratory syndrome virus NJ-1106R, for example, the medicine can be a live virus vaccine.
The medicine prepared from the porcine reproductive and respiratory syndrome virus NJ-1106R can treat or prevent the body temperature rise, the mental appetite decline, the cough, the asthma or the wild virosis caused by PRRSV infection and also can prevent the pulmonary sarcoidosis caused by PRRSV infection.
The medicine can promote pigs to produce PRRSV immune antibodies.
Claims (9)
1. The porcine reproductive and respiratory syndrome virus is characterized in that the porcine reproductive and respiratory syndrome virus NJ-1106R strain has the preservation number of: CGMCC No.12008.
2. A porcine reproductive and respiratory syndrome virus, characterized by the following amino acid positions: amino acid position is based on VR-2332 strain,
(1) ORF1a: amino acid 122 is His, amino acid 151 is Ser, amino acid 780 is Asn, amino acid 799 is Asn, amino acid 1152 is Pro, amino acid 1664 is Gly, and amino acid 2109 is Phe;
(2) ORF1b:656 amino acid Leu,991 amino acid Ala;
(3) GP2: amino acid 10 is Phe and amino acid 51 is Val;
(4) GP3: amino acid 85 is Thr;
(5) GP4: amino acid 160 is Gly;
(6) GP5: amino acid 55 is Phe and amino acid 64 is Ala;
(7) n: amino acid 48 is Thr and amino acid 118 is Ile.
3. Use of a porcine reproductive and respiratory syndrome virus according to claim 1 or 2 in the manufacture of a medicament for the treatment or prophylaxis of PRRSV infection.
4. The use according to claim 4, characterized in that the porcine reproductive and respiratory syndrome virus according to claim 1 or 2 is used in the preparation of a medicament for preventing infection by HP-PRRSV strain or NADC 30-like strain.
5. Use according to claim 4 or 5, characterized in that the medicament is a vaccine.
6. The use according to claim 4 or 5, characterized in that the medicament comprises live porcine reproductive and respiratory syndrome virus.
7. The use according to claim 4 or 5, characterized in that the porcine reproductive and respiratory syndrome virus is used for the preparation of a medicament for the treatment or prevention of elevated body temperature, decreased appetite, coughing, asthma or avirus blood disorder caused by PRRSV infection.
8. The use according to claim 4 or 5, characterized in that the use of porcine reproductive and respiratory syndrome virus in the manufacture of a medicament for the prevention of pulmonary sarcoidosis caused by PRRSV infection.
9. The use according to claim 4 or 5, characterized in that the use of porcine reproductive and respiratory syndrome virus in the manufacture of a medicament for the production of PRRSV immune antibodies.
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