Porcine reproductive and respiratory syndrome NADC30-like attenuated mutant strain and application thereof

Abstract

The invention belongs to the technical field of veterinary biological products, and particularly relates to a porcine reproductive and respiratory syndrome NADC30-like attenuated mutant strain and application thereof. The invention provides a porcine reproductive and respiratory syndrome NADC30-like attenuated mutant strain, namely an attenuated mutant strain AHFY F140 obtained by continuously transmitting a porcine reproductive and respiratory syndrome NADC30-like virulent AHFY strain on a Mac145 cell for 160 generations for attenuation, and the strain is high in proliferation capacity, good in safety and immune efficacy, and capable of being used for preparing a porcine reproductive and respiratory syndrome NADC30-like attenuated mutant strain for treating the porcine reproductive and respiratory syndrome NADC30-like virulent AHFY strain for treating the porcine reproductive and respiratory syndrome NADC30-like virulent AHFY. The prepared live vaccine can effectively prevent the porcine reproductive and respiratory syndrome caused by infection of the NADC30-liked strain on pigs. The vaccine provided by the invention has the advantages of fast immune efficacy generation, long protection period and the like, and can obviously shorten the immune blank area, reduce the immune frequency and improve the economic benefit.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and specifically relates to a porcine reproductive and respiratory syndrome NADC30-like attenuated mutant strain and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped positive-strand RNA virus that is widely prevalent in pig-raising countries around the world. It can be divided into two genotypes based on genomic and antigenic diversity, namely the European type (PRRSV-1) and the North American type (PRRSV-2). Since the isolation of PRRSV in my country in 1996, most of the reported prevalent strains belong to the PRRSV-2 type, including the classical type, the highly pathogenic type (HP-PRRSV), and the NADC30-like and NADC34-like types. At present, there are multiple PRRSV strains in my country's pig herds at the same time, with NADC30-like being the main prevalent strain.

[0003] Porcine reproductive and respiratory syndrome (PRRS) caused by PRRSV, commonly known as "blue ear disease", is a highly immunosuppressive disease that can cause reproductive or respiratory disorders and persistent infection in pigs. Its clinical symptoms depend on the pig's growth stage, immune status, secondary or co-infected pathogens, environmental conditions and management level. It mainly causes reproductive disorders in sows such as abortion, premature birth, and birth of weak piglets, as well as respiratory system disorders and growth retardation in piglets and fattening pigs. It is a highly contagious disease that seriously endangers the pig industry.

[0004] Vaccine immunization is one of the effective strategies for PRRS prevention and control. The main PRRS attenuated vaccines used in my country include Ingelvac PRRS mLV, CH-1R and R98 strains obtained by in vitro passage of the classic PRRSV2 strain, and JXA1-R, HuN4-F112, TJM-F92 and GDr180 strains attenuated by passage of HP-PRRSV strains. Existing PRRS attenuated vaccine immunization can effectively prevent and control infection with homologous PRRSV wild strains, but its cross-protection is poor. At present, my country has not yet successfully developed an attenuated vaccine strain for NADC30-like infection. Summary of the invention

[0005] To solve the above problems, the present invention provides a porcine reproductive and respiratory virus, which was deposited in the General Microbiology Center of China Culture Collection Administration on December 12, 2023, with the address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.27955.

[0006] Furthermore, the nucleotide sequence of the virus is shown in SEQ ID NO:1.

[0007] The present invention also provides the use of the virus in preparing vaccines.

[0008] The present invention also provides the use of the virus in preparing medicines for preventing or treating porcine reproductive and respiratory syndrome.

[0009] The present invention also provides a vaccine, comprising an antigen and a heat-resistant freeze-drying protective agent. The antigen is the living virus.

[0010] The present invention also provides the use of the vaccine in preparing a medicine for preventing porcine reproductive and respiratory syndrome caused by PRRSV NADC30-like strain.

[0011] The present invention has the following beneficial effects:

[0012] The whole genome sequencing analysis of the porcine reproductive and respiratory syndrome NADC30-like attenuated mutant strain AHFY F140 of the present invention showed that a stable natural double gene deletion occurred in the NSP2 region. After immunization by neck muscle injection in pigs, the clinical indicators of porcine reproductive and respiratory syndrome were observed to be safe. Mac145 cells used for strain proliferation can be well propagated by conventional methods, and the virus titer harvested after inoculation and culture is as high as 10 8.70 TCID 50 / mL and above, far higher than the current routine culture titer of live vaccine strains in China 10 7.0 TCID 50 / mL. The live vaccine prepared with the attenuated mutant strain AHFY F140 of porcine reproductive and respiratory syndrome NADC30-like can effectively prevent the occurrence or spread of NADC30-like porcine blue ear disease. After one immunization, the protection rate can reach 100% for the strong strain AHFY strain of porcine reproductive and respiratory syndrome NADC30-like and other similar types of attack protection. The immune efficacy is compared with other similar vaccines sold in the domestic market, and it is found that the vaccine of the present invention has fast immune efficacy and a long protection period (protection period of more than 6 months), which can significantly shorten the immune blank area, reduce the number of immunizations, and improve economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 Pathogenic effects of AHFY F1 and F140 on Marc-145 cells; A shows the cytopathic effect, aggregation and shedding of Marc-145 cells after 48 hours of infection with AHFY F1; B shows the cytopathic effect, aggregation and shedding of Marc-145 cells after 48 hours of infection with AHFY F140; C shows the uninfected control Marc-145 cells.

[0015] Figure 2 The dynamic changes of body temperature of weaned piglets infected with the virulent strains of F1 and F140 of AHFY;

[0016] Figure 3 Anatomical lesions of lung tissues of weaned piglets infected with the virulent AHFY F1 strain and the attenuated F140 mutant strain; A shows dark brown consolidation and meaty changes in the lungs of AHFY F1-infected pigs; B shows normal red lungs of AHFY F140-infected pigs. DETAILED DESCRIPTION

[0017] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0018] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0021] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0022] Example 1. Screening of the highly virulent AHFY strain of porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-like

[0023] 1. Screening of the highly virulent AHFY strain of PRRSV NADC30-like

[0024] In 2022, a large-scale pig farm in Anhui Province suddenly broke out in sows and weaned piglets, with an incidence rate of about 20% and a mortality rate of about 10%. Through clinical symptoms and pathological anatomy observations, the clinical symptoms of sick piglets were fever, mental depression, loss of appetite, dyspnea, sometimes abdominal breathing, unwillingness to walk or slow walking, cyanosis of peripheral organs such as ears and limbs, dark purple, and obvious blue-purple patches on the skin. Autopsy revealed that the sick pigs had a large amount of effusion in the chest cavity and pericardium, and enlarged lymph nodes; the lungs were dark red in appearance, and dark red patchy congestion and dark red hemorrhages were visible on the surface of the lungs. Serum and lungs were collected for PRRSV, classical swine fever virus (CSFV) antibody and virus antigen testing, and it was determined that the epidemic in the pig farm was caused by infection with the NADC30-like PRRSV strain.

[0025] The present invention fully grinds the fresh lung tissue samples of diseased pigs, treats with double antibodies (penicillin and streptomycin), centrifuges at 12000 rpm / min and 4°C for 10 min, and filters and sterilizes with a 0.22 μm microporous filter membrane. Then, the samples are tested by PCR / RT-PCR for PCV2, pseudorabies virus (PRV), PRRSV, CSFV, porcine parvovirus (PPV), etc., and the results are confirmed to be positive for PRRSV NADC30-like strain. The supernatant of the treated samples is inoculated into Marc145 cells for separation and culture, and finally a high-purity PRRSV NADC30-like strain is obtained, which is named as AHFY strain.

[0026] 2. Identification of the physicochemical and biological characteristics of the highly virulent AHFY strain of PRRSV NADC30-like

[0027] (1) Cytopathic properties: The PRRSV NADC30-like AHFY strain can cause obvious characteristic cytopathic effects in Marc145 cells. Cell lesions can be observed 48 hours after virus infection, manifested by cell shrinkage and aggregation. Obvious cytopathic effects can be observed 96 hours after infection, manifested by cell aggregation and shedding.

[0028] (2) Virus growth curve The growth curve of the PRRSV NADC30-like AHFY strain isolate on Marc-145 cells showed that the virus titer was the lowest at 12 hours after infection, which was 10 2.33 TCID 50 / mL; thereafter, the virus titer increased rapidly until 48h, reaching a maximum of 10 4.43 TCID 50 / mL; from 48h to 120h, the virus titer remained at a high level, the proliferation performance was good, and tended to be stable.

[0029] (3) Whole genome sequencing analysis showed that the homology between the PRRSV NADC30-likeAHFY isolate and other known strains ranged from 60.2% to 89.9%, proving that it was a new strain.

[0030] (4) In the animal regression test, 5 / 5 of the pigs in the challenge group showed obvious PRRSV viremia, mental depression, loss of appetite, drooling, persistent high fever, difficulty breathing, sometimes abdominal breathing, unwillingness to walk or walking slowly, cyanosis of peripheral organs such as ears and limbs, dark purple, and obvious blue-purple patches on the skin; 21 days after the challenge, the pigs were killed and dissected, and multiple lymph nodes in the upper respiratory tract (mandibular lymph nodes, hilar lymph nodes and anterior vena cava lymph nodes) were found to be enlarged, congested, and congested. The lungs were consolidating, meaty, congested, and congested, with hemorrhages, and dark red in appearance. Dark red patchy congestion and dark red hemorrhages were seen on the surface of the lungs, proving that the PRRSV NADC30-like AHFY strain is a highly virulent strain.

[0031] The specific experimental methods are as follows:

[0032] (1) Cytopathic effect: Samples identified as positive for PRRSV NADC30-like AHFY strain were filtered through a 0.22 μm microporous filter, and 300 μL were taken and inoculated on Marc-145 cells grown into a monolayer. The cells were adsorbed in a 37°C 5% CO2 incubator for 1 hour, the supernatant was discarded, and the cells were washed twice with PBS. DMEM culture medium containing 2% newborn calf serum was added and cultured in a 37°C 5% CO2 incubator. A blank control was set up at the same time. The cells were cultured for 48 hours to 96 hours, and the cytopathic effect (CPE) was observed. 48 hours after virus infection, the cells began to become lesions, manifested as cell shrinkage and aggregation; after 96 hours, obvious cytopathic effects were observed, manifested as cell aggregation and shedding, while the control cells were normal. When the CPE reached about 80%, the virus solution was collected. For details, see Figure 1 A.

[0033] (2) Virus growth curve drawing: The virus liquid collected at 12, 24, 48, 60, 72, 84, 96, 108, and 120 hours after the PRRSV NADC30-like AHFY strain was inoculated into a monolayer of Marc-145 cells was used to determine the virus content. A 10-fold serial dilution was performed using cell maintenance medium (DMEM containing 2% newborn calf serum), and 10 －1 , 10 －2 , 10 －3 , 10 －4 , 10 －5 Five dilutions were inoculated into well-growing monolayer Marc-145 cells (96-well cell plate), and 8 wells were inoculated for each dilution, with 0.1 mL per well. Virus positive control wells and cell negative control wells were set up at the same time. The cells were cultured and observed at 37°C for 7 days. Those with CPE were considered infected, and the virus titer (TCID 50 The results showed that the virus titer was lowest at 12h after infecting the cells, which was 102.33 TCID 50 / mL; thereafter, the virus titer increased rapidly until 48h, reaching a maximum of 10 4.43 TCID 50 / mL; from 48h to 120h, the virus titer remained at a high level, the proliferation performance was good, and tended to be stable.

[0034] (3) Whole genome sequencing analysis The Clustal W alignment method of MegAlign software was used to compare the nucleotide homology of the whole genome of the AHFY F1 generation original strain and the representative PRRSV strain. The results showed that the homology between the AHFY strain and the same type of NADC30 strain prevalent in the United States and China ranged from 87.88% to 89.94%; the homology with the HP-PRRSV strain was about 84.7%; the homology with the classical PRRSV strain was about 85.0%; the homology with the American lineage 5 strain was about 84.6%; and the lowest homology with the European strain was about 60.2%. The PRRSV NADC30-like AHFY isolated strain was proved to be a new PRRSV NADC30-like virus strain.

[0035] (4) Animal regression test: 10 healthy susceptible piglets (negative for PRRSV ELISA antibody and PRRSV antigen PCR) at 21 days of age were randomly divided into 2 groups, 5 pigs in each group. One group was challenged with the F1 virus culture of the PRRSV NADC30-like AHFY strain (10 5.0 TCID 50 / mL), 1mL was injected into each head and neck muscle; the two groups were inoculated with cell culture medium only through the same route; clinical observation was performed for 21 days after inoculation. During the period, it was found that 5 / 5 of the pigs in the challenge group had obvious PRRSV viremia, and the mental state of the pigs was always poor according to clinical observation. The body temperature rose to above 40°C, of ​​which 4 pigs rose to 41°C and had a high fever. They had loss of appetite, drooling, and obvious weight loss or decrease. They had difficulty breathing, sometimes abdominal breathing, unwillingness to walk or walked slowly, cyanosis of peripheral organs such as ears and limbs, and dark purple color. Obvious blue-purple patches appeared on the skin. The pigs were killed and dissected 21 days after the challenge, and multiple lymph nodes in the upper respiratory tract (mandibular lymph nodes, hilar lymph nodes, and anterior vena cava lymph nodes) were enlarged, congested, and congested. The lungs were consolidated, meaty, congested, congested, and had bleeding spots. The appearance was dark red, and dark red patchy congestion and dark red bleeding clots were visible on the surface of the lungs, proving that the PRRSV NADC30-like AHFY strain was a strong strain.

[0036] (5) Pure and sterile growth, free of mycoplasma and exogenous virus contamination.

[0037] Example 2: Preparation of PRRSV NADC30-like AHFY strain attenuated mutant vaccine strain

[0038] 1.PRRSV NADC30-like virulent AHFY strain was continuously propagated to 160 generations in Marc-145 cells

[0039] The PRRSV NADC30-like virulent AHFY strain F0 was inoculated into Marc-145 cells grown into a monolayer at a 1% inoculation ratio according to conventional culture methods, and the cell shrinkage, aggregation and shedding and other cytopathic effects (CPE) were observed for 48h to 96h. When the CPE reached about 80%, the virus liquid was collected and recorded as the F1 generation. This was continued to the F160 generation, and the viruses of each generation were frozen and stored in a -80°C refrigerator.

[0040] 2. Identification of the physicochemical and biological characteristics of PRRSV NADC30-like AHFY F140 strain

[0041] (1) Cytopathic properties The PRRSV NADC30-likeAHFY F140 strain can cause obvious characteristic cytopathic effects on Marc-145 cells. Cell lesions can be observed 24 hours after virus infection, manifested as cell shrinkage and aggregation. Obvious cytopathic effects can be observed 96 hours after infection, manifested as cell aggregation and shedding. For details, see Figure 1 B.

[0042] (2) Virus content determination The results of virus content determination of PRRSV NADC30-like AHFY F120, F130, F140, F150, and F160 strains on Marc-145 cells showed that the virus titer was 10 8.10～8.70 TCID 50 / mL, has reached a relatively high level (the conventional vaccine toxin is generally 10 7.0 TCID 50 / mL), and has been maintained at a high level, with excellent proliferation performance and tending to be stable.

[0043] (3) Whole genome sequencing analysis PRRSV NADC30-like AHFY F0, F10, F20, F30, F40, F50, F60, F70, F80, F90, F100, F110, F120, F130, F140, F150, F160 strains whole genome sequence comparison analysis found that the strain had a stable 30bp and 579bp large fragment natural double gene deletion in the NSP2 gene (PRRSV virulence-related gene) since the F90 generation. The deletion sites are located at positions 2402-2431 and 2509-3087 of the viral genome sequence, and the deletion has been stable until the F160 generation. The total length of the genomic DNA of this strain is 14413bp, and its nucleotide sequence is SEQ ID NO: 1. The homology with known domestic and foreign PRRSV NADC30-like epidemic strains is 88.50% to 90.80%, indicating that the obtained PRRSV NADC30-likeAHFY F90 to F160 naturally propagated double gene deletion strains are a new PRRSV candidate vaccine strain. In order to ensure the genetic stability of the vaccine during use, the PRRSV NADC30-likeAHFY F140 generation was used as a vaccine candidate strain.

[0044] (4) Animal safety test PRRSV NADC30-like AHFY F140 strain was tested at 10 7.0 TCID 50 / mL, 5 / 5 of the pigs had no obvious adverse reactions after injection, and their spirit, feeding, drinking, breathing and body temperature were normal, and the body temperature was maintained within 40.0℃. There were no clinical symptoms on the body surface, and anatomical observation showed no obvious enlargement, congestion and congestion of multiple lymph nodes in the upper respiratory tract (mandibular lymph nodes, hilar lymph nodes and anterior vena cava lymph nodes). The lungs were normal, without obvious consolidation, meaty changes, congestion, congestion, hemorrhages and other lesions, and the lungs were normal flesh red in appearance. It is proved that the PRRSV NADC30-likeAHFYF140 strain has become a weak mutant strain and can be used as a vaccine candidate strain.

[0045] The specific experimental methods are as follows:

[0046] (1) The cytopathic effect is the same as the cytopathic effect steps of the above-mentioned strong strains.

[0047] (2) Virus content determination The PRRSV NADC30-like AHFY F120, F130, F140, F150, and F160 strains were inoculated into monolayer Marc-145 cells and then the virus content was determined. A 10-fold serial dilution was performed using cell maintenance medium (DMEM containing 2% newborn calf serum, purchased from GIBCO). 10－5 , 10 －6 , 10 －7 , 10 －8 , 10 －9 Five dilutions were inoculated into well-growing monolayer Marc-145 cells (96-well cell plate), and 8 wells were inoculated for each dilution, with 0.1 mL per well. Virus positive control wells and cell negative control wells were set up at the same time. The cells were cultured and observed at 37°C for 7 days. Those with CPE were considered infected, and the virus titer (TCID 50 / mL). The results showed that the virus titers of PRRSV NADC30-like AHFY F120, F130, F140, F150, and F160 strains were 10 8.10 , 10 8.50 , 10 8.70 , 10 8.70 , 10 8.70 TCID 50 / mL, has reached an extremely high level (the conventional vaccine toxin is generally 10 7.0 TCID 50 / mL), and has been maintained at a high level, with excellent proliferation performance and tending to be stable.

[0048] (3) Whole genome sequencing analysis The virus fluid of PRRSV NADC30-like AHFY F0, F10 F20, F30, F40, F50, F60, F70, F80, F90, F100, F110, F120, F130, F140, F150 and F160 strains was used to extract nucleic acids of each generation using a virus extraction kit according to the conventional virus nucleic acid extraction method, and the whole genome sequencing was performed using the second-generation sequencing method. The whole genome sequence alignment analysis was performed using MegAlign software. It was found that the NSP2 gene (PRRSV virulence-related gene) of this passaged strain had a stable natural double gene deletion of 30 bp and 579 bp since the F90 generation. The deletion sites were located at positions 2402-2431 and 2509-3087 of the viral genome sequence, and the deletion was stable until the F160 generation. The full length of the genomic DNA of the strain is 14413 bp, and its nucleotide sequence is SEQ ID NO: 1. The homology with the known domestic and foreign PRRSV NADC30 epidemic strains is 88.50% to 90.80%, indicating that the obtained PRRSV NADC30-likeAHFY F90 to F160 generation naturally propagated double gene deletion strain is a new PRRSV candidate vaccine strain. In order to ensure the genetic stability of the vaccine during use, the PRRSV NADC30-likeAHFY F140 generation is used as a vaccine candidate strain.

[0049] (4) Animal safety test Five healthy susceptible piglets (both PRRSV ELISA antibody and PRRSV antigen PCR tests were negative) aged 21 days were randomly divided into two groups, with 5 pigs in each group. One group was injected with 1 mL of PRRSV NADC30-like AHFYF140 virus solution into the neck muscle for 10 5.0 TCID 50 / mL / head; another group was injected with 1mL of PRRSV NADC30-likeAHFYF1 generation virulent virus solution into the neck muscle, 10 5.0 TCID 50 / mL / head, serving as the original virulent control group. Clinical observation was conducted 21 days after vaccination. In the PRRSV NADC30-like AHFY F140 strain group, 5 / 5 pigs showed no obvious adverse reactions after injection. Their spirits, feeding, drinking, breathing, and body temperature were normal. The body temperature was maintained below 40.0℃. There were no clinical symptoms on the body surface. Anatomical observation showed no obvious enlargement, congestion, or congestion of multiple lymph nodes in the upper respiratory tract (mandibular lymph nodes, hilar lymph nodes, and anterior vena cava lymph nodes). The lungs were normal without obvious lesions such as consolidation, meaty changes, congestion, congestion, and hemorrhages. The lungs appeared normal flesh-red. Another group of PRRSV NADC30-like AHFY F1 virulent control group injected with the neck muscle showed the same changes in body temperature, clinical symptoms, and pathological changes as those in the virulent screening and identification. The chart of daily rectal temperature changes in piglets infected with the AHFY F140 attenuated mutant strain and the F1 virulent strain (see Figure 2 ) and lung tissue anatomy (see Figure 3 , 3A: The lungs of AHFYF1 challenged pigs were dark brown and fleshy; 3B: The lungs of AHFYF140 challenged pigs were ruddy and normal). This indicates that the AHFY F140 strain has become a weak mutant strain and can be used as a vaccine candidate strain.

[0050] (4) Pure and sterile growth, free of mycoplasma and exogenous virus contamination.

[0051] The virus strain was sent for preservation at the China Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on November 9, 2023, with the preservation number: CGMMCCNo.27955.

[0052] Example 3. Production and application effect of PRRSV NADC30-like freeze-dried live vaccine (AHFY F140 strain)

[0053] 1. Production of PRRSV NADC30-like freeze-dried live vaccine (AHFY F140 strain)

[0054] 1.1 Preparation of PRRSV NADC30-like virus solution (AHFYF140 strain)

[0055] 1.1.1Resuscitation and culture of Marc-145 cells: Take out the frozen Marc-145 cells (purchased from the ATCC cell bank in the United States) from the liquid nitrogen and quickly put them in a 37°C water bath to thaw them quickly. Transfer the cells in each cryotube to a centrifuge tube containing 5mL of cell growth medium, centrifuge at 500r / min for 5min, discard the supernatant, resuspend the cells with 10mL of cell growth medium (MEM nutrient solution containing 10% calf serum, 1% L-glutamine, 100IU / mL penicillin, and 80μg / mL streptomycin), transfer the cells to a disposable sterile cell culture bottle, blow and evenly, and place it in a 37°C, 5% CO2 incubator for static culture. After a good monolayer is formed for 48h to 72h, discard the growth medium, wash the cell surface twice with PBS, digest the cells with cell digestion solution (0.25% trypsin-0.02% EDTA), and when a pinpoint-sized gap appears on the cell surface, aspirate and discard the cell digestion solution, tap the cell bottle, and the cells will fall off evenly along the bottle wall. At this time, use a pipette to absorb the cell growth medium, gently blow and disperse the cells, so that the cells are dispersed into single cells, and expand the dispersed cell suspension at a ratio of 1:3 to 1:4.

[0056] 1.1.2 Virus proliferation: When the cells have covered the monolayer flask wall, inoculate the PRRSV NADC30-like AHFY F140 strain onto the cell monolayer, supplement with cell maintenance medium (DMEM nutrient solution containing 2% calf serum, 100 IU / mL penicillin, 80 μg / mL streptomycin, pH 7.4-7.6), and culture at 37°C. Observe the cytopathic effect every day. When the cytopathic effect reaches 80%, harvest the virus culture medium and store it in a -20°C refrigerator for later use.

[0057] 1.2 The harvested virus liquid was subjected to sterility test and virus content determination, and the qualified virus liquid was mixed with the prepared heat-resistant freeze-drying protective agent at a volume ratio of 7:1. Penicillin and streptomycin were added in proportion to make the final concentrations of penicillin 100 IU / mL and streptomycin 80 μg / mL, respectively. The mixture was shaken thoroughly, quantitatively packaged, and rapidly freeze-dried in a vacuum.

[0058] 1.3 Put the packaged virus liquid into the freeze-drying box, select the pre-freezing temperature of -40℃, the low temperature holding time during pre-freezing is 2h, and the pre-freezing is controlled at -15℃ in the early stage. Quickly freeze the product after releasing the desorption heat: the product temperature in the sublimation stage is -33℃, the plate temperature is set to -2℃, and the sublimation time is 14-17h; the desorption temperature is 26℃, the desorption time is 4h, and the whole freeze-drying process is 17h-27h. Put the stopper out of the box to obtain the PRRSV NADC30-like freeze-dried live vaccine (AHFY F140 strain).

[0059] 1.4 Determination of virus content before and after freeze-drying The finished vaccine was diluted to 10% with cell maintenance medium. -4 , 10 - 5, 10 - 6. Three dilutions were inoculated into 96-well cell plates of Marc-145 cells that had grown into a monolayer. Each dilution was inoculated into 6 wells, with 0.1 mL per well. The cells were cultured in an incubator containing 5% CO2 and 37°C for 5 to 7 days. The TCID was calculated using the Reed-Muench method. 50 The virus content of each portion should be ≥10 5.0 TCID 50 , and compared with the virus content in the control group. The results are shown in Table 1.

[0060] Table 1 Virus content of live vaccine before and after freeze-drying (TCID 50 / head) changes

[0061] Group Before freeze-drying After freeze-drying Example 1 Vaccine Group <![CDATA[10 6.0 ]]> <![CDATA[10 5.9 ]]> Example 2 Vaccine Group <![CDATA[10 6.1 ]]> <![CDATA[10 6.0 ]]> Example 3 Vaccine Group <![CDATA[10 6.2 ]]> <![CDATA[10 6.0 ]]> Conventional vaccine control group <![CDATA[10 6.0 ]]> <![CDATA[10 5.5 ]]>

[0062] As shown in Table 1, the virus content loss of the live vaccine prepared by using the heat-resistant protective agent of the present invention before and after freeze-drying is small, and does not exceed 0.2 titers. This shows that the heat-resistant protective agent provided by the present invention has a better protective effect on the virus than conventional protective agents.

[0063] 2. Vaccine safety and efficacy testing

[0064] 2.1 Safety test for piglets 10 healthy susceptible piglets (both negative for PRRSV ELISA antibody and PRRSV antigen PCR) at 21 days of age were randomly divided into 2 groups, 5 in each group, and the PRRSV NADC30-like freeze-dried live vaccine (AHFY F140 strain) of the present invention and the commercially available highly pathogenic PRRSV conventional live vaccine (GDr180 strain) of the control group were injected with a one-time overdose of the neck muscle, 1.0 mL / head (10 doses). After injection of the AHFY F140 strain vaccine group, 5 / 5 of the pigs had no obvious adverse reactions, and their spirits, feeding, drinking, breathing, body surface, and body temperature were all normal, and the body temperature was maintained at 39.1-39.9°C; after injection of the GD180 strain vaccine group, 4 / 5 of the pigs had no obvious adverse reactions, spirits, feeding, drinking, breathing, body surface, and body temperature were all normal, and the body temperature was maintained at 39.3-40.1°C, and one pig had a transient fever and shortness of breath. The results are shown in Table 2.

[0065] Table 2 Results of the safety test of two vaccines on piglets

[0066]

[0067]

[0068] *Note: "4 / 5 normal" means that one pig in the control group injected with conventional vaccines had a transient respiratory distress reaction.

[0069] The test results show that the freeze-dried AHFY F140 strain vaccine group of the present invention is superior to the conventional vaccine GDr180 strain control group.

[0070] 2.2 Immunization efficacy test for piglets

[0071] Fifteen 21-day-old healthy susceptible piglets (both PRRSV ELISA antibody and PRRSV antigen PCR tests were negative) were randomly divided into three groups, A, B, and C, with 5 piglets in each group, wherein group A was an AHFY F140 vaccine immunization group, group B was an AHFY F140 vaccine immunization group, group B was an AHFY F140 vaccine immunization group, and group C was a saline injection control group. All were injected intramuscularly in the neck, 1.0 dose / head. 28 days after the first immunization, the virus culture fluid (10 5.0 TCID 50 / mL) were used to attack the virus in each group, and 1mL was injected into each head and neck muscle; clinical observation was continued for 21 days after the attack. The results showed that 5 / 5 pigs in the normal saline control group had obvious PRRSV viremia, and the pigs were in poor mental state during clinical observation. The body temperature rose to above 40°C, of ​​which 4 pigs rose to 41°C and had a high fever; loss of appetite, salivation, difficulty breathing, abdominal breathing, unwillingness to walk or walking slowly, cyanosis of the ears and limbs and other peripheral organs, dark purple, and obvious blue-purple patches on the skin; 21 days after the attack, the pigs were killed and dissected, and multiple lymph nodes in the upper respiratory tract (mandibular lymph nodes, hilar lymph nodes and anterior vena cava lymph nodes) were enlarged, congested, and congested, and the lungs were consolidating, meaty, congested, congested, and had bleeding spots. The appearance was dark red, and dark red patchy congestion and dark red bleeding clots were visible on the surface of the lungs. After the AHFY F140 vaccine immunization group, 5 / 5 had no obvious clinical symptoms of PRRSV, and the autopsy showed no obvious lesions in 5 / 5 tissues and organs 21 days after the virus attack. 4 / 5 of the GDr180 vaccine immunization group had no obvious clinical symptoms of PRRSV, and the autopsy showed no obvious lesions in 4 / 5 tissues and organs 21 days after the virus attack, and 1 / 5 had typical clinical symptoms and lesions of PRRSV. The above results show that the AHFY F140 vaccine immunization group can provide complete immune protection, while the GDr180 vaccine immunization group can only provide partial protection. The specific results are shown in Table 3.

[0072] Table 3: PRRSVAHFY F140 live vaccine efficacy test results

[0073]

[0074]

[0075] 2.3 Immune cross protection test

[0076] 40 healthy susceptible piglets (both PRRSV ELISA antibody and PRRSV antigen PCR tests were negative) at 21 days of age were randomly divided into groups 1, 2, 3, and 4, with 10 piglets in each group. Five piglets in each group were randomly selected for neck muscle injection of the live vaccine prepared by the AHFY F140 strain of the present invention, 1.0 dose / pig, and the remaining 5 piglets were used as blank controls. HB2 strain, YT5 strain, and SX strain of each NADC30-like virulent strain isolated in the laboratory were used for neck muscle injection 28 days after the first immunization, and the challenge dose was 10 5.0 TCID 50 / head, and observed continuously for 21 days after the challenge, and the incidence and mortality of the experimental piglets were recorded. After the challenge, none of the piglets in the AHFY F140 immunization groups became ill or died, and all were protected; 5 / 5 of the piglets in the control groups became ill. The results show that the live vaccine prepared by the AHFYF140 strain can resist the attack of the same type of NADC30-like strong strain and play a protective role. The specific results are shown in Table 4.

[0077] Table 4 The protection results of AHFY F140 live vaccine against other NADC30-like virulent strains

[0078]

[0079]

[0080] The above results show that the live vaccine prepared by the PRRS V AHFY F140 strain of the present invention can effectively prevent porcine blue ear disease caused by the PRRSV NADC30-like strain.

[0081] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A porcine reproductive and respiratory virus, characterized in that The strain was deposited in the General Microbiology Center of China Culture Collection Administration on December 12, 2023, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.27955.

2. The virus according to claim 1, characterized in that The nucleotide sequence of the virus is shown in SEQ ID NO:

1.

3. Use of the virus as claimed in claim 1 in preparing a vaccine.

4. Use of the virus as claimed in claim 1 in the preparation of a medicament for preventing or treating porcine reproductive and respiratory syndrome.

5. A vaccine, characterized in that It comprises an antigen and a heat-resistant freeze-drying protective agent; the antigen is the living virus as claimed in claim 1.

6. Use of the vaccine as claimed in claim 5 in the preparation of a medicament for preventing porcine reproductive and respiratory syndrome caused by PRRSV NADC30-like strain.

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