A porcine reproductive and respiratory syndrome virus HB03R strain of NADC30 class attenuated strain, vaccine and application thereof

Abstract

This invention belongs to the field of biotechnology and discloses a NADC30 type porcine reproductive and respiratory syndrome virus (PRRSV) strain HB03R, its vaccine, and its application. The invention uses NADC30 type PRRSV strain HB03 as the parent strain, and obtains an attenuated strain through cell passage to 100 generations for purification. Compared with the parent strain HB03, the HB03R strain has one site of 11 consecutive amino acid deletions and 21 site-directed mutations at the genomic amino acid level. Based on this HB03R strain, a high-immunoprotection, stable, high-yield, and safe attenuated vaccine against NADC30 type PRRSV strain HB03R for emergency preventative vaccination is prepared. This attenuated strain has high safety; high-dose and repeated-dose immunizations with the invented attenuated vaccine have no effect on the clinical manifestations of piglets.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and discloses a NADC30 type porcine reproductive and respiratory syndrome virus HB03R strain, its vaccine, and its application. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is a serious viral infectious disease affecting the global swine industry, caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The main clinical symptoms include reproductive disorders in pregnant sows (abortion, premature birth, stillbirth, and mummified fetuses, etc.) and respiratory diseases in pigs of all ages, which can lead to secondary infections. PRRSV belongs to the Arteritisviridae family and the Arteritisvirus genus. It is an enveloped, non-segmented, single-stranded positive-sense RNA virus. Based on differences in viral genome and antigenicity, PRRSV is classified into European type (genotype 1) and North American type (genotype 2).

[0003] Since 2012, genotype 2 strains with high homology to the US NADC30 strain have been isolated in China. Due to their close genetic evolutionary relationship with the NADC30 strain, these viruses are currently collectively referred to as NADC30-like PRRSV strains in China. The hallmark gene characteristic of these strains is the deletion of 131 discontinuous amino acids in the NSP2 protein, resulting in significant genomic variation and classifying them as moderately virulent PRRSVs. Furthermore, these strains readily recombine with other PRRSV strains, leading to rapid changes in their pathogenicity and further complicating the control of PRRS. Since 2014, the prevalence of NADC30-like PRRSV strains in my country has shown an intensifying trend, with clinical detection rates increasing year by year. They have become the main circulating strain in China after classic strains and highly pathogenic strains (HP-PRRSV), posing new challenges to pig farming in my country.

[0004] Vaccination is a common measure for controlling PRRS. Existing PRRS vaccines mainly include inactivated vaccines, live attenuated vaccines, and novel genetically engineered vaccines. Unfortunately, research shows that existing commercially available PRRSV vaccines offer very limited protection against NADC30-like strains, further contributing to the rapid spread of these strains. Therefore, there is an urgent need to develop a vaccine with stronger protection against NADC30-like strains to control their prevalence.

[0005] Attenuated live vaccines offer multiple advantages, including multiple immunization routes, small immunization doses, long duration of immunity, no need for adjuvants, and rapid delivery of immune cells to the respiratory and digestive mucosa after immunization. These cells interact with pathogens to provide immune protection, preventing further infection and invasion. Furthermore, their immunomodulatory effects also contribute to pathogen elimination. Therefore, developing a safe and reliable attenuated live vaccine against NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) is of significant practical importance for the control of currently prevalent NADC30-like PRRSV strains. Summary of the Invention

[0006] In view of the current lack of NADC30 porcine reproductive and respiratory syndrome virus (PRRSV) attenuated vaccines with good immunization efficacy and safety in the market, the purpose of this invention is to provide a NADC30 porcine reproductive and respiratory syndrome virus (PRRSV) HB03R strain attenuated vaccine and its preparation method, so as to achieve the production of a vaccine with high immunization protection rate, high yield, good stability, low cost and safety through an efficient and practical preparation method.

[0007] A live attenuated strain of NADC30 porcine reproductive and respiratory syndrome virus, HB03R, was deposited at the China Center for Type Culture Collection on March 24, 2026, with accession number CCTCC NO: V202631.

[0008] A live attenuated NADC30 porcine reproductive and respiratory syndrome virus vaccine, the active ingredient of which is the live attenuated HB03R strain of NADC30 porcine reproductive and respiratory syndrome virus as described in claim 1.

[0009] NADC30 type porcine reproductive and respiratory syndrome virus attenuated vaccine, comprising a protein and a pharmaceutically acceptable carrier, wherein the protein comprises an amino acid sequence as shown in SEQ ID NO:43.

[0010] Furthermore, the attenuated live vaccine is a freeze-dried vaccine, which is made by mixing the vaccine preparation virus liquid and the freeze-drying protectant at a ratio of 1:1 (v / v), aseptically dispensing, and freeze-drying. The freeze-drying protectant is formulated as follows: 25g gelatin, 40g sucrose, 20g enzymatically hydrolyzed casein, 15g tryptone, 4g dipotassium hydrogen phosphate, 0.56g potassium dihydrogen phosphate, dissolved in an appropriate amount of water for injection, and then brought to a final volume of 500ml. The mixture is then autoclaved at 121℃ for 30 minutes and stored at 37℃.

[0011] Furthermore, the viral solution used for seed preparation is prepared using the HB03R strain described in claim 1 as the seed virus, and the viral titer of the viral solution is not less than 10. 7.0 TCID50 / ml; Each dose of the freeze-dried vaccine product contains 10 HB03R strain virus. 6.0 TCID 50 / ml.

[0012] A method for preparing the NADC30 class porcine reproductive and respiratory syndrome virus attenuated vaccine includes the following steps: S1. Preparation of virus solution for seedling production: Using the HB03R strain as seed virus, qualified virus solution for seedling production is obtained through cell proliferation, virus inoculation, culture and harvesting, filtration and purification and semi-finished product inspection. S2. Vaccine preparation: The qualified vaccine virus solution is slowly thawed at 4°C and mixed evenly with the pre-cooled sterile freeze-drying protectant at a volume ratio of 1:1 to obtain a vaccine semi-finished product mixture. S3. Aseptic dispensing and freeze-drying: The vaccine semi-finished product mixture is aseptically and quantitatively dispensed into freeze-drying bottles. After freeze-drying, it is stored at low temperature to obtain the attenuated live vaccine product. S4. Finished Product Inspection: The freeze-dried vaccine product undergoes sterility testing, mycoplasma testing, exogenous virus testing, and virus titer determination. Once all indicators meet the requirements of the "Veterinary Biological Products Quality Standard of the People's Republic of China", it is considered a qualified vaccine.

[0013] The application of the NADC30 type porcine reproductive and respiratory syndrome virus attenuated strain HB03R, wherein the HB03R strain is used to prepare an attenuated vaccine to prevent piglets from being infected with NADC30 type porcine reproductive and respiratory syndrome virus.

[0014] The application of the NADC30 type porcine reproductive and respiratory syndrome virus attenuated vaccine, wherein the attenuated vaccine is used to prevent piglets from being infected with NADC30 type porcine reproductive and respiratory syndrome virus, the method of administration is intramuscular injection, the dosage is 1 ml per piglet, and 28-day-old PRRSV antigen and antibody double negative piglets are suitable for vaccination.

[0015] Furthermore, the attenuated vaccine exhibits good immunoprotective efficacy against the homologous NADC30 PRRSV HB03 strain and a certain degree of immunoprotective efficacy against the heterologous NADC30 PRRSV E3 strain. Piglets can produce specific ELISA antibodies 14 days after immunization, with an immunoprotective rate of ≥60%. It can effectively inhibit viral replication in piglets, reduce pathological damage to lung tissue, and has no obvious adverse reactions.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This protocol uses a cell-passaged attenuated strain of the currently popular NADC30-like PRRSV strain as the seed virus for vaccine preparation, ensuring the high immunoprotective efficacy of the vaccine. 2. The attenuated strain prepared by this method has good stability. The NADC30-like PRRSV HB03R attenuated strain cultivated maintained good stability after cloning and purification. Small vacuoles within 1 mm were selected for cloning and purification. When preparing attenuated seedlings, the diameter and morphological characteristics of the phagocytic spots were detected three times, which maintained the characteristics of small vacuoles. 3. This method uses Marc-145 cells in suspension culture to prepare viral solution for vaccine production, which ensures high-efficiency vaccine production; 4. The attenuated strain prepared in this method has high safety, and the attenuated vaccine invented by high-dose and repeated-dose immunization has no effect on the clinical manifestations of piglets.

[0017] This plan will provide a material basis and technical support for controlling the spread of NADC30 type porcine reproductive and respiratory syndrome virus strains in clinical practice and reducing the pressure of PRRS prevention and control, and has broad application prospects. Attached Figure Description

[0018] Figure 1 Cytopathic effects and RT-PCR identification images of NADC30 porcine reproductive and respiratory syndrome virus strain HB03 inoculated into Marc-145 cells. In the images, A is the uninoculated blank cell control; B is the cytopathic effect after virus inoculation; C is the RT-PCR identification image. M: DNA Marker DL2000; 1: Sample; 2: Negative control; 3: Positive control. Figure 2 Results of gene mutation analysis of NADC30 porcine reproductive and respiratory syndrome virus strain HB03R; Figure 3 The results of body temperature monitoring in the virulence comparison test of NADC30 porcine reproductive and respiratory syndrome virus strain HB03R and its parent strain (HB03); Figure 4 For the virulence comparison test of NADC30 porcine reproductive and respiratory syndrome virus strain HB03R and its parent strain (HB03), pathological autopsy and HE staining of tissue sections are shown. (A is lung tissue autopsy, B is lung tissue section HE staining). Figure 5 The ELISA antibody results for the efficacy test of the NADC30 porcine reproductive and respiratory syndrome virus HB03R strain attenuated vaccine. Figure 6 The results of body temperature monitoring in the efficacy test of the NADC30 porcine reproductive and respiratory syndrome virus HB03R strain attenuated vaccine; Figure 7 The results of the efficacy test of the NADC30 porcine reproductive and respiratory syndrome virus HB03R strain attenuated vaccine are viremia and tissue load. A represents viremia and B represents tissue load. Figure 8 The images show pathological autopsy and HE staining of tissue sections from a live attenuated NADC30 porcine reproductive and respiratory syndrome virus (HB03R) vaccine efficacy test. A is an autopsy of lung tissue, and B is a lung tissue section stained with HE. Detailed Implementation

[0019] This invention is achieved through the following technical solution: Example 1: Screening and generation of NADC30 porcine reproductive and respiratory syndrome virus HB03R strain attenuated vaccine 1. Isolation and identification of the virus strain The NADC30 porcine reproductive and respiratory syndrome virus (PRRSV) HB03R strain attenuated vaccine described in this embodiment has an active ingredient that is an attenuated strain of NADC30 porcine reproductive and respiratory syndrome virus HB03 obtained through cell passage attenuation. The parent strain HB03 of this attenuated strain was obtained through the following cell culture isolation method combined with molecular biological identification: 1.1 Pathological sample collection Tissue samples were collected from pigs exhibiting typical symptoms of porcine reproductive and respiratory syndrome (PRRS) (fever, cough, lethargy, and loss of appetite) at a pig farm in Hubei Province. The samples were washed three times with sterile physiological saline, and sterile DMEM culture medium was added at a ratio of 1:5 (w / v). The samples were then homogenized in an ice bath using a sterile grinder. The samples were subjected to three freeze-thaw cycles (frozen at -80℃ for 30 min and thawed in a 37℃ water bath), and centrifuged at 4℃ and 8000 rpm for 20 min. The supernatant was collected and filtered through a 0.22 μm sterile filter to obtain the treated solution, which was stored at -80℃ for later use.

[0020] 1.2 Cell Culture and Virus Isolation After reviving and culturing Marc-145 cells, the above-mentioned pathogen treatment solution was seeded into a monolayer of Marc-145 cells at a ratio of 10% (v / v). The cells were incubated at 37°C for 1.5 h, with the culture flask gently shaken once every 30 min to ensure that the pathogen treatment solution was evenly applied to the cells. After the adsorption was completed, the unadsorbed pathogen treatment solution was discarded, and DMEM maintenance medium containing 2% fetal bovine serum was added. The cells were then incubated at 37°C in a 5% CO2 incubator. The cytopathic effect (CPE) was observed under an inverted microscope daily for 7 consecutive days.

[0021] If typical PRRSV cytopathic effects (cell shrinkage, rounding, detachment, and plaque formation) appear after 3-5 days of culture, the cell culture is collected, subjected to three freeze-thaw cycles, and the supernatant is collected by centrifugation. This is the primary isolated strain. If no cytopathic effects appear, the culture is blindly passaged for three generations. If no cytopathic effects are found, it is determined that no PRRSV has been isolated. In this example, typical cytopathic effects appeared on the 4th day after inoculation with the pathogen treatment solution. The primary isolated strain was collected, named as a candidate strain, and stored at -80°C for later use.

[0022] 1.3 RT-PCR identification and strain nomenclature Total RNA was extracted from the above candidate strains using a kit, and cDNA was synthesized by reverse transcription using the extracted total RNA as a template.

[0023] PCR identification primers were designed targeting the PRRSV NSP2 protein gene, with a target fragment of 1465 bp. The primer sequences are as follows: The upstream primer sequence is PRRSV-NSP2F: 5'-TTGAGTGTGCTATGGCTGACGTC-3'. The downstream primer sequence is PRRSV-NSP2R: 5'-TCAAAGGAGGTGTCTAACACGC-3'. PCR amplification reaction system (25 μL): 2 μL cDNA template, 1 μL upstream primer (10 μmol / L), 1 μL downstream primer (10 μmol / L), 12.5 μL 2×PCR Mix, 8.5 μL sterile double-distilled water; PCR amplification conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 35 cycles; 72℃ final extension for 10 min, and 4℃ to terminate the reaction.

[0024] After PCR amplification, 10 μL of the amplification product was taken and subjected to 1.5% agarose gel electrophoresis. After electrophoresis, the product was observed in a gel imaging system. If a specific band of about 1465 bp appeared and was consistent with the expected target fragment size, it was determined to be PRRSV positive. At the same time, a blank control group (sterile double-distilled water to replace cDNA template) and a positive control group (cDNA of a known NADC30 class PRRSV strain) were set up to ensure the reliability of the identification results.

[0025] In this embodiment, the RT-PCR amplification products of the candidate strain showed a clear 1465bp specific band after electrophoresis, consistent with the band in the positive control group, while the blank control group showed no band; combined with cytopathic characteristics ( Figure 1 The results of identification by A and B and RT-PCR (in Chinese) Figure 1 The candidate strain was confirmed by the Chinese Center for Type Culture Collection (CCTCC) as NADC30 porcine reproductive and respiratory syndrome virus (PRRSV), and officially named NADC30 porcine PRRSV HB03 strain, which is the parent strain for subsequent attenuation through passage. It was deposited at the China Center for Type Culture Collection on March 24, 2026, with accession number CCTCC NO: V202631.

[0026] 2. Virus screening Using the HB03 strain obtained through isolation and identification as the parental strain, the attenuated and genetically stable strains were screened through continuous passage in Marc-145 cells for attenuation, plaque purification, and whole-genome sequencing analysis. Virulence was then verified through animal experiments, and the candidate attenuated vaccine strain HB03R was finally determined. The specific steps are as follows: 2.1 In vitro passage attenuation After isolating the NADC30-like PRRSV HB03 strain, it was continuously passaged in adherent Marc-145 cells, with plaque purification and whole-genome sequencing performed every 10 passages. The strain from the 100th passage was selected as the cell-passaged attenuated strain of NADC30-like porcine reproductive and respiratory syndrome virus HB03, and named NADC30-like PRRSV HB03R strain.

[0027] Table 1. Propagation titers of NADC30-like PRRSV HB03 strain at different generations 2.2 Genome characteristics of the virus strain Total RNA was extracted from HB03R strain (100 generations) and its parental strain HB03, reverse transcribed into cDNA, and then sequenced into a whole genome. Comparison and analysis of the amino acid sequences of both strains revealed the following hallmark characteristics of HB03R: 11 consecutive deletions and 21 mutations were found in the entire genome of HB03R compared to its parental strain HB03. Specific characteristics are as follows: (1) Amino acid deficiency characteristics: The 11 consecutive amino acid deletions are located at positions 187-197 of the amino acid sequence encoded by the non-structural protein 2 (NSP2) gene, and this deletion site is a hallmark deletion of the HB03R strain. (2) Amino acid mutation characteristics: The 21 amino acid mutations are distributed in NSP1-3, NSP9-10, and structural proteins ORF2-5. The specific mutation sites and amino acid changes are as follows: NSP1β: Methionine (M) at position 71 is mutated to isoleucine (I), denoted as M71I, with a total of 1 mutation; NSP2: There are a total of 6 mutations, namely, proline (P) at position 511 is mutated to leucine (L) (P511L), aspartic acid (D) at position 550 is mutated to glycine (G) (D550G), isoleucine (I) at position 562 is mutated to valine (V) (I562V), methionine (M) at position 594 is mutated to threonine (T) (M594T), arginine (R) at position 630 is mutated to methionine (M) (R630M), and proline (P) at position 768 is mutated to serine (S) (P768S); NSP3: Valine (V) at position 107 is mutated to isoleucine (I), denoted as V107I, with a total of 1 mutation; NSP9: Tyrosine (Y) at position 144 is mutated to histidine (H), denoted as Y144H, with a total of 1 mutation; NSP10: There are two mutations, namely, the 13th serine (S) is mutated to leucine (L) (S13L) and the 285th asparagine (N) is mutated to aspartic acid (D) (N285D). ORF2a: There are two mutations, namely, the 78th tyrosine (Y) is mutated to phenylalanine (F) (Y78F) and the 137th serine (S) is mutated to threonine (T) (S137T). ORF2b: Threonine (T) at position 56 is mutated to serine (S), denoted as T56S, with a total of 1 mutation; ORF3: There are 3 mutations, namely, proline (P) at position 55 is mutated to arginine (R) (P55R), histidine (H) at position 79 is mutated to tyrosine (Y) (H79Y), and isoleucine (I) at position 237 is mutated to threonine (T) (I237T); ORF4: There are 3 mutations, namely, asparagine (N) at position 33 is mutated to aspartic acid (D) (N33D), isoleucine (I) at position 124 is mutated to valine (V) (I124V), and threonine (T) at position 158 is mutated to serine (S) (T158S); ORF5: Glutamic acid (E) at position 162 is mutated to lysine (K), denoted as E162K, for a total of 1 mutation.

[0028] Further analysis revealed that the 11 consecutive amino acid deletions and 21 site-directed mutations were mainly distributed in viral replication-related functional regions (NSPs) and immunogenicity-related structural regions (ORFs) (the amino acid sequence of this genome is shown in SEQ ID NO:43, and the nucleotide sequence encoding this amino acid is shown in SEQ ID NO:44). It is speculated that this deletion-mutation combination is the core molecular basis for the attenuation of virulence, genetic stability, and good immunogenicity of the HB03R strain. Furthermore, when the HB03R strain was passaged to the 150th generation, the genomic amino acid mutation situation was completely consistent with that of the 100th generation, with no new mutations or reversion mutations. This indicates that the attenuated strain has fully adapted to the Marc-145 cell culture environment, can be stably passaged, and has no genetic basis for virulence reversion. The statistical results of gene mutations during passage are shown in the figure below. Figure 2 As shown.

[0029] Specifically, the gene sequence and amino acid sequence number of strain HB03 are shown in Table 2.

[0030] Table 2 2.3 Toxicity of the Toxin 2.3.1 Fifteen 28-day-old PRRSV antigen- and antibody-negative piglets, also negative for other major pathogens (swine fever virus, pseudorabies virus, and African swine fever virus), were randomly divided into three groups of five piglets each. The specific groupings are as follows: Group 1 (Parental Virus Control Group): Piglets were administered 2 ml of parental NADC30-like PRRSV HB03 strain virus via intranasal drip and intramuscular injection (virus content: 1×10⁻⁶). 6.0 TCID 50 / ml); Group 2 (Attenuated Virus Experimental Group): Piglets were given 2 ml of passaged attenuated NADC30-like PRRSVHB03R strain virus solution via nasal drip and intramuscular injection (virus content: 1×10⁻⁶). 6.0 TCID 50 / ml); Group 3 (blank control group): Piglets were inoculated with 4 ml of DMEM culture medium in the same manner.

[0031] Body temperature and clinical manifestations of each group were recorded on the designated date. Twenty-one days after the challenge, all piglets were euthanized and subjected to pathological examination.

[0032] 2.3.2 Test Results: (1) Clinical manifestations: Piglets in Group 1 (parental toxin control group) exhibited symptoms of porcine reproductive and respiratory syndrome, such as fever, cough, lethargy, and decreased appetite. Piglets in Group 2 (attenuated toxin experimental group) and Group 3 (blank control group) did not exhibit fever, cough, lethargy, or anorexia. Body temperature monitoring results were as follows. Figure 3 As shown.

[0033] (2) Pathological changes: In Group 1 (parental toxicity control group), solidification was observed in the lung tissue of piglets. HE staining of lung tissue sections showed varying degrees of alveolar epithelial cell proliferation, extensive inflammatory cell infiltration, and widening of alveolar septa. However, no lesions were observed in the pathological autopsy and HE staining of lung tissue sections of piglets in Group 2 (attenuated toxicity experimental group) and Group 3 (blank control group). Figure 4 ).

[0034] 2.4 Toxicity Reversion Test 2.4.1 Experimental Method: The passaged attenuated NADC30-like PRRSV HB03R strain was subjected to a 4×10⁻⁶ saturation rate. 7.5 TCID 50Piglets were inoculated with a dose of [dose / ml], and viral load was detected using the probe-based IRT-qPCR method and cell isolation method. The results showed that viral load was high in lung and lymph node tissues 7-10 days post-inoculation, and began to decrease on day 14. Therefore, we used a mixed homogenate of lung and lymph node tissues collected 10 days post-inoculation as tissue virus for passage experiments in pigs. Body temperature and clinical observation were performed after each passage of tissue virus inoculation, and the virus was passaged continuously in pigs for a total of 5 generations.

[0035] 2.4.2 Experimental Results: No clinical symptoms of PRRS (elevated body temperature and / or respiratory symptoms) were observed in any of the pigs inoculated from tissue virus P1 to tissue virus P5 passages. No lung lesions were found in pathological necropsy and HE staining of tissue sections, proving that the attenuated virus of NADC30-like PRRSV HB03R cell line will not revert to virulence after 5 passages in pigs.

[0036] The above results indicate that, compared with the parental NADC30-like PRRSV HB03 strain, the passaged attenuated NADC30-like PRRSV HB03R strain exhibits significantly reduced virulence. It is non-pathogenic in piglets after inoculation, does not cause typical clinical symptoms of porcine reproductive and respiratory syndrome (PRRS) or pathological damage to lung tissue, and demonstrates good safety. It fully meets the virulence requirements for attenuated vaccine candidate strains and can be used as a candidate strain for NADC30-type PRRS virus attenuated vaccines in subsequent vaccine preparation.

[0037] Example 2: Preparation and testing of NADC30 type porcine reproductive and respiratory syndrome virus HB03R strain attenuated vaccine This embodiment aims to prepare a NADC30 class porcine reproductive and respiratory syndrome virus HB03R strain attenuated vaccine based on the HB03R strain attenuated virus obtained in Example 1, including the following steps: 1. Preparation of virus solution for vaccine production 1.1 Cell proliferation: 1.1.1 Cell resuscitation and passage Remove the frozen Marc-145 cells from the liquid nitrogen tank, thaw them rapidly in a 37°C water bath, transfer them to centrifuge tubes, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells in the cell growth medium, and incubate them in a 37°C, 5% CO2 incubator. After 48-72 hours, when the cells have grown into a monolayer, digest them with 0.25% trypsin and passage them at a 1:3 ratio to expand the culture.

[0038] 1.1.2 Preparation of primary seed cells The Marc-145 cells expanded from the above culture were transferred to a roller bottle, the rotation speed was adjusted to 10 r / h, and the cells were cultured at 37°C. When the cells reached a confluent monolayer (48-72 hours), the cells were digested with 0.25% trypsin and passaged at a ratio of 1:3 for further culture.

[0039] 1.1.3 Preparation of Secondary Seed Cells The expanded Marc-145 cells in roller bottles were digested with 0.25% trypsin, counted, and then divided into groups of 4 × 10⁻⁶. 5 ~5×10 5 Cells were inoculated into a bioreactor containing 5 g / L microcarriers at a density of cells / ml. The bioreactor was set at a temperature of 37℃, a rotation speed of 50 r / min, dissolved oxygen of 50%, and pH of 7.2 for suspension culture.

[0040] 1.2 Virus inoculation: When the cells on the surface of the microcarriers in the bioreactor have grown to a good monolayer (72 hours), the growth medium in the tank is drained, cell maintenance medium is added, and HB03R strain virus is inoculated at a ratio of 0.5% (v / v). The bioreactor temperature is set at 37℃, the rotation speed at 50 r / min, the dissolved oxygen at 50%, and the pH at 7.0, and the culture continues.

[0041] 1.3 Observation and Virus Fluid Harvesting: After inoculation, samples are taken every 6 hours starting from 18 hours to observe cytopathic effects. When about 80% of the cells show cytopathic effects (24-36 hours), the viral fluid is harvested. The viral fluid is then frozen and thawed at -15°C and filtered through a filter cartridge to obtain a semi-finished product.

[0042] 1.4 Inspection of semi-finished products The above-mentioned semi-finished product (filtered virus solution) was subjected to sterility testing, mycoplasma testing, and exogenous virus testing. It met all the indicators specified in the "Veterinary Biological Products Quality Standard of the People's Republic of China", and the virus titer was not less than 10. 7.0 TCID 50 When the concentration is 1 ml, it is used as a virus solution for vaccine preparation and stored at -15°C or below for later use.

[0043] 2. Vaccine preparation and freeze-drying 2.1 Seedling distribution The prepared semi-finished product, which meets all the required indicators, is mixed evenly with the freeze-drying protectant at a 1:1 volume ratio, and then aseptically dispensed into quantitative containers. The freeze-drying protectant is formulated as follows: 25g gelatin, 40g sucrose, 20g enzymatically hydrolyzed casein, 15g tryptone, 4g dipotassium hydrogen phosphate, 0.56g potassium dihydrogen phosphate, dissolved in an appropriate amount of water for injection, and brought to a final volume of 500ml. The mixture is then autoclaved at 121℃ for 30 minutes and stored at 37℃.

[0044] 2.2 Freeze-drying After being dispensed, the vaccine is freeze-dried in a low-temperature freeze dryer, then capped, sealed, and labeled to produce the NADC30 type porcine reproductive and respiratory syndrome virus HB03R strain attenuated vaccine, which should be stored at 2-8℃.

[0045] 3. Safety trials of attenuated live vaccines 3.1 Selection and grouping of experimental animals Twenty 28-day-old piglets were selected, exhibiting negative results for both PRRSV antigen and antibody, and also negative for antigens and antibodies against other major pathogens (swine fever virus, pseudorabies virus, and African swine fever virus). They were randomly divided into two groups of 10 piglets each. There were no significant differences in weight or health status between the groups. The specific groupings are as follows: Group 1 (Immunization Group): Each piglet was intramuscularly injected with 10 doses of the NADC30-like PRRSV HB03R strain attenuated vaccine prepared in this embodiment (10 doses / piglet). 6.0 TCID 50 / dose, injection volume 1mL / head); Group 2 (Blank Control Group): Piglets were not immunized by injection and were raised in the same environment as the immunized piglets, with the same feed, water and feeding management, serving as a blank control.

[0046] Observation continued for 21 days, with temperature monitoring for 14 days.

[0047] 3.2 Experimental Results and Analysis Compared with the blank control group, the immunized group showed no fever, no cough, wheezing, lethargy, or anorexia, and no abnormalities in feed and water intake. No adverse reactions were observed at the injection site or throughout the body. All pigs survived, indicating that the attenuated live vaccine is highly safe for immunized piglets.

[0048] 4. Potency trials of attenuated live vaccines This study aims to verify the immunoprotective effect of the HB03R strain attenuated vaccine against homologous NADC30 PRRSV (HB03 strain) and heterologous NADC30 PRRSV (E3 strain). The immunogenicity and cross-protective ability of the vaccine were evaluated by detecting antibody levels, viremia, tissue viral load, and pathological changes after vaccination. The specific steps are as follows: 4.1 Selection and grouping of experimental animals Twenty-five 28-day-old piglets that were negative for both PRRSV antigen and antibody, and also negative for other major pathogens (swine fever virus, pseudorabies virus, and African swine fever virus) antigen and antibody, were randomly divided into five groups of five piglets each. There were no significant differences in weight or health status among the groups. Specific grouping and treatment details are as follows: Groups 1 and 2 were the immunization groups, groups 3 and 4 were the challenge control groups, and group 5 was the blank control group.

[0049] Group 1 (Homologous Immunization Group): Each piglet was intramuscularly injected with one dose of the HB03R strain attenuated live vaccine prepared in this embodiment (containing 10 mmol / L of virus). 6.0 TCID 50 / dose, injection volume 1ml / head); Group 2 (Heterologous Immunization Group): Each piglet was intramuscularly injected with one dose of the HB03R strain attenuated vaccine prepared in this embodiment. The vaccination method and dosage were the same as those of Group 1. Group 3 (Homologous challenge control group): No vaccine was administered, serving as a blank control for challenge with HB03 strain; Group 4 (heterogeneous challenge control group): No vaccine was administered, serving as a blank control for E3 strain challenge; Group 5 (blank control group): No vaccine was administered or challenged, serving as a blank control throughout the entire process.

[0050] 4.2 Vaccination and Challenge Piglets in groups 1 and 2 were routinely fed after vaccination. Twenty-eight days post-immunization, serum PRRSV-specific antibody levels were measured to confirm that the vaccine had induced an effective immune response. Afterward, challenge experiments were conducted. Piglets in groups 1 and 3: Inoculated with 2 ml of P2 generation NADC30-like PRRSVHB03 strain virus solution (homogeneous strain, virus content: 1×10⁻⁶) via a combined nasal and intramuscular injection route. 6.0 TCID 50 / ml), for a total of 4 ml / head; Piglets in groups 2 and 4 were inoculated with 2 ml of P2 generation NADC30-like PRRSVE3 strain virus solution (heterologous strain, virus content: 1×10⁻⁶) via a combination of intranasal and intramuscular injection. 6.0 TCID 50 / ml), for a total of 4 ml / head; Group 5 blank control piglets: Inoculated with 4 mL / head of sterile DMEM medium in the same way (nasal drops + intramuscular injection), and the rest of the feeding and management were the same as other groups.

[0051] Note: The P2 generation NADC30-like PRRSV E3 strain (heterologous strain) used is a clinically isolated NADC30-like PRRSV epidemic strain. It was identified by whole-genome sequencing and showed 86.5% homology with the HB03 strain. It was used to verify the cross-protective ability of the vaccine.

[0052] The experimental period was 28 days post-immunization + 21 days post-challenge, with sample collection and indicator testing conducted throughout the process, as detailed below: (1) Blood sample collection: Blood samples were collected from the anterior vena cava of three piglets randomly selected from each group at 0 (the day of vaccination), 7, 14, 21 and 28 days after immunization, and 3, 5, 7, 10, 14 and 21 days after challenge. Serum was separated and used to detect PRRSV-specific antibodies and viremia (viral load) in vivo. (2) Clinical indicator observation: After the challenge, the body temperature of piglets was measured at regular intervals every day (rectal temperature measurement) and the changes in body temperature were recorded; at the same time, the clinical manifestations of each group of piglets were observed and recorded, including mental state, appetite, respiratory state, presence or absence of typical PRRS symptoms such as coughing and panting, until 21 days after the challenge. (3) Pathological examination: 21 days after the challenge, all piglets were euthanized for pathological examination and assessment of the viral replication level in the tissues.

[0053] 4.3 Test Results: (1) Antibody level: Specific ELISA antibody detection showed that all piglets in Group 1 (homologous immunization group) and Group 2 (heterologous immunization group) were positive for specific ELISA antibodies 14 days after immunization. Figure 5 (A and B); while no PRRSV-specific antibodies were detected in piglets in groups 3, 4, and 5 at any time point after immunization, indicating that the HB03R strain attenuated vaccine can rapidly induce a specific immune response in piglets and produce high levels of protective antibodies.

[0054] (2) Clinical manifestations: After the challenge, the piglets in Group 1 (homologous immunization group) did not have symptoms of fever, and did not have typical clinical symptoms of PRRS such as wheezing, coughing, lethargy, and decreased appetite. Their growth was good. In Group 2 (heterologous immunization group), two piglets had a body temperature higher than 40.5℃ (lasting for 1-2 days and recovering on their own), while the other piglets did not have fever or any typical clinical symptoms of PRRS. Piglets in Group 3 (homologous challenge control group) had elevated body temperature exceeding 40.0℃ on the second day after challenge, and their body temperature was ≥40.5℃ on the fifth, seventh, and eighth days after challenge. They also exhibited varying degrees of PRRS symptoms (coughing, lethargy, and decreased appetite).

[0055] In Group 4 (heterogeneous challenge control group), the piglets' body temperature began to rise on the second day after the challenge, and they showed fever symptoms of ≥40.5℃ for 3 consecutive days from the 5th to the 7th day after the challenge. They also showed varying degrees of PRRS symptoms (coughing, lethargy and decreased appetite).

[0056] Group 5 (blank control group) piglets showed no abnormal clinical signs throughout the process, and their body temperature remained normal. Body temperature monitoring results were as follows: Figure 6 As shown.

[0057] (3) Viremia and viral load in tissues: qRT-PCR detection showed that the viral load in the blood of piglets in groups 3 and 4 (challenge control group) reached its peak on day 5 after challenge, with an average value of 10. 5.848 copies / ul and 10 6.642 copies / ul, then gradually decrease. In piglets of Group 1 (homologous immunization group) and Group 2 (heterologous immunization group), the viral load in the blood was significantly lower than that in the corresponding challenge control group at all time points after challenge, and the differences were statistically significant. The viral load in Group 1 (homologous immunization group) was 10% lower than that in Group 3 (homologous challenge control group). 1.820 copies -10 3.432 The viral load in group 2 (heterologous immunity group) was 10 copies lower than that in group 4 (heterologous challenge control group). 1.90 copies -10 4.118 copies.

[0058] The viral load levels in each group's tissues were consistent with the viral load in the blood. The viral loads in the lungs, spleen, submandibular lymph nodes, and inguinal lymph nodes of both immunization groups (Group 1 and Group 2) were significantly lower than those of the two challenge control groups (Group 3 and Group 4). In summary, compared with the challenge control groups, the viral replication levels in piglets in both immunization groups were significantly lower than those in the corresponding challenge control groups. Figure 7 This indicates that the HB03R strain attenuated vaccine can effectively inhibit the replication of homologous and heterologous NADC30 class PRRSV in piglets.

[0059] (4) Pathological changes: Pathological autopsy and HE staining showed that no lesions were found in the lung tissue of piglets in Group 1 (homologous immunization group), Group 2 (heterologous immunization group), and Group 5 (blank control group). However, in piglets in Group 3 (homologous challenge control group) and Group 4 (heterologous challenge control group), lung tissue showed consolidation, and HE staining of tissue sections showed different degrees of alveolar epithelial cell proliferation, extensive inflammatory cell infiltration, and widening of alveolar septa. Figure 8 ).

[0060] (5) Immunoprotective effect: The first group (homologous immunization group) of piglets achieved a protection rate of 5 / 5, and the second group (heterologous immunization group) of piglets achieved a protection rate of 3 / 5 (Table 2), indicating that the vaccine has a good protective effect against homologous strains and a certain protective effect against heterologous strains.

[0061] The above results indicate that the attenuated NADC30 porcine reproductive and respiratory syndrome virus (PRRSV) strain HB03R prepared in this embodiment has a stable preparation process, meets quality standards, and is highly safe. It also exhibits good immunoprotective efficacy against homologous NADC30-like PRRSV strains and a certain degree of immunoprotective efficacy against heterologous NADC30-like PRRSV strains. It can effectively inhibit viral replication in vivo and reduce pathological damage, and can be used for clinical prevention of NADC30 porcine reproductive and respiratory syndrome virus infection.

[0062] Table 2. Protection rate of immunized piglets at 28 days of age. The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A NADC30 type porcine reproductive and respiratory syndrome virus attenuated strain HB03R, characterized in that, The attenuated strain of NADC30 porcine reproductive and respiratory syndrome virus was named NADC30 porcine reproductive and respiratory syndrome virus HB03R strain and was deposited at the China Center for Type Culture Collection on March 24, 2026, with accession number CCTCC NO: V202631.

2. A NADC30 type attenuated live vaccine for porcine reproductive and respiratory syndrome virus, characterized in that, The active ingredient is the attenuated strain HB03R of NADC30 porcine reproductive and respiratory syndrome virus as described in claim 1.

3. The attenuated vaccine according to claim 2, characterized in that, It comprises a protein and a pharmaceutically acceptable carrier, wherein the protein comprises an amino acid sequence as shown in SEQ ID NO:

43.

4. The attenuated vaccine according to claim 2, characterized in that, The attenuated live vaccine is a freeze-dried vaccine, which is made by mixing the vaccine preparation virus liquid and the freeze-drying protectant at a ratio of 1:1 (v / v), aseptically dispensing, and freeze-drying. The freeze-drying protectant is formulated as follows: 25g gelatin, 40g sucrose, 20g enzymatically hydrolyzed casein, 15g tryptone, 4g dipotassium hydrogen phosphate, 0.56g potassium dihydrogen phosphate, dissolved in an appropriate amount of water for injection, and then brought to a final volume of 500ml. The mixture is then autoclaved at 121℃ for 30 minutes and stored at 37℃.

5. The attenuated vaccine according to claim 4, characterized in that, The viral solution used for seed preparation is prepared using the HB03R strain described in claim 1 as the seed virus, and the viral titer of the viral solution is not less than 10. 7.0 TCID 50 / ml; Each dose of the freeze-dried vaccine product contains 10 HB03R strain virus. 6.0 TCID 50 / ml.

6. A method for preparing a NADC30 type porcine reproductive and respiratory syndrome virus attenuated vaccine according to any one of claims 2 to 5, characterized in that, Includes the following steps: S1. Preparation of virus solution for seedling production: Using the HB03R strain described in claim 1 as the seed virus, qualified virus solution for seedling production is obtained through cell proliferation, virus inoculation, culture and harvesting, filtration and purification and semi-finished product inspection. S2. Vaccine preparation: The qualified vaccine virus solution is slowly thawed at 4°C and mixed evenly with the pre-cooled sterile freeze-drying protectant at a volume ratio of 1:1 to obtain a vaccine semi-finished product mixture. S3. Aseptic dispensing and freeze-drying: The vaccine semi-finished product mixture is aseptically and quantitatively dispensed into freeze-drying bottles. After freeze-drying, it is stored at low temperature to obtain the attenuated live vaccine product. S4. Finished Product Inspection: The freeze-dried vaccine product undergoes sterility testing, mycoplasma testing, exogenous virus testing, and virus titer determination. Once all indicators meet the requirements of the "Veterinary Biological Products Quality Standard of the People's Republic of China", it is considered a qualified vaccine.

7. The application of the NADC30 type porcine reproductive and respiratory syndrome virus attenuated strain HB03R as described in claim 1, characterized in that, The HB03R strain was used to prepare an attenuated vaccine to prevent piglets from being infected with NADC30 porcine reproductive and respiratory syndrome virus.

8. The application of a NADC30 type porcine reproductive and respiratory syndrome virus attenuated vaccine according to any one of claims 2 to 5, characterized in that, The attenuated vaccine is used to prevent piglets from being infected with NADC30 porcine reproductive and respiratory syndrome virus. The administration method is intramuscular injection, and the dosage is 1 ml per piglet. Piglets that are negative for both PRRSV antigen and antibody at 28 days of age are suitable for vaccination.

9. The application according to claim 8, characterized in that, The attenuated vaccine has good immunoprotective efficacy against the homologous NADC30 PRRSV HB03 strain and a certain degree of immunoprotective efficacy against the heterologous NADC30 PRRSV E3 strain. Piglets can produce specific ELISA antibodies 14 days after immunization, with an immunoprotective rate of ≥60%. It can effectively inhibit viral replication in piglets, reduce pathological damage to lung tissue, and has no obvious adverse reactions.

Images