Attenuated porcine reproductive and respiratory syndrome virus strain, identification kit, preparation method and application thereof

Abstract

The present invention provides an attenuated strain of porcine reproductive and respiratory syndrome virus, an identification kit, a preparation method and an application thereof, and belongs to the technical field of virus prevention and control. The present invention is to provide a recombinant attenuated vaccine strain of porcine reproductive and respiratory syndrome virus, and the vaccine strain can effectively distinguish between wild strain infection and vaccine strain immunity. The present invention provides an attenuated strain H18-R of porcine reproductive and respiratory syndrome virus, the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus, the deposit number of which is CGMCC NO.46203, and the deposit date is October 17, 2024. The H18-R vaccine is safe and non-pathogenic to pigs after immunization, and can effectively induce the body to produce an immune response to resist the attack of porcine reproductive and respiratory syndrome virus, providing good protection for immunized pigs.

Description

Technical Field

[0001] This invention belongs to the field of virus prevention and control technology, specifically relating to an attenuated strain of porcine reproductive and respiratory syndrome virus, an identification kit, its preparation method, and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a serious viral infectious disease that threatens the global swine industry. Commonly known as "blue ear disease," it is characterized by reproductive disorders in pregnant sows and respiratory symptoms in piglets. PRRS viruses belong to the order Nidovirales, family Arteriviridae, and genus Betaarterivirus, and are further divided into Betaarterivirus suid 1 (formerly the European type) and Betaarterivirus suid 2 (formerly the North American type). PRRS is a single-stranded positive-sense RNA virus with a genome approximately 15 kb long. It has a cap structure upstream of the 5' untranslated region and a Poly(A) tail downstream of the 3' untranslated region. Its genome encodes at least 11 open reading frames (ORFs): ORF1a, ORF1b, ORF2a, ORF2b, ORF3-7, ORF5a, and NSP2(TF). ORF1 constitutes the largest proportion of the viral genome (approximately 80%), encoding a replicase complex. This complex is translated via a ribosomal frameshift mechanism to produce polyproteins pp1a and pp1ab. ORF1a is then cleaved by three proteases to produce at least 16 nonstructural proteins (NSPs), with NSP2 exhibiting the greatest variation. The disease was first discovered in American pigs in 1987 and subsequently spread worldwide. In 1996, Guo Baoqing et al. first isolated PRRSV in China. Since then, PRRSV has been prevalent in my country, continuously mutating and recombinizing, causing multiple PRRS outbreaks. Currently, the prevalence of PRRSV in my country has mainly gone through three stages: the first stage was dominated by classic strains, represented by CH-1a; the second stage was dominated by highly pathogenic PRRSV (HP-PRRSV) strains, represented by JXA1, HuN4, and TJ; and the third stage is dominated by NADC30-like PRRSV. Since 2015, the L1 lineage NADC30PRRSV has gradually become the dominant strain in my country.

[0003] Vaccination is an effective means of preventing and controlling PRRS. Commercially available PRRSV vaccines in China mainly include inactivated vaccines, live attenuated vaccines, and genetically engineered vaccines, primarily targeting the classical strain and HP-PRRSV. These vaccines have played a positive role in preventing PRRSV, but as the virus continues to mutate, existing commercially available vaccine strains cannot provide complete immune protection against NADC30-like PRRSV. Therefore, it is necessary to develop more effective vaccine strains. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant attenuated vaccine strain of porcine reproductive and respiratory syndrome virus, which can effectively distinguish between wild-type virus infection and vaccine-induced immunity.

[0005] This invention provides an attenuated strain H18-R of arterivirus porcine reproductive and respiratory syndrome virus (PRRSV), which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.46203 and deposit date of October 17, 2024.

[0006] The present invention provides a vaccine for porcine reproductive and respiratory syndrome virus, the vaccine comprising the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus as described in claim 1.

[0007] Furthermore, the vaccine also includes a pharmaceutical carrier or pharmaceutically acceptable excipients.

[0008] This invention provides the application of the above-mentioned attenuated strain H18-R of porcine reproductive and respiratory syndrome virus in the preparation of vaccines or drugs for the prevention of porcine reproductive and respiratory syndrome (PRRS).

[0009] This invention provides an ELISA kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and identifying attenuated PRSV strain H18-R. The kit contains the characteristic deletion protein of the attenuated PRSV strain H18-R, the amino acid sequence of which is SEQ ID NO.2.

[0010] To further specify, the kit shown includes a first antibody and a second antibody, wherein the first antibody comprises PRRS-positive serum and the second antibody is HRP-labeled goat anti-pig IgG (H+L).

[0011] This invention provides the application of the above-mentioned attenuated strain H18-R of porcine reproductive and respiratory syndrome virus, the protein with the amino acid sequence shown in SEQ ID NO.2, the microbial cell carrying the protein shown in SEQ ID NO.2, the coding gene SEQ ID NO.2 encoding the protein shown in SEQ ID NO.2, and the recombinant vector containing the coding gene SEQ ID NO.1 in the preparation of a diagnostic and detection kit for porcine reproductive and respiratory syndrome virus.

[0012] The present invention provides an antigenic peptide for Reproductive and Respiratory Syndrome Virus (RSV), the amino acid sequence of which is shown in SEQ ID NO.2.

[0013] The present invention provides a nucleotide encoding the above-mentioned antigenic peptide.

[0014] This invention provides a recombinant vector or recombinant microbial cell containing the above-mentioned nucleotides for the application in the preparation of an ELISA detection kit for detecting porcine reproductive and respiratory syndrome virus and for identifying and distinguishing attenuated porcine reproductive and respiratory syndrome virus strain H18-R.

[0015] Beneficial Effects: In one aspect of the present invention, a recombinant attenuated vaccine strain for porcine reproductive and respiratory syndrome (PRRS) is provided. The parent strain of the recombinant attenuated vaccine strain is a naturally recombinant PRRS virus, whose antigenic structural proteins are derived from lineage 1 and lineage 8 strains, respectively, and have been artificially passaged and domesticated to become the attenuated vaccine strain H18-R. This H18-R vaccine is safe and non-pathogenic to pigs after immunization, and can effectively induce an immune response in the body to resist the attack of PRRS virus, providing good protection for immunized pigs.

[0016] In another aspect, this invention provides a serological differential diagnostic method for distinguishing between the aforementioned recombinant attenuated porcine reproductive and respiratory syndrome (PRRS) vaccine strain and naturally infected wild-type strains. Previously, it was difficult to differentiate between vaccine-immunized pigs and naturally infected pigs with wild-type strains when using attenuated vaccine strains. Therefore, establishing a serological or etiological differential diagnostic method capable of distinguishing between vaccine-immunized pigs and wild-type infected pigs is crucial. Based on the unique deletion characteristics of the attenuated vaccine strain H18-R NSP2, this invention uses the 169 amino acids of the deleted NSP2 protein expressed in prokaryotes as a coating antigen to establish an ELISA antibody detection method. This method yields negative results for serum from H18-R strain-immunized pigs, and positive results for serum from pigs infected with the parent strain, PRRSV wild-type strain, and commercially available PRRS vaccine-immunized pigs. It also shows no cross-reactivity with positive sera from other pathogens (ASFV, CSFV, PRV, PEDV, PPV, and PCV-2). This ELISA method has good specificity and sensitivity, and can effectively distinguish between pigs naturally infected with wild-type strains and those immunized with vaccine strains, meeting the clinical needs for differential diagnosis between vaccine-immunized strains and natural wild-type strains.

[0017] [Bio-Collection Information]: An attenuated strain H18-R of porcine reproductive and respiratory syndrome virus (PRRSV), classified and named Arterivirus, with accession number CGMCC NO.46203, deposited on October 17, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0018] Figure 1 Recombinant analysis of the genome sequence of the attenuated vaccine strain H18-R;

[0019] Figure 2 Levels of PRRSV N protein antibodies in serum of piglets immunized with attenuated vaccine strain H18-R;

[0020] Figure 3 Clinical symptom scores of piglets after immunization with attenuated vaccine strain H18-R;

[0021] Figure 4 Changes in body temperature of piglets after immunization with attenuated vaccine strain H18-R;

[0022] Figure 5 Serum viral load in piglets after immunization with attenuated vaccine strain H18-R following challenge;

[0023] Figure 6 Expression, purification, and identification of the characteristic deletion protein D169 of the attenuated vaccine strain H18-R. Lane M is the molecular weight standard for proteins; (a): 1: pET-24a, empty vector control, induced expression, whole bacterial culture; 2: pET-24a-D169, uninduced expression, whole bacterial culture; 3: pET-24a-D169, induced expression, whole bacterial culture; 4: pET-24a-D169, induced expression, supernatant; 5: pET-24a-D169, induced expression, precipitation; (b): 1: SDS-PAGE verification of purified D169; (c): 1: Western Blot verification of purified D169 (His-tagged antibody, 1:10000 dilution); (d): 1: Western Blot verification of purified D169 (PRRS-positive serum, 1:100 dilution).

[0024] Figure 7 Test results of the D169 protein-based ELISA kit; (a) specificity test; (b) sensitivity test;

[0025] Figure 8 The D169 protein-based ELISA kit was used to differentiate and diagnose PRRSV-positive sera from different sources: (a) positive sera from pigs infected with the parent strain of the recombinant attenuated vaccine; (b) positive sera from pigs immunized with commercially available live attenuated vaccines in China; (c) positive sera from pigs infected with clinical wild-type virus; and (d) sera from pigs immunized with the attenuated vaccine strain H18-R (IDEXX PRRSVX3 test showed positive PRRSV N protein antibody). Detailed Implementation

[0026] Example 1. Domestication and Identification of PRRSV Attenuated Vaccine Strain H18-R

[0027] 1. Materials and Methods

[0028] 1.1 Virus strains and cells

[0029] PRRSV H18 strain (full name HeB-108 strain, GenBank accession no. MN046224.1) was isolated and preserved in our laboratory. Monkey embryonic kidney epithelial cells Marc-145 were preserved in our laboratory.

[0030] 1.2 Continuous subculturing and domestication of PRRSV H18 strain

[0031] Marc-145 cells were seeded in T-25 cell culture flasks. When the cell confluence reached 80%, the culture medium was replaced with 2% FBS DMEM cell maintenance medium. 500 μL of the virus-containing cell culture supernatant was thawed at room temperature, centrifuged at 2000 rpm for 2 min to remove cell debris, and then seeded into Marc-145 cells. The cells were then incubated at 37°C in a 5% CO2 cell culture incubator. Two days after inoculation, the cells and supernatant were harvested and subjected to a freeze-thaw cycle at -80°C. The harvested cell supernatant was used as parent virus to reinfect Marc-145 cells. This inoculation was repeated in Marc-145 cells until passage 130, and the attenuated strain from passage 130 was named H18-R.

[0032] 1.3 PRRSV H18-R strain whole genome amplification and sequencing

[0033] Primers were designed using Oligo 6.0 to cover the entire viral genome (Table 1). The entire genome was amplified in 12 segments, each approximately 1.4 kb in size, with a 100 bp overlap between segments.

[0034] Collect 140 μL of H18 cell supernatant and extract viral RNA using the QIAamp Viral RNA Mini Kit, following the manufacturer's instructions. Use PrimeScript... TM The first-strand cDNA was synthesized using the II 1st Strand cDNA Synthesis Kit. The specific procedure was as follows: 1 μL of Random 6mers (50 μM), 1 μL of dNTP Mixture (10 mM each), and 8 μL of template RNA were added. The reaction mixture was incubated at 65°C, followed by rapid cooling on ice. Then, 4 μL of 5×PrimeScript II Buffer, 1 μL of RNase Inhibitor (40 U / μL), 1 μL of PrimeScript II RTase (200 U / μL), and RNase-Free dH2O were added to the denatured reaction mixture to a final volume of 20 μL. The reaction conditions were: 30°C for 5 min, 42°C for 1 h, and 70°C for 15 min.

[0035] Using cDNA as a template, high-fidelity DNA polymerase was used to amplify various target fragments. The amplification system consisted of: 10 μL 5×Q5 reaction buffer, 1 μL 10 mM dNTPs, 2.5 μL each of 10 μM forward and reverse primers, 0.5 μL Q5 high-fidelity DNA polymerase, 2 μL template cDNA, and ddH2O to a final volume of 50 μL. The reaction conditions were: 98℃ for 30 s; 98℃ for 10 s, 53℃–60℃ for 30 s, 72℃ for 1 min and 30 s, for 30 cycles; 72℃ for 2 min, and storage at 4℃. The amplification products were observed by 1% agarose gel electrophoresis. After gel purification, the target fragments were sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. Sequencing results were analyzed using Lasergene software for multiple sequence alignment, and the viral sequence was determined by combining the sequencing peak diagram.

[0036] Table 1 Primers for viral whole genome amplification

[0037]

[0038]

[0039] 1.4 Recombination Analysis

[0040] Representative strains from each branch of PRRSV were selected as reference strains, including L1 branch NADC30 (GenBank accession no. MH500776), L2 branch XW008 (GenBank accession no. KF724404), L3 branch MD001 (GenBank accession no. KP998431), L4 branch EDRD-1 (GenBank accession no. AB288356), L5 branch VR-2332 (GenBank accession no. AY150564), L6 branch P129 (GenBank accession no. AF494042), L7 branch SP (GenBank accession no. AF184212), L8 branch JXA1 (GenBank accession no. EF112445), and L9 branch MN30100 (GenBank accession no. EF536000). After aligning the reference sequence and the target sequence with MEGA 7, Simplot (v3.5.1) was used to perform similarity plotting to determine the recombination signal and recombination location.

[0041] 2. Results

[0042] After inoculating Marc-145 cells with H18, obvious cytopathic effects such as cell shrinkage, rounding, and aggregation appeared 2 days later. These cytopathic effects became increasingly pronounced with each passage. The cell strain was passaged to its 130th generation and named H18-R.

[0043] To obtain the complete H18-R genome sequence, the viral genome was amplified in 12 segments. Agarose gel electrophoresis showed that the 12 segments amplified with single bands, each approximately 1.4 kb in size, consistent with expectations. These segments were then assembled to obtain the complete H18-R genome sequence, which is 14,486 bp in size (excluding polyA). Comparison of the full-length H18-R genome with reference strains of each PRRSV lineage revealed that H18-R is a naturally recombinant strain with lineage 1 as the primary parent and lineage 8 as the secondary parent. Figure 1 ).

[0044] During the domestication process, a partial amino acid deletion occurred in the H18-R genome, located in the NSP2 coding region from 1271 to 1795 bp. The nucleotide sequence of the deleted portion is shown in SEQ ID NO. 1. Bioinformatics software analysis showed that the deleted amino acid sequence (SEQ ID NO: 2) had a high antigenicity index. This deleted sequence can not only distinguish H18-R from the parental strain H18 and wild-type strains, but also differentiate H18-R from other vaccine strains, thus possessing diagnostic value.

[0045] Nucleotide sequence (SEQ ID NO:1):

[0046] GGGACTCTGGCGTGCCGGGATGAGCTCCTCGATTTGTCTGCATCTTCGCAGACTGAAT

[0047] ACGAGGCTTCCCCTTTGGCGTTACCGCAGAGTGAGGACGCCCTGGCGATGAGGAGGC

[0048] AAGAAGCTGAGGAAACCCTGAGCGAAGCCTCGGGTGTGCCGGACGACATCAGATTGA

[0049] CACCCGTGTCGTCAAGCAGCTCCCTGTCAAGCGTAGAGATTACACGCCCAAAATACTC

[0050] AGCTCAAGCCATCATTGACACAGGTGGGCCCTGTTGTGGGCACCTCCAAGAGTTAAAG

[0051] GGGAAATACCTCGATGTGATGCGTGAGGGCTGTGATGCCACCAAGCTCGATGATCCTG

[0052] CCACGCAAGAATGGCTTTCTCGCATGTGGGACGGAGTGGACATGTTAACCTGGCGGA

[0053] GTACGTCCATTTTTCAAGCGCCTTTCATCTTGGCTGATAAGTTCAAATCTCTCCCGAAGATGATACTCGAAACACCACCGCCCTACCCTTGCGGGTTTGTGATG.

[0054] Example 2. Immunoprotective Experiment of Porcine Reproductive and Respiratory Syndrome Virus Recombinant Attenuated Vaccine Strain H18-R

[0055] 1. Materials and Methods

[0056] 1.1 Grouping of experimental animals and immunization with vaccines

[0057] Six 28-day-old piglets that were negative for both PRRSV antigen and antibody and for classical swine fever virus (CSFV), pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2) antigen were randomly divided into a vaccine-immunized group and a non-immunized control group, with three piglets in each group. The two groups were kept in isolation.

[0058] 1.2 Immunization and Challenge Experiments of Recombinant Attenuated Vaccine Strain

[0059] The vaccinated pigs were administered PRRSV H18-R strain via intramuscular injection in the neck, at a dose of 1 mL / head (10 5.0 TCID50 / mL). The unimmunized control group was injected with DMEM medium via the same inoculation method and dosage. On day 28 post-vaccination, all experimental pigs were injected intranasally and intramuscularly with 3 mL (10 TCID50 / mL) of the virulent PRRSV H18 strain. 5.0 TCID50 / mL).

[0060] 1.3 Body temperature measurement and clinical symptom observation of experimental pigs

[0061] After immunization with the recombinant attenuated vaccine, the clinical manifestations of the experimental pigs were observed daily. Following inoculation with the virulent H18 strain, clinical symptoms were observed and quantified daily, and body temperature was measured daily until 21 days after challenge.

[0062] 1.4 Detection of PRRSV-specific antibodies in experimental porcine serum

[0063] Serum samples were collected from all experimental pigs on days 0, 7, 14, and 21 after immunization with the recombinant attenuated vaccine strain, and the PRRSV N protein level in the serum was detected using the IDEX XPRRSV antibody detection kit.

[0064] 1.5 Detection of viral load in experimental pig serum

[0065] Serum was collected from all experimental pigs on days 7, 10, 14 and 21 after inoculation with the virulent H18 strain. The viral load in the serum was quantified using the universal PRRSV-2RT-qPCR method established in our laboratory.

[0066] 2. Results

[0067] Immunoprotection experiments were conducted on 28-day-old piglets using a recombinant attenuated vaccine strain. Fourteen days post-immunization, serum PRRSV N protein antibodies in the pigs became seropositive (3 / 3), and antibody levels continued to increase thereafter. Figure 2 This indicates that the H18-R strain has good immunogenicity and can effectively activate the body to produce a humoral immune response. Within 28 days after vaccination, no abnormalities such as fever or respiratory symptoms were observed in the vaccinated pigs, indicating that the H18-R strain, after in vitro passage and domestication, has no pathogenicity in pigs and has good safety. After challenging all experimental pigs with the parent virulent strain, clinical symptoms began to appear in the unvaccinated control group on day 1 post-challenge and disappeared by day 15 post-challenge. The clinical symptoms included fever, cough, runny nose, and other respiratory symptoms. Figure 3 In the unvaccinated control group, pigs showed a significant increase in body temperature 2–11 days after challenge, with all three experimental pigs exhibiting fever (temperature >40.0℃) lasting 2–7 days. In contrast, the vaccine-immunized group showed no fever, respiratory symptoms, or other clinical manifestations within 21 days after challenge. Figure 4 This indicates that the H18-R strain can effectively resist the challenge of the parent virulent strain and effectively reduce the morbidity rate of piglets. Serum samples were collected on days 7, 10, 14, and 21 after challenge to detect viral load. The viral load in the non-immunized control group was the highest on day 7 after challenge, followed by a gradual decrease in viremia. The viremia in the H18-R immunized group remained at a low level on day 21 after challenge, significantly lower than that in the non-immunized control group. Figure 5 The results indicate that the H18-R strain can significantly reduce viremia in challenged pigs and has a good immune protective effect.

[0068] Example 3. Establishment and application of an ELISA method for the differential diagnosis of attenuated vaccine strain H18-R.

[0069] 1. Materials and Methods

[0070] 1.1 Plasmids and serum clinical samples

[0071] Serum from pigs immunized with vaccine strain H18-R, serum from pigs infected with parental strain H18, positive and negative sera for porcine reproductive and respiratory syndrome (PRRS), specific pathogen-free (SPF) swine serum, and positive sera for classical swine fever (CSF), porcine epidemic diarrhea (PED), pseudorabies (PR), porcine parvovirus infection (PPI), and porcine circovirus type 2 (PCV2) were all preserved in our laboratory; positive sera for African swine fever (ASF) were provided by the National African Swine Fever Specialized Laboratory; all clinical samples used in this experiment were preserved in our laboratory. The pET-24a plasmid was preserved in our laboratory.

[0072] 1.2 Structural and antigenic analysis of proteins encoded by the deleted regions

[0073] Based on the alignment results of the H18-R vaccine strain sequence with the full-length PRRSV-2 NSP2 sequence (n=1242) in GenBank and the unique deletion characteristics of its NSP2 region, a corresponding protein expression coding sequence was designed. The secondary structure and antigenic epitopes were predicted using Protean software in DNASTAR. Based on indicators such as Antigenic Index and Hydrohobicity Plot, the antigenicity as a protein antigen and its potential for soluble expression were preliminarily assessed. Furthermore, its tertiary structure was predicted using Phyre2 software, and its spatial conformation was preliminarily predicted. The protein expression coding sequence designed based on the NSP2 deletion region of the H18-R strain was codon-optimized and synthesized by Beijing Ruiboxingke Biotechnology Co., Ltd., and named D169 (SEQ ID NO:2).

[0074] Amino acid sequence (SEQ ID NO:2):

[0075] GTLACRDELLDLSASSQTEYEASPLALPQSEDALAMRRQEAEETLSEASGVPDDIRLTPVSS

[0076] SSSLSSVEITRPKYSAQAIIDTGGPCCGHLQELKGKYLDVMREACDATKLDDPATQEWLSR MWDGVDMLTWRSTSIFQAPFILADKFKSLPKMILETPPPYPCGFVM.

[0077] Construction of 1.3pET-24a-D169 prokaryotic expression vector

[0078] After culturing the synthetic bacterial strain, the plasmid was extracted and simultaneously digested with Nde I and Xho I at 37℃ for 3 hours. The target fragment and the vector fragment were then ligated, and the recombinant plasmid was named pET-24a-D169.

[0079] 1.3 Prokaryotic expression, purification and identification of the target protein D169

[0080] The expression strain was induced to express D169 protein at small doses under different temperatures (16℃ and 37℃) and IPTG concentrations (0.1, 0.3, 0.5, and 1.0 mM) to determine the optimal expression conditions (37℃, 1 mM IPTG). Subsequently, large-scale expression was induced, and high-purity D169 protein was obtained using nickel column affinity chromatography, with protein concentration determined by the BCA method. Western blotting was then performed using mouse His-tag mAb and PRRS-positive serum as primary antibodies, and goat anti-mouse IgG (H+L) and goat anti-pig IgG (H+L) as secondary antibodies.

[0081] 1.4 Determine the optimal antigen coating concentration and optimal serum dilution

[0082] D169 protein concentration was determined according to the BCA method. Gradual dilutions (20 μg / mL to 0.02 μg / mL) were performed using coating buffer (carbonate buffer, pH adjusted to 9.6), 100 μL per well, and incubated overnight at 4°C. PRRS positive and negative sera were serially diluted 2-fold (1:25 to 1:400) with serum diluent, 100 μL per well, and incubated at 37°C for 1 h.

[0083] 1.5 Optimization of reaction conditions

[0084] The reaction conditions were optimized by considering factors such as the type of blocking buffer (5% skim milk, 1% and 2% BSA, 10% FBS), the type of serum diluent (PBS, 1% and 2% BSA, 10% FBS, 0.5% casein diluent), the serum incubation time (30, 45, 60, and 120 min), the enzyme-labeled antibody dilution (1:1000–1:16000, 2-fold serial dilution), the enzyme-labeled antibody incubation time (30, 45, 60, and 120 min), and the substrate color development time (5, 10, 15, and 20 min). The reactivity of PRRS positive and negative sera under different conditions was examined, the OD450nm value was measured, and the P / N ratio was calculated. The optimal reaction conditions were determined by comprehensive consideration.

[0085] 1.6 ROC analysis to determine critical values

[0086] For 84 animal serum samples from clinical trials (67 PRRS-positive and 17 PRRS-negative sera as determined by the IDEXX kit), OD450nm values ​​were measured using the optimized D169-ELISA method. The S / P ratio of the serum samples under this method was calculated as (OD450nm value of the test sample - mean OD450nm value of the negative control) / (mean OD450nm value of the positive control - mean OD450nm value of the negative control). Based on the OD450nm and S / P values, ROC (Receiver Operating Characteristic Curve) curve analysis was performed using SPSS (Statistical Product and Service Solutions) software. According to the sensitivity and specificity of the D169-ELISA method, the cut-off values ​​for OD450nm and S / P values ​​after statistical processing were obtained.

[0087] 1.7 Specificity, sensitivity and repeatability tests

[0088] Following the established D169-ELISA method, serum from pigs immunized with the H18-R vaccine strain and serum from pigs infected with its parent strain H18, as well as PRRS, ASF, CSF, PR, PED, PPI, and PCV2 positive sera, were simultaneously tested. A negative control (SPF pig serum) was established to assess its specificity. PRRS positive sera were serially diluted at 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, and simultaneously detected using this method and the IDEXX kit to assess its sensitivity. D169 protein purified from the same and different batches was coated onto ELISA plates, and 10 PRRS positive sera were tested using this method, with each serum sample tested three times. Statistical analysis was performed on the results, and the coefficient of variation of the S / P values ​​was calculated to assess its reproducibility.

[0089] 1.8 Clinical Serum Conformity Test and Preliminary Application

[0090] 175 clinical serum samples (139 samples from wild-type infected pigs and 36 samples from pigs immunized with other vaccine strains) were simultaneously tested using the D169-ELISA method and the IDEXX kit, and the concordance rate between the two methods was calculated based on the results. Further tests were performed on pig serum infected with parental strain HeB-108 (0, 7, 14, 21, 28, 35, 42, 49 dpi), pig serum immunized with commercially available PRRS vaccine strains (HuN4-F112, GDr180, JXA1-R, PC, VR-2332 strains) (0, 7, 14, 21, 28 dpi), 8 samples of clinical wild-type infected pig serum, and pig serum immunized with vaccine strain H18-R (0, 7, 14, 21, 28 dpi). The sensitivity and differential diagnostic potential of this method in clinical application were preliminarily analyzed according to the cutoff value determination method.

[0091] 2. Results

[0092] By varying the temperature and IPTG concentration, it was found that the highest overall expression level of the target protein D169 was achieved when cultured at 37℃ with 1 mM IPTG and a shaking speed of 200 r / min for 5 h, and the highest expression level was observed in the supernatant after sonication. SDS-PAGE analysis showed a distinct protein band at approximately 24 kDa. Figure 6 a). The target protein with high purity was obtained by nickel column affinity chromatography. Figure 6 b) The protein concentration was determined to be 0.045 g / L using the BCA method. The purified target protein was then identified by Western blotting using a mouse His-tag mAb as the primary antibody. Figure 6 c) and using PRRS-positive serum as the primary antibody ( Figure 6d) The specificity was good, indicating that D169 was correctly expressed and had good reactivity.

[0093] D169 protein was serially diluted with coating buffer, and PRRS positive and negative sera were serially diluted with serum diluent. The optimal coating concentration was determined to be 2.5 μg / mL based on OD450 nm and P / N values, and the optimal serum dilution was 1:50 (Table 2). Through exploration and optimization of various reaction conditions, combined with the OD450 nm and P / N values ​​of positive and negative sera, the following blocking solutions were ultimately determined: 5% skim milk, 37℃ for 2 h; 0.5% casein diluent, room temperature for 45 min; enzyme-labeled antibody dilution of 1:1000, room temperature for 60 min; and TMB chromogenic solution, room temperature for 15 min.

[0094] Table 2 determines the optimal antigen coating concentration and optimal serum dilution.

[0095]

[0096]

[0097] Note: P represents positive serum OD. 450nm Value, N represents negative serum OD 450nm The values ​​are shown in the underlined section, which represents the reaction value corresponding to the optimal conditions.

[0098] Eighty-four clinical serum samples were tested using this method (the IDEXX kit showed 67 positive and 17 negative samples). The S / P ratio was calculated based on the results, and ROC curve statistical analysis was performed. The results showed that the cutoff value for positive and negative serology was 0.525; an S / P value ≥ 0.525 was considered positive, and an S / P value < 0.525 was considered negative. Similarly, the cutoff value for positive and negative serology at the corresponding OD450nm value was 0.159.

[0099] The established D169-ELISA method was used to detect serum from pigs immunized with vaccine strain H18-R, serum from pigs infected with its parent strain H18, PRRS-positive serum, and serum positive for other swine diseases (ASF, CSF, PR, PED, PPI, and PCV-2) (SPF pig serum was used as a negative control). Only serum from pigs infected with the parent strain and PRRS-positive serum showed positive results; all others were negative. Figure 7 a) The target protein D169, as the coating antigen, does not cross-react with sera from other swine diseases, nor with sera from swine immunized with vaccine strain H18-R. This indicates that the method has good specificity and can be used for the differential diagnosis of immunization with vaccine strain H18-R and other PRRSV infections.

[0100] The established D169-ELISA method was used to detect PRRS-positive serum diluted 2-fold. When the positive serum was diluted 1:800, the S / P value was 0.60, indicating that it was still positive. Figure 7 (b) Meanwhile, the results from the IDEXX kit were used as a control, and the sensitivity of the two methods was found to be similar (the sensitivity of the IDEXX kit was 1:1600), further demonstrating the high sensitivity of this method. ELISA plates were coated with D169 target protein prepared in the same and different batches, and the D169-ELISA method was used to detect 10 PRRS sera with different antibody levels three times each. Statistical analysis of the results using SPSS software revealed that the intra-batch mean coefficient of variation was 3.13%, all less than 5%; the inter-batch mean coefficient of variation was 6.90%, all less than 10% (Table 3), indicating that this method has good reproducibility.

[0101] Table 3. Repeatability Tests for the D169-ELISA Method

[0102]

[0103]

[0104] 175 clinical serum samples (139 samples of swine serum infected with wild-type virus strain and 36 samples of swine serum immunized with other vaccine strains) were simultaneously tested using the established D169-ELISA method and the IDEXX PRRSV antibody detection kit. The results are shown in Table 4. The positive rate of this method was 69.71% (122 / 175), while the positive rate of the IDEXX kit was 76.57% (134 / 175), with a positive concordance rate of 89.55% (120 / 134). The negative rate of this method was 30.29% (53 / 175), while the negative rate of the IDEXX kit was 23.43% (41 / 175), with a negative concordance rate of 95.12% (39 / 41). The overall concordance rate was 90.86% (159 / 175). The above results indicate that the detection results of this method for serum antibody levels in pigs infected with PRRSV other than vaccine strain H18-R are close to those of the IDEXX kit, and the method has high sensitivity and reliability for clinical application.

[0105] Table 4 Comparison of detection results of serum samples using the IDEXX kit and the D169-ELISA method

[0106] IDEXX reagent kit D169-ELISA method Compliance rate Number of negative samples 41 53(39*) 95.12％ Number of positive samples 134 122(120*) 89.55％ total 175 175(159*) 90.86％

[0107] Note: The number marked with an asterisk (*) in parentheses indicates the number of samples for which the two methods yielded consistent results.

[0108] Serum samples from pigs infected with parental strains of known clinical background were tested using the D169-ELISA method at 0, 7, 14, 21, 28, 35, 42, and 49 days post-infection. Results showed that D169 antibodies began to appear in pigs at 21 days post-infection and were still detectable at 49 days post-infection. Figure 8 a). This method was used to detect D169 antibodies in swine serum immunized with five currently commercially available vaccines (HuN4-F112, GDr180, JXA1-R, PC, and VR-2332 strains) at 0, 7, 14, 21, and 28 days post-immunization. The results showed that D169 antibodies could be detected in the swine serum immunized with these commercial vaccines after 21 days. Figure 8 b). This method was used to detect D169 antibodies in 8 clinical wild-type infected pig serum samples. The results showed that D169 antibodies were detected in all samples, and the OD450nm value and S / P value were both high. Figure 8 c). Specifically, this method was used to detect H18-R strain immunized swine serum (0, 7, 14, 21, 28 dpi), and all results were negative. Figure 8 d). The above results further demonstrate that this method can detect antibodies against the D169 protein encoded by the missing region in porcine serum infected with wild-type strains other than vaccine strain H18-R and other vaccine strains, and can be used for clinical differential diagnosis.

Claims

1. An attenuated strain H18-R of arterivirus porcine reproductive and respiratory syndrome virus, characterized in that: It is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the accession number CGMCC NO.46203 and the deposit date October 17, 2024. The NSP2 coding region of the attenuated strain H18-R continuously lacks nucleotides 1271-1795 relative to the HeB-108 strain, and the nucleotide sequence of the missing part is shown in SEQ ID NO.

1. The attenuated strain H18-R belongs to the NADC30-like strain of the L1 lineage.

2. A porcine reproductive and respiratory syndrome virus vaccine, characterized in that: The vaccine comprises the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus according to claim 1.

3. The vaccine according to claim 2, characterized in that The vaccine further comprises a pharmaceutical carrier or a pharmaceutically acceptable excipient.

4. Use of the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus according to claim 1 in the preparation of a vaccine or medicine for preventing blue ear disease in pigs.

5. An ELISA test kit for detecting porcine reproductive and respiratory syndrome virus and distinguishing the attenuated porcine reproductive and respiratory syndrome virus strain H18-R, characterized in that: The kit contains a protein with an amino acid sequence as shown in SEQ ID NO.

2.

6. The ELISA detection kit according to claim 5, characterized in that The kit includes a primary antibody and a secondary antibody, wherein the primary antibody includes PRRS positive serum and the secondary antibody is HRP-labeled goat anti-swine IgG (H+L).

7. Use of the attenuated strain H18-R of porcine reproductive and respiratory syndrome virus according to claim 1, the protein with an amino acid sequence as shown in SEQ ID NO. 2, the microbial cell carrying the protein shown in SEQ ID NO. 2, the gene encoding the protein shown in SEQ ID NO. 2 as SEQ ID NO. 1, and the recombinant vector containing the gene encoding SEQ ID NO. 1 in the preparation of a diagnostic and detection kit for porcine reproductive and respiratory syndrome virus.

8. An antigenic peptide of reproductive and respiratory syndrome virus, characterized in that: The amino acid sequence of the antigen peptide is shown in SEQ ID NO.

2.

9. A nucleotide encoding the antigenic peptide according to claim 8.

10. Use of a recombinant vector or recombinant microbial cell containing the nucleotide according to claim 9 in preparing an ELISA detection kit for detecting porcine reproductive and respiratory syndrome virus and identifying and distinguishing the attenuated porcine reproductive and respiratory syndrome virus strain H18-R.

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