Porcine reproductive and respiratory syndrome virus-like nadc34 strain, isolation and identification method thereof, infection model construction and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a porcine reproductive and respiratory syndrome virus NADC34 strain and a separation and identification method, an infection model construction and application thereof. The method innovatively integrates key links such as screening of epidemic strains with sufficient pathogenicity, standardization of virus preparation, determination of the best infection route and optimization of the most suitable attack dose, firstly determines a 'nose drop and muscle injection combined' route and a '4x10 5.0 TCID 50 ' dose per animal as core standardization parameters of the model, and establishes a comprehensive quantitative evaluation system matched with the parameters, covering body temperature kinetics, virusemia dynamics, multi-organ virus load and systematic pathological observation, so as to systematically solve technical bottlenecks such as lack of the strain-specific evaluation model, insufficient standardization of the existing model and inconsistency of evaluation indexes.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a porcine reproductive and respiratory syndrome virus strain NADC34, its isolation and identification method, infection model construction and application. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is a major global infectious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRSV), resulting in continuous and enormous economic losses to the pig farming industry. Since the disease was first reported in China in 1996, PRRSV has continued to evolve in my country due to its extremely high genetic variation rate and frequent recombination events, giving rise to numerous strains with complex genotypes and lineages, varying pathogenicity and transmission characteristics, posing unprecedented and severe challenges to the effective prevention and control of the disease.

[0003] In recent years, the NADC34-like PRRSV strain has gradually become prevalent in my country and North America, becoming one of the main circulating PRRSV lineages. This strain was first reported in North America, then entered my country through genetic evolution, and was rapidly detected in multiple provinces, showing a continuous spread. The NADC34-like strain has unique genetic characteristics and often recombines with locally prevalent strains, further enriching its genetic diversity and increasing the difficulty of prevention and control. Epidemiological surveys show that the infection rate of this strain in pig herds is increasing year by year, and existing commercial vaccines have limited cross-protective effects, posing new challenges to PRRS prevention and control in my country.

[0004] Currently, the construction and application of challenge models for PRRSV mainly rely on early classic strains (such as VR-2332), highly pathogenic strains (such as JXA1, lineage 8.7), or major circulating strains from previous years. However, as the virus continues to evolve, these traditional models have revealed significant technical defects and limitations when used to study emerging NADC34-like strains: 1. Insufficient timeliness and representativeness of the strains: The strains used in the existing models have generational gaps in genetic evolution compared to the currently prevalent NADC34-like strains. This strain has a unique genetic background, which makes it impossible for challenge models based on older strains to realistically simulate the infection dynamics, tissue tropism, and pathogenic process of NADC34-like strains in pigs.

[0005] 2. Lack of Model Standardization and Evaluation System: For NADC34-like strains, there is a lack of unified standardized parameters for constructing challenge models (such as challenge dose, route, animal age, and health status) and objective evaluation indicators. Existing studies mostly use wild-isolated strains, whose in vivo passage history and virulence stability are unclear, resulting in poor reproducibility and low comparability between different studies, and failing to provide a consistent benchmark for key applications such as vaccine efficacy evaluation.

[0006] 3. Difficulty in meeting the needs of precise prevention and control research and development: The lack of a dedicated model that accurately reflects the real-world infection characteristics of NADC34-like strains severely restricts the research and development process for evaluating the immunization efficacy of specific vaccines against this strain, screening novel antiviral drugs, and elucidating the pathogenic mechanism. Protection data obtained from existing models may not be able to predict the actual effectiveness of vaccines against NADC34 strains.

[0007] In addition, most existing commercial vaccines target traditional PRRSV strains (such as highly pathogenic PRRSV), and have limited cross-protective effects against NADC34-like PRRSV. There is an urgent need to develop prevention and control technologies adapted to NADC34-like PRRSV, and standardized strains, standardized isolation and identification methods, and stable infection models are the foundation for the development of prevention and control technologies.

[0008] Therefore, screening and isolating a stable NADC34-like strain with typical pathogenic characteristics, and establishing standardized isolation and identification methods and standardized infection models are of great significance for the epidemiological monitoring, vaccine development, drug screening and disease control of NADC34 PRRSV, and are also technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0009] The core of this invention lies in providing a standardized method for constructing a challenge model of porcine reproductive and respiratory syndrome virus (PRRSV) NADC34-like strains. This method successfully constructs a stable and reproducible animal infection model by selecting a currently prevalent and representative NADC34-like strain and systematically establishing a complete set of standardized parameters, including experimental animal standards, challenge dosage and route, and key observation indicators. The model constructed by this invention not only realistically simulates the natural infection process of this strain, enabling in-depth exploration of its pathogenic mechanism, but more importantly, it provides a crucial and universally applicable experimental benchmark and evaluation system for objectively comparing and evaluating the effectiveness of vaccine candidates, antiviral drugs, or treatment regimens against this strain. Thus, it becomes a key link from basic research to effective prevention and control applications.

[0010] To solve the above problems, the present invention adopts the following technical solution: First, a porcine reproductive and respiratory syndrome virus (PRRSV) strain NADC34 was proposed. This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:V202630 and deposit date of March 24, 2026.

[0011] A method for isolating and identifying PRRSV-like NADC34 strains was also proposed, including the following steps: (1) Virus isolation: Take lung tissue from pigs suspected of having PRRS-like NADC34, homogenize and centrifuge, take the supernatant, inoculate it into well-grown porcine alveolar macrophages (PAMs), culture for 72 hours to observe cytopathic effects, and if no cytopathic effects are observed, pass it blindly for 3 generations. (2) Gene amplification and sequencing: Total RNA was extracted from positive viral fluid, and RT-PCR amplification was performed using Nsp2 gene and GP5 specific primers. After purification of the amplification product, Sanger sequencing was performed. (3) Genetic evolution analysis and identification: The gene sequence obtained by sequencing was compared with the PRRSV reference strain sequence in the NCBI database for homology comparison. The genetic evolution tree was constructed using the neighbor-joining method with MEGA software to determine that the strain type was a NADC34-like strain. (4) Virus cloning and purification: The isolated and identified NADC34 strain was purified by three rounds of plaque cloning and recorded as generation P0. It was then inoculated with PAMs and passaged three times, recorded as generation P3 virus. TCID was then measured. 50 For use in subsequent research.

[0012] Furthermore, in step (2), primers for the Nsp2 and GP5 genes are designed as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; the RT-PCR reaction system has a total volume of 25 μL, including 1 μL of One Step Enzyme Mix, 12.5 μL of 2X One-Step Reaction Solution (dye plus), 1 μL each of forward and reverse primers, and 2 μL of Total RNA. The remaining volume is made up with RNase-free water; the PCR reaction program is as follows: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 2 min; 35 cycles (denaturation at 94℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 2 min); final extension at 72℃ for 5 min, and storage at 4℃.

[0013] This invention also proposes an application of the aforementioned PRRSV-like NADC34 strain in constructing a PRRSV infection model.

[0014] This invention also proposes a method for constructing an infection model of PRRSV-like NADC34 strain, comprising the following steps: (1) Screening of experimental animals: Healthy piglets that were negative for porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine pseudorabies virus, classical swine fever virus, and mycoplasma hyopneumoniae antigen and antibody were screened and then fed acclimatized for use. (2) Preparation of challenge strain: The PRRSV-type NADC34 strain P3 generation virus solution was diluted with 1640 medium to prepare the challenge working solution, and it was prepared and used immediately. (3) Virus challenge procedure: A combined nasal drop and intramuscular injection approach was used for virus challenge, with a total inoculation volume of 4 mL (virus content 10) per piglet. 5.0 TCID 50 / ml), of which 2mL is administered via nasal drops and 2mL via intramuscular injection in the neck, and the challenge dose is 4×10 5.0 TCID 50 / head; (4) Model monitoring: After the virus attack, the model was continuously monitored for 21 days to verify whether it was successfully constructed.

[0015] Furthermore, in step (2), the preparation method of the PRRSV-like NADC34 strain P3 generation virus solution is as follows: the cloned and purified PRRSV-like NADC34 strain is inoculated into PAMs, and the P3 generation virus solution is obtained by continuous passage for 3 generations. The virus titer is determined to be not less than 10. 5 TCID 50 / ml.

[0016] Furthermore, in step (4), the criteria for successful model construction are as follows: piglets exhibit high fever lasting more than 3 days after challenge, with a body temperature ≥40.5℃; the viral load in whole blood reaches its peak at 7-12 days and remains detectable on day 21; typical lesions such as dark red consolidation and interstitial widening of the lungs are visible upon necropsy; histopathological examination reveals characteristic interstitial pneumonia of PRRSV; and high viral load can be detected in target organ tissues, with a viral load not less than 10. 4.0 copies / μL.

[0017] Furthermore, in step (4), the monitoring indicators include: body temperature monitoring, whole blood viremia monitoring, gross lung lesion observation, histopathological examination, and tissue viral load detection.

[0018] Furthermore, in step (4), both whole blood viremia monitoring and tissue viral load detection were performed using PRRSV-specific real-time qRT-PCR. Primers and probes were designed for the ORF6 gene, such as SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. qRT-PCR reaction system: total volume 20μL, specific components are 2X One Step RT-qPCR Buffer II (probe) 10μL, Pro Taq HS DNA Polymerase 0.4μL, EVO M-MLV RTase Enzyme Mix II 0.4μL, forward and reverse primers (10μM) 1μL each, probe (10μM) 0.4μL, template RNA 5μL, and the remaining volume is made up to 20μL with RNase-free water; The reaction program was set as follows: reverse transcription at 42℃ for 5 min (to reverse transcribe RNA into cDNA); pre-denaturation at 95℃ for 30 s; followed by cycles of 95℃ for 5 s and 60℃ for 30 s, for a total of 40 cycles.

[0019] Finally, this invention also proposes the application of the PRRSV-like NADC34 strain infection model constructed by the method in the evaluation of PRRSV vaccine potency and the screening of antiviral drugs.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention successfully constructed a complete and standardized animal challenge model for PRRSV-like NADC34 strains. Its core advantage lies in its precise response to the urgent needs of current epidemic prevention and control, and its systematic solution to long-standing key technical bottlenecks in this field, specifically in the following aspects: 1. Timeliness, representativeness, and clear pathogenicity: The model was constructed directly using currently prevalent NADC34-like strains. The selected specific isolates have clear pathogenicity and can stably induce high fever (>40.5℃) and high-level persistent viremia (peak value >10). 4.0 Significant characteristics such as copies / μL and typical interstitial pneumonia were observed, effectively overcoming the shortcomings of some strains in similar studies that had weak infection indicators and inconsistent model success due to insufficient virulence, thus ensuring the reliability and discriminative power of the model.

[0021] 2. High standardization and strong reproducibility: Through systematic experiments, the optimal challenge route (combined nasal and intramuscular injection) and core challenge dose (4 × 10⁻⁶) were established, including the challenge strain (specific isolate), experimental animal standards (healthy piglets negative for porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine pseudorabies virus, classical swine fever virus, and Mycoplasma hyopneumoniae antigen and antibody), the optimal challenge route, and the core challenge dose. 5.0 TCID 50 The model incorporates a complete set of key standardized parameters, including the head, and has a clear and controllable construction process, which greatly improves the reproducibility and comparability of results across different laboratories and studies.

[0022] 3. Scientifically optimized challenge route for comprehensive infection simulation: The established "combined nasal drops and intramuscular injection" challenge route, after comparative verification, is significantly superior to single routes in inducing persistent high fever, establishing stable and high-level viremia, and forming widespread high-titer infections in multiple tissues such as the lungs, spleen, and lymph nodes. It can more comprehensively and realistically simulate the natural infection process of the virus.

[0023] 4. The evaluation system is comprehensive, objective, and quantitative: A multi-dimensional, quantitative evaluation index integrating clinical symptoms (body temperature), virology (dynamic viremia and multi-tissue viral load), and pathology (gross lesions and histopathology) has been established. This system can comprehensively and objectively quantify the degree of infection and the effectiveness of immune or therapeutic interventions, providing a solid and consistent quantitative benchmark for research and development evaluation.

[0024] 5. Clear application orientation and high practical value: This model is designed specifically to address the challenges of controlling the current NADC34-like strain. It can be used directly and accurately to evaluate the efficacy of specific vaccines, antiviral drugs and treatment regimens against this prevalent strain. It effectively overcomes the prediction bias that may occur when using old models for evaluation and accelerates the research and development process of precision prevention and control products. Attached Figure Description

[0025] Figure 1 This is a phylogenetic tree of the PRRSV strain isolated in this invention and the reference strain based on the GP5 gene nucleotide sequence; Figure 2 A phylogenetic tree of the PRRSV strain isolated in this invention and a reference strain based on the Nsp2 gene nucleotide sequence; Figure 3 This is an agarose gel electrophoresis image of the RT-PCR amplification products of the Nsp2 and GP5 genes of the PRRSV strain isolated in this invention; in the image, lane 1 M is the DNA molecular weight standard, lanes 2 and 3 are the samples to be tested, and the target band size is about 800 bp. Figure 4 Figures showing changes in body temperature, viremia, and tissue viral load in pigs under different PRRSV challenge routes; Figure 5 Figures showing changes in body temperature, viremia, and tissue viral load in pigs under different PRRSV challenge doses; Figure 6 Figure 1 shows the results of body temperature changes, viremia, and tissue viral load measurements in pigs after PRRSV immunization challenge. Detailed Implementation

[0026] Example 1: Source of the strain This invention successfully isolated a strain of porcine reproductive and respiratory syndrome virus (PRRSV) from clinical tissues of pigs infected with PRRSV in Henan Province. Through genetic evolutionary analysis and genome comparison (e.g.,...), the strain was further analyzed. Figure 1 , Figure 2 The strain was identified as a NADC34-type PRRSV strain and named JLCC-2022. After propagation by inoculating PAMs with this strain, its viral titer was found to be no less than 10. 5 TCID 50 / ml.

[0027] Example 2: Isolation and Identification of PRRSV-type NADC34 Strains 1. Processing of clinical tissues and preparation of crude virus extract Lung tissue with typical lesions was collected from a pig farm in Henan Province suspected of having an outbreak of swine reproductive and respiratory syndrome. Approximately 0.5g of sample was precisely taken from the boundary between diseased and normal tissue and placed in a sterile centrifuge tube. 1mL of sterile PBS buffer was added and thoroughly mixed. The centrifuge tube was then placed in a tissue homogenizer and homogenized at 60Hz for 1 min, with 5-second intervals, repeated 4 times to obtain the tissue homogenate.

[0028] The homogenate was subjected to freeze-thaw treatment: freezing at -20℃ and thawing at room temperature constituted one cycle, which was repeated three times to fully release the virus in the tissue. After freeze-thaw, the homogenate was centrifuged at 4℃ and 12000rpm / min for 5min, and the supernatant was collected. The supernatant was then filtered through a 0.22μm sterile filter membrane for sterilization. The sterile tissue homogenate filtrate was collected as the inoculation material for virus isolation and was temporarily stored at 4℃ for later use.

[0029] 2. Virus isolation and culture Resuscitated PAMs were transferred to T25 culture flasks. After 24 hours, the T25 culture flasks containing dense monolayers of porcine alveolar macrophages were removed, the culture medium was discarded, and the cells were gently washed once with sterile PBS buffer, discarding the wash solution to avoid damaging the cells. Subsequently, 1 mL of the above-mentioned sterile tissue homogenate filtrate was inoculated, and the culture flasks were placed in a 37°C, 5% CO2 incubator for virus adsorption for 2 hours. After adsorption, the inoculum in the culture flasks was removed, and 5 mL of 1640 cell maintenance medium containing 2% fetal bovine serum was added. The flasks were then incubated at 37°C, 5% CO2. During culture, the cytopathic effect (CPE) was observed daily under a microscope, and the time of lesion appearance and typical lesion characteristics were recorded.

[0030] After approximately 72 hours of culture, the cells exhibited typical CPE. The cells and supernatant in the culture flask were collected and transferred to a sterile centrifuge tube. The collected material was subjected to two freeze-thaw cycles at -70°C and centrifuged at 4°C and 12,000 rpm for 5 minutes. The supernatant was collected. If no CPE was observed, the cells were blindly passaged for three consecutive generations, and the supernatant was collected for subsequent identification experiments.

[0031] 3. Identification and genetic evolution analysis of viruses 3.1 Target gene amplification, electrophoresis detection and product purification (1) Viral RNA extraction Take 200 μL of virus solution and extract and purify total viral RNA according to the instructions of the viral nucleic acid extraction and purification kit from Nanjing Zhongke Bayer Medical Technology Co., Ltd. The obtained RNA template is stored at -80℃ for later use.

[0032] Specific primers were used for amplification of key fragments of the viral genome using specific primers for the NSP2 and GP5 genes and the EVOM-MLVOneStepRT-qPCRKit (dyeplus) from Aikerui Biotechnology Co., Ltd.

[0033] The primers used to amplify a portion of the Nsp2 gene are: Nsp2-F:5'-ACCTCCTTTGATTGGGATGTTGTG-3' (SEQ ID NO:1); Nsp2-R: 5'-ATGATGGCTTGAGCTGAGTA-3' (SEQ ID NO: 2).

[0034] The primers used to amplify part of the GP5 gene are: GP5-F:5'-GGCGACCGTTTTAGCCTGTCTT-3' (SEQ ID NO:3); GP5-R: 5'-ATCATTATTGGCGTGTAGGTG-3' (SEQ ID NO: 4).

[0035] The reaction system (25 μL) consisted of: 1 μL One-Step Enzyme Mix, 12.5 μL 2X One-Step Reaction Solution (dye plus), 1 μL each of forward and reverse primers (10 μM), 2 μL Total RNA, and 7.5 μL RNase-free water.

[0036] PCR reaction program: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 2 min; 35 cycles of 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 2 min; final extension at 72℃ for 5 min; and storage of amplified products at 4℃.

[0037] For agarose gel electrophoresis detection, take 5 μL of PCR amplification product, add an appropriate amount of loading buffer, mix well, and then load the sample into a 1% agarose gel for electrophoresis detection. After electrophoresis, observe the bands under a gel imaging system and record the size and position of the specific bands.

[0038] like Figure 3 As shown, the target gene amplification results are as follows. 1% agarose gel electrophoresis shows that the PCR amplification product has a clear, single, specific band at about 800bp, indicating that the PRRSV target gene fragment has been successfully amplified.

[0039] The target band was purified using the OMEGA EZNA® Gel Extraction Kit to obtain the purified PCR product.

[0040] 3.2 Sequencing and Evolutionary Analysis The purified Nsp2 and GP5 gene PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for Sanger sequencing to obtain the gene nucleotide sequences.

[0041] Sequence alignment: The nucleotide sequences of the Nsp2 and GP5 genes obtained from sequencing were uploaded to the NCBI database, and the BLAST tool was used to perform homology comparison with the published PRRSV reference strain sequences in the database.

[0042] The phylogenetic tree was constructed using MEGA software with the neighbor-joining method. The Bootstrap value was set to 1000 (repeated 1000 times). Cluster analysis was performed between this isolate and PRRSV reference strains of different lineages to construct the phylogenetic tree.

[0043] To determine the strain type, the gene lineage and evolutionary relationship of this isolate are determined based on the sequence homology comparison results and the clustering of the phylogenetic tree.

[0044] Sequence and phylogenetic analysis results: NCBI BLAST sequence alignment showed that the Nsp2 and GP5 genes of this isolate were highly homologous to the NADC34 type PRRSV reference strain; phylogenetic analysis showed that this isolate clustered with the NADC34 type PRRSV strain in the same branch, thus identifying this isolate as a PRRSV-like NADC34 strain (e.g., Figure 1 , Figure 2 (As shown).

[0045] 3.3 Virus cloning and purification Take the inoculation supernatant that shows obvious cytopathic effects and is correctly identified, and perform a series of 10-fold serial dilutions with cell maintenance medium (1640 culture medium containing 2% bovine serum). Take 10... -2 ~10 -7 Porcine alveolar macrophages that had grown to a monolayer and for which the growth medium had been discarded were inoculated into 6-well cell culture plates at different dilutions, 500 μl per well; after adsorption at 37°C for 2 hours, the virus solution was discarded; a 2% low-melting-point agarose solution was prepared and placed in a water bath at 40°C–50°C for later use; the above agarose was mixed with 2× phenol red-free 1640 maintenance medium at a ratio of 1:1 and added to each culture well, 2 ml per well; after cooling and solidification, the culture plate was inverted and cultured at 37°C with 5% CO2.

[0046] Observe the lesions daily, and perform a second covering when obvious lesions appear: Take the mixture prepared as described above, add neutral red to a final concentration of 0.002%, add 2 ml of the mixture to each culture well, allow it to cool and solidify to form a second covering layer, and incubate the culture plate upside down at 37°C and 5% CO2. Observe under a microscope within 48 hours, pick at least 3 empty plaques and add them to 200 μL of cell maintenance medium, repeating freeze-thaw twice; inoculate porcine alveolar macrophages that have formed a dense monolayer after thawing and whose growth medium has been discarded into 24-well cell culture plates, adsorb at 37°C for 2 hours, add 800 μL of cell maintenance medium, and incubate at 37°C and 5% CO2. When obvious lesions appear in the cells, harvest the cell culture, freeze-thaw twice at -70°C, and then identify and determine the virus content by RT-PCR. Perform continuous cloning and purification of the virus solution for 3 generations using the above method, and record it as the P0 generation virus solution. Passage the virus solution with PAMs 3 times and determine the TCID. 50 This is designated as the P3 generation virus for subsequent research.

[0047] Example 3: Preservation of Viruses The PRRSV-like NADC34 strain (named JLCC-2022 strain) isolated and identified in this invention was prepared into freeze-dried virus and deposited at the China Center for Type Culture Collection (CCTCC). Classification and naming: Porcine reproductive and respiratory syndrome virus (PRRSV-JLCC-2022). Deposit address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Deposit date: March 24, 2026. Deposit number: CCTCCNO: V202630. Deposit period: at least 30 years.

[0048] Example 4: Determination of titer of PRRSV-type NADC34 strain (TCID) 50 Law) 1. Serial dilution of virus solution (10-fold series) Take the PRRSV-like NADC34 strain P3 progeny obtained in Example 2, take a sterile 1.5 mL centrifuge tube, and perform a 10-fold serial dilution using cell maintenance medium containing 2% fetal bovine serum. Obtain 10 -¹ Up to 10 -6 The product was serially diluted 10-fold, with strict aseptic technique throughout.

[0049] 2.96-well plate cell plating and virus inoculation After resuscitating PAMs, adjust the concentration to an appropriate level and seed them directly into 96-well cell culture plates at a sufficient number of cells per well, ensuring that the cells immediately cover the bottom of the wells and form a dense monolayer. Incubate the culture plates at 37°C and 5% CO2 for 12-24 hours. Once the cells have fully adhered to the plates and recovered to their physiological state, discard the original culture medium.

[0050] The PRRSV strain to be tested was serially diluted 10-fold using 1640 cell maintenance medium containing 2% fetal bovine serum, and 10 were selected. -2 Up to 10 -6 Five consecutive dilutions were prepared. 100 μL of virus solution from each dilution was inoculated into each of the above-mentioned 96-well plates, with eight replicates per dilution. A normal cell control group (8 wells) was also established, with only 100 μL of the above-mentioned cell maintenance medium added to each well.

[0051] The inoculated culture plates were placed in a 37°C, 5% CO2 incubator for 5 consecutive days. The occurrence of cell-specific pathological effects (CPE) in each group was observed and recorded daily using an inverted microscope. Finally, the Reed-Muench method was used to calculate the median tissue culture infectious dose (TCID) of this PRRSV strain. 50 / mL).

[0052] Experimental results show that the PRRSV-like NADC34 strain P3 generation virus fluid isolated in this invention, calculated by the Reed-Muench method, has an infectivity titer of not less than 10. 5 TCID 50 / mL indicates that this strain has good proliferation capacity and infectivity on PAMs.

[0053] Example 5: Determination of the Infection Pathway 1. Screening of experimental pigs Eight-week-old piglets from a pig farm in Hubei Province were selected and tested for major pathogens and related antibodies. A total of 20 healthy piglets were selected, and they were negative for porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine pseudorabies virus, classical swine fever virus, and mycoplasma hyopneumoniae antigen and antibody.

[0054] 2. Experimental grouping and challenge procedures 2.1 Experimental animals and grouping: Twenty PRRSV-negative piglets aged 8 weeks after screening were randomly divided into 4 groups.

[0055] 2.2 Virus strain and dosage: The virus strain used for challenge was the PRRSV-like NADC34 strain P3 generation virus solution isolated and identified in Example 1. Using 1640 medium, the virus stock solution was prepared to contain 10... 5.0 TCID 50 / ml working solution, the challenge dose was set at 4×10 5.0 TCID 50 / head.

[0056] 2.3. Virus attack operation Group A (nasal drop group): Using a sterile syringe, 2 mL of virus solution containing the prescribed viral dose was slowly dripped into the nasal cavity through each nostril, for a total of 4 mL (total dose 4 × 10⁻⁶). 5.0 / head).

[0057] Group B (intramuscular injection group): 4 mL of viral fluid containing the prescribed viral dose was injected into the neck muscle (total dose 4 × 10⁻⁶). 5.0 / head).

[0058] Group C (combined nasal and intramuscular injection challenge group): The above-mentioned nasal (2mL) and intramuscular (2mL) procedures were performed simultaneously, for a total of 4mL of virus solution inoculated (total dose 4×10). 5.0 / head).

[0059] Group D (blank control group): The same volume of sterile PBS buffer (2 mL each) was injected into the bilateral nasal cavity and the neck intramuscularly.

[0060] 2.4 Clinical and Sample Collection Monitoring Monitoring was conducted for 21 days after the virus challenge.

[0061] 2.4.1 Body temperature monitoring: Rectal body temperature was measured daily for 21 consecutive days after the challenge.

[0062] 2.4.2 Viremia monitoring: Blood samples were collected from the anterior vena cava on days 2, 5, 7, 9, 11, 14, 18, and 21 after viral challenge. The viral RNA load in whole blood was detected by qRT-PCR. A standard curve was established using standards with known copy numbers. The Ct values ​​of the samples were substituted into the linear regression equation to calculate the viral RNA copy number (copies / μL). The viral nucleic acid copy number was also recorded.

[0063] The reaction system for quantitative real-time qRT-qPCR is as follows: (1) Extract RNA from whole blood (method as in Example 2) (2) The reaction system was constructed according to the instructions of the EVO M-MLV One Step RT-qPCR Kit II (probe) from Acrel Biotech Co., Ltd. Specific primers and probes targeting porcine reproductive and respiratory syndrome virus were used for detection; their sequences are as follows: Upstream primer M-qF:5'-TTGCTAGGCCGCAAGTAC-3' (SEQ ID NO:5); Downstream primer M-qR:5'-ACGCCGGACGACAAATGC-3' (SEQ ID NO:6); Probe MP:5'-FAM-CTGGCCCCTGCCCACCAC-BHQ1-3' (SEQ ID NO:7); The total reaction volume was 20 μL, and the specific components were: 10 μL of 2X One Step RT-qPCR Buffer II (probe), 0.4 μL of Pro Taq HS DNA Polymerase, 0.4 μL of EVO M-MLV RTase Enzyme Mix II, 1 μL each of forward and reverse primers (10 μM), 0.4 μL of probe (10 μM), 5 μL of template RNA, and the remaining volume was made up to 20 μL with RNase-free water.

[0064] (3) Reaction program setup Reverse transcription at 42℃ for 5 min (to reverse transcribe RNA into cDNA); pre-denaturation at 95℃ for 30 s; followed by cycles of 95℃ for 5 s and 60℃ for 30 s, for a total of 40 cycles.

[0065] (4) Experimental control setup A negative control (using RNase-free water instead of template RNA) and a positive control (a known PRRSV positive viral solution) were set up.

[0066] 2.4.3 Pathological examination: All surviving animals were necropsy on day 21 after challenge.

[0067] 2.4.4 Tissue viral load detection: Lung and inguinal lymph nodes were collected, homogenized, and then the viral RNA load in the tissues was detected by qRT-PCR.

[0068] 2.4.5 Qualitative Evaluation of Lung Lesions: Autopsy was performed on day 21 post-infection to evaluate lung lesions. First, lesions were observed in each group of lungs, and the presence and distribution of gross lesions such as dark red consolidation and interstitial widening on the surface of the lung lobes were systematically recorded; this result served as an important preliminary evaluation indicator. Subsequently, samples were taken and fixed from typical lesion areas and their junctions, and paraffin embedding, sectioning, HE staining, and subsequent full-field digital section scanning were performed by Guangzhou Maike Biotechnology Co., Ltd. Based on the high-resolution digital section images provided by the company, the researchers conducted a final histopathological verification and qualitative determination under double-blind conditions to assess the presence of characteristic interstitial pneumonia lesions (widening of alveolar septa with inflammatory cell infiltration).

[0069] 3. Experimental Results 3.1 Body temperature monitoring results like Figure 4 As shown, the combined nasal and intramuscular injection group (Group C) exhibited the most significant and sustained febrile response. The incidence and duration of high fever (>40.5℃) in this group were higher or longer than those in the single nasal injection group (Group A) or the single intramuscular injection group (Group B). Although single-route groups could induce fever, the severity and stability of the febrile response were not as good as those in the combined route. No abnormal fever was observed in the blank control group.

[0070] 3.2 Results of pancytemic viremia like Figure 4 As shown, dynamic monitoring of whole blood viral load (qRT-PCR) after challenge indicated that the combined nasal and intramuscular injection approach (Group C) was the most advantageous in establishing high-level, persistent viremia. This group achieved a high viral load (10T) as early as day 7 post-challenge. 4.56 (copies / μL), and maintained a high level (10 copies / μL) even in the later monitoring period (day 18). 3.27 The viral load was highest at 10 copies / μL, demonstrating the most stable and sustained dynamics of viremia. In contrast, the nasal drop group (Group A) and the intramuscular injection group (Group B) experienced viral load increases from day 2 to day 7 post-challenge (with a maximum of 10 copies / μL in Group B). 4.25 The viral load decreased more rapidly than in the combined challenge group (copies / μL), but the peak viral load, persistence, and stability were all lower than in the combined challenge group. The blank control group showed negative results at all time points.

[0071] 3.3 Histopathological Results Gross lesion observation showed that the proportion of individuals with typical lung lesions such as visual consolidation after viral challenge was 2 / 5, 3 / 5, and 4 / 5 in the nasal drop group, intramuscular injection group, and combined nasal drop and intramuscular injection group, respectively. In the blank control group (Group D), all animals had pink lungs with soft and uniform texture, and no visible lesions such as consolidation, hemorrhage, or interstitial widening were observed.

[0072] Further histopathological (HE staining) results showed that the lung tissue of animals in the combined nasal and intramuscular injection challenge group (Group C) exhibited typical PRRSV interstitial pneumonia lesions, including significant widening of alveolar septa and characteristic changes such as extensive infiltration of lymphocytes and macrophages within the alveolar spaces and septa. While some pathological changes were also observed in the nasal injection group (Group A) and the intramuscular injection group (Group B), the degree and extent of the lesions were less pronounced than in Group C, showing focal, mild alveolar septal thickening and a small amount of inflammatory cell infiltration. In the blank control group, the alveolar structure was clear and intact, no alveolar septal thickening was observed, no inflammatory exudate was found in the alveolar spaces, and no pathological changes such as lymphocyte or macrophage infiltration were observed.

[0073] 3.4 Detection of viral load in tissues like Figure 4 As shown, based on the viral load (qRT-PCR) results of tissue samples examined on day 21 post-challenge, the combined intranasal and intramuscular challenge group (Group C) induced the highest levels of viral infection in multiple target organs. In lung tissue, the viral load in the combined challenge group (10-1) was significantly lower. 5.29 The number of copies / g was significantly higher in the nasal drop group than in the 10-copies / g group. 4.70 copies / g) and intramuscular injection group (10 ... 4.56 copies / g). In the spleen and inguinal lymph nodes, the viral load in the combined challenge group (10 copies / g). 4.87 copies / g, 10 5.20 The copies / g ratio was also higher than in the other two single-pathway groups. All tissue samples in the blank control group tested negative.

[0074] 4. Conclusion The above results indicate that, when challenged with the PRRSV-like NADC34 strain isolated using this patent, the combined nasal and intramuscular injection approach (Group C) most comprehensively and stably simulates the core pathogenic process of the virus on lung tissue, manifesting as typical and significantly severe interstitial pneumonia lesions. While the single nasal injection approach (Group A) or intramuscular injection approach (Group B) can also induce some pathological changes, the extent and severity of the lesions are not as significant as in the combined challenge group. Therefore, the combined challenge approach is more advantageous in simulating lung pathological damage caused by the PRRSV-like NADC34 strain, providing the optimal choice for constructing a standardized challenge model.

[0075] Example 6: Determination of the Infection Dosage This embodiment is based on the combined intranasal and intramuscular injection challenge route determined in Example 5. By setting three gradient challenge doses (low, medium, and high), the induction effects of different doses on clinical symptoms, viremia dynamics, tissue viral load, and pathological damage in PRRSV-negative piglets were compared. The optimal challenge dose that can stably, efficiently, and economically construct a PRRSV-like NADC34 strain piglet infection model was screened out, providing a standardized basis for infection dosage for subsequent vaccine potency evaluation and antiviral drug screening.

[0076] 1. Screening of experimental pigs The method is the same as in Example 5, and 20 PRRSV-negative 8-week-old piglets were selected.

[0077] 2. Experimental grouping and challenge procedures 2.1. Experimental Animals and Grouping Twenty 8-week-old PRRSV-negative piglets selected using the same method as in Example 5 were randomly divided into four groups of five piglets each: a low-dose challenge group, a medium-dose challenge group, a high-dose challenge group, and a blank control group.

[0078] 2.2 Preparation of challenge strains and dosage The challenge strain was the PRRSV-like NADC34 strain P3 generation virus fluid isolated and identified above. The virus stock solution was prepared into virus-containing solutions of 10- cells using cell maintenance medium. 4.0 10 5.0 10 6.0 TCID 50 / mL of attack working solution.

[0079] 2.3 Virus attack operation All challenge groups used the combined nasal drop + intramuscular injection challenge route determined in Example 5, while the blank control group underwent an equal volume of sterile PBS for simulated inoculation. The total inoculation volume was uniformly 4 mL (2 mL nasal drops + 2 mL intramuscular injection), and the procedure was as follows: Low-dose challenge group: vaccinated 10 4.0 TCID 50 / mL of challenge working solution, total dose 4×10 4.0 TCID 50 / head; Medium-dose challenge group: vaccinated 10 5.0 TCID 50 / mL of challenge working solution, total dose 4×10 5.0 TCID 50 / head; High-dose challenge group: vaccinated with 10 6.0 TCID 50 / mL of challenge working solution, total dose 4×10 6.0 TCID 50 / head; Blank control group: 2 mL of sterile PBS was instilled through the nasal cavity and 2 mL of sterile PBS was injected intramuscularly in the neck, for a total inoculation volume of 4 mL, with no virus inoculation.

[0080] 2.4 Clinical and Sample Collection After the challenge, continuous monitoring was conducted for 21 days. The monitoring methods, sample collection time, and processing methods were the same as in Example 5.

[0081] 3. Experimental Results 3.1 Body temperature monitoring results like Figure 5 As shown, both the high-dose and medium-dose challenge groups induced significant hyperthermia. Following challenge, the body temperature of animals in both groups rose rapidly. In the high-dose group, the vast majority of individuals maintained a hyperthermia above 40.5℃ from day 1 until day 21 post-challenge. The medium-dose group also exhibited significant hyperthermia, with peak body temperatures reaching 41.1℃, which persisted for several days. Although some individuals experienced slight fluctuations during the mid-infection period, the hyperthermia effectively simulated infection characteristics. While a rise in body temperature was observed in the low-dose challenge group, only a few individuals briefly reached 40.5℃, indicating a weaker and less persistent fever response. The body temperature of the blank control group remained within the normal physiological range (39.3-40℃).

[0082] 3.2 Results of pancytemic viremia like Figure 5 As shown, dynamic monitoring of whole blood viral load (qRT-PCR) after challenge revealed that both the medium-dose and high-dose challenge groups rapidly established high and stable levels of viremia. The total viral load in the medium-dose group was approximately 10 on day 2 post-challenge. 3.32 The viral load initially increased by 10 copies / μL, then steadily rose, remaining at a high level from day 7 to 12. It was still detectable in the later stages of infection (day 21). 2.95 The viremia dynamics in the high-dose group were similar to those in the medium-dose group, with slightly higher overall viral load at each time point, but without significant difference. Although viremia was detectable in the low-dose group, the overall viral load at each time point was significantly lower than that in the medium- and high-dose groups, and the decrease was faster. All time points in the blank control group were negative. These results indicate that the medium-dose (10 copies / μL) group... 5.0 TCID 50 ( / head) is sufficient to establish a systemic viral infection comparable to high doses and is sustained and stable.

[0083] 3.3 Histopathological Examination Results Gross lesion observation showed that the proportion of individuals with typical lung lesions such as visual consolidation after challenge was 2 / 5, 4 / 5, and 5 / 5 in the low-dose challenge group, medium-dose challenge group, and high-dose challenge group, respectively. In the blank control group (group D), all animals had pink lungs with soft and uniform texture, and no visible lesions such as consolidation, hemorrhage, or interstitial widening were observed.

[0084] Further histopathological (HE staining) results showed that the lung tissues of animals in both the medium-dose and high-dose groups exhibited typical PRRSV interstitial pneumonia lesions, including significant widening of alveolar septa and characteristic changes such as extensive infiltration of lymphocytes and macrophages within the alveolar spaces and septa. The severity of the lesions was comparable in both groups. While some pathological changes were also observed in the low-dose group, the extent and severity of the lesions were less than in the medium- and high-dose groups, manifesting as focal, mild alveolar septal thickening and a small amount of inflammatory cell infiltration. In the blank control group, the alveolar structure was clear and intact, with no thickening of the alveolar septa, no inflammatory exudate within the alveolar spaces, and no pathological changes such as lymphocyte or macrophage infiltration.

[0085] 3.4 Results of viral load testing like Figure 5 As shown, based on the viral load (qRT-PCR) results of tissues examined on day 21 post-infection, the infection levels in major target organs showed a dose-dependent relationship between different dose groups. In lung tissue, the medium-dose group (10... 5.01 copies / g) and high-dose group (10 copies / g) 5.09 The infection level (copies / g) was significantly higher in the low-dose group than in the low-dose group (10 copies / g). 4.29 copies / g). In spleen tissue, viral load (10 copies / g) in the medium and high dose groups. 4.30 copies / g, 10 4.68 The number of copies / g was also significantly higher in the low-dose group (10 copies / g). 3.86 Copies / g). The same was observed in the inguinal lymph nodes in the medium-dose group (mean 10). 4.91 copies / g) and high-dose group (10 ... 5.14 The loading (copies / g) was higher in the low-dose group than in the low-dose group (10 copies / g). 4.07 (Copies / g). All tissue samples in the blank control group tested negative.

[0086] 4. Conclusion Based on comprehensive indicators such as clinical symptoms, dynamic viremia, and tissue viral load, this embodiment determines to use 10 5.0 TCID 50A medium dose was administered via a combination of nasal drops and intramuscular injection for challenge. This dose stably induced a typical high fever response (body temperature >40.5℃), with viral load reaching its peak (10⁻⁶) on days 7-12 post-challenge. 4.72 The viral load was [number of copies / μL], and maintained a sustained and stable viremia until the late stage of infection (detectable on day 21). Tissue viral load analysis showed that the medium-dose group achieved a level of infection in the lungs comparable to the high-dose group (mean values ​​of 10 [copies / μL]). 5.01 With 10 5.09 copies / g), in the spleen (10 copies / g), 4.30 copies / g) and inguinal lymph nodes (10 copies / g) 4.91 It also caused significant systemic and local infections in copies / g and successfully induced typical PRRSV interstitial pneumonia lesions.

[0087] In summary, this attack strategy "4×10" 5.0 TCID 50 " / head (2mL nasal drops + 2mL intramuscular injection)" can stably, effectively and economically establish a piglet infection model of PRRSV-like NADC34 strain, which is suitable for subsequent vaccine or drug evaluation.

[0088] Example 7: Preparation of a non-commercial PRRSV attenuated vaccine strain for immunization evaluation The non-commercial attenuated live vaccine used in the subsequent immune challenge experiments of this invention was obtained by selecting and attenuating the parental strain PRRSV JLCC-2022, which was preserved in Example 3 of this invention, through continuous passage in cells. The specific preparation and attenuation process of this attenuated vaccine strain are as follows: The revived parental strain JLCC-2022 was inoculated into well-grown Marc-145 cells and continuously passaged in DMEM medium containing 2% fetal bovine serum at 37°C and 5% CO2. Cytopathic effect (CPE) was observed at each passage. When 70%-80% typical CPE was observed, the virus was released through repeated freeze-thaw cycles. The supernatant was collected by centrifugation and inoculated into the next generation of cells at a 0.5% MOI. After continuous passage to the 100th generation, a stable, adapted cell line was obtained and labeled JLCC-2022-F100.

[0089] To verify the safety of the aforementioned high-generation passaged strain JLCC-2022-F100, the method was the same as in Example 5, screening 10 PRRSV-negative 4-week-old piglets. (The last sentence appears to be incomplete and possibly refers to a separate study.) 5.0 TCID 50 The dose is administered via a single intramuscular injection per head.

[0090] Experimental results: 1. Body temperature and clinical symptoms: After 28 days of continuous observation following immunization, the body temperature of all piglets remained within the normal range, and no sustained fever above 40.0℃ typical of virulent strain infection was observed; in addition, the piglets' mental state and feed intake were normal, and no respiratory distress or cyanosis was observed.

[0091] 2. The RT-qPCR method described in Example 5 was used to periodically test blood samples from piglets and tissue samples from necropsy. Results showed that throughout the observation period, only extremely low levels of transient viral nucleic acid were detected in the blood of inoculated piglets. Viral colonization and proliferation were not detected in key target organs such as the lungs, spleen, and inguinal lymph nodes, and the viral load in tissues remained below the detection threshold. Furthermore, necropsy and histopathological observation confirmed that this passaged strain did not cause any substantial pathological damage to the organism.

[0092] Example 8 Model Validation – Immune Challenge Test This embodiment is based on the "nasal drops + intramuscular injection combined attack route" determined in Embodiment 5 and the "4×10" method determined in Embodiment 6. 5.0 TCID 50 " / head challenge dose", constructing a standardized model for immune challenge experiments of PRRSV-like NADC34 strains.

[0093] 1. Screening of experimental pigs The method is the same as in Example 5, and 15 PRRSV-negative 4-week-old piglets were selected.

[0094] 2. Experimental Design and Immunization 2.1 Experimental grouping: 15 piglets were randomly divided into 3 groups of 5 piglets each. Group A (Immunization and Challenge Group): Administered NADC34 live attenuated vaccine (1 dose / head), and challenged 28 days after vaccination.

[0095] Group B (challenge control group): No immunization was performed, only challenge was performed, serving as a control for challenge infection.

[0096] Group C (blank control group): No immunization or challenge was performed; only an equal volume of sterile PBS was injected to monitor the baseline status of the animals.

[0097] In this embodiment, an attenuated vaccine strain (JLCC-2022-F100) with a genetic background completely identical to the challenge strain was used for immunization to verify the evaluation capability of the JLCC-2022 infection model constructed in this invention. The vaccine strain JLCC-2022-F100 was prepared according to the method described in Example 7.

[0098] 3.2 Immunization Program Immunization group: Group A animals were immunized with NADC34 attenuated live vaccine (1 dose intramuscularly). Control group: Groups B and C were injected with the same volume of sterile PBS buffer at the same time.

[0099] 4. Virus Attack Challenge 4.1 Timing of the attack On day 28 post-immunization, when the vaccine had completed the induction of the immune response, the experimental pigs in groups A and B were challenged with the virus.

[0100] 4.2 Virus attack operation Using the PRRSV-like NADC34 strain P3 generation virus fluid isolated and identified in Example 1, the challenge dose (4 × 10⁻⁶) was strictly followed according to the challenge dose determined in Example 6. 5.0 TCID 50 / head, total inoculation volume 4mL) (2mL nasal drops + 2mL intramuscular injection) and the challenge route determined in Example 5 (nasal drops + intramuscular injection) were used to challenge animals in groups A and B. Group C was inoculated with an equal volume of sterile PBS buffer.

[0101] 5. Monitoring Indicators and Methods After the challenge, continuous monitoring was conducted for 21 days. The monitoring methods, sample collection time, and processing methods were the same as in Example 5.

[0102] 6. Test Results 6.1 Body temperature monitoring results: like Figure 6 As shown, in the immune challenge validation experiment, the challenged control group (unimmunized) animals exhibited typical PRRSV infection fever characteristics after challenge, characterized by persistent high fever (body temperature >40.5℃) for several days, lasting for approximately 10 days, with the highest individual peak reaching 41.4℃. In contrast, the immune challenge group animals showed significantly reduced fever, with only brief, mild increases in body temperature, without developing persistent high fever, and their body temperature rapidly returned to normal levels (not exceeding 40.0℃) in the later stages of infection. The blank control group maintained normal body temperature throughout, showed no fever response, and had normal mental state, food and water intake.

[0103] 6.2 Pancytemia: like Figure 6 As shown, based on the dynamic monitoring results of whole blood viral load (qRT-PCR) after challenge, the challenge control group (Group B) showed high viremia on day 2 after challenge, and reached peak viral load on day 12 (10⁻⁶). 4.3 (copies / μL), and remained at a high level thereafter, remaining stable until day 21 (10 copies / μL). 2.9 The viral replication in the immune challenge group (Group A) was significantly suppressed, with a peak viral load of (10 copies / μL). 3.2The viral load in whole blood of most animals was significantly lower than that in the challenge control group, and after day 14 post-challenge, the viral load had decreased to below the detection limit or to very low levels (10-1 μL). 0.90 The blank control group (Group C) had negative results at all time points.

[0104] 6.3 Histopathology: Gross observation revealed that, at necropsy on day 21 post-infection, 4 / 5 of the animals in the challenge control group (Group B) showed varying degrees of visible lung lesions, characterized by scattered or confluent dark red to brownish-red consolidation areas in multiple lobes, with a firm texture and significantly widened interstitium. In the immune challenge group (Group A), only a few animals (1 / 5) showed a small amount of focal light red consolidation areas in their lungs. In the blank control group (Group C), the lungs were pink in color, uniform in texture, and showed no abnormalities.

[0105] Further histopathological examination (HE staining) confirmed that in the challenge control group (Group B), all animals exhibited typical PRRSV interstitial pneumonia lesions, including significantly widened alveolar septa, extensive infiltration of lymphocytes and macrophages, and inflammatory exudate in some alveolar spaces. In the immune challenge group (Group A), only a few animals exhibiting gross lesions showed mild, localized interstitial thickening and a small amount of inflammatory cell infiltration; the lung tissue morphology of the remaining animals was basically normal. No related pathological changes were observed in the blank control group (Group C).

[0106] 6.4 Organ viral load: like Figure 6 As shown, based on the viral load (qRT-PCR) results of tissues examined on day 21 post-challenge, the viral load in the challenge control group (Group B) was significantly higher than that in the immune challenge group (Group A) in all major target organs. In the lungs, the viral load in the challenge control group (10T) was significantly lower than that in the immune challenge group (Group A). 4.86 The number of copies / g was significantly higher in the immune challenge group (10 copies / g) than in the immune challenge group (10 copies 3.35 copies / g). In the spleen, the viral load in the challenge control group (10 copies / g). 4.46 The number of copies / g was also higher than that of the immune challenge group (10 copies / g). 3.41 Copies / g). In the inguinal lymph nodes, the challenge control group also maintained a higher viral load (10 copies / g). 4.92 The viral load was 10 copies / g, while the viral load in the immune challenge group was at a lower level (10 copies / g). 3.56 (Copies / g). All tissue samples in the blank control group tested negative.

[0107] 7. Conclusion This embodiment successfully verified the efficacy of the "nasal drops + intramuscular injection combined with viral challenge" approach through an immune challenge experiment, using a 4×10... 5.0 TCID50 The PRRSV-like NADC34 strain piglet infection model constructed using the " / head challenge dose" exhibits excellent stability, sensitivity, and discriminative power, and can serve as a reliable benchmark tool for evaluating vaccine and drug efficacy. Specific validation conclusions are as follows: 1. The model exhibits good stability. The unimmunized challenge control group (Group B) animals comprehensively and stably simulate the natural infection process and pathogenic characteristics of the PRRSV-like NADC34 strain. Specifically, the challenge control group (Group B) animals developed a persistent high fever (body temperature >40.5℃, with an individual peak of 41.4℃) for approximately 7 days, establishing and maintaining a high level of viremia (peak whole blood viral load reaching 10). 4.33 (Copies / g, still detectable until day 21), inducing high viral loads in multiple target organs such as the lungs, spleen, and lymph nodes (e.g., up to 10 copies / g in lung tissue). 4.86 (copies / g), and caused typical macroscopic and microscopic pathological changes in interstitial pneumonia.

[0108] 2. The model exhibits excellent sensitivity and discrimination. In contrast, the clinical symptoms (fever), viral replication level (viremia and tissue load), and pathological damage of animals immunized with the vaccine (Group A animals immunized with PRRSV attenuated live vaccine) were significantly alleviated or suppressed. The blank control group C showed no abnormalities throughout the process, further demonstrating that this model can clearly and objectively distinguish the difference between "immune protection" and "non-immune protection" and can effectively evaluate the actual protective effect of vaccine immunization against NADC34 strain challenge.

[0109] 3. The model is highly practical. The standardized model has a clear operation and high reproducibility. The challenge protocol is clear and the judgment criteria are clear. It can provide a unified and reliable animal infection model to support subsequent research on PRRSV-like NADC34 strain-related vaccine development, vaccine potency evaluation, and antiviral drug screening. It has high scientific research and application value.

Claims

1. A porcine reproductive and respiratory syndrome virus (PRRSV) NADC34 strain, characterized in that, This strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:V202630 and deposit date of March 24, 2026.

2. A method for isolating and identifying PRRSV-like NADC34 strains, characterized in that, Includes the following steps: (1) Virus isolation: Take lung tissue from pigs suspected of having PRRS-like NADC34, homogenize and centrifuge, take the supernatant, inoculate it into well-grown porcine alveolar macrophages (PAMs), culture for 72 hours to observe cytopathic effects, and if no cytopathic effects are observed, pass it blindly for 3 generations. (2) Gene amplification and sequencing: Total RNA was extracted from positive viral fluid, and RT-PCR amplification was performed using Nsp2 gene and GP5 specific primers. After purification of the amplification product, Sanger sequencing was performed. (3) Genetic evolution analysis and identification: The gene sequence obtained by sequencing was compared with the PRRSV reference strain sequence in the NCBI database for homology comparison. The genetic evolution tree was constructed using the neighbor-joining method with MEGA software to determine that the strain type was a NADC34-like strain. (4) Virus cloning and purification: The isolated and identified NADC34 strain was purified by three rounds of plaque cloning and recorded as generation P0. It was then inoculated with PAMs and passaged three times, recorded as generation P3 virus. TCID was then measured. 50 For use in subsequent research.

3. The method for isolating and identifying PRRSV-type NADC34 strains according to claim 2, characterized in that, In step (2), primers were designed for the Nsp2 and GP5 genes as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:

4. The RT-PCR reaction system consisted of a total volume of 25 μL, including 1 μL of One Step Enzyme Mix, 12.5 μL of 2X One-Step Reaction Solution dye plus, 1 μL each of forward and reverse primers, and 2 μL of Total RNA. The remaining volume was made up with RNase-free water. The PCR reaction program was as follows: reverse transcription at 50℃ for 30 min; pre-denaturation at 94℃ for 2 min. Perform 35 cycles, each cycle consisting of: 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 2min; final extension at 72℃ for 5min, and storage at 4℃.

4. The application of the PRRSV-like NADC34 strain of claim 1 in constructing a PRRSV infection model.

5. A method for constructing an infection model of PRRSV-like NADC34 strain, characterized in that, Includes the following steps: (1) Screening of experimental animals: Healthy piglets that were negative for porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine pseudorabies virus, classical swine fever virus, and mycoplasma hyopneumoniae antigen and antibody were screened and then fed acclimatized for use. (2) Preparation of challenge strain: The PRRSV-type NADC34 strain P3 generation virus solution described in claim 1 was diluted with 1640 medium to prepare the challenge working solution, and was prepared and used immediately. (3) Virus challenge procedure: A combined nasal drop and intramuscular injection approach was used for virus challenge. The total inoculation volume of virus for each piglet was 4 mL, and the virus content was 10. 5.0 TCID 50 / ml, of which 2mL is administered via nasal drops and 2mL via intramuscular injection in the neck; the challenge dose is 4×10⁻⁶. 5.0 TCID 50 / head; (4) Model monitoring: After the virus attack, the model was continuously monitored for 21 days to verify whether it was successfully constructed.

6. The construction method according to claim 5, characterized in that, In step (2), the preparation method of P3 generation virus solution of PRRSV-like NADC34 strain is as follows: the cloned and purified PRRSV-like NADC34 strain is inoculated into PAMs, and the virus solution is obtained by continuous passage for 3 generations. The virus titer is determined to be not less than 10. 5 TCID 50 / ml.

7. The construction method according to claim 5, characterized in that, In step (4), the criteria for successful model construction are as follows: piglets exhibit high fever lasting more than 3 days after challenge, with a body temperature ≥40.5℃; the viral load in whole blood reaches its peak at 7-12 days and remains detectable on day 21; typical lesions such as dark red consolidation and interstitial widening of the lungs are visible upon necropsy; histopathological examination reveals characteristic interstitial pneumonia of PRRSV; and high viral load is detectable in target organ tissues, with a viral load not less than 10. 4.0 copies / μL.

8. The construction method according to claim 5, characterized in that, In step (4), the monitoring indicators include: body temperature monitoring, whole blood viremia monitoring, gross lung lesion observation, histopathological examination, and tissue viral load detection.

9. The construction method according to claim 8, characterized in that, In step (4), whole blood viremia monitoring and tissue viral load detection were performed using PRRSV-specific real-time qRT-PCR. Primers and probes were designed for the ORF6 gene, such as SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:

7. qRT-PCR reaction system: Total volume 20 μL, specific components are: 2X One Step RT-qPCR Buffer II probe 10 μL, Pro Taq HS DNA Polymerase 0.4 μL, EVO M-MLV RTase Enzyme Mix II 0.4 μL, forward and reverse primer concentration 10 μM, volume 1 μL each, probe concentration 10 μM, volume 0.4 μL, template RNA 5 μL, and the remaining volume is made up to 20 μL with RNase-free water; The reaction program was set as follows: reverse transcription at 42℃ for 5 min; pre-denaturation at 95℃ for 30 s; followed by cycles of 95℃ for 5 s and 60℃ for 30 s, for a total of 40 cycles.

10. The application of the PRRSV-like NADC34 strain infection model constructed by any one of claims 5-9 in the evaluation of PRRSV vaccine potency and screening of antiviral drugs.

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