This study was conducted in accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The Committee on the Ethics of Animal Experiments of the Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS) reviewed and approved the protocols. Six-week-old BALB/c mice were provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. (experimental animal license number: SYXK (Jing) 2012–0024). PRRSV immunization experiments using these 6-week-old BALB/c mice were conducted within the animal biosafety level 2 facilities of the HVRI of the CAAS (approval number SY-2020-MI-123).

The HP-PRRSV strain HuN4, HP-PRRSV vaccine (HuN4-F112, TJM-F92, JXA1-R), classical PRRSV vaccine CH-1R and MLV, NADC30-like PRRSV SDHS53, NADC34-like PRRSV TZJ1341, European PRRSV (VP046 BIS and DV) and recombinant SDHS53 N protein (240 μg/ml) used in this study were retained at the HVRI of the CAAS. Porcine parvovirus (PPV), porcine circovirus type 2 (PCV-2), classical swine fever virus (CSFV), transmissible gastroenteritis virus (TGEV), porcine pseudorabies virus (PRV), and porcine epidemic diarrhea virus (PEDV) were collected at the HVRI. Latex microspheres (diameter = 300 nm) were purchased from Hangzhou Xiaoque Technology Co., Ltd. (Hangzhou, China). MES, EDC·HCl, and NHS were procured from Sigma (Sigma, St. Louis, MO, United States). Nitrocellulose (NC) membranes (Millipore 180) were obtained from Millipore (Billerica, United States). Polyester membranes were purchased from Pall Corporation (NY, United States), and absorbent paper and PVC sheets were procured from Shanghai Jinbiao Biotechnology Co., Ltd. (Shanghai, China).

Female BALB/c mice aged 6 weeks were immunized intraperitoneally three times at 2 week intervals with SDHS53 (10^7.0 median tissue culture infectious dose (TCID₅₀)/ml) in Freund’s complete adjuvant (Sigma, St. Louis, MO, United States) for the first immunization and in Freund’s incomplete adjuvant for the other two immunizations and then boosted intraperitoneally with virus only. Three days after the final booster injection, the mice were euthanized by carbon dioxide asphyxiation, and spleen cells were then fused with SP2/0 cells using 50% (v/v) polyethylene glycol (Sigma, St. Louis, MO, United States). The fused cells were successively cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL Co., Ltd., United States) containing HAT (Sigma, St. Louis, MO, United States), in DMEM containing HT (Sigma, St. Louis, MO, United States) and then in DMEM supplemented only with 20% fetal bovine serum (HyClone Laboratories Inc., South Logan, UT, United States). Hybridoma cell lines secreting antibodies against PRRSV were screened and subcloned at least three times by limiting dilution. The hybridoma culture supernatants were screened for antibodies using an indirect immunofluorescence assay (IFA). Antibodies that bound to PRRSV but failed to bind MARC-145 cells were considered positive for PRRSV. Stable cells were injected into the abdominal cavities of BALB/c mice that had been pretreated with Freund’s incomplete adjuvant. Approximately 1 week later, the ascites were harvested, and the mAbs were purified from the ascites using an antibody purification kit (Protein G Resin, GenScript, Nanjing, China) per the manufacturer’s instructions. The isotype of the produced mAb was determined with a Pierce Rapid ELISA Mouse mAb Isotyping Kit (Thermo Scientific, MA, United States) according to the manufacturer’s instructions.

Transient transfection was performed to identify the structural proteins that bound to the mAbs. 293T cells were transiently transfected with the eukaryotic expression vector p3 × FLAG-CMV-SDHS53-GP2, GP3, GP4, GP5, M, or N using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) according to the manufacturer’s protocol. p3 × FLAG-CMV-transfected cells were used as a negative control. The cells were harvested, and IFA was performed as described above.

The ORF7 gene of SDHS53 was divided into two overlapping fragments: N1 and N2. Specific primers (Table 1) were used to amplify these fragments. The PCR products amplified from these two fragments were cloned separately into the pGEX-6p-1 expression vector and used to transform E. coli BL21 (DE3) cells (Tiangen, Beijing, China) in which the corresponding proteins were expressed. The recombinant proteins were identified by SDS-PAGE and Western blotting as described above. Upon the identification of bound polypeptides, N2 of SDHS53 was divided into several segments with N-terminal truncations, and these fragments were identified by SDS-PAGE and Western blotting as described above.

The amino acid sequences of representative PRRSV N proteins (classical PRRSV, HP-PRRSV, lineage 3 PRRSV, NADC30-like strains, NADC34-like strains, and EU-PRRSV) were aligned using DNASTAR MegAlign software. Protein structure models of the SDHS53-N protein were predicted using the SWISS-MODEL online website.¹ The three-dimensional (3D) structure data were analyzed using PyMOL software (Hu et al., 2016).

The optimal antibody concentrations for the test lines, control lines, and conjugation with the latex microsphere solution were determined as previously described (Shi et al., 2021), with some modifications. First, the latex microspheres were activated. Briefly, the latex microspheres were washed twice in double-distilled water, and 0.9 ml of 0.1 mol/l MES (pH 6.5), 0.1 ml of 20 mg/ml EDC·HCl, and 0.075 ml of 20 mg/ml NHS were then added successively to each 0.2 ml of latex microspheres. The mixture was shaken for 2 h and then centrifuged at 8000 × g for 10 min. The precipitate was resuspended in 0.1 mol/l boric acid buffer (pH 7.2), and the mixture was shaken for 15 min after ultrasonic homogenization. This washing procedure was repeated twice.

Subsequently, 0.5 ml of activated latex microspheres was mixed with 0.5 ml of N protein-specific mAb 1H4, shaken for 2 h, and then centrifuged at 10000 × g for 25 min. The precipitate was resuspended using 1 ml of 0.5% (w/v) casein to remove potential excess reactivity. The mixture was shaken for 15 min after ultrasonic homogenization. This blocking procedure was repeated twice.

The ICS (Figure 1A) includes four components: a sample pad, a conjugate pad, an NC membrane, and an absorbent pad (Figure 1B). The NC membrane was incubated with two antibodies, mAb 1H4 and goat anti-mouse IgG, dissolved in PBS to generate the test and control lines, respectively. The conjugate pad, which is composed of a polyester membrane, was treated with the latex microsphere-mAb 1H4 conjugate by spraying using an XYZ3050 Dispense Workstation at 3 μl/cm and then dried for 1 h at 37°C. mAb 1H4 and goat anti-mouse IgG were diluted with PBS to final concentrations of 2.0 and 1.0 mg/ml, respectively. The XYZ3050 Dispense Workstation (BioDot, Inc., Sky Park, Irvine, CA, United States) was used to spray both antibodies, and the NC membrane was then dried for 1 h at 37°C.

The test strips were assembled and used as described by Wang et al. (2019). The sample pad, conjugate pad, NC membrane, absorbent pad, and PVC sheet were assembled in sequence. The sample pad and the conjugate pad overlapped by 2 mm with one end of the conjugate pad; the other end of the conjugate pad overlapped (2 mm) with one end of the NC membrane underneath the conjugate pad; and the absorbent pad overlapped by 2 mm with the other side of the NC membrane. Subsequently, the whole plate was cut into 3-mm-wide strips, and these strips were assembled in strip cassettes with desiccant.

During the testing process, 100 μl of liquid sample was dropped onto the sample pad. When the sample liquid reached the conjugate pad, PRRSV reacted with the latex microsphere-1H4 conjugate to form an antigen-latex microsphere-1H4 complex. The complex then traveled through the NC membrane via capillary action and then reacted with mAb 1H4 on the test line, resulting in a dark red band. Conversely, in samples lacking PRRSV, the free conjugate that could not bind to the sample continued to travel to the control line. At the control line, the goat anti-mouse IgG antibody reacted with mAb 1H4 on the complex, and a dark red band appeared. The results could be observed within 8 min at room temperature. Two bands appeared with the positive samples (one on the test line and one on the control line), whereas only one band appeared (on the control line) with the negative samples, and no appearance of a band indicated that the test was invalid (Figure 1C).

Common porcine viruses, including EU-PRRSV, PPV, PCV-2, CSFV, TGEV, PRV, and PEDV, were tested to evaluate the specificity of the ICS. PRRSV HuN4 and SDHS53 were used as positive controls. All virus types were used at the same dose (2 × 10^4.0 TCID₅₀/ml).

To evaluate the sensitivity of the ICS, 10^6.0 TCID₅₀/ml PRRSV HuN4 and recombinant SDHS53 N protein (240 μg/ml) were serially diluted in PBST and PBS, respectively, and 100 μl of each dilution was used for the ICS test. The sensitivity was determined by finding the minimum dilution concentration that yielded a positive result. In contrast, the sensitivity of the strip was evaluated by comparing the detection of the same sample with that of PCR using the following primer sequences targeting ORF5: forward 5′-AATGTGTCAGGCGTCGTGGCT-3′ and reverse 5′-CAGAATGTACTTGCGGCCTAGC-3′. In brief, DMEM was used to dilute HuN4 (10^6.0 TCID₅₀/ml) from 10⁻¹ to 10⁻⁶, and viral RNA was extracted from 140 μl of the solution using a TIANamp Virus RNA Kit (Tiangen, Beijing, China). The ORF5 gene containing its flanking regions was amplified using the abovementioned primers. Thermal cycling was performed as follows: one cycle of 95°C for 5 min; 30 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min 30 s; and a final cycle of 72°C for 10 min. The PCR products were electrophoresed on a 1% agarose gel. The PCR product (1,107 bp) was sequenced by Comate Bioscience Co., Ltd. (Changchun, China).

To evaluate the applicability of the ICS, HuN4, HuN4-F112, TJM-F92, JXA1-R, CH-1R, MLV, SDHS53, TZJ1341, VP046 BIS, and DV were used for the ICS test.

To ascertain the reproducibility and stability of the ICS during storage at room temperature (RT), 3 batches of strips (batch numbers: 202101, 202102, and 202103) were used for the detection of PRRSV (10 virus samples were used to evaluate the applicability of the ICS were selected). All strips from each batch were stored at RT and used for two tests of the samples at a 6-month interval.

One hundred eight clinical serum and lung tissue samples from pigs with suspected PRRSV infection in Northeast China were collected and tested for PRRSV using the ICS test and PCR. The coincidence rate of the two methods was then calculated.

One mAb, 1H4, was generated by IFA detection using SDHS53 as the antigen source. The isotype of mAb 1H4 was identified with a Pierce Rapid ELISA Mouse mAb Isotyping Kit as IgG1. mAb 1H4 specifically recognized the N protein expressed by transiently transfected cells (Figure 2A) and not other structural proteins (data not shown). Western blotting showed that the mAb specifically recognized the 15-kDa protein bands of PRRSV (Figure 2B).

The Western blot and IFA results described above confirmed that mAb 1H4 recognized the N protein. Two overlapping fragments (N1 and N2) from the ORF7 gene of SDHS53 were prepared by PCR and cloned into an expression vector, pGEX-6p-1, for expression as GST fusion proteins. After nucleotide sequencing, the recombinant protein fragments encoded by these constructs were expressed in E. coli BL21 (DE3) cells and identified by SDS-PAGE and Western blotting using mAb 1H4. The results showed that mAb 1H4 recognized SDHS53-N2 (aa 56 ~ 123). The N2 fragment was then serially truncated from the N-terminus, and SDHS53-N27 (aa 107 ~ 123) was recognized by mAb 1H4 (Figure 2C).

Multiple sequence alignment of representative PRRSV N proteins showed that the epitope (¹⁰⁷PTHHTVRLIRVTASPSA¹²³) recognized by mAb 1H4 was highly conserved among the HP-PRRSV strains and classical PRRSV strains, whereas several substitutions were present in the other PRRSVs (Figure 3A). Visualization of the predicted 3D structure, which was based on simulated data from the SWISS-MODEL online server, showed that the C-terminus of the N protein (aa 68 ~ 118) existed in the form of a homodimer (Figure 3B). Subsequently, the 3D structure data were analyzed using PyMOL software, and the results revealed that the epitope (aa 107 ~ 123) recognized by mAb 1H4 was fully exposed on the surface (aa 107 ~ 118) of each N protein monomer (Figure 3C).

The assay specificity was determined using EU-PRRSV, PPV, PCV-2, CSFV, TGEV, PRV, and PEDV. As demonstrated by the strips shown in Figure 4, PRRSV was positive at the test line, and the other viruses were negative, which suggested that the ICS exhibited good specificity.

To determine the sensitivity of the ICS, serial dilutions of PRRSV HuN4 (10^6.0 TCID₅₀/ml) were prepared in PBST and added to the ICS sample pad. The results showed that the limit of detection of the ICS was 5 × 10^2.0 TCID₅₀/ml (Figure 5A). To evaluate the PCR sensitivity, DMEM was used to dilute HuN4 (10^6.0 TCID₅₀/ml) from 10⁻¹ to 10⁻⁶, and RT-PCR was performed. The minimum virus amount was 10^3.0 TCID₅₀/ml (Figure 5B). The recombinant SDHS53 N protein (240 μg/ml) was serially diluted in PBST to evaluate the sensitivity of the ICS, and the results showed that the minimum detection limit was 15 ng/ml (Figure 5C). The above results showed that the sensitivity of the ICS was very high at both the TCID₅₀ level and the protein level.

To evaluate the applicability of the ICS, the ICS test was performed using HuN4, HuN4-F112, TJM-F92, JXA1-R, CH-1R, MLV, SDHS53, TZJ1341, VP046 BIS, and DV. The results showed that the ICS could detect all types of NA-PRRSVs (Figure 6), whereas EU-PRRSVs were not detected, which greatly broadens the applicability of the ICS.

To determine its reproducibility and stability, the ICS assay was performed using a different batch of strips after storage at RT for 0 and 6 months. As shown in Table 2, 8 of the 10 tested samples were detected as positive, and 2 of the 10 tested samples were detected as negative. Identical results were observed with strips from the same batch and different batches after different storage times.

PCR and the ICS were compared with respect to their ability to detect PRRSV in 108 porcine clinical samples. The PCR results identified 46 positive and 62 negative samples, whereas the ICS assay results identified 40 positive samples and 68 negative samples (Table 3). The positive rates of the two methods were 42.59 and 37.03%, respectively. The ICS assay was shown to exhibit relatively high specificity and sensitivity (90.32 and 73.91%, respectively), and relatively high positive predictive value (PPV) and negative predictive value (NPV; 85 and 82.35%, respectively). The overall coincidence rate between the test strip and PCR method was 83.33%, which indicated that the ICS method established in this study can be used for the detection of clinical samples.