Cells, virus and antibodies
Marc-145 cells were grown in Dulbecco's modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. The modified live vaccine HuN4-F112 strain, the infectious clones of HuN4-F112 and HuN4-F112-C5, were generated in our previous study (Tian et al. 2009; Zhao et al. 2015). Positive pig serum against HP-PRRSV HuN4-F112, NADC30-like PRRSV HeB108 strain, classical swine fever virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine circovirus type 2, pseudorabies virus, and porcine parvovirus, as well as a monoclonal antibody (3F7) against PRRSV, were obtained from our laboratory (Wang et al. 2014).
Dataset and alignment of NSP2 from PRRSV-2
In our previous study, two amino acid residues at positions 585–586 in the NSP2 of PRRSV HeB108 were deleted during viral passage in Marc-145 cells (unpublished data), indicating that these or nearby residues can be used as markers for next-generation DIVA vaccines. To identify the deletions in circulating PRRSV and MLV strains, all 907 NSP2 full-length sequences of PRRSV-2 available from GenBank in 2020 were downloaded, followed by amino acid sequence alignment and insertion/deletion (indel) analysis using DNASTAR software v7.1.0 (DNASTAR, Madison, WI, USA).
Immunoreactivity of m1B and m2B peptides
Indirect enzyme-linked immunosorbent assay (ELISA) was used to evaluate the immunoreactivity of m1B and m2B peptides. All peptides employed in the study were synthesized by GL Biochem, Ltd. (Shanghai, China). ELISA plates were coated with m1B or m2B peptides alone or in combination at a dose of 10 µg/well by incubation in carbonate-bicarbonate buffer (pH 9.6) at 4 °C overnight. The plates were then blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBST) for 1 h at 37 °C. After washing thrice with PBST, 100 μL PRRSV-positive pig serum (1:40 dilution) was added to the wells; the plates were incubated followed by incubation at 37 °C for 1 h, washed again, and then incubated with horseradish peroxidase-conjugated rabbit anti-pig IgG (1:40,000 dilution; Sigma-Aldrich, St. Louis, MO, USA) in PBST at 37 °C for 1 h. Finally, the plates were washed and incubated with 100 μL/well of 3,3',5,5'-tetramethylbenzidine (Invitrogen) for 15 min. The reaction was stopped with 2 M H2SO4 (100 μL/well), and the results were read at 450 nm.
Assessment of specificity
The specificity of 1B2B-ELISA indirect ELISA was examined using the antisera of the six porcine viruses mentioned above to assess the degree of assay cross-reactivity.
Immunoreactivity of m1B and m2B truncated peptides
To identify the immunodominant antigen regions in the m1B and m2B peptides, seven overlapping peptides (m1B1–m1B7) spanning the m1B region were designed. Each peptide was 16 residues long, and the overlapping region between two adjacent peptides spanned eight residues (Table 1). Similarly, seven overlapping peptides (m2B1–m2B7) were designed and synthesized to probe the m2B region. The resulting m1B1–m1B7 and m2B1–m2B7 peptides were used as coating antigens in ELISA, and their reactivity to PRRSV-positive serum was detected as described above.
Generation of PRRSV m1B or m2B deletion mutants
Plasmids harboring m1B or m2B deletion mutants were constructed using HuN4-F112 as a template and specifically designed primers (Table 2); a plasmid harboring the m1B and m2B double-deletion mutant was constructed using HuN4-F112-C5 as a template and specifically designed primers. The native m1B and m2B genes in the HuN4-F112 infectious clone were replaced with their deletion variants via overlap PCR amplification using ultra-fidelity DNA polymerase (TaKaRa, Shiga, Japan) as described previously (Yu et al. 2014), followed by digestion with restriction enzymes (Fse I and Nhe I) and ligation. The deletions were confirmed by DNA sequencing. The plasmids were transfected into Marc-145 cells using Xtreme GENE-HP DNA transfection reagent (Roche Applied Science, Basel, Switzerland) as described previously (Zhao et al. 2018).
At 5 days post-transfection, the cells were assayed by indirect immunofluorescence (Gao et al. 2016). Marc-145 cells were incubated with a monoclonal antibody against the M protein of PRRSV and stained with fluorescein isothiocyanate-labeled anti-mouse IgG.
The supernatant was recovered and passaged twice in Marc-145 cells and identified by reverse transcription PCR. Viral RNA from rHuN4-F112, rHuN4-F112-m1B, rHuN4-F112-m2B, rHuN4-F112-C5, and rHuN4-F112-C5-m1B-m2B were extracted using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Reverse transcription reactions were performed at 2 °C for 10 min and 42 °C for 1 h using the M-MLV Reverse Transcription Polymerase System (TaKaRa) and then amplified with B-900-F/B-900-R primers (Table 2), and the PCR products were sent for DNA sequencing. Viral rescue was quantified in Marc-145 cells.
Growth kinetics of deletion-marker viruses in Marc-145 cells
To monitor the effect of deletion on viral replication, Marc-145 cells were grown to 80% confluence in a six-well plate and then infected with the third-passage viruses (rHuN4-F112-C5 and rHuN4-F112-C5-m1B-m2B) at a multiplicity of infection of 0.01. The supernatant was harvested at 0, 24, 48, 72, and 96 h post-inoculation. After RNA extraction and reverse transcription, the viral copy numbers at different time points were determined by quantitative reverse transcription PCR as described previously (Wei et al. 2008). All samples were tested thrice and the replication kinetic curve was drawn using GraphPad software (GraphPad, Inc., La Jolla, CA, USA). Measured values were expressed as the mean ± standard deviation. Significance was assessed using Student’s t-test at P < 0.05.