Single amino acid substitution in RdRp reduces viral recombination frequency of NADC30-like porcine reproductive and respiratory syndrome virus type 2

Fig 1. Location of ribavirin-resistant mutation in the RdRp and helicase of PRRSV-2. (A) The ribavirin-resistant mutations in the nsp9 (RdRp) and nsp10 (helicase). The residues at positions 286, 360, 491, and 544 in nsp9. The residues at positions 62, 205, 327, and 406 in nsp10. (B) Alignment of nsp9 and nsp10 amino acid sequences revealed the conservation of different mutation sites. Colors indicate the locations of mutations screened with ribavirin.

Fig 2. Identification of the ribavirin-resistant PRRSV-2 mutants. (A) Identification by immunofluorescence assay. Recombinant and parental plasmids (4 μg) were transfected into Marc-145 cells. At 5 d post-transfection, the transfected cells were fixed and stained with the monoclonal antibody 3F7 against PRRSV-2, followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG. Scale bar, 400 μm. (B) Genetic stability of the mutation sites in the P3 and P10 generations.

Fig 3. Growth kinetics of the ribavirin-resistant PRRSV-2 mutants. Growth properties of HuN4‑derived (A–D) and HeB108‑derived (E–H) ribavirin‑resistant mutants in Marc‑145 cells in the absence (-Ribavirin) or presence (+Ribavirin) of 300 μM ribavirin. Confluent Marc‑145 cell monolayers in 24-well plates were infected with the indicated ribavirin‑resistant mutants at an MOI of 1. Each data point represents the mean value of three replicates with SD. The symbols in panels A–H denote the statistical significance of differences between groups: *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Specifically, in panels A–D: *, HuN4 vs. R-HuN4-A286T, #, HuN4 vs. R-HuN4-D491N, &, HuN4 vs. R-HuN4-T205I, δ, HuN4 vs. R-HuN4-T544I, Ψ, HuN4 vs. R-HuN4-TNII. In panels E–H: *, HeB108 vs. VGSS, #, HeB108 vs. R-HeB108-G327S, &, HeB108 vs. R-HeB108-N406S, δ, HeB108 vs. S62G, Ψ, HeB108 vs. I360V.

Fig 4. Clinical symptoms of piglets infected with R-HeB108-VGSS, R-HuN4-TNII, and their parental viruses. (A) Rectal temperatures of piglets. The cut-off value of fever was set at 40.0 ℃. The percent survival (B), clinical sign scores of piglets (C), average daily weight gain rate (D), and thymus/body weight (g/kg) ratio (E), were compared between the infected and uninfected groups. *, P < 0.05, ***, P < 0.001, significant difference between the infected and uninfected groups. The data are presented as mean ± SD.

Fig 5. Pathological lesion of the lung and thymus. (A–E) Lungs of infected and uninfected piglets, consolidation was observed in the lungs of infected piglets. (F–J) Histopathological examination of the lungs of infected piglets. Massive lymphomononuclear cell infiltration was observed in HuN4 infected piglets. (K–O) Thymus atrophy caused by R-HuN4 and R-HeB108 was less severe than that caused by parental viruses HuN4 and HeB108.

Fig 6. Antibody kinetics and viral distribution in tissues, serum, or swabs. Serum, nasal, and anal swabs were collected at 0, 3, 7, 10, 14, and 21 dpi, whereas tissue samples were collected at 21 dpi. (A) PRRSV-2-specific antibodies were measured using the IDEXX ELISA kit. Serum was confirmed to be positive when the S/P ratio>0.4. (B) Viral loads in different tissues. (C) Viral load in serum. Viral shedding in nasal swabs (D) and anal swabs (E). *, P < 0.05, significant difference between the R-HeB108-infected and HeB108-infected groups. The data are presented as mean ± SD.

Fig 7. Mutation frequency and genetic stability analysis of R-HeB108-VGSS and R-HuN4-TNII. The mutation frequencies of R-HeB108 (A) and R-HuN4 (B) and their parental viruses were determined by collecting serum samples at 10 dpi and 21 dpi, and lung tissue samples at 21 dpi, from infected piglets. Approximately 50 monoclonal colonies were sequenced for each sample of ORF3 or ORF5. The mean mutation frequencies (number of nucleotide mutations per 10,000 nucleotides sequenced) ± SD represent the averages of all replicates. The same pattern of reduced mutation frequency in the polymerase mutant compared to the parental virus was observed across all replicates. (C) The stability of the mutation site was detected at 21 dpi in infected piglets. All mutation sites can exist stably. **, P < 0.01, significant difference between the mutant and parental virus.

Fig 8. Competition assay of mutations in vitro. (A) R-HeB108-I360V, R-HeB108-S62G, R-HeB108-G327S, R-HeB108-N406S, or R-HeB108-VGSS mutants were mixed with the parental virus HeB108 at a ratio of 9:1, 1:1, or 1:9, respectively; (B) R-HuN4-A286T, R-HuN4-D491N, R-HuN4-T544I, R-HuN4-T205I, or R-HuN4-TNII mutants were mixed with the parental virus HuN4 at a ratio of 9:1, 1:1, or 1:9, respectively. The mixed mutant virus and the parental virus were inoculated into Marc-145 cells at an MOI of 0.1, and the progeny viruses were passaged in Marc-145 cells for 5 passages. Subsequently, the nsp9 or nsp10 region was sequenced. The abundance of each competitor was measured as the height of the nucleotide peak for the mutant or the parental virus in sequencing chromatograms. *, represents the mutation site.

Fig 9. Viral sensitivity to ribavirin or 5-FU. HeB108 and its ribavirin-associated mutants were inoculated into Marc-145 cells that were pre-treated with ribavirin (A) or 5-FU (B) at an MOI of 0.01 for 72 h. HuN4 and its ribavirin-associated mutants were inoculated into Marc-145 cells that were pre-treated with ribavirin (C) or 5-FU (D). Cells without ribavirin or 5-FU treatment served as a control to evaluate the virus titers. Viral genomic RNA level was determined by RT-qPCR. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 10. The recombination frequency of mutant viruses in PAMs. (A–D) The recombination frequency of R-HeB108 mutant virus was detected in co-infection assay with HuN4. (E–H) The recombination frequency of R-HuN4 mutant virus co-infected with HeB108. The experiments were performed in four replicates.

Fig 11. Nanopore sequencing analysis after co-infection of Marc-145 cells with R-HeB108-I360V and R-HuN4. Sequencing depth analysis of the co-infection of the parental virus HeB108 (A) and R-HeB108-I360V mutant virus (B) with HuN4 strain. Analysis of recombination frequency between HeB108 (C) and R-HeB108-I360V (D). (E) Length distribution of viral reads longer than 1 kb. (F) Schematic diagram illustrating the mapping of recombinant reads to the viral genome. Each recombinant RNA molecule is aligned to the HuN4 reference genome, with gene boundaries annotated in gray below. Sequences are colored according to their parental origin (red, HeB108; blue, HuN4). Horizontal black dotted lines indicate deleted regions, and vertical black dashed lines denote the positions of genomic transcription regulatory sequences (TRSs).