Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a major infectious disease that poses a severe threat to the global swine industry. To investigate the role of miRNAs in the infection and susceptibility of PRRSV, four miRNA libraries were constructed and sequenced from PRRSV-infected and mock-infected of Tibetan pigs and Large White pigs at 7 days post-infection. A novel miRNA, miR-novel-80, was differentially expressed between PRRSV-infected and mock-infected porcine alveolar macrophages from 2 pig breeds. Importantly, the over-expression of miR-novel-80 inhibited the replication of a PRRSV-1 strain and multiple lineages (L1, L5, and L8) of PRRSV-2 strains in a dose-dependent manner. Bioinformatic predictions and experimental validation demonstrated that miR-novel-80 restricts viral replication through a dual antiviral mechanism. Directly, it targets the PRRSV nsp1-coding region within the ORF1a to suppress viral proliferation. Indirectly, miR-novel-80 specifically down-regulates the expression of host factor CXXC finger protein 4 (CXXC4). This reduction relieves the suppression of the Wnt/β-catenin signaling pathway, which in turn activates NF-κB-dependent innate immune responses to further inhibit PRRSV infection. Collectively, this study investigates the biological characteristics of miR-novel-80 and unveils its underlying molecular mechanisms in restricting PRRSV infection in vitro. However, its biological function and anti-PRRSV therapeutic effect in vivo need further investigation.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), poses a serious threat to the global swine industry. Although modified live virus (MLV) vaccines have been widely used in the field for PRRS prevention for decades, the safety and efficacy of these vaccines have long been controversial. Here, we report a rare recombination pattern in China: the emergence of a novel NADC30-like PRRSV strain recombined from two MLV-like strains. Genome comparative analysis reveals that the SCMS2025 isolate has a non-continuous 136-amino acid deletion in the NSP2 protein and shares the highest nucleotide identity of 87.6% with lineage 5 (L5) strains. Phylogenetic analysis showed that SCMS2025 was classified into L1 (NADC30-like) strains based on ORF5 genotyping, whereas it belonged to a single branch between L1 and L5 strains based on the complete genomic sequences. Strikingly, genomic recombination analysis revealed that the newly emerged PRRSV isolate likely resulted from complex recombination events between NADC30-like and two MLV-like strains (RespPRRS MLV and TJbd14-1 MLV-like strains). Furthermore, SCMS2025 infection caused transient overt clinical signs followed by rapid recovery, indicating that the novel PRRSV isolate is a low pathogenic strain. Notably, all SCMS2025-inoculated piglets remained seronegative for PRRSV-specific antibodies throughout the entire 14-day observation period, suggesting a delayed onset of the host humoral immune response. Our study provides evidence for the ongoing evolution of PRRSV through inter lineage recombination and highlights the urgent need for safe and effective vaccines.