The role of immune checkpoint molecules in PRRSV-2-induced immune modulation: insights from comparative evaluation including NADC34-like PRRSV.

The emergence of genetically diverse PRRSV-2 lineages, including the NADC34-like virus, presents significant challenges to the swine industry due to their variable pathogenicity and impact on immune modulation. However, the precise mechanisms underlying virus-induced immune modulation remain poorly understood. This study investigated the immunopathological characteristics of the Korean NADC34-like PRRSV strain JBNU-22-N01 in comparison with the NADC30-like strain PJ73 and the prototype strain VR2332 in a weaned piglet model. All PRRSV-2 strains exhibited moderate pathogenicity without mortality, following a similar infection course characterized by interstitial pneumonia, cytokine cascades (e.g., IFN-α, IFN-γ, IL-1β, IL-12p40, IL-10, CCL2, CCL5, CCL8, and CXCL10), and upregulation of interferon-stimulated genes [e.g., ISG12(A)]. Flow cytometry analysis revealed significant alterations in the composition of bronchoalveolar lavage (BAL) cell populations, including a decreased frequency of alveolar macrophages and an increased proportion of infiltrating immune cells (monocyte-derived cells, T cells, and NK cells). However, the majority of infiltrated CD4 and CD8 T cells exhibited minimal expression of effector cytokines (e.g., IFN-γ), suggesting a phenotypically naïve-like or functionally unresponsive state. The mRNA expression levels of BAL cells revealed significant upregulation of immune checkpoint molecules (e.g., PD1, PDL1, CTLA4, LAG3, and IDO1), indicating immune regulation potentially mediated through cell-to-cell communication. Notably, IDO1 expression was modestly associated with increased systemic Kyn/Trp ratios, supporting the hypothesis that the kynurenine pathway may contribute to systemic immune modulation during PRRSV infection. From a strain-specific perspective, JBNU-22-N01 displayed a faster replication rate, resulting in an accelerated and heightened infection process with stronger immune responses. It uniquely induced robust expression of immune checkpoint molecules and enhanced IDO1 protein production in bronchoalveolar lavage cells, highlighting the immunomodulatory potential of NADC34-like PRRSV. These findings offer insights into both general and strain-specific immunopathogenesis of PRRSV-2 and support the development of better treatments.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen affecting the swine industry; however, its immune-related mechanisms remain incompletely understood. Here, we analyzed how three genetically distinct PRRSV strains, including the globally prevalent NADC34-like strain, interact with the immune system in piglets. Our results showed that PRRSV induces severe lung inflammation accompanied by immune cell infiltration. However, many infiltrating immune cells remained inactive, likely due to increased expression of immune-suppressive molecules. Among these, the enzyme indoleamine 2,3-dioxygenase-1 (IDO1) was notably upregulated, activating a metabolic pathway linked to immune regulation and suppression. The NADC34-like strain displayed a faster replication rate, leading to more rapid immune responses and stronger suppression compared with the other strains. These findings provide new insights into how PRRSV manipulates the immune system and suggest potential targets for improved prevention and treatment strategies.