Porcine respiratory coronavirus as a model for acute respiratory disease: mechanisms of different infection outcomes
Porcine respiratory coronavirus (PRCV) is a naturally occurring pneumotropic coronavirus in the pig, providing a valuable large animal model to study acute respiratory disease. PRCV pathogenesis and the resulting immune response was investigated in pigs, the natural large animal host. We compared two strains, ISU-1 and 135, which induced differing levels of pathology in the respiratory tract to elucidate the mechanisms leading to mild or severe disease. The 135 strain induced greater pathology which was associated with higher viral load and stronger spike-specific antibody and T cell responses. In contrast, the ISU-1 strain triggered mild pathology with a more balanced immune response and greater abundance of T regulatory cells. A higher frequency of putative T follicular helper cells was observed in animals infected with strain 135 at 11 days post-infection. Single-cell RNA-sequencing of bronchoalveolar lavage revealed differential gene expression in B and T cells between animals infected with 135 and ISU-1 at 1 day post infection. These genes were associated with cell adhesion, migration, and immune regulation. Along with increased IL-6 and IL-12 production, these data indicate that heightened inflammatory responses to the 135 strain may contribute to pronounced pneumonia. Among BAL immune cell populations, B cells and plasma cells exhibited the most gene expression divergence between pigs infected with different PRCV strains, highlighting their role in maintaining immune homeostasis in the respiratory tract. These findings indicate the potential of the PRCV model for studying coronavirus induced respiratory disease and identifying mechanisms that determine infection outcomes. Overall design: For scRNA-seq two group of animals were used. First group received low pathogenic PRCV ISU-1 and second group received high pathogenic PRCV 135 intranasally. 4 animals from each group were humanly culled at day 1 and 20 post infection and subsequently bronchoalveolar lavage were obtained. To account for the strong domination of macrophages in BAL cell preparations, we sorted major leukocyte to reresent equal proportion of each major cell types. In brief, cryopreserved BAL cells were thawed and labelled with antibodies against CD4 (clone 74-12-4, PerCP-Cy5.5-conjugated, BD Biosciences), CD8ß (clone PPT23, FITC-conjugated, Bio-Rad), CD3 (clone PPT3, APC-conjugated, Southern Biotech), CD16 (clone G7, AF647-conjugated, Bio-Rad), CD172a (clone 74-22-15, PE-conjugated, Bio-Rad) and Fixable Viability Dye eFluor780 (ThermoFisher) and sorted on a FACSAria UIII (BD Biosciences). This allowed the sorting of CD4+ (CD4 T cells), CD8ß+ (CD8 T cells), CD16+CD172a+ (Macrophages) and CD3-CD16- (B cells) cells.  12,000 of each cell types were collected. Cells from each fraction were processed for partitioning and barcoding using a 10x Genomics Chromium iX Controller.