Transcriptional profiling reveals differential effect of in vivo porcine reproductive and respiratory syndrome virus infection on infected and bystander pulmonary alveolar macrophage cells. Transcriptional profiling reveals differential effect of in vivo porcine reproductive and respiratory syndrome virus infection on infected and bystander pulmonary alveolar macrophage cells

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a serious threat to the swine industry worldwide for many years. PRRSV has a high tropism for swine pulmonary alveolar macrophages (PAM), an essential innate immune cell. Here, we used RNA-sequencing approach to examine the transcriptional profile of in vivo infected and bystander PAM in the lung of PRRSV-infected swine at 7 days post-infection (dpi). We utilize the PRRSV virion stably expressing EGFP reporter gene to discriminate infected and bystander PAM cells. This work will allow us to differentiate the direct and indirect effect of infection at the cellular level. Firstly, average ~4.2% of reads from infected, while only ~0.06 % reads from bystander cells found mapping to the FL12-EGFP genome. Though most differentially expressed genes (DEGs) are shared between infected and bystander PAM compared to PAM from mock-infected animals, infected cells produce more inflammatory cytokines, interferon signatures, and antiviral immune genes than bystander cells. Importantly, upregulation of NFKB inhibitors (NFKBI) (NFKBIA, NFKBIZ, and TNFAIP3) and T cell exhaustion genes (PD-L1 (CD274), PRDM1, and IL10) was exclusively observed in infected cells. In-situ staining of lung tissue using NFKBI RNA probes confirms temporal upregulation of these immune regulators. Both NFKBI and T-cell exhaustion are prominent immune escape mechanism noted in a several viral infections, however, their role is unexplored in PRRSV pathogenesis. Collectively, these results reveal distinctive transcriptional changes occur within infected and bystander PAM and unique upregulation of NFKBI and T-cell exhaustion pathway in infected PAM indicates a strategy employ by PRRSV to subverts host immune responses. Overall design: For both control (mock) and infected pigs the transcriptome of the Pulmonary alveolar macrophages (PAM) was sequenced; infected pigs were further divided into a bystander group and a FL12-EFGP+ group. Each group had three biological replicates each.