PD-1 expression has been previously described as being induced on specific cell subsets in response to viral or bacterial infection [20] . Thus, we first determined the status of PD-1 expression at 72 h after MHV-3 infection (10 PFU) by immunohistochemical techniques. PD-1-positive cells were observed in tissues from the thymus, spleen, lymph nodes and liver. Cellular expression was localized to the cell membrane and in the cytoplasma while was completely absent from the nuclear compartment. PD-1-positive cells were distributed throughout the medulla and cortex of the thymus and lymph nodes. In the spleen, PD-1-positive cells were restricted to the germinal center under normal conditions, but extended to the red pulp after infection. In infected liver, more PD-1-positive cells were present in the portal and parenchymal areas, as opposed to the relatively low presence of PD-1-positive cells in only the portal area in phosphate-buffered saline (PBS) treated-mice (Fig. 1A) . The amount of PD-1-positive cells in the different organs of infected and control mice were counted and compared, results showed that the number of positive cells was significantly higher in infected mice (Fig. 1B) . Furthermore, FACS analysis revealed that PD-1 expression was enhanced on multiple subsets of immune cells, including the CD4 + and CD8 + T cells, NK1.1 + NK cells and CD68 + macrophages (Fig. 1C) . PD-1positive cells were also observed in the lung, heart and kidney, however, the numbers of PD-1 positive cells in these tissues did not significantly increase in response to MHV-3 infection (Fig. S1 ).