Fig. 2. HTNV can infect primary human CD8⁺ T cells in vitro and promote their production of cytotoxic factors.

a Confocal microscopy identification of HTNV NPs in CD8⁺ T cells infected with HTNV. Positively selected CD8⁺ T cells from a healthy donor were infected with HTNV, collected at 0, 48, 72, and 96 h post infection, stained with either anti-HTNV-NP or anti-CD8a antibodies, and counterstained with DAPI (blue, top panel). Single-positive cells are either infected (HTNV NP, green, second panel) or CD8⁺ T cells (red, third panel); double-positive cells constitute an overlaid image of a single-positive cell and appear orange (bottom panel). (n = 3 cell cultures per experiment). b Identification of HTNV infection of CD8⁺ T cells (NP⁺CD8⁺) among PBMCs from an HFRS patient (H35-2). HTNV NP (green) or CD8 (red) single-positive cells, as well as NP⁺CD8⁺ double-positive (orange) cells, are shown. A normal control sample from an uninfected subject (N1) was included for comparison. c At 0, 24, 48, 72, and 96 h post infection, viral loads were measured by the detection of the HTNV s segment gene using quantitative real-time PCR, and β-actin was used as a housekeeping gene for normalization. (n = 9 cell cultures per experiment). The amounts of granzyme A, granzyme B, and perforin in culture supernatants were measured by ELISA. Values are expressed as the mean ± SEM from three independent experiments. One-way ANOVA with a post hoc Tukey test was used for multiple comparisons. (n = 3 cell cultures per experiment). d Culture supernatants from CD8⁺ T cells infected with HTNV were collected at 0, 48, 72, 96 h post infection and used to inoculate Vero E6 cells, which were stained for HTNV NP (red) after 7 days of culture. Scale bar = 10 μm. (n = 3 cell cultures per experiment).