HTNV infection of CD8+ T cells is associated with disease progression in HFRS patients

Abstract

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8⁺ T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8⁺ T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8⁺ T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8⁺ T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies.

Fig. 1. Characterization of virally infected T lymphocytes from HRFS patients at various stages of the disease.

a Classification of disease stages of HFRS patients. b Representative FACS plots of viral infection (HTNV-NP positive) of CD4⁺ and CD8⁺ T cells in PBMC samples obtained from HFRS patient No. 10 on days 10, 13, 19, and 23 after symptom onset. c Comparison of the percentages of CD8⁺ T cells (top panel) and CD4⁺ T cells (bottom panel) between the acute and convalescent phases of HFRS. (Acute, n = 62; Conv., n = 60). d Comparison between the frequency of HTNV-infected CD8⁺ T cells and CD4⁺ T cells during the acute and convalescent phases of HFRS. Each point represents a single sample; red indicates the acute phase and blues the convalescent phase. Groups were compared using a two-tailed unpaired Student’s t-test. (Acute, n = 62; Conv., n = 60).

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).

Fig. 3. Ultrastructural identification of HTNV virions at different stages of the viral life cycle in primary human CD8⁺ T cells.

CD8⁺ T cells were infected with HTNV for 72 h and then fixed with 2% glutaraldehyde and analyzed by TEM. a Representative images of uninfected CD8⁺ T cells (I); representative images at high magnification revealed the presence of virus-like particles contained within multivesicular bodies (MBVs) (II and III, yellow arrows); the presence of vesicles secreted from the T cells (IV, black arrows). b Distribution of vesicle structures containing virion particles in the cytoplasm of HTNV-infected CD8⁺ T cells. Viral entry occurred first by virion attachment to the cell surface (I). Nucleocapsids are assembled in the vesicles, and some microvesicles formed in the vesicles (II). The nucleocapsids budded into the smooth vesicles and acquired viral envelopes (III). These nucleocapsids were not yet enclosed by an envelope, as shown by the light-colored structure in the core. As the nucleocapsids replicated, after which the ribosomes attached to the surface of the vesicles decreased initially and gradually disappeared completely (IV). At 96 h after infection, CD8⁺ T cell necrosis was detected, along with an increase in mature viral particles in the cytoplasm (V). The red arrows represent the virus. c Schematic illustration of the life cycle of hantaviruses in primary CD8⁺ T cells. Steps 1–5 correspond to the same stages in b.

Fig. 4. Correlations between HTNV-infected CD8⁺ T cells and clinical indices.

a Comparisons of white blood cell (WBC), lymphocyte (LYMPH), neutrophil (NEUT), and platelet (PLT) counts and levels of perforin, granzyme A (GZMA), and granzyme B (GZMB) between patients at the acute and convalescent phases of HFRS. Groups were compared using a two-tailed unpaired t-test. (Acute, n = 62; Conv., n = 60). b Correlations between CD8⁺ T cells or HTNV-infected CD8⁺ T cells and WBC, LYMPH, NEUT, PLT, perforin, GZMA, and GZMB. Correlations with P < 0.05 are indicated with an r value that ranged from −1 to 1. Negative correlations are shown in blue and positive correlations in red (n = 122). c Receiver operating characteristic curve (ROC) and area under the curve (AUC) analyses of the prognostic values of various clinical parameters for HFRS stages. The PLT count had the highest diagnostic value (AUC = 0.9149), followed by secreted GMZB (AUC = 0.8386) and perforin (AUC = 0.7448) levels, the WBC count (AUC = 0.7615) and LYMH count (AUC = 0.7111), and then the percentage of CD8⁺ T cells (AUC = 0.7195) or HTNV-infected CD8⁺ T cells (AUC = 0.7). (Acute, n = 62; Conv., n = 60).