Limitations of a proper SFTSV mouse model using human C-type lectin receptors

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with a human mortality rate of up to 30%, posing a significant threat to public health. However, the lack of suitable research models has impeded the development of effective human vaccines. In this study, we engineered transgenic mice (3xTg) using a novel construct that simultaneously expresses three C-type Lectin receptors, identified as critical SFTSV entry receptors. While this construct substantially enhanced viral binding and infection in BJAB cells, the 3xTg mice exhibited only limited SFTSV replication in the lymph nodes and spleen, without significant impacts on morbidity or mortality. These findings highlight that the overexpression of entry receptors alone is insufficient to fully recapitulate human SFTSV infection in mice. Moreover, our results reveal that the introduction of multiple entry receptors does not necessarily translate to enhanced infection efficacy. This underscores the need for further investigation into the interplay between SFTSV entry mechanisms and host factors to develop more robust mouse models. Advancing such models will be crucial for unraveling the pathogenesis of SFTS pathology and improving strategies for its prevention and treatment in humans.

Keywords: DC-SIGN; DC-SIGNR; LSECtin; severe fever thrombocytopenia syndrome virus; transgenic mice.

FIGURE 1

Generation of a transgene construct containing three SFTSV receptors and its expression. (A) Schematic illustration showing the strategy for generating the transgene construct of pCAG-hDC-SIGN-hDC-SIGNR-hLSECtin (pCAG-3xSFTSVR). pCAG, indicated by the bright blue arrow, contains a CMV enhancer element, chicken beta-actin promoter, and the splice acceptor of the rabbit beta-globin. Human DC-SIGN is colored yellow, human DC-SIGNR is green and human LSECtin is red. P2A is colored ocean blue, and T2A is colored sky blue. The tri-cistronic construct in a pCAG backbone with 2A peptides expresses human DC-SIGN, human DC-SIGNR, and human LSECtin. (B–E) Tri-cistronic construct expression test. Constructs were transfected into the HEK 293T cells, then the cells were collected 30 h after transfection for (B) western blot analysis and (C–E) fixed 24 h after transfection for immunocytochemistry. Nuclei were stained by DAPI. Scale bar = 20 μm. (F–K) Comparative analysis of SFTSV infectivity in transfected BJAB cells (MOI = 1, n = 3). Cells were transfected with various vectors: control pmCherry-C1 (F–K, Red), pCAG-hDCSIGN (F,I, green), pCAG-hDCSIGNR (G,J, green), pCAG-hLSECtin (H,K, green) or pCAG-3xSFTSVR (F–K, blue), and incubated for 48 h. Transfected cells were infected with GFP-expressing SFTSV and then analyzed by flow cytometry after 24 h post-infection. Cells were stained with anti-DCSIGN antibody (F), anti-DCSIGNR antibody (G), or anti-LSECtin antibody (H). **P > 0.01, ***p < 0.001, ****p < 0.0001. A two-way ANOVA was performed to compare variables between groups.

FIGURE 2

Expression of three SFTSV receptors in 3xTG mouse. (A–C) RT-PCR analysis demonstrated the relative mRNA levels of hDC-SIGN, hDC-SIGNR, and hLSECtin in the thymus, spleen, lung, liver, kidney, heart, brain, and small intestine of wildtype (WT, n = 1) and 3xTG mice (n = 1). (D) Western blot analysis revealed the expression of hDC-SIGN, hDC-SIGNR, and hLSECtin in the thymus, spleen, lung, liver, kidney, heart, brain, and small intestine of wildtype (WT, n = 1) and 3xTG (n = 1) mice. (E–G) Immunohistochemistry staining showed increased expression of the three human SFTSV receptors in the spleen, kidney, and liver of 3xTG mouse (n = 1) compared to WT mouse (n = 1). The scale bar for the spleen is 100 μm; for the kidney and liver, it is 50 μm.

FIGURE 3

Low Susceptibility of 3xTG Mice to SFTSV Infection. Mice were subcutaneously infected with 10⁵ FFU of SFTSV. (A and B) Survival probability (A) and body weight (B) were monitored in young (n = 6) and aged (n = 5) 3xTG mice, along with young (n = 6) and aged (n = 5) WT C57BL/6J and IFNAR KO mice (n = 5), after SFTSV infection. (C,D) Viral titers in the serum (C), spleen, and lymph nodes (D) were quantified in young (n = 3) and aged (n = 4) 3xTG mice, young (n = 3) and aged (n = 4) WT C56BL/6J mice, and IFNAR KO mice (n = 3) 3 days post-infection. (E) Platelet counts were measured in the same group at the same time points. *p < 0.05, ***P > 0.001, ****p < 0.0001. Two-way ANOVA was performed to compare the variables between groups [(C,D)].