Heterozygous TLR3 Mutation in Patients with Hantavirus Encephalitis

Abstract

Puumala hantavirus (PUUV) hemorrhagic fever with renal syndrome (HFRS) is common in Northern Europe; this infection is usually self-limited and severe complications are uncommon. PUUV and other hantaviruses, however, can rarely cause encephalitis. The pathogenesis of these rare and severe events is unknown. In this study, we explored the possibility that genetic defects in innate anti-viral immunity, as analogous to Toll-like receptor 3 (TLR3) mutations seen in HSV-1 encephalitis, may explain PUUV encephalitis. We completed exome sequencing of seven adult patients with encephalitis or encephalomyelitis during acute PUUV infection. We found heterozygosity for the TLR3 p.L742F novel variant in two of the seven unrelated patients (29%, p = 0.0195). TLR3-deficient P2.1 fibrosarcoma cell line and SV40-immortalized fibroblasts (SV40-fibroblasts) from patient skin expressing mutant or wild-type TLR3 were tested functionally. The TLR3 p.L742F allele displayed low poly(I:C)-stimulated cytokine induction when expressed in P2.1 cells. SV40-fibroblasts from three healthy controls produced increasing levels of IFN-λ and IL-6 after 24 h of stimulation with increasing concentrations of poly(I:C), whereas the production of the cytokines was impaired in TLR3 L742F/WT patient SV40-fibroblasts. Heterozygous TLR3 mutation may underlie not only HSV-1 encephalitis but also PUUV hantavirus encephalitis. Such possibility should be further explored in encephalitis caused by these and other hantaviruses.

Keywords: Toll-like receptor 3; central nervous system infections; encephalitis; genetic diseases; hantavirus; primary immunodeficiency diseases.

Fig. 1

TLR3 mRNA expression levels were determined by RT-qPCR, as normalized to wild-type (WT) relative expression levels, in P2.1 TLR3-deficient fibrosarcoma cells without transfection (NT) or transfected with empty vector (EV), HA-tagged TLR3 WT, L742F (p.Leu742Phe), R867Q (p.Arg867Gln), or E746X (a). IFNL1 (IL29) induction by poly(I:C) stimulation, as normalized to wild-type (WT) fold induction level, in P2.1 cells not transfected (P2.1) or stably transfected with empty vector (P2.1+EV), HA-tagged TLR3 WT, L742F, R867Q, or E746X (b). Production of IL-29 (c and d), and IL-6 (e and f) in SV40-fibroblasts from three healthy controls (C1, C2, C3), P1, and a TLR3−/− HSE patient, 24 h after stimulation with 1, 5, or 25 μg/ml poly(I:C) (c and e), or with 25 μg/ml poly(I:C) in the presence of lipofectamine (poly(I:C)+lipo; d and f), or lipofectamine alone, as assessed by ELISA. Schematic structure of the human TLR3 gene and protein, featuring the leader sequence (L), leucine-rich repeats (LRRs) of the ectodomain, transmembrane domain (TM), linker region (LR), and Toll/IL-1 receptor (TIR) domain. Roman numerals indicate the coding exons. Previously reported mutations found in patients with HSE patients (E110K, L297V, L360P, P554S, G743D, R811I, R867Q), severe influenza (F303S, P554S, P680L) or Varicella zoster virus infection (L199F, R867X), that have been previously experimentally characterized (F303S, L360P, P554S, P680L, G743D, R811I, R867Q, R867X) or not (E110K, L199F, L297V) are shown in blue. The L742F mutation found in the two patients with complicated Puumala hantavirus infection is shown in red (g)