Débora Silva Pinto

Brazil

Characterization of Saint Louis encephalitis virus (SLEV) neuroinfection in central nervous system (CNS) human cell models and IFNAR -/- mice.

Débora Silva Pinto1; Déborah Pereira Pina da Silva1,2; Luan Rocha Lima1; Sharton Vinícius Antunes Coelho1; Luciana Barros de Arruda1.

1. Universidade Federal do Rio de Janeiro- UFRJ;
2. Universidade Federal do Estado do Rio de Janeiro

Abstract

Background

Saint Louis Encephalitis Virus (SLEV) is an Orthoflavivirus transmitted mainly by Culex mosquitoes, which infection may cause neurological symptoms such as meningitis and encephalitis. Despite recent detections in humans and animal reservoirs across the Americas, SLEV pathogenesis remains poorly understood. Here, we investigated the effects of SLEV infection on the CNS using in vitro and in vivo models

Methods

Human cell lines of brain microvascular endothelial cells (HBMECs), microglia (C20), astrocyte (FHA) and neuroblastoma (SH-SY5Y) were used as in vitro models to characterize the permissive cell types in vitro. The cells were mock-treated or infected with SLEV, and the infection was followed up to 72 hours post-infection (h.p.i). Cell viability was measured by LDH release. Infectious viral particles were quantified by plaque assay, and viral RNA was measured in the cell lysates and culture medium by RT-qPCR. As an in vivo model, IFNAR-deficient C57BL/6 (IFNAR-/-) mice were intravenously infected with different SLEV doses, and clinical signs and mortality were daily monitored.

Results

All tested CNS-derived cell lines were susceptible to SLEV infection, showing time-dependent increase in intra- and extracellular viral RNA expression. However, the magnitude of infectious particle release (PFU), cytopathic effect (CPE), and infection kinetics varied across cell types. HBMECs and SH-SY5Y showed a progressive PFU production until 72 h.p.i., followed by cell death. Infection of FHA cells resulted in altered cell morphology and death at 48 h.p.i in both conditions, concurrent with a significant reduction in PFU release on SLEV-infected cells after 48h.p.i. In C20 microglia, PFU levels peaked at 24 h.p.i. and did not increase thereafter, and cell death was detected from 72 h.p.i. In vivo, IFNAR⁻/⁻ mice infected with the highest inoculum showed weight loss by 3 days post-infection (d.p.i.) and succumbed by 5 d.p.i. All animals presented neurological symptoms and exhibited high viral loads in the brain, confirming SLEV neurotropism.

Conclusions

These findings demonstrate that brain endothelial cells, neurons, astrocytes, and microglia are permissive to SLEV infection in vitro, although with distinct replication dynamics and CPE patterns. In addition, detection of high viral loads in the brains of IFNAR⁻/⁻ mice following systemic inoculation demonstrates that SLEV is neuroinvasive in immunodeficient mice, supporting hematogenous dissemination as a route for CNS entry.