Characterization Of Usutu Virus Replication And Associated Immune Response In Vitro 2308012

Abstract Introduction Usutu virus (USUV), a mosquito-borne flavivirus, is an emerging public health concern capable of causing neurological disease. However, the cellular mechanisms driving USUV neuropathogenesis remain unclear. Different neural and endothelial cell types may vary in their susceptibility to infection and virus-induced damage. This project investigates early cellular responses to USUV in four models: Vero C1008 (E6), SK-N-SH (human neuronal cell line), primary human brain cortical astrocytes (HBCA), and primary human brain microvascular endothelial cells (HBMVE). Determining which cell types support viral replication or exhibit greater cytotoxicity will provide insight into how USUV impacts the central nervous system. Methods Cells were infected with USUV at an MOI of 1, and pellets and supernatants were collected at different time points. Plaque assays quantified infectious titers, and RTq-PCR measured host immune responses. The CellTiter 96 Aqueous One Solution assay assessed USUV-induced cell damage relative to controls. Results USUV infection was successfully established in the four cells lines. Infected cultures showed morphological changes such as rounding, shrinkage, and detachment, with VERO E6 exhibiting the strongest cytopathic effect. Plaque assays confirmed infectious virus in all cell types, with higher titers in VERO E6 and HBCA cells. Cytotoxicity assays showed reduced viability in infected wells, especially in more permissive cells. Infection also induced strong immune activation, marked by increased cytokine, chemokine, and interferon RNA levels. RNA-seq of infected and mock-treated cells provided an unbiased view of virus-induced cellular reprogramming and highlighted pathways that may contribute to cell-type vulnerability and potential therapeutic targets. Conclusion Together, this work establishes a framework for understanding USUV interactions with brain-associated cell types and supports future studies on the mechanisms driving USUV-related neurological injury. Funding Source This work was supported by GSU Institutional Funds. Topic Categories Viral Immunology (VIR)