OPTIMIZATION OF LENTIVIRAL TRANSDUCTION FOR EBOLA VP40 EXPRESSION IN A HARD-TO-TRANSDUCE HUMAN CELL LINE

A. ABSTRACT Ebola virus (EBOV) causes severe hemorrhagic fever with high case fatality rates, yet research on the viral life cycle is severely constrained by the requirement for Biosafety Level 4 (BSL-4) containment. While BSL-2 surrogate models exist, current plasmid-based overexpression systems generate non-physiological protein levels that distort the stoichiometry required for authentic viral assembly. This thesis establishes and validates a quantitative lentiviral delivery platform for the EBOV matrix protein VP40 in Huh-7 cells, a disease-relevant hepatocyte model that is notoriously refractory to transfection. By systematically optimizing transduction parameters, specifically identifying the critical synergy between cationic polymers (Polybrene) and mechanical sedimentation (spinoculation), this study overcame the intrinsic entry barriers of the hepatic host. The optimized protocol enabled the reproducible, dose-dependent expression of VP40, effectively bridging the gap between artifact-prone overexpression and restrictive infection models. Although high-load transduction revealed a cell-type specific cytotoxicity that defines the therapeutic window of the system, the platform successfully prioritizes delivery fidelity over raw abundance. This tunable BSL-2 model provides a standardized scaffold for dissecting concentration-dependent matrix assembly, investigating liver-specific host factors, and conducting medium-throughput screens for assembly inhibitors.