Construction of a CMV promoter-driven Langat virus cDNA clone and reporter viruses

Langat virus (LGTV) is a tick-borne member of the Flaviviridae family and a biosafety level 2 surrogate for studying tick-borne encephalitis virus (TBEV) replication and pathogenesis. Here, we report the construction of a plasmid encoding a cytomegalovirus (CMV) promoter-driven LGTV cDNA that initiates infection following direct transfection of mammalian cells. Incorporation of three introns eliminated viral cDNA-associated toxicity in bacteria, enabling stable propagation of the full-length plasmid. Transfection of this construct resulted in high-level production of infectious LGTV, which exhibited robust replication kinetics, though slightly slower growth compared to a patient-derived isolate. We further engineered mCherry and Gaussia luciferase reporter versions of the clone, which yielded viruses expressing high levels of their respective reporters while retaining efficient replication. These LGTV infectious clones provide versatile tools for investigating viral replication, gene function, and pathogenesis, and may facilitate screening for antiviral inhibitors. Highlights Single-plasmid CMV-driven system launches infectious Langat virus (LGTV) Three introns stabilize full-length LGTV cDNA in E. coli DNA transfection yields high-titer infectious virus in mammalian cells Reporter LGTVs enable fluorescent and luminescent infection readouts Provides a versatile platform for LGTV genetics and pathogenesis studies