Fluorescent Reporters for Tracking Serotype-Specific Dengue Virus Infection

Abstract

Dengue virus (DENV) is the most common arthropod-borne virus globally, accounting for 400 million infections each year in over 100 countries. Caused by four distinct serotypes (DENV1-4) with increasing co-circulation in recent decades, dengue poses a unique immunological challenge given the possibility for mixed infections to occur. Despite decades of research, fundamental questions about how these serotypes interact at the cellular level during mixed infections remain unresolved. Existing methods cannot simultaneously provide serotype specificity, single-cell resolution, and live infection dynamics. Studies have also been largely confined to mosquito cell lines or patient samples with unknown infection history, making controlled investigation in human-relevant cell types difficult. In this thesis, we developed and validated two molecular tools to enable serotype-specific tracking of DENV infection in human hepatocytes. Using Circular Polymerase Extension Reaction, we generated mScarlet- and mGreenLantern-expressing reporter viruses for all four DENV serotypes by inserting spectrally orthogonal fluorescent proteins between the E and NS1 genes of infectious cDNA clones. We performed coinfection experiments using fluorescent DENV2 reporter pairs at defined multiplicities of infection, and population dynamics were analyzed by flow cytometry. We additionally developed a genome-modification-free approach, validating an ER-tethered GFP reporter that undergoes nuclear translocation when a linker sequence containing the capsid cleavage site is recognized by the NS2B/3 viral protease. Reporter viruses reliably tracked infection dynamics, and within-serotype coinfection experiments demonstrated that relative population abundance is tunable by inoculum dose. The ER-tethered reporter produced robust nuclear translocation in response to infection, while substitution with a heterologous cleavage sequence led to a six-hour delay, reflecting sequence specificity for NS2B/3 substrate recognition. These data have established a modular approach for studying DENV coinfections as well as inter-serotype protease activity, with possible extensions to the broader Flaviviridae family.