Replication-competent adenovirus reporters utilizing endogenous viral expression architecture.

Adenoviruses are double-stranded DNA viruses widely used as platforms for vaccines, oncolytics, and gene delivery. However, tools for studying adenoviral gene expression in real time during infection remain limited. Here, we describe a set of fluorescent and bioluminescent reporter viruses built using the modular AdenoBuilder reverse genetics system and informed by high-resolution maps of Ad5 transcription. These reporters utilize endogenous early and late transcriptional units, enabling visualization of viral gene expression without exogenous promoters or splicing elements. These model viruses replicate with kinetics nearly indistinguishable from wild-type virus and have enabled real-time fluorescent imaging as well as longitudinal bioluminescent sampling from the same infected samples. Together, this next generation of adenovirus reporters provides a modular and tractable platform for studying viral gene regulation and replication dynamics in real time, with broad applications for basic research and high-throughput screening. IMPORTANCE: This research provides powerful new tools to rapidly study adenovirus gene expression and replication. By integrating fluorescent and secreted luciferase reporters into native viral regulatory elements, we enable real-time, non-destructive tracking of early and late stage infection in living cells. These modular reporters are compatible with a wide range of genetic and chemical perturbations, allowing researchers to investigate the function of specific viral genes, host interactions, and the impact of host genes and antiviral compounds. Importantly, the high-throughput nature of these systems overcomes limitations of traditional plaque assays to quantify viral replication dynamics. Our work will allow for the rapid creation of both novel infectious and replication-incompetent viral vectors.