Modified Project Summary/Abstract Section Viral Pathogenesis & Persistence Core E supports CFAR investigators and their innovative projects while maintaining strong capabilities to assist researchers with more traditional basic and translational virology research. The core combines expertise from virologists at both Case and Pitt to promote research on HIV-1 pathogenesis and persistence by RUSTBELT CFAR investigators. Core E offers researchers innovative services to measure HIV-1 persistence using assays that identify and quantify latent, expressed, and inducible HIV-1, along with pioneering ex vivo methods to evaluate the effectiveness of novel Latency Reversal Agents (LRA) and other curative strategies in fully autologous systems, enabling more detailed characterization of HIV-1 reservoir dynamics and clonality. Case and Pitt also have complementary systems to quantify HIV-1 persistence, having originally developed ultrasensitive single-copy quantitative PCR (qPCR) assays targeting HIV-1 gag/pol and creating novel techniques to measure HIV-1 env RNA transcripts through induced transcription–based sequencing. These methods have been continuously improved and expanded to now quantify intact versus defective proviral DNA and are automated for sensitive detection of HIV-1 RNA in plasma or culture supernatants. Core E also provides advanced viral outgrowth assays to assess inducible, infectious HIV-1 using new approaches, such as employing a fully autologous system that meets or exceeds the gold standard in the field. Progress in automation has increased throughput, consistency, and quality of near full-length HIV-1 sequencing, while new technologies now enable integration site analysis from the same sample at a single-cell level. Additionally, Core E offers sophisticated imaging platforms to study HIV-1 infection at single virion resolution, including real-time live cell imaging. The focus of Core E on cutting-edge virology complements our second laboratory core, Core F (Systems Biology & Biostatistics), which emphasizes host studies through single host cell genomics and immunology. The combined capabilities of Core F’s transcriptomic and proteomic analyses with the validated HIV latency and reversal models from Core E facilitate applying these detailed techniques to advanced studies in basic virology, prevention, treatment, and cure research. Core E will also support HIV prevention efforts in coordination with Clinical Sciences Core D and the EHE SWG2. The core plans to work closely with samples from Uganda Core C, currently one of two national sites for HIV drug resistance testing in Uganda, to transfer biological samples to the US for virological studies on different viral Clades. Most importantly, Core E will dedicate its efforts to helping CFAR investigators obtain critical data for their projects.