PROJECT SUMMARY: The United States is in the midst of an enormous opioid epidemic. In 2015, more than 52,000 American’s died due to a drug overdose, over 60% of which were associated with prescription or illicit opioid use. Given that injection drug use remains the major risk factor for HCV transmission, concomitantly the US has observed a striking >150% increase in the incidence of HCV between 2010-2013, and a 364% increase in new HCV infections between 2006 and 2012 in four Appalachian states, most notably in rural areas. As such, there is urgent need to more strategically detect, prevent, treat and control HCV on a national level. Under the parent award we built a high-quality, high-throughput HCV next-generation sequencing (NGS) platform that enabled the generation of HCV HVR1 sequences from over 1,000 patient samples derived from 8 rural study sites, along with an in-house informatics pipeline capable of validating phylogenetic clustering identified by the CDC GHOST laboratory. Transmission network analysis of the NGS data showed that the rate of clustering (likely direct transmission) varied considerably across study sites ranging from 10% to 42%. Across all geographical sites, genotype 1a (57%) was the most common followed by genotype 3a (30%), 2b (9%), 1b (4%) and mixed genotypes represented less than 5% of cases. The implementation of the GHOST HCV molecular surveillance technology illustrates that genomic surveillance of HCV in rural communities is feasible and suggest underlying factors may be influencing the size of transmission networks across sites. Under the parent award we also examined the feasibility of using dried blood spot (DBS) collection for this application. A pilot study comprising 14 serum samples revealed that viral populations within DBS were genetically indistinguishable from viral populations derived from plasma, demonstrating that DBS can capture comparable transmission networks and viral diversity observed from the traditional collection of serum samples. Thus, DBS can augment traditional HCV surveillance approaches as a practical and cost-effective alternative to frozen plasma in resource-limited settings such as rural populations. Due to the substantial impact of the SARS-CoV-2 pandemic upon both our laboratory work at the Ragon Institute, and upon our UH3 collaborators providing specimens for our work, many of the initial goals of our application were not completed. This Administrative Supplement to our U24 application proposes to support the generation and analysis of additional HCV NGS data for the CDC’s GHOST center to identify HCV transmission links among persons who inject drugs (PWID); validate the application of dried blood spots for the collection and generation of transmission links by NGS data; manage the collection and storage of serum samples from HIV- and HCV- infected participants from the clinical research sites under RFA-DA-17-014; and ship specimens to the CDC for syphilis testing and phylogenet...