PROJECT SUMMARY/ABSTRACT (Project 2) Rotavirus (RV) infection kills 200,000 children annually. Rotaviruses are non-enveloped, triple-layered, icosahedral viruses with dsRNA genomes composed of 11 separate segments. RVs are ubiquitous, highly infectious and cause severe diarrheal diseases in the young of most mammalian species including humans. RV remains the primary cause of acute life-threatening diarrhea in infants and young children under the age of four. RVs can also afflict elderly, immunocompromised, and healthy adults but disease is generally less severe. Despite the availability of several safe and effective vaccines, RV causes 114 million diarrhea episodes, 24 million outpatient visits, 2.4 million hospitalizations, and approximately 200,000 deaths in young children annually. Currently available licensed vaccines have limited efficacy (<60%) in most parts of the less-developed world. The lack of highly effective RV vaccines for the third world is due, at least in part, to an incomplete understanding of RV interactions with the enteric immune system and gut. Using primary small bowel enteroids, we previously made several seminal discoveries on the mechanisms that regulate RV host range restriction (HRR), neutralization, and spread across the intestine. Our goal is to build on these successes to understand key features of RV biology, pathogenesis, and immunity. We will take advantage of recent technological advances including: i) use of human donor-derived small bowel enteroids to model human RV infection in and through the gut; ii) development of an efficient reverse genetics (RG) system to modify RV genomes; and iii) development of a system to differentiate functional microfold (M) cells in ileum organoids. This project has the following three Aims: 1) We will use our optimized RG system to generate targeted genetic reassortants between human and select animal RVs and examine their replication and abilities to inhibit innate immune responses in human organoids. We expect to identify the genetic basis of human RV HRR, which will prove useful to guide the rational design of third-generation RV vaccines. 2) We will perform competition blocking analysis and crystallographic examination of mAb/RV protein interactions to elucidate the structural basis of human RV neutralization in the human gut and use a novel genome-wide CRISPR-Cas9 screening approach to identify the RV-specific binding dependency factors on the enterocyte surface. 3) We will employ genetic tools (e.g., CRISPR-Cas9 deletion of STAT1) or pharmacological inhibition of IFN signaling (i.e., ruxolitinib) to determine whether disabling innate immunity renders M cells susceptible to RV infection. Finally, we will examine whether the M cells function as critical entry conduits to widespread epithelial cell infection via creating basolateral cell surface access. These RV studies and findings should also be broadly relevant to other enteric pathogens and non-infectious intestinal infl...