Leveraging primary cell and organoid culture to identify and map the human virome - Despite years of study, human susceptibility to chronic disease is still not fully understood, compelling the continued search for modifiable risk factors. One previously hidden variable in human health is the human virome. Advances in genome sequencing technology have begun to reveal a vast array of viruses that reside within the body without causing overt symptoms, and early evidence indicates that this collection of viruses can drive distinct health states which impact susceptibility to many common diseases, ranging from autoimmunity to cardiovascular disease to cancer. Understanding how the virome influences human health will lead to new strategies to prevent and treat common, impactful diseases. However, the first step towards uncovering these connections is being able to accurately observe, characterize, and quantify the human virome. The goal of this project is to develop new strategies to characterize the human virome by tackling major technological limitations in the field. While advances in next generation sequencing (NGS) technology have fueled exciting discoveries in the human virome research, there are clear knowledge gaps, which include lack of information about (1) eukaryotic cell infecting viruses, (2) RNA viruses, and (3) the virome within human tissues, including the cells and locations in which viruses of the virome reside. Here we will address these knowledge gaps by developing new methods to interrogate the human virome, focusing on the human tonsil. We chose the tonsil, a secondary lymphoid organ, due to the likely importance of the virome of lymphoid organs in shaping immune responses, the availability of human tonsil tissue for research, and the high detection rate of eukaryotic viruses in tonsils, supporting feasibility. This project will pioneer two approaches to address knowledge gaps in the field. First, we will enhance detection of the eukaryotic cell- infecting virome by amplifying viruses using epithelial and tonsil organoid cultures. Based on prior work, we expect that viruses infecting these tissue types will be prevalent in human tonsils, including RNA respiratory viruses, which predominantly replicate in epithelial cells and enter the tonsil during acute infection, but can persist long-term post-acute infection, and DNA viruses that infect lymphocytes, the predominant cell type in tonsils. We will enhance detection of viruses that replicate in epithelia in Aim 1 using airway epithelial organoid cultures, and viruses that replicate in tonsil cells using tonsil organoid culture in Aim 2. Second, we will map viral sequences to host cells or spatial locations within the tonsil using single cell sequencing (ScSeq) and spatial transcriptomics (ST) using a novel strategy. Data from Aims 1 and 2 will reveal viral RNAs present in each tonsil donor, including genomes of RNA viruses and viral mRNA from biologically active DNA viruses. In Aim 3, we will use this information to create custom primers to amplify viral reads during ScSeq and ST library preparation, allowing us to identify the host cells and spatial locations of viral reads within tonsil tissue. Together these Aims will advance methods to observe and understand the tissue-resident human virome.
