Concentration and single virion analysis of the virome

ABSTRACT Clinical virology is predicated on the workflow of analyzing small sample volumes (to minimize patient discomfort, storage, and fit legacy infrastructure) for a single analyte, i.e., virus, with utmost precision. The human virome project (HVP) does not share these restrictions. The HVP priority tissues of urine, stool, saliva, and blood can be acquired from healthy people in a 50 – 500 ml sample size. For instance, the Red Cross makes normal plasma available in 500 ml bags, and NGS allows for the characterization of all human viruses in a single reaction. This proposal responds to RFA-RM-23-018 with the development of two novel tools: AIM 1, a virus purification pipeline that utilizes larger volumes to achieve < 1 genome copy/ ml sensitivity; AIM 2, a single virion detection pipeline to provide an accurate denominator of how many intact virions a given NGS sequence set is representing. We hypothesize that Viral Direct Counts (VDC) akin to bacterial direct counts will become the essential parameter for integrating the studies in the HVP.