Project Summary A substantial portion of patients with liver disease, ranging from 5% to 30%, have unknown causes beyond the established etiologies. Unknown etiology is observed across a wide array of clinical phenotypes in liver disease, such as acute liver failure (ALF), hepatitis, cirrhosis, and liver cancer. These are collectively referred to as cryptogenic liver disease (CLD). It has long been hypothesized there exist additional human viruses that cause CLD. In our recent serum virome study, we identified a 387-nt DNA fragment (GenBank MW468091), named Seq260, from 1 of 9 CLD patients. In a series of experiments of gene-walking, enzymatic digestion, and rolling circle amplification and analyses, we have demonstrated that Seq260 is a linear single-stranded DNA. We screened Seq260 in 409 subjects, including healthy blood donors (n=200), hepatitis C virus infection (n=100), Acute liver failure (ALF) patients with indeterminate etiology (n=50), and liver transplantation (LT) patients with (n=45) and without known etiology (n=14). Seq260 was detected in 5 CLD patients (1 ALF and 4 LT) and 1 LT patient with nonalcoholic steatohepatitis (NASH)-associated cirrhosis. One patient had Seq260 quantifiable in liver, showing a titer in the liver 7.74 times higher than that in serum (2.4x106 copies/g vs. 3.1x105 copies/mL). Machine learning analysis reached a high score (likelihood) of Seq260 being a eukaryotic viral sequence. Aggregately, these data lead to our hypothesis that Seq260 represents an unrecognized human virus with liver tropism. To determine if Seq260 represents a novel hepatitis virus, we bring about a research plan in the current proposal that consists of three major experiments. First, we will screen Seq260 in CLD patients as well as the controls. We have been granted access to patient specimens from two NIH- sponsored clinical trials, ALF study group (ALFSG), and the adult-to-adult living donor liver transplantation cohort study (A2ALL). Unknown etiology accounted for 5.5% and 29.5% respectively in the ALFSG and A2ALL. Seq260 copy numbers will be quantitated in both serum and liver in Seq260-positive patients with liver tissue available. Second, we will determine the full genome of the putative virus containing Seq260. Finally, we will evaluate antibody responses in virus-positive patients and the controls. A peptide-based serological test will be developed for the putative virus. Peptides will be individually assessed for their specificity and sensitivity in two virus-positive patients with large volumes of serum available. Selected peptides will then be combined to ELISA tests for the measurement of antibody (IgG and IgM) responses in virus-positive and virus-negative patients. Taken together, the proposed study will characterize a novel human virus and understand its etiological link to liver disease from a clinical aspect. It will expand our knowledge of the human virome as well as the etiology of liver disease without a known cause.