Hepatotropic viruses from human plasma using deep sequencing: a new generation diagnostic tool. human metagenome

Many viruses have been associated with liver diseases; however, their detection in patients suffering from hepatic disorders has been restricted to specific diagnostic tools such as PCR and serological assays. We conducted an unbiased metagenomics survey in plasma filtrates from patients affected by several hepatic disorders, including hepatitis B and C, autoimmune hepatitis (AIH) and non-alcoholic steatohepatitis (NASH). Pooled plasma from four patients that tested negative for hepatotropic viruses was used as negative control. RNA and DNA libraries prepared from enriched viral particles were constructed and sequenced to catalog viruses present in plasma. Hepatitis viruses were readily detected at high coverage in three out of four samples with such diagnosis, while abundant GB virus C was found in the plasma derived from a patient initially diagnosed with hepatitis C. Torque teno virus sequences were found at high frequency in AIH and NASH samples, but were absent in hepatitis samples. After classification of sequences into human, endogenous retroviruses, bacteria, phage and virus taxa, a substantial fraction of reads in each library, ranging from 35% to 72%, remained unclassified. Unknown reads, altogether with virus, phage and endogenous retroviruses reads were assembled into scaffolds and then analyzed by BLASTx using the non-redundant protein databases. We assembled the full genome of a circovirus and extensive scaffolds that encoded proteins similar to those from mimiviruses, among others. We confirmed the presence of the circovirus and the known hepatitis viruses in our samples by PCR. Thus, we have established a reliable protocol for the identification of viruses in plasma that could also be adaptable to urine, bile, saliva and other body fluids.