Abstract
We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.
Highlights
Techniques such as cell culture, electron microscopy and serology have been instrumental in discovering unknown viruses
Plasma samples were obtained from patients with autoimmune hepatitis (AIH), chronic hepatitis B virus (HBV) infection, chronic hepatitis C virus (HCV) infection, non-alcoholic steatohepatitis (NASH), and healthy subjects (NOR); each RNA library was named by diagnosis and a suffix ‘D’ was added for each DNA library
We performed a taxonomic classification of reads into human, bacteria, phage, human endogenous retroviruses (HERV), viruses, and unknown categories (Supplemental Tables S1–S14)
Summary
Techniques such as cell culture, electron microscopy and serology have been instrumental in discovering unknown viruses These procedures led to the identification of the viruses causing hepatitis A and B [1,2], acquired immune deficiency syndrome [3], and acute respiratory syndrome [4]. An unbiased approach has been used to identify viruses by DNAse treatment of serum, followed by amplification and sequencing of the nucleic acids protected in virions [8] Using this method, was hepatitis B virus (HBV) identified in the serum of an infant with acute hepatitis B, but so too were two species of bovine parvoviruses, possibly because bovine serum was used to dilute the experimental samples [8]
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