Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs

Abstract

Background: Next-generation metagenomic sequencing (NGMS) has opened new frontiers in microbial discovery but has been clinically characterized in only a few settings.

Objective: To explore the plasma virome of persons who inject drugs and to characterize the sensitivity and accuracy of NGMS compared with quantitative clinical standards.

Design: Longitudinal and cross-sectional studies.

Setting: A clinical trial (ClinicalTrials.gov: NCT01285050) and a well-characterized cohort study of persons who have injected drugs.

Participants: Persons co-infected with hepatitis C virus (HCV) and HIV.

Measurements: Viral nucleic acid in plasma by NGMS and quantitative polymerase chain reaction (PCR).

Results: Next-generation metagenomic sequencing generated a total of 600 million reads, which included the expected HIV and HCV RNA sequences. HIV and HCV reads were consistently identified only when samples contained more than 10 000 copies/mL or IU/mL, respectively, as determined by quantitative PCR. A novel RNA virus, human hepegivirus-1 (HHpgV-1), was also detected by NGMS in 4 samples from 2 persons in the clinical trial. Through use of a quantitative PCR assay for HHpgV-1, infection was also detected in 17 (10.9%) of 156 members of a cohort of persons who injected drugs. In these persons, HHpgV-1 viremia persisted for a median of at least 4538 days and was associated with detection of other bloodborne viruses, such as HCV RNA and SEN virus D.

Limitation: The medical importance of HHpgV-1 infection is unknown.

Conclusion: Although NGMS is insensitive for detection of viruses with relatively low plasma nucleic acid concentrations, it may have broad potential for discovery of new viral infections of possible medical importance, such as HHpgV-1.

Primary funding source: National Institutes of Health.

Fig 1. Correlation of plasma viremia with NGMS viral reads

HIV and HCV plasma viral RNA quantities, measured by quantitative PCR assays were correlated with NGMS viral sequence read number, expressed as viral reads per million mapped reads. Although the 2 measurments were highly correlated, of 12 samples with PCR determined viremia levels less than <10000 copies or IU/mL, HIV or HCV reads were only detected in 4 (33%). One of the samples with undetectable HIV viremia by quantittive PCR also had no NGMS reads that mapped to HIV and therefore was not included in the analysis. Markers below the dashed line indicate that no HIV or HCV reads were detectable for those samples. HCV=hepatitis C virus;NGMS = next -generation metaenomic sequencing;PCR=polymerase chain reaction.

Fig 2. Sequence and Phylogenetic Analysis of ISV-1

a) The NGMS reads were large enough to permit construction of a contiguous sequence minus the untranslated regions. More than 90% of the reads used for the assembly mapped back to the genome; showing an average coverage of 57-fold at time point 2. b) Phylogenetically, the NS5B nucleotide sequence was grouped with NS5B sequences from previously identified HHpgV-1.Phylogenetic reconstruction was done on MEGA7 using the Jukes-Cantor model and maximum likelihood algorithm with 1000 bootstrap replicates. Only bootstrap values >80 % are shown. S: nucleocapsid; E1 and E2: envelope glycoproteins; X: protein of unknown function; NS2, NS3, NS4A, NS4B, NS5A, and NS5B:non-structural proteins.HHpgV-1 GenBank IDs: KT427413.1, KT427408.1, KT427407.1, KU159665.1, KU159664.1, KT427414.1, KT427412.1, KT427411.1, KT427410.1, KT427409.1, and KT439329.1. HCV GenBank ID:KX621472. The genomic arragement of HHpgV-1 was modified from Berg and colleagues (6)