Discovery of a Novel Human Pegivirus in Blood Associated with Hepatitis C Virus Co-Infection

Abstract

Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93-94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.

Fig 1. Discovery and whole-genome characterization of HPgV-2.

(A) Three NGS reads with remote amino acid homology (<60%) to SPgV-A / GBV-A, of which two were overlapping, were detected in a plasma sample from a patient with HCV infection who died of abdominal sepsis (the index case). (B) The initial set of contigs generated from de novo assembly of HPgV-2 reads that were identified using BLASTx alignment to other pegivirus genomes. (C) Gap closure using PCR followed by Sanger sequencing. (D) Coverage plot showing mapping of the initial NGS data to the nearly complete (>98%) assembled draft genome. (E) Coverage plot showing mapping of the NGS data from a subsequent sequencing run to the complete HPgV-2 genome, after the 5' and 3' ends were recovered using RACE [20]. (F) Genomic arrangement of HPgV-2. Putative cleavage sites within the polyprotein are indicated with black triangles (structural proteins) or hollow triangles (non-structural proteins). Arrows denote predicted N-linked (red) or O-linked (blue) glycosylation sites.

Fig 2. Phylogenetic analysis of HPgV-2 relative to other pegiviruses and hepaciviruses.

(A) Pairwise amino acid identity plots comparing HPgV-2 (UC0125.US) with other representative pegiviruses (red) and hepaciviruses (blue). The sliding window size is 50 nt. (B) Phylogenetic trees of the NS3 (left) and NS5B (right) proteins were constructed for 10 newly sequenced HPgV-2 strains (boldface red), representative hepaciviruses, and all fully sequenced pegiviruses in the NCBI nt database except for members of the simian pegivirus clade, for which 5 representative strains are shown (triangle). Each tree is rooted with yellow fever virus (YFV) as an outgroup.