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

<div><p>Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the <i>Hepacivirus</i> and <i>Pegivirus</i> genera, respectively, of the family <i>Flaviviridae</i>. 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.</p></div

Prevalence of HPgV-2 RNA in HCV and non-HCV infected groups.

<p>*P-value of differences in HPgV-2 RNA (top) and antibody (bottom) prevalence between reference and comparison groups. Index case (HCV Ab+ /NAT+) has been excluded from totals screened.</p><p>Prevalence of HPgV-2 RNA in HCV and non-HCV infected groups.</p

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

<p><b>(A)</b> 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>(B)</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.</p

Molecular and Serologic Characterization of HPgV-2 Strains

<p>Abbreviations: NA, not applicable; NT, not tested.</p><p>*These two samples are from the same individual with bleed dates 26 days apart (sample ABT0030P.US is the earlier sample).</p><p>**These two samples are from the same individual with bleed dates 60 days apart (sample ABT0035P.US is the earlier sample).</p><p>^†Italicized numbers show elevated signals, but below the cutoff value.</p><p>Molecular and Serologic Characterization of HPgV-2 Strains</p

Discovery and whole-genome characterization of HPgV-2.

<p><b>(A)</b> 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>(B)</b> The initial set of contigs generated from <i>de novo</i> assembly of HPgV-2 reads that were identified using BLASTx alignment to other pegivirus genomes. <b>(C)</b> Gap closure using PCR followed by Sanger sequencing. <b>(D)</b> Coverage plot showing mapping of the initial NGS data to the nearly complete (>98%) assembled draft genome. (<b>E)</b> 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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005325#ppat.1005325.ref020" target="_blank">20</a>]. (<b>F)</b> 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.</p