Virome Analysis of Transfusion Recipients Reveals a Novel Human Virus That Shares Genomic Features with Hepaciviruses and Pegiviruses

Abstract

To investigate the transmission of novel infectious agents by blood transfusion, we studied changes in the virome composition of blood transfusion recipients pre- and posttransfusion. Using this approach, we detected and genetically characterized a novel human virus, human hepegivirus 1 (HHpgV-1), that shares features with hepatitis C virus (HCV) and human pegivirus (HPgV; formerly called GB virus C or hepatitis G virus). HCV and HPgV belong to the genera Hepacivirus and Pegivirus of the family Flaviviridae. HHpgV-1 was found in serum samples from two blood transfusion recipients and two hemophilia patients who had received plasma-derived clotting factor concentrates. In the former, the virus was detected only in the posttransfusion samples, indicating blood-borne transmission. Both hemophiliacs were persistently viremic over periods of at least 201 and 1,981 days. The 5' untranslated region (UTR) of HHpgV-1 contained a type IV internal ribosome entry site (IRES), structurally similar to although highly divergent in sequence from that of HCV and other hepaciviruses. However, phylogenetic analysis of nonstructural genes (NS3 and NS5B) showed that HHpgV-1 forms a branch within the pegivirus clade distinct from HPgV and homologs infecting other mammalian species. In common with some pegivirus variants infecting rodents and bats, the HHpgV-1 genome encodes a short, highly basic protein upstream of E1, potentially possessing a core-like function in packaging RNA during assembly. Identification of this new human virus, HHpgV-1, expands our knowledge of the range of genome configurations of these viruses and may lead to a reevaluation of the original criteria by which the genera Hepacivirus and Pegivirus are defined.

Importance: More than 30 million blood components are transfused annually in the United States alone. Surveillance for infectious agents in the blood supply is key to ensuring the safety of this critical resource for medicine and public health. Here, we report the identification of a new and highly diverse HCV/GB virus (GBV)-like virus from human serum samples. This new virus, human hepegivirus 1 (HHpgV-1), was found in serum samples from blood transfusion recipients, indicating its potential for transmission via transfusion products. We also found persistent long-term HHpgV-1 viremia in two hemophilia patients. HHpgV-1 is unique because it shares genetic similarity with both highly pathogenic HCV and the apparently nonpathogenic HPgV (GBV-C). Our results add to the list of human viruses and provide data to develop reagents to study virus transmission and disease association and for interrupting virus transmission and new human infections.

FIG 1

Virome analysis of TTVS pre- and posttransfusion samples. (Top) Statistical analysis of total virus sequences (left), anellovirus sequences (middle), and virus-like unclassified sequences (right) in pre- and posttransfusion samples. (Bottom) Metagenomic binning of all viruses sequences in pre- and posttransfusion samples from twelve TTVS subjects. Red arrows represent pre- and posttransfusion sample pairs that were similar in amount of total virus sequences, and purple arrows represent pre- and posttransfusion sample pairs where posttransfusion samples had significantly larger amounts of total virus sequences.

FIG 2

Genome organization and polyprotein cleavage sites of HHpgV-1. The positions of cleavage sites of the viral NS2 (white triangles) and NS3 protease (grey triangles) were predicted by alignment and homology to sites in the nonstructural genes previously characterized in other pegiviruses and hepaciviruses. Cleavage sites in structural gene regions (black triangles) were independently predicted using the SignalP 4.1 server. Predicted N-linked glycosylation sites in envelope proteins are depicted by vertical arrows; the single predicted O-linked glycosylation site is indicated with a blue arrow. The amino acid sequence of the predicted Y protein is shown above the genome diagram. Charged residues and cysteines are in color.

FIG 3

Maximum-likelihood phylogenetic analysis of the NS3 and NS5B genes of HHpgV-1 (tt790) and available complete genome sequences of other hepaciviruses and pegiviruses infecting different mammalian species. Phylogenetic analysis of each data set used 100 bootstrap resamplings to determine robustness of grouping; values are shown on branches. Abbreviations: HcV, hepacivirus; PgV, pegivirus; OWM, Old World monkey; NWM, New World monkey; R, rodent; B, bat; Bo, bovine; NPHV, nonprimate hepacivirus. Lineage numbers are shown in parentheses.

FIG 4

Amino acid sequence divergence between HHpgV-1 and other pegiviruses using a 240-nucleotide fragments with 24-nucleotide increments across the virus alignment (midpoint plotted on the x axis). Within-species distances for human and simian pegiviruses were included for comparison. The divergence scan was numbered using the AF121950 HPgV reference sequence. OWM, Old World monkey; NWP, New World primate; NWM, New World monkey.

FIG 5

Predicted RNA secondary structure of the 5′ UTR, based on structural mapping of the HHpgV-1 sequence to hepacivirus sequences based on an initial seed match in domain IIIe. Stem-loops for domains I and II were predicted by Mfold.

FIG 6

ML phylogenetic analysis of partial NS3 sequences of HHpgV-1 variants found in different human serum samples using a rodent pegivirus sequence as the outgroup. Phylogenetic analysis used 100 bootstrap resamplings to determine robustness of grouping. HHpgV-1 variants found in serum samples from the same patients are labeled on the right (tt, TTVS; m, MHCS).