Antibodies to the Novel Human Pegivirus 2 Are Associated with Active and Resolved Infections

Abstract

A novel blood-borne human pegivirus (HPgV), HPgV-2, was recently identified in hepatitis C virus (HCV)-infected individuals and individuals who had received multiple transfusions. Robust serological assays capable of detecting antibodies in HPgV-2-infected individuals are needed to establish global seroprevalence rates and potential disease associations. The two objectives of this study were to determine the utility of mammalian cell-expressed HPgV-2 E2 glycoprotein or bacterium-expressed nonstructural protein 4AB (NS4AB) in detecting past or present infections and to compare the total prevalence (antibody and RNA positive) of HPgV-2 with that of the other human pegivirus, HPgV-1 (GB virus C [GBV-C]). HPgV-2 E2 antibodies were detected in 13 (92.86%) of 14 HPgV-2-viremic cases, and NS4AB antibodies were detected in 8 (57.14%) of 14 cases. The HPgV-2 seroprevalence was significantly higher (P < 0.0001) among HCV-infected individuals (3.31% [24 of 726 samples]) than among non-HCV-infected individuals (0.30% [4 of 1,348 samples]). Of 31 anti-E2-positive samples, 22 had supplemental supporting data; 12 samples were HPgV-2 RNA positive and 10 nonviremic samples were antibody positive for peptides or NS4AB. The total prevalence of HPgV-1 (35.00%) was significantly higher than that of HPgV-2 (1.33%) in all populations tested (P < 0.0001). For HPgV-1, codetection of antibodies to E2 and RNA was infrequent (5.88%). In contrast, antibodies to E2 were detected in most HPgV-2-viremic individuals (92.86%), as is observed among individuals chronically infected with HCV, most of whom are antibody positive for HCV E2. Our studies indicate that HPgV-2 circulates with HCV and displays a profile similar to the serological profile of HCV-infected persons, although the pathogenicity of this virus has yet to be established.

FIG 1

Expression and purification of the NS4AB recombinant protein. (A) Design of the NS4AB recombinant protein. The NS4A sequence is italicized, and the sequence representing peptide 16 (P16) is underlined. The expected size of the recombinant protein is 54 kDa. AA, amino acids. (B) Western blot of fractions from the Ni+ column purification of the NS4AB recombinant antigen, detected using an anti-His antibody. Lane 1, induced cytoplasmic lysate; lane 2, flowthrough fraction; lane 3, wash 1; lane 4, wash 2; lane 5, eluted protein.

FIG 2

Design, purification, and characterization of HPgV-2 E2 and HPgV-1 E2. (A) Design of the E2 expression plasmids. The ectodomain of each E2 protein was expressed in a mammalian expression vector containing a leader sequence. For the 314-amino acid (AA) HPgV-2 E2, the expected size was 35.2 kDa; for the 315-amino acid HPgV-1 E2, the expected size was 33.6 kDa. Predicted N- and O-linked glycosylation sites are indicated. (B) Western blot of fractions from the Ni+ column purification of HPgV-2 E2 and HPgV-1 E2, using an anti-His antibody. Lanes 1 to 5, HPgV-2 E2; lane 1, input; lane 2, flowthrough fraction; lane 3, wash 1; lane 4, wash 2; lane 5, eluted protein; lanes 6 to 10, HPgV-1 E2; lane 6, input; lane 7, flowthrough fraction; lane 8, wash 1; lane 9, wash 2; lane 10, elute. (C) Western blot of samples, using an anti-His antibody, after PNGase F deglycosylation of purified proteins. One or 10 μg of purified HPgV-2 E2 or HPgV-1 E2 was treated with PNGase F. Lanes 1 to 3, HPgV-2 E2; lane 1, untreated protein; lane 2, 1 μg treated with PNGase F; lane 3, 10 μg treated with PNGase F; lanes 4 to 6, HPgV-1 E2; lane 4, untreated protein; lane 5, 1 μg treated with PNGase F; lane 6, 10 μg treated with PNGase F. (D) Immunofluorescence of COS-7 cells transfected with HPgV-2 E2 or HPgV-1 E2 expression constructs. (Top) HPgV-2 E2. (Middle) HPgV-1 E2. (Bottom) no-DNA control. Magnification for all panels, ×20.

FIG 3

Distribution of S/CO values for anti-HPgV-2 E2 reactivity. Numbers of samples are plotted on the y axes, and the corresponding S/CO values are plotted on the x axes. Dashed vertical lines, cutoff values of the population-based median plus 7 SDs; values greater than 1 were considered positive. *, positive samples (S/CO of >1); accompanying numbers indicate the numbers of samples. The x axis for the HCV data set was modified to show samples with greater S/CO values.