Background: GB Virus C (GBV-C)-HIV co-infection is associated with prolonged survival in vivo, and with diminished HIV replication in vitro. GBV-C replication is inefficient in vitro, and most culture systems use GBV-C positive serum to infect PHA/IL-2 stimulated PBMCs. The site of GBV-C replication has not been clearly identified. We studied GBV-C replication sites in vitro and ex vivo, and determined if T-cell activation is required for GBV-C replication.

Methods: GBV-C RNA was detected by RT-PCR and quantified by real-time PCR. GBV-C infected PBMC cultures from GBV-C positive donors were cultured with and without IL-2 (5%) and PHA (5mcg/mL). GBV-C infected PBMC cultures were also compared with GBV-C infected cultures enriched for CD4, CD8, B cells or monocytes (prepared from the same donor). The purity of enriched cell populations was determined by FACS. Finally, PBMCs from HIV-GBV-C co-infected subjects were fixed, sorted, and the concentration of GBV-C RNA per 50,000 cells was measured in specific cellular subpopulations.

Results: GBV-C replication in PBMCs was reproducibly higher when cultured without the addition of IL-2 and PHA. GBV-C replication increased in GBV-C infected donor PBMCs following CD8 depletion; whereas no increase in replication occurred in CD8 depleted healthy donor PBMCs were infected with GBV-C, suggesting a GBV-C-specific CD8 response in the infected donor. GBV-C replication was identified in all cell populations studied, but was greatest in the enriched B-cell cultures which were > PBMCs > CD4 = CD8. Monocytes did not appear to produce significant GBV-C RNA. Enriched cell populations were 82% pure (B cells), 96% (CD4 and CD8 T-cells), and 71% (monocytes). GBV-C in cells from 9 HIV-GBV-C co-infected subjects found an equal amount of GBV-C RNA in CD4 and CD8 cells, with variable amounts in B-cells. GBV-C was usually absent in monocytes.

Conclusions: GBV-C is a pan-lymphotropic virus and is not CD4-restricted, similar to a related pestivirus, BVDV. Although most published studies utilize PHA/IL-2 stimulated PBMCs for culture, GBV-C replication was actually inhibited by PHA/IL-2. GBV-C can be cultured ex vivo in PBMCs as well as in enriched populations of B-cells, CD4 cells, and CD8 cells; and it appears that CD8 cells influence virus growth in PBMC’S. Improved cell culture systems should facilitate understanding of the mechanism of action of GBV-C induced HIV inhibition.