In vitro infection of human peripheral blood mononuclear cells by GB virusC hepatitis G virus

GB virus C (GBV-C), also known as hepatitis G Virus, is a recently discover ed flavivirus-like RNA agent with unclear pathogenic implications. To inves tigate whether human peripheral blood mononuclear cells (PBMC) are suscepti ble to in vitro GBV-C infection, we have incubated PBMC from four healthy b lood donors with a human GBV-C RNA-positive serum. By means of (i) strand-s pecific reverse transcription-PCR, cloning, and sequencing; (ii) sucrose ul tracentrifugation and RNase sensitivity assays; (iii) fluorescent in situ h ybridization; and (iv) Western blot analysis. it has been demonstrated that GBV-C is able to infect in vitro cells and replicate for as long as 30 day s under the conditions developed in our cell culture system. The concentrat ion of GBV-C RNA increased during the second and third weeks of culture. Th e titers of the genomic strand were 10 times higher than the titers of the antigenomic strand. In addition, the same predominant GBV-C sequence was fo und in all PBMC cultures and in the in vivo-GBV-C-infected PBMC isolated fr om the donor of the inoculum, GBV-C-specific fluorescent in situ hybridizat ion signals were confined to the cytoplasm of cells at different times duri ng the culture period. Finally, evidence obtained by sucrose ultracentrifug ation, RNase sensitivity assays, and Western blot analysis of the culture s upernatants suggests that viral particles are released from in vitro-GBV-C- infected PBMC, In conclusion, our study has demonstrated, for the first tim e, GBV-C replication in human lymphoid cells under experimental in vitro in fection conditions.