Existence of distinct GB virus C/hepatitis G virus variants with different tropism

Abstract

To study the existence of GB virus C/hepatitis G virus (GBV-C/HGV) variants with different tropism, we have analyzed the heterogeneity and quasispecies composition of GBV-C/HGV isolated from in vitro-infected peripheral blood mononuclear cells (PBMC) and from sera, livers, and PBMC from two chronically infected patients. For this purpose, the GBV-C/HGV 5' noncoding region (5'NCR) was amplified by reverse transcription-PCR and the amplified products were cloned and sequenced. These analyses showed that the master 5'NCR sequences isolated from the in vitro-infected PBMC and from the PBMC isolated from the patient whose serum was used as the inoculum were identical but different from that of the inoculum. Furthermore, phylogenetic analysis revealed that all PBMC sequences grouped together into a branch which was separate from those of the inoculum. For one of the two chronically infected patients, all the sequences from the PBMC and one from the liver clustered into a single branch while the sequences from the serum and all the other liver sequences grouped together in the other branch. For the other patient, the sequences from the serum and PBMC and three sequences from the liver grouped together into one branch, while the remaining five sequences from the liver were separated in a different cluster. In conclusion, our results support the existence of different GBV-C/HGV variants with different tissue tropism.

FIG. 1

Nucleotide alignment of the master sequences recovered from the inoculum (I), the PBMC of the patient whose serum was used as the inoculum (IP), the PBMC from the four healthy donors infected in vitro with GBV-C/HGV (D1 through D4), and the pool of cells from the four donors infected in vitro with GBV-C/HGV (PP).

FIG. 2

Unrooted neighbor-joining tree constructed with the 5′NCR nucleotide sequences from the in vitro-infected PBMC. The isolates are designated I (inoculum), IP (PBMC of the patient whose serum was used as the inoculum), D1 through D4 (PBMC from the four healthy donors infected in vitro with GBV-C/HGV), or PP (pool of cells from the four donors infected in vitro), followed by the clone number and, in parentheses, the number of clones bearing the same sequence. Bootstrap values are shown in the nodes of the tree.

FIG. 3

Histograms of the frequency of each evolutionary distance in the GBV-C/HGV 5′NCR sequences from serum, liver, and PBMC specimens of the two patients with GBV-C/HGV infection.

FIG. 4

Unrooted neighbor-joining trees constructed with the 5′NCR nucleotide sequences of GBV-C/HGV isolated from serum, liver, and PBMC samples of patients 1 (a) and 2 (b). The clones are designated P1 or P2 (indicating those isolated from patient 1 or 2, respectively), plus S, L, or P (indicating the source as serum, liver, or PBMC, respectively), followed by the clone number and, in parentheses, the number of clones with identical sequences in each compartments. The bootstrap values are given in the nodes of the trees.

FIG. 4

Unrooted neighbor-joining trees constructed with the 5′NCR nucleotide sequences of GBV-C/HGV isolated from serum, liver, and PBMC samples of patients 1 (a) and 2 (b). The clones are designated P1 or P2 (indicating those isolated from patient 1 or 2, respectively), plus S, L, or P (indicating the source as serum, liver, or PBMC, respectively), followed by the clone number and, in parentheses, the number of clones with identical sequences in each compartments. The bootstrap values are given in the nodes of the trees.