Characterization of long-term cultures of hepatitis C virus
Journal of Virology
The human T-and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted. Hepatitis C virus (HCV) is a major cause of
... r cause of posttransfusion hepatitis, which often leads to liver cirrhosis and cancer. HCV is a worldwide health problem. Development of vaccines and other antiviral therapies is awaited. To develop these, cell culture systems for propagating HCV are needed, but the levels of virus replication reported up to now have been too low (3, 5, 9, 10, 14, 15, 18) . This paper deals with the serial transmission of HCV in human lymphatic cells to obtain cell culture-adapted HCV. The amount of HCV contained in the inoculum used, H77 (1), was determined by reverse transcription (RT)-PCR to be 10 7 genomes per ml and was determined by chimpanzee inoculation (6) to be 10 6.5 50% infectious doses per ml. The HCV virions were determined to be free of antibodies by immunoprecipitation with anti-human immunoglobulin (7). Two independent transmission series (experiments 1 and 2) were carried out for both HPBMa10-2 (20) and Daudi cells (18). Two milliliters of the cell suspension (5 ϫ 10 5 cells per ml) was mixed with 20 l of undiluted H77. After incubation for 2 h at 37ЊC, the cells were washed twice and resuspended in the medium. An aliquot was harvested for RT-PCR analysis immediately after adsorption. The rest of the cells were subcultured every 3 to 4 days for 2 weeks, and then transmission of the virus to uninfected cells was started. For serial transmission in HPBMa cells, we performed alternate cocultivations with drugresistant HPBMa cells (17) . As Daudi cells became unhealthy after HCV infection, probably because of interferon activity induced by the infection (18) , HCV-positive cultures were maintained by adding fresh Daudi cells after removing the old medium (which contained interferons) at each passage. For the detection of HCV in the cultures, a 100-l portion of the culture medium or a pellet of approximately 10 5 cells was used. Detailed procedures for RNA extraction, the RT-PCR of various regions of HCV, and sequencing were previously described (14). The designs of the primers for the nested PCR were based on the published sequence of H77 (8) and used to amplify the 5Ј noncoding region (nucleotide [nt] 63 to 301), hypervariable region (HVR; nt 1429 to 1618), E2-NS1 region (nt 1466 to 1994), and NS5 region (nt 8755 to 8929). In inoculum H77, the HVR showed the highest degree of heterogeneity (8, 11), and 13 different HVR sequences were found in our assay, with H1-1 being the major HVR sequence present ( Fig. 1 and 2 , column Inoc.). Four of these 13 sequences were recovered from the sera of chimpanzees infected for 9 to 10 days (16) with the same inoculum, suggesting that these four variants were infectious in vivo ( Fig. 1 and 2 , column Ch.). Sequences of HCV maintained in the HPBMa10-2 and Daudi cell cultures were monitored from day 0 to 298 postinoculation. In experiment 2 with HPBMa10-2 cells, seven different HVR sequences were recovered immediately after adsorption, but on day 67 only three sequences (H1-1, H1-2, and H1-3) were recovered, and on day 221 only H1-2 remained. A similar phenomenon was observed in experiment 1; again only H1-2 persisted in the culture (Fig. 1) . In the two experiments with Daudi cells, only H1-1 was detected after adsorption, but beyond day 21 only H1-2 remained (Fig. 2) . We examined a wider region of E2-NS1 (amino acid [aa] position 384 to 542) for the Daudi cell culture (Fig. 3) . The 12 clones examined in the inoculum were all different from one another, but 60 to 182 days after infection, E13, E14, E15, and E16, which were present in undetectably minor quantities in the inoculum, persisted in the culture. These clones had a histidine at aa 480 instead of leucine or proline as in the other, aborted, clones. The nucleotide sequence in the HVRs of E13, E15, and E16 was the same as that of H1-2 ( Fig. 1 and 2) . Figure 4 shows a similar analysis of the NS5 region. Four different sequences were detected in the inoculum, with the sequence in the majority being NS5-1. On day 196 in HPBMa10-2 or day 192 in Daudi cells, only NS5-7, which was present in undetectably minor quantities in the inoculum, remained. Figure 5 shows data obtained for the 5Ј noncoding region. Five different sequences from the inoculum were amplified, but on day 193 in HPBMa10-2 cells, only NC-7 persisted, and on day 308 in Daudi cells, NC-7, NC-8, and NC-9 persisted. Direct sequencing showed that the sequence with an A residue at nt 107, characteristic of these three clones, became the most common at 16 days after infection (data not shown).