Timing of rapid increase in viral titers coincides with differentiation of cells. Similar trends were observed when JFH-FBS was used instead of JFH-HS; however, initial viral titers were lower (not shown). After 21 days, viral titers in HS-cultured cells reached a plateau. CP-690550 order We were able to achieve continuous production of viral titers of ∼108 RNA copies/mL for at least 105 days using HCV JFH-1 (Fig. 6F). We were able to infect cells before differentiation, as well as cells that were fully differentiated. Eventually,

similar titers were reached using either method (Fig. 6F). We have also tried to infect differentiated cells with 35 different patient sera (genotypes 1-6), but infection was not detected in any of those cultures (using RNA titering). Previously, only extensive adaptation of JFH-1 resulted in production of high viral titers, and this typically resulted in induction of cell death.[14] To examine whether we had also produced tissue culture adaptations, we have sequenced a JFH-HS viral variant after 24 days of culture

and only could confirm a single mutation in NS2, at nucleotide position 2925 (A to G), resulting in a Q to R change. Additionally, we detected three mixed positions in NS5a, but none of these mutations resulted in an amino acid change. Last, we investigated LY2109761 molecular weight whether the biophysical properties of the virus produced by cells in HS media were different from virus produced by cells in FBS media. We investigated viral stability, viral density, ApoB association, and specific infectivity (Fig. 7). We wanted to determine whether a change in viral half-life of JFH-HS, compared to JFH-FBS, could be a contributing factor to the increased viral titers. At 4°C, both viral variants were stable. However, at 37°C, the half-life of JFH-FBS was 10-14 hours, whereas the half-life of the JFH-HS variant was approximately 75 hours (Fig. 7A). We found that virus produced by cells in HS media shifts toward a lower density on a sucrose gradient. Virus produced in FBS media had a median density of JFH of 1.16 g/mL, consistent with previous reports.[15] However, virus produced in HS media had a median density of 1.09 g/mL. In addition, a peak with a very low

density appeared (Fig. find more 7B). Overall, 35% of the virus produced in FBS media had a density lower than 1.16 g/mL, whereas 75% of the virus produced in HS media had a density lower than 1.16g/mL (Fig. 7C). The low density of virus produced by cells in HS media is more consistent with the density of virus derived from patients and chimeric mice.[16] HCV in patients has been consistently shown to be associated with ApoB[4, 17]; however, previous reports have shown that virus produced in culture is not associated with ApoB, but instead with apolipoprotein E.[18] We determined whether HCV produced in HS media was associated with ApoB (Fig. 7D). Consistent with previous reports, approximately 5% of the JFH-FBS virus variant was associated with ApoB.