Dynamic behavior of hepatitis C virus quasispecies in a long-term culture of the three-dimensional radial-flow bioreactor system

Strain-dependent viral dynamics and virus-cell interactions in a novel in vitro system supporting the life cycle of blood-borne hepatitis C virus #

“…PCR products were then purified and cloned using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). Ten recombinant clones were randomly isolated for each PCR product and sequenced as described 16…”

Three-dimensional cell culture models for investigating human viruses

“…A similar result has been found in the RFB system following transfection of FLC4 cells with a dicistronic HCV genome derived from genotype 1b, as well as in the 3D/TGP system using Huh-7 cells (Murakami et al, 2006). Furthermore, research has demonstrated that a long-term culture of the 3D RFB system provides a potential platform for investigating HCV dynamics, as well as examining the therapeutic effects of interferon alpha in this 3D culture model (Murakami et al, 2008).…”

“…Nickerson et al, 2007 Several 3D models can monitor and control physiological conditions: temperature, pH, oxygen concentration, metabolites and growth factors. Murakami et al, 2008;Li and Cui., 2014;Worthington et al, 2015 Limitations References…”

“…Culture conditions can automatically be controlled. Temperature, pH and oxygen concentration in the conditioning vessel are continuously monitored by PC and conditioned by mass flow controller (Murakami et al, 2008). The RFB-derived 3D models have mainly been used for hepatitis C virus (HCV).…”

Cellular models for the screening and development of anti-hepatitis C virus agents

“…PH5CH and FLC4 cell lines, but not HuS-E, were shown to produce virions in the extracellular medium for a long period (4 to 8 weeks) after infection. Hyper variable region quasispecies analysis in FLC4 cells versus inoculum revealed a different pannel of sequences in the cellular viral RNA as compared with the inoculum RNA, suggesting adaptation of HCV to the conditions prevailing in these cells Murakami et al, 2008). When FLC4 cells were transfected with HCV H77 (1a) RNA and cultured in a bioreactor, HCV RNA was detected in the extracellular medium almost two months posttransfection and remained at a level of 10 5 -10 6 copies/day for another three months, consistent with the relative amount of core protein (13pg/day).…”