We show that a dicistronic hepatitis C virus (HCV) genome of genotype 1b supports the production and secretion of infectious HCV particles in two independent three-dimensional (3D) culture systems, the radial-flow bioreactor and the thermoreversible gelation polymer (TGP), but not in monolayer cultures. Immunoreactive enveloped particles, which are 50-60 nm in diameter and are surrounded by membrane-like structures, are observed in the culture medium as well as at the endoplasmic reticulum membranes and in dilated cytoplasmic cisternae in spheroids of Huh-7 cells. Infection of HCV particles is neutralized by anti-E2 antibody or patient sera that interfere with E2 binding to human cells. Finally, the utility of the 3D-TGP culture system for the evaluation of antiviral drugs is shown. We conclude that the replicon-based 3D culture system allows the production of infectious HCV particles. This system is a valuable tool in studies of HCV morphogenesis in a natural host cell environment.

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Mechanism of formation of a laminin-apatite composite layer on the surface of an ethylene-vinyl alcohol copolymer (EVOH) using a liquid phase coating process was investigated by transmission electron microscopy (TEM). In this coating process, an EVOH substrate is alternately dipped in calcium and phosphate solutions, and then immersed in a laminin-containing calcium phosphate (LCP) solution. From the results obtained by the present study, formation of the laminin-apatite composite layer on EVOH is likely to proceed via the following events. By the alternate dipping process, particulate amorphous calcium phosphate, which is a precursor of apatite, was deposited onto the EVOH surface. When the specimen was subsequently immersed in the LCP solution, the amorphous calcium phosphate on the specimen transformed itself into needle-like apatite crystal, and then grew into a layer. During this process, laminin molecules contained in the LCP solution were incorporated into a matrix of the apatite crystals to produce a laminin-apatite composite layer on the EVOH surface.

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