IN-VITRO PROPAGATION AND PRODUCTION OF HEPATITIS-E VIRUS FROM IN VIVO-INFECTED PRIMARY MACAQUE HEPATOCYTES

Hepatitis E virus (HEV) is responsible for sporadic cases as well as l arge epidemics of acute viral hepatitis in many developing countries. The nucleotide sequence of HEV appears to be unique among known viruse s and thus may represent a prototype human pathogen in a novel class o f single-stranded, positive-sense RNA viruses. To facilitate further s tudies of the biology of HEV, a tissue culture system using a serum-fr ee medium formulation has been developed to propagate the virus in vit ro. Hepatocytes were isolated from livers of cynomolgus macaques exper imentally infected with a HEV (Burma strain) inoculum and maintained i n long-term cultures. Using a highly strand-specific RT-PCR assay, bot h the positive-sense and the negative replicative strands of HEV RNA w ere detected in these hepatocytes throughout the course of the experim ents. Positive-strand genomic RNA was also detected in the culture med ium, suggesting the production and secretion of HEV virus particles. T he virus particles were successfully concentrated 200-fold from the me dium using ultrafiltration, and they could be observed by immunoelectr on microscopy using anti-HEV-positive immune serum. These results demo nstrate the capacity of this hepatocyte culture system to replicate HE V in vitro, thus providing an experimental means to study the replicat ive process of the virus. (C) 1996 Academic Press, Inc.