In VitroPropagation and Production of Hepatitis E Virus fromin Vivo-Infected Primary Macaque Hepatocytes

AbstractHepatitis E virus (HEV) is responsible for sporadic cases as well as large epidemics of acute viral hepatitis in many developing countries. The nucleotide sequence of HEV appears to be unique among known viruses and thus may represent a prototype human pathogen in a novel class of single-stranded, positive-sense RNA viruses. To facilitate further studies of the biology of HEV, a tissue culture system using a serum-free medium formulation has been developed to propagate the virusin vitro.Hepatocytes were isolated from livers of cynomolgus macaques experimentally infected with a HEV (Burma strain) inoculum and maintained in long-term cultures. Using a highly strand-specific RT–PCR assay, both the positive-sense and the negative replicative strands of HEV RNA were detected in these hepatocytes throughout the course of the experiments. Positive-strand genomic RNA was also detected in the culture medium, suggesting the production and secretion of HEV virus particles. The virus particles were successfully concentrated 200-fold from the medium using ultrafiltration, and they could be observed by immunoelectron microscopy using anti-HEV-positive immune serum. These results demonstrate the capacity of this hepatocyte culture system to replicate HEVin vitro,thus providing an experimental means to study the replicative process of the virus