IN-VITRO INFECTION AND REPLICATION OF HEPATITIS-E VIRUS IN PRIMARY CYNOMOLGUS MACAQUE HEPATOCYTES

An in vitro model was developed to replicate hepatitis E virus (HEV) i n normal primary cynomolgus macaque hepatocytes using a hormonally def ined, serum-free medium formulation. Primary hepatocytes were infected in tissue culture following isolation by collagenase treatment of liv er wedge biopsy material. Viral replication was monitored by a highly strand-specific reverse transcription-polymerase chain reaction (RT-PC R) assay, which could detect the positive-and negative-strands of HEV RNA independently in a sensitive and specific manner. Several infectio us HEV (Burma strain) inocula were titered by this RT-PCR assay, and a minimum effective infectious dose was determined. Appearance of newly replicated virus was demonstrated by detection of both strands of HEV RNA in experimentally infected hepatocytes as well as the genomic pos itive-strand viral RNA in the culture medium. Infectivity of the virus particles present in the media was confirmed by serial passage and re plication of the virus in culture. Using this in vitro infection syste m, a neutralization assay was developed to assess the ability of anti- HN antibodies to block virus infection of liver cells. Results present ed in this report represent the first in vitro demonstration of a neut ralizing anti-HEV antibody directed against the ORF2-encoded putative capsid protein. (C) 1997 Academic Press.