Tam A W, White R, Yarbough P O, Murphy B J, McAtee C P, Lanford R E, Fuerst T R
Department of Molecular Virology, Genelabs Technologies, Redwood City, California 94063, USA.
Virology. 1997 Nov 10;238(1):94-102. doi: 10.1006/viro.1997.8817.
An in vitro model was developed to replicate hepatitis E virus (HEV) in normal primary cynomolgus macaque hepatocytes using a hormonally defined, serum-free medium formulation. Primary hepatocytes were infected in tissue culture following isolation by collagenase treatment of liver wedge biopsy material. Viral replication was monitored by a highly strand-specific reverse transcription-polymerase chain reaction (RT-PCR) assay, which could detect the positive- and negative-strands of HEV RNA independently in a sensitive and specific manner. Several infectious 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 positive-strand viral RNA in the culture medium. Infectivity of the virus particles present in the media was confirmed by serial passage and replication of the virus in culture. Using this in vitro infection system, a neutralization assay was developed to assess the ability of anti-HEV antibodies to block virus infection of liver cells. Results presented in this report represent the first in vitro demonstration of a neutralizing anti-HEV antibody directed against the ORF2-encoded putative capsid protein.
利用一种激素定义的无血清培养基配方，开发了一种体外模型，用于在正常原代食蟹猴肝细胞中复制戊型肝炎病毒（HEV）。通过胶原酶处理肝楔形活检材料分离出原代肝细胞后，将其在组织培养中进行感染。通过一种高度链特异性逆转录聚合酶链反应（RT-PCR）检测法监测病毒复制，该检测法能够以灵敏且特异的方式独立检测HEV RNA的正链和负链。通过这种RT-PCR检测法对几种传染性HEV（缅甸株）接种物进行了滴定，并确定了最小有效感染剂量。通过在实验感染的肝细胞中检测HEV RNA的两条链以及培养基中的基因组正链病毒RNA，证实了新复制病毒的出现。通过病毒在培养中的连续传代和复制，证实了培养基中存在的病毒颗粒的感染性。利用这种体外感染系统，开发了一种中和试验，以评估抗HEV抗体阻断肝细胞病毒感染的能力。本报告中呈现的结果代表了针对ORF2编码的假定衣壳蛋白的中和抗HEV抗体的首次体外证明。
