In VitroInfection and Replication of Hepatitis E Virus in Primary Cynomolgus Macaque Hepatocytes

Tam, Albert W.; White, Robert G.; Yarbough, Patrice O.; Murphy, Brendan; McAtee, C. Patrick; Lanford, Robert E.; Fuerst, Thomas R.

doi:10.1006/viro.1997.8817

Cited by 61 publications

(34 citation statements)

References 17 publications

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“…Furthermore, the high-titered anti-ORF3 antibody used in the immunofluorescence assays did not neutralize wild-type virions (data not shown) in agreement with a previous report that anti-ORF3 failed to neutralize HEV in a cynomolgus primary hepatocyte culture system (18). These data strongly suggest that the protein encoded by ORF3 is either not present in or is not an essential component of virions.…”

Section: Figsupporting

confidence: 90%

ORF3 Protein of Hepatitis E Virus Is Not Required for Replication, Virion Assembly, or Infection of Hepatoma Cells In Vitro

Emerson

Nguyen

Torian

et al. 2006

J Virol

120

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A subclone of Huh-7 cells that could be relatively efficiently transfected and infected with hepatitis E virus was identified. Following transfection, infectious virus was produced but remained predominantly cell associated. Intracellular virus, recovered by lysis of transfected cells, infected naïve cells. This in vitro-produced virus appeared to be antigenically identical to virus isolated from clinical samples. Lysates from cells transfected with mutant viral genomes unable to synthesize ORF3 protein contained infectious virions that were similar in number, thermostability, and sedimentation characteristics to those in lysates transfected with wild-type viral genomes. Therefore, in contrast to its requirement in vivo, ORF3 protein is not required for infection of Huh-7 cells or production of infectious virus in vitro.Hepatitis E was first recognized as a unique disease in 1980 (reviewed in reference 15). The first complete hepatitis E virus (HEV) sequence, obtained in 1991, demonstrated that the virion contained a 7.2-kb positive-strand RNA genome composed of three open reading frames (ORFs), each in a different frame. ORF1 encodes enzymes needed for genomic replication, including an RNA-dependent RNA polymerase and the methyl-and guanylyltransferases (13). Recently, it was demonstrated that ORF2 and ORF3 proteins are encoded by a bicistronic subgenomic RNA that is also capped (9). ORF2 encodes the capsid protein, and ORF3 encodes a protein of unknown function that contains only 114 amino acids (9) rather than the 123 reported previously. The virus does not contain a lipid envelope and is the sole member of the newly created genus Hepevirus (2).An efficient cell culture system for HEV is not available, and nonhuman primates are the smallest animal models; as a result, much of the molecular biology of this virus remains to be described. Hepatitis E infection is generally acquired by ingestion of water contaminated with sewage (19). The primary site of infection is not known, but virus replicates in the liver and causes acute hepatitis which does not progress to chronicity. Viremia can occur, and infectious virus is shed in feces.In the absence of a productive cell culture system, attempts to define the molecular biology of HEV have depended on two different approaches. In the first approach, individual viral proteins were expressed at high levels from plasmid vectors. Data from this approach suggested that a subpopulation of the ORF2 protein is glycosylated (19,22) and that ORF2 protein and ORF3 protein interact with each other (20); ORF3 protein was phosphorylated under these conditions (23). On the basis of other expression studies, ORF3 protein was also proposed to be a regulatory protein that interacts with cellular proteins in the mitogen-activated protein kinase signaling pathway (11). Finally, ORF3 protein was proposed to expedite the export of ␣ 1 -microglobulin from the liver (21). Because these systems utilized nonphysiological concentrations and combinations of viral proteins, their biological relev...

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Section: Figsupporting

confidence: 90%

ORF3 Protein of Hepatitis E Virus Is Not Required for Replication, Virion Assembly, or Infection of Hepatoma Cells In Vitro

Emerson

Nguyen

Torian

et al. 2006

J Virol

120

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“…Since HEV is presumably transmitted by the fecal-oral route, it is unclear how the virus reaches the liver, and an extrahepatic site(s) of replication would be a possible explanation (2,37). Primary hepatocytes are the only known sites of HEV replication (43,44). In a preliminary study with naturally infected pigs, we found that positive-strand HEV RNA was detectable in a number of tissues, even after viremia was cleared (X. J. Meng et al, unpublished data).…”

Section: Discussionmentioning

confidence: 99%

Evidence of Extrahepatic Sites of Replication of the Hepatitis E Virus in a Swine Model

et al. 2001

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Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. Our recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication and pathogenesis in a swine model. In an early study, we experimentally infected specificpathogen-free pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were inoculated intravenously with swine HEV, 19 pigs (group 2) were inoculated with the US-2 strain of human HEV, and 17 pigs (group 3) were used as uninoculated controls. The clinical and pathological findings have been previously reported. In this expanded study, we aim to identify the potential extrahepatic sites of HEV replication using the swine model. Two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days postinoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive-strand HEV RNA in each tissue collected during necropsy at different DPI. A negative-strand-specific RT-PCR was standardized and used to detect the replicative, negative strand of HEV RNA from tissues that tested positive for the positive-strand RNA. As expected, positive-strand HEV RNA was detected in almost every type of tissue at some time point during the viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine HEV-and human HEVinoculated pigs. However, replicative, negative-strand HEV RNA was detected primarily in the small intestines, lymph nodes, colons, and livers. Our results indicate that HEV replicates in tissues other than the liver. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV.Hepatitis E virus (HEV), the causative agent of hepatitis E, has been recognized as a major cause of enterically transmitted non-A, non-B hepatitis in many developing countries (1,30,37,39). Transmission of the virus occurs primarily by the fecal-oral route through contaminated drinking water in areas with poor sanitation. The disease affects mainly young adults, with a reported mortality rate of up to 25% for pregnant women (10, 14, 37, 39). In the United States, sporadic cases of acute hepatitis E without known risk factors have been documented and anti-HEV antibodies have been detected in a significant proportion of healthy individuals (21,28,32,37,39,45). HEV is a positive, single-stranded RNA virus without an envelope. The genome, which is about 7.5 kb in size, contains three open reading frames (ORFs) and a short 5Ј and 3Ј nontranslated region (13,18,31,38). ORF 1 is...

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“…HEV cannot be cultured routinely, although it has recently been propagated in primary macaque hepatocytes (33,34) and a virus resembling HEV has been cultured in A549 cells (13). As a result, studies on HEV protein synthesis, processing, and assembly have been limited to heterologous gene expression systems.…”

Section: Hismentioning

confidence: 99%

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

Tyagi

Surjit

Lal

2005

J Virol

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Hepatitis E virus (HEV), a human plus-stranded RNA virus, contains three open reading frames (ORF).Of these, ORF1 encodes the viral nonstructural polyprotein, ORF2 encodes the major capsid protein, and ORF3 codes for a phosphoprotein of undefined function. Recently, using the yeast two-hybrid system to screen a human cDNA liver library, we have isolated and characterized AMBP (␣ 1 -microglobulin/bikunin precursor), which specifically interacts with the ORF3 protein of HEV. The ORF3 protein expedites the processing and secretion of ␣ 1 -microglobulin. When checked individually for interaction, the second processed protein from AMBP, bikunin, strongly interacted with the full-length ORF3 protein. This protein-protein interaction has been validated by immunoprecipitation in both COS-1 and Huh7 cells and by His 6 pull-down assays. In dual-labeling immunofluorescent staining, followed by fluorescence microscopy of transfected human liver cells, ORF3 colocalized with endogenously expressed bikunin. Finally, a 41-amino-acid C-terminal region of ORF3 has been found to be responsible for interacting with bikunin. The importance of this virus-host protein-protein interaction, with reference to the viral life cycle, has been discussed.Hepatitis E is an acute disease endemic in many countries throughout developing parts of the world, in particular on the continents of Africa and Asia, where it causes epidemics and sporadic infections. The causative agent, hepatitis E virus (HEV), is transmitted via the fecal-oral route, predominantly through contaminated water (7,10,25,26). HEV is a plusstranded RNA virus with a 7.2-kb genome containing three open reading frames (ORF), ORF1, ORF2, and ORF3, encoding three different proteins (20,32,35). ORF1 (5,079 bp) is at the 5Ј end of the genome and is predicted to code for the putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (1,20,27,35). In the absence of a reliable in vitro culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken. ORF2 and ORF3 have been expressed in Escherichia coli, animal cells, baculovirus, and yeast and in vitro in a coupled transcription-translation system (12,15,17,22,23). ORF2, the major capsid protein for HEV, is synthesized as a precursor and is processed through signal sequence cleavage into the mature protein, which is capable of selfassociation (18, 36). ORF3 encodes a small 13.5-kDa phosphoprotein that is expressed intracellularly, associates with the cytoskeleton, and shows no major processing (3, 42). The ORF3 protein dimerizes using a 43-amino-acid region, interacts with SH3 domains, and activates mitogen-activated protein kinase (21, 37). The phosphorylated form of the ORF3 protein has also been shown to interact with the nonglycosylated form of the major capsid protein, ORF2 (38). These properties of ORF3 clearly indicate that this protein may have multiple roles in HEV pathogenesis. Rece...

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In VitroInfection and Replication of Hepatitis E Virus in Primary Cynomolgus Macaque Hepatocytes

Cited by 61 publications

References 17 publications

ORF3 Protein of Hepatitis E Virus Is Not Required for Replication, Virion Assembly, or Infection of Hepatoma Cells In Vitro

ORF3 Protein of Hepatitis E Virus Is Not Required for Replication, Virion Assembly, or Infection of Hepatoma Cells In Vitro

Evidence of Extrahepatic Sites of Replication of the Hepatitis E Virus in a Swine Model

The 41-Amino-Acid C-Terminal Region of the Hepatitis E Virus ORF3 Protein Interacts with Bikunin, a Kunitz-Type Serine Protease Inhibitor

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