Abstract

We isolated subclones of Huh7 cells and demonstrated that they differed greatly in their ability to be transfected with hepatitis E virus (HEV) genomes or to be infected with HEV virions. We used one of the subclones to confirm that monoclonal antibodies presumed to be neutralizing antibodies based on indirect evidence did indeed neutralize HEV. We found that transfection of this subclone resulted in production of virions that could be extracted from the cells and used to infect new cells. Therefore, this system was utilized to study recombinant viral mutants. In particular, we demonstrated that ORF3 protein is not a structural component of virions: ORF3 protein was not required for infection, replication, or assembly of HEV in cultured Huh7 cells and virions produced in the absence of ORF3 protein could not be distinquished from those made in its presence. We demonstrated that the HEV replication strategy involves synthesis of a single subgenomic RNA which is a bicistronic messenger RNA that encodes both the ORF3 protein and the capsid protein. We identified heat shock cognate protein 70 as a participant in the process by which HEV enters into Huh7 cells.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000596-16
Application #
7301884
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2006
Total Cost
Indirect Cost

  Institution

Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code

  Publications

Graff, Judith; Zhou, Yi-Hua; Torian, Udana et al. (2008) Mutations within potential glycosylation sites in the capsid protein of hepatitis E virus prevent the formation of infectious virus particles. J Virol 82:1185-94
Purcell, R H; Emerson, S U (2008) Hepatitis E: an emerging awareness of an old disease. J Hepatol 48:494-503
Yu, Claro; Zimmerman, Carl; Stone, Roger et al. (2007) Using improved technology for filter paper-based blood collection to survey wild Sika deer for antibodies to hepatitis E virus. J Virol Methods 142:143-50
Zhou, Yi-Hua; Chen, Zhaochun; Purcell, Robert H et al. (2007) Positive reactions on Western blots do not necessarily indicate the epitopes on antigens are continuous. Immunol Cell Biol 85:73-8
Stoszek, Sonia K; Engle, Ronald E; Abdel-Hamid, Mohamed et al. (2006) Hepatitis E antibody seroconversion without disease in highly endemic rural Egyptian communities. Trans R Soc Trop Med Hyg 100:89-94
Chen, Zhaochun; Earl, Patricia; Americo, Jeffrey et al. (2006) Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus. Proc Natl Acad Sci U S A 103:1882-7
Chen, Zhaochun; Moayeri, Mahtab; Zhou, Yi-Hua et al. (2006) Efficient neutralization of anthrax toxin by chimpanzee monoclonal antibodies against protective antigen. J Infect Dis 193:625-33
Graff, Judith; Torian, Udana; Nguyen, Hanh et al. (2006) A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. J Virol 80:5919-26
Meky, Fatma A; Stoszek, Sonia K; Abdel-Hamid, Mohamed et al. (2006) Active surveillance for acute viral hepatitis in rural villages in the Nile Delta. Clin Infect Dis 42:628-33
Emerson, Suzanne U; Clemente-Casares, Pilar; Moiduddin, Nasser et al. (2006) Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay. J Gen Virol 87:697-704

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