Mammalian expression of the Hepatitis E Virus (HEV) proteome for the establishment of diagnostic immunofluorescence tests (IFTs).

OSTERMAN, A.; VIZOSO PINTO, M. G.; HAAS, J.; PETER, J.; NITSCHKO, H.; BAIKER, A.

Leipzig, Alemania

Jornada; 19. Jahrestagung der Gesellschaft für Virologie; 2009

Sociedad alemana de Virología

HEV is a major cause of acute hepatitis in developing countries in Africa, Asia and the MiddleEast. Interestingly, an increasing number of sporadic cases of HEV infections in industrialized countries is reported, also acquired as zoonoses from domestic livestock. The National Institutes of Health classified Hepatitis E as anemerging disease and the Robert Koch-Institute registered a rising incidence in Germany by 94% over the last twoyears. Despite the increasing relevance of this pathogen in clinical virology, the performance of most commerciallyavailable tests is poor in terms of sensitivity as well as specificity. The aim of our project is the development ofa new diagnostic test system for the confirmation of HEV infections. The genome of HEV encodes three openreading frames (ORFs). Two of them - ORF2, the capsid protein, and ORF3, a protein of controversially discussedfunction - are used within commercial test systems. The largest ORF1 (poly-) protein, however, is not part of currentassay formats.In conclusion we constructed a Gateway® compatible library of all HEV ORFs. More than 90% of theHEV-proteome was recombinantly expressed in E.coli. To account for correct post-translational modifications andretention of potential epitopes we also established a transient mammalian expression system for the respective 18HEV ORFs and ORF fragments utilizing HeLa cells and a Gateway® compatible, N-terminally His-tagged, pCR3based expression vector. We could demonstrate mammalian expression of HEV proteins by an anti-His antibodyin different subcellular compartments. These results provide the basis for novel IFTs to analyse and confirm HEVinfections.