Seroreactive Hepatitis E Virus (HEV) protein is suitable for a diagnostic immunofluorescence test protocol

OSTERMAN, A.; VIZOSO PINTO, M. G.; HAASE, R.; NITSCHKO, H.; BAIKER, A.

Zeilitzheim, Alemania

Workshop; Clinical Virology 3rd WORKSHOP. German Society of Virology (GfV); 2009

Sociedad alemana de Virología

The Hepatitis E Virus was named after its enteric route of transmission and propensity to causeepidemics in regions of poor sanitation and hygiene in the developing world. Over the last years increasingnumbers of autochthonous cases, zoonotic spread and chronic infections has led to a different understandingand new interests in this pathogen (Aggarwal et al., J. Gastroenterol. Hepatol., 2009). The 7.2 kb HEV genomeis polyadenylated and flanked by two NTRs. Three sub-genomic mRNAs encode for three open reading frames(ORFs): ORF1 is translated to a functional polyprotein, ORF2 to the structural protein and ORF3 to a smallphosphoprotein with uncertain function. The ORF1 polyprotein is posttranslationally processed into differentfunctional proteins with methyltransferase, protease, helicase and polymerase domains.Westernblot, Line Immunoassay,ELISA or EIA are currently widely used test formats to serologically detect HEV infections. However suchassays are mainly based on bacterially expressed fragments of ORF2 and ORF3 of the virus and are known toshow low clinical performance. Therefore the aim of our project is the development of new tools for serologicaldiagnosis of HEV with the following advantages: (i) the tools are based on the recombinant expression of all three(poly-) proteins; (ii) a mammalian expression system offers the possibility of correct posttranslational modificationsand retention of potential epitopes. From a synthesized full length HEV genotype 1 cDNA-bank we constructed apDONR207 based complete HEV gene library. 17 respective HEV ORFs and ORF fragments were subcloned in aHis-tagged mammalian expression vector using the Gateway® technology (Invitrogen, Karlsruhe, Germany). Aftertransfection to HeLa, Huh7 and 293TT cells we could visualize transient expression of all 17 recombinant proteinsby fluorescence microscopy. The localizations of the proteins in different subcellular compartments were identicalin all tested cell lines. Two HEV RNA positive patient sera showed reactivity of IgG to two HEV proteins expressedin 293TT cells. One of the reactive proteins was used to establish a new HEV IFT protocol suitable for standarddiagnostic procedures.