title: Heterologous gene expression by infectious and replicon vectors derived from tick-borne encephalitis virus and direct comparison of this flavivirus system with an alphavirus replicon.
abstract: The flavivirus tick-borne encephaltis virus (TBEV) was established as a vector system for heterologous gene expression. The variable region of the genomic 3' non-coding region was replaced by an expression cassette consisting of the reporter gene enhanced green fluorescent protein (EGFP) under the translational control of an internal ribosomal entry site element, both in the context of an infectious virus genome and of a replicon lacking the genes of the surface proteins prM/M and E. The expression level and the stability of expression were measured by fluorescence-activated cell-sorting analysis and compared to an established alphavirus replicon vector derived from Venezuelan equine encephaltis virus (VEEV), expressing EGFP under the control of its natural subgenomic promoter. On the first day, the alphavirus replicon exhibited an approximately 180-fold higher expression level than the flavivirus replicon, but this difference decreased to about 20- and 10-fold on days 2 and 3, respectively. Four to six days post-transfection, foreign gene expression by the VEEV replicon vanished almost completely, due to extensive cell killing. In contrast, in the case of the TBEV replicon, the percentage of positive cells and the amount of EGFP expression exhibited only a moderate decline over a time period of almost 4 weeks. The infectious TBEV vector expressed less EGFP than the TBEV replicon at all times. Significant expression from the infectious vector was maintained for four cell-culture passages. The results indicate that the VEEV vector is superior with respect to achieving high expression levels, but the TBEV system may be advantageous for applications that require a moderate, but more enduring, gene expression.
authors.affiliation: Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
authors.forename: Rainer
authors.initials: R
authors.lastname: Gehrke
authors.affiliation: Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
authors.forename: Franz X
authors.initials: FX
authors.lastname: Heinz
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Davis
authors.affiliation: Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
authors.forename: Christian W
authors.initials: CW
authors.lastname: Mandl
mesh_terms: D000524:Alphavirus / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004666:Encephalitis Virus, Venezuelan Equine / Q000235:genetics / Q000378:metabolism; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D005416:Flavivirus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D012093:Replicon
chemical_list: D049452:Green Fluorescent Proteins