In vitro search for alternative promoters to the human immediate early cytomegalovirus (IE-CMV) to express the G gene of viral haemorrhagic septicemia virus (VHSV) in fish epithelial cells

Abstract

Present DNA vaccines against fish rhabdoviruses require intramuscular injection (fish-to-fish vaccination) of their G-protein gene under the control of the human immediate early cytomegalovirus (IE-CMV) promoter, while immersion delivery (mass DNA vaccination), for instance, by using fish epithelial-specific promoters, would be more practical for aquaculture. To find fish epithelial-specific promoters alternative to the IE-CMV, a comparative study of the effectiveness of different fish promoters constitutively expressing the G gene of the viral haemorrhagic septicemia virus (VHSV) in the epithelial papulosum cyprini (EPC) cell line was performed. The study included MCV1.4 (an alternative IE-CMV promoter version), AE6 (a version of the carp β-actin promoter), long terminal repeats (LTR) of zebrafish or walleye retroviruses, trout Mx1, carp myosin-heavy-chain and flatfish pleurocidin promoters and salmonid sleeping beauty (SB)/medaka Tol2 transposon repeats. The G-protein expression in transfected EPC cells was studied by estimating the number of cells expressing the G-protein in their membrane and the average expression level per cell. In addition, in an attempt to reduce their sizes, some regions of the MCV1.4 and AE6 promoters were deleted and expression levels compared to those observed for full-length promoters. Since both zebrafish LTR and carp AE6 promoters were the most effective regulatory sequences for expressing the VHSV G-protein in EPC cells, these sequences might be candidates for new DNA vaccine vectors for fish epithelial tissues avoiding the IE-CMV promoter. Furthermore, known transcription factor binding sites (TFBS) common to most of the fish G-expressing promoters, might enable the future design of fully synthetic or hybrid promoters with improved efficacy of VHSV G-protein expression in epithelial fish cells.