Cell culture evaluation of the Semliki Forest virus expression system as anovel approach for antigen delivery and expression in fish

Heterologous gene expression by Semliki Forest virus (SFV) expression vecto rs was investigated in fish cell culture. Experiments performed using an in fectious strain of SFV, replication-defective SFV particles, and recombinan t SFV RNA constructs encoding the Escherchia coli LacZ or firefly luciferas e reporter genes indicated that levels of SFV-mediated expression in fish c ells were dependent on cell type and temperature. Maximal expression levels were observed in the two salmonid-derived cell lines CHSE-214 and F95/9 at 25 degrees C and 20 degrees C. As the temperature was lowered to 15 degree s C or below, levels of reporter gene expression were reduced up to 1000-fo ld, indicating that the SFV replication complex functioned inefficiently at low temperatures. The ability of SFV expression systems to function in fis h cells was further investigated by analyzing the expression of the protect ive VP2 antigen of infectious pancreatic necrosis virus (IPNV) from the var ious constructs, including a novel DNA-based SFV plasmid. The VP2 protein p roduced in CHSE-214 and F95/9 cells transfected or infected with the recomb inant SFV-IPNV VP2 constructs appeared to be synthesized in an antigenicall y correct form, as evidenced by the ability to react with several conformat ion-dependent IPNV-specific monoclonal antibodies. Whether the temperature- restricted replication and expression displayed by SFV-based constructs in fish cell culture also occurs in vivo remains to be determined.