New tools to study RNA interference to fish viruses: Fish cell lines permanently expressing siRNAs targeting the viral polymerase of viral hemorrhagic septicemia virus.

摘要

Previous studies have indicated that low transfection efficiency can be a major problem when gene inhibition by the use of small interfering RNAs (siRNAs) is attempted in fish cells. This may especially be true when targeting genes of viruses which are fast replicating and which can still infect cells that have not been transfected with the antiviral siRNAs. To increase the amount of antiviral siRNAs per cell a different strategy than transfection was taken here. Thus, we describe carp epithelioma papulosum cyprinid (EPC) cell clones expressing siRNAs designed to target the L polymerase gene of the viral hemorrhagic septicemia virus (VHSV), a rhabdovirus affecting fish. Eight siRNA sequences were first designed, synthesized and screened for inhibition of in vitro VHSV infectivity. Small hairpin (sh) DNAs corresponding to three selected siRNAs were then cloned into pRNA-CMV3.1/puro plasmids, transfected into EPC cells and transformed clones were obtained by puromycin selection. Sequence-specific interference with VHSV could only be observed with EPC clones transformed with a mixture of the three shDNAs, rather than with those clones obtained with individual sh DNAs. However, interference was not specific for VHSV as infection with an heterologous fish rhabdovirus, was also reduced to a similar extent. It was shown that this reduction was not due to an Mx response in the transformed cell clones. Here, we discuss some of the possible reasons for such data and future work directions. EPC clones stably expressing rhabdoviral specific siRNA sequences could be a strategy to further investigate the use of RNA interference for targeting costly fish pathogenic viruses.