Inhibition of rabies virus propagation in mouse neuroblastoma cells by an intrabody against the viral phosphoprotein

Rabies virus (RABV) is highly neurotropic and causes acute infection of the central nervous system. Death can be averted by prompt post-exposure prophylaxis; however, after clinical symptoms appear, the mortality rate is almost 100% and no reliable treatment is available. In this study, we investigated whether intracellular immunization using single-chain variable fragments (scFvs) against RABV phosphoprotein (RABV-P) could inhibit RABV propagation in neuronal cells. Of four scFv clones derived from an scFv phage-displayed library, scFv-P19 showed extremely high transfection efficiency and stable expression in mouse neuroblastoma (MNA) cells. The intracellular affinity and inhibition of RABV propagation were investigated using RABV-infected MNA cells pretransfected with the scFv-P19 gene. The specific interaction between scFv and RABV-P was confirmed by an immunoprecipitation assay and an indirect immunofluorescence assay showing that these molecules colocalized in the cytoplasm. Measurements of the spread of RABV in a culture well and the virus titer in the supernatant showed that RABV inhibition peaked 3 days after infection, at 98.6% and 99.9% inhibition, respectively. Although the mechanism of RABV inhibition by scFv-P19 is not clear, this scFv-based intracellular immunization could be a candidate for future RABV therapeutic studies if combined with appropriate delivery and application systems.