title: Inhibition of rabies virus propagation in mouse neuroblastoma cells by an intrabody against the viral phosphoprotein.
abstract: 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.
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Toyama, Shinjuku, Tokyo, Japan. ykaku@nih.go.jp
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kaku
authors.forename: Akira
authors.initials: A
authors.lastname: Noguchi
authors.forename: Kozue
authors.initials: K
authors.lastname: Hotta
authors.forename: Akio
authors.initials: A
authors.lastname: Yamada
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inoue
mesh_terms: D000818:Animals; D000918:Antibody Specificity / Q000235:genetics; D045744:Cell Line, Tumor; D003593:Cytoplasm; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D047468:Immunoprecipitation; D042541:Intracellular Space / Q000276:immunology; D051379:Mice; D009447:Neuroblastoma / Q000821:virology*; D010750:Phosphoproteins / Q000276:immunology*; D056990:Post-Exposure Prophylaxis; D011820:Rabies virus / Q000187:drug effects* / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D014764:Viral Proteins / Q000276:immunology*; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D010750:Phosphoproteins; D057127:Single-Chain Antibodies; D014764:Viral Proteins