title: Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers.
abstract: Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.
authors.affiliation: Department of Microbiology and Center for Fish Disease Research, Oregon State University, Corvallis, OR, USA. hearnma@science.oregonstate.edu
authors.forename: M
authors.initials: M
authors.lastname: Alonso
authors.forename: D A
authors.initials: DA
authors.lastname: Stein
authors.forename: E
authors.initials: E
authors.lastname: Thomann
authors.forename: H M
authors.initials: HM
authors.lastname: Moulton
authors.forename: J C
authors.initials: JC
authors.lastname: Leong
authors.forename: P
authors.initials: P
authors.lastname: Iversen
authors.forename: D V
authors.initials: DV
authors.lastname: Mourich
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D020029:Base Pairing; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D029142:Infectious hematopoietic necrosis virus / Q000187:drug effects* / Q000235:genetics*; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D060172:Morpholinos; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D009025:Morpholines; D060172:Morpholinos; D010455:Peptides; D012367:RNA, Viral