Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers.

TitleInhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers.
Publication TypeJournal Article
Year of Publication2005
AuthorsAlonso M, Stein DA, Thomann E, Moulton HM, Leong JC, Iversen P, Mourich DV
JournalJournal of fish diseases
Volume28
Issue7
Pagination399-410
Date Published2005 Jul
ISSN0140-7775
KeywordsAnimals, Antiviral Agents, Base Pairing, Base Sequence, Blotting, Western, Cell Line, Dose-Response Relationship, Drug, Flow Cytometry, Infectious hematopoietic necrosis virus, Microscopy, Fluorescence, Morpholines, Morpholinos, Oncorhynchus mykiss, Peptides, RNA, Viral
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.

DOI10.1111/j.1365-2761.2005.00641.x
Alternate JournalJ. Fish Dis.