Suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers.
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a contagious disease characterized by reproductive failure in sows and respiratory disease in piglets. This infectious disease results in significant losses in the swine industry and specific anti-PRRSV drugs are needed. In this study, we evaluated a novel class of antisense compounds, peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs), for their ability to suppress PRRSV replication in cell culture. P-PMOs are analogs of single-stranded DNA and contain a modified backbone that confers highly specific binding to RNA and resistance to nucleases. Of six P-PMOs tested, one ('5UP1'), with sequence complementary to the 5'-terminal 21 nucleotides of the PRRSV genome, was found to be highly effective at reducing PRRSV replication in a specific and dose-dependent manner in CRL11171 cells in culture. 5UP1 treatment generated up to a 4.5log reduction in infectious PRRSV yield, while a control P-PMO had no effect on viral titer. Immunofluorescence assay with an anti-PRRSV monoclonal antibody confirmed the titer observations. The sequence-specificity of 5UP1 effect was confirmed in part by a cell-free luciferase reporter assay system, which showed that 5UP1-mediated inhibition of translation decreased if the target-RNA contained mispairings in relation to the 5UP1 P-PMO. Real-time RT-PCR showed that the production of PRRSV negative-sense RNA was reduced if 5UP1 was added to cells at up to 6h post-virus inoculation. Cell viability assays detected no cytotoxicity of 5UP1 within the concentration-range of this study. These results indicate that P-PMO 5UP1 has potential as an anti-PRRSV agent.
Zhang
Yan-Jin
YJ
Center for Pediatric Research, Eastern Virginia Medical School, Norfolk, VA 23510, USA. zhangyj@umd.edu
Stein
David A
DA
Fan
Su-Min
SM
Wang
Kai-Yu
KY
Kroeker
Andrew D
AD
Meng
Xiang-Jin
XJ
Iversen
Patrick L
PL
Matson
David O
DO
D020121:5' Untranslated Regions; D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary; D009025:Morpholines / Q000494:pharmacology*; D060172:Morpholinos; D019318:Porcine Reproductive and Respiratory Syndrome / Q000517:prevention & control*; D019316:Porcine respiratory and reproductive syndrome virus / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D013552:Swine; D014779:Virus Replication / Q000187:drug effects
D020121:5' Untranslated Regions; D009025:Morpholines; D060172:Morpholinos; D012367:RNA, Viral