title: Transduction of hematopoietic stem cells to stimulate RNA interference against feline infectious peritonitis.
abstract: Objectives The goals of the study were: (1) to develop and evaluate non-replicating lentivirus vectors coding for feline coronavirus (FCoV)-specific micro (mi)RNA as a potential antiviral therapy for feline infectious peritonitis (FIP); (2) to assess the feasibility of transducing hematopoietic stem cells (HSCs) with ex vivo introduction of the miRNA-expressing lentivirus vector; and (3) to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. Methods HSCs were obtained from feline bone marrow and replicated in vitro. Three lentiviruses were constructed, each expressing a different anti-FCoV miRNA. HSCs were stably transduced with the miRNA-expressing lentivirus vector that produced the most effective viral inhibition in a feline cell line. The effectiveness of the transduction and the expression of anti-FCoV miRNA were tested by infecting the HSCs with two different strains of FCoV. The inhibition of coronavirus replication was determined by relative quantification of the inhibition of intracellular viral genomic RNA synthesis using real-time, reverse-transcription PCR. The assessment of virus replication inhibition was determined via titration of extracellular virus using the TCID50assay. Results Inhibition of FCoV was most significant in feline cells expressing miRNA-L2 that targeted the viral leader sequence, 48 h postinfection. miRNA-L2 expression in stably transduced HSCs resulted in 90% and 92% reductions in FIPV WSU 79-1146 genomic RNA synthesis and extracellular virus production, respectively, as well as 74% and 80% reduction in FECV WSU 79-1683 genomic RNA synthesis and extracellular virus production, respectively, as compared with an infected negative control sample producing non-targeting miRNA. Conclusions and relevance These preliminary results show that genetic modification of HSCs for constitutive production of anti-coronavirus miRNA will reduce FCoV replication.
authors.affiliation: 1 Infectious Diseases, Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia, Tifton, GA, USA.|2 Department of Virology, Faculty of Veterinary Medicine, University of Sadat, Sadat City, Egypt.
authors.forename: Eman A
authors.initials: EA
authors.lastname: Anis
authors.affiliation: 3 Large Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA.
authors.forename: Madhu
authors.initials: M
authors.lastname: Dhar
authors.affiliation: 4 Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA.
authors.forename: Alfred M
authors.initials: AM
authors.lastname: Legendre
authors.affiliation: 1 Infectious Diseases, Veterinary Diagnostic and Investigational Laboratory, College of Veterinary Medicine, University of Georgia, Tifton, GA, USA.
authors.forename: Rebecca P
authors.initials: RP
authors.lastname: Wilkes
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use*; D002415:Cats; D016765:Coronavirus, Feline / Q000235:genetics*; D016766:Feline Infectious Peritonitis / Q000188:drug therapy / Q000821:virology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology / Q000627:therapeutic use*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D014161:Transduction, Genetic / Q000662:veterinary; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D034741:RNA, Small Interfering