In vitro effects of the active metabolite of leflunomide, A77 1726, on feline herpesvirus-1.
OBJECTIVE
To determine whether the active metabolite of leflunomide, A77 1726 (A77), inhibits replication of feline herpesvirus-1 (FHV-1) in cell culture.


METHODS
Crandell Rees feline kidney (CRFK) cell cultures.


METHODS
Cell cultures were inoculated with FHV-1 and treated simultaneously with concentrations of A77 ranging from 0 to 200microM. The antiviral effect of A77 was determined by use of conventional plaque reduction assays. The effect of A77 on viral load was determined via real-time PCR analysis, and transmission electron microscopy was used to evaluate the effect of A77 on viral morphology. To determine whether the antiviral effect was attributable to alterations in CRFK cell viability and number, CRFK cells were treated with various concentrations of A77 and stained with Annexin V and propidium iodide to assess apoptosis and a mitochondrial function assay was used to determine cell viability.


RESULTS
Concentrations of A77 > or = 20microM were associated with substantial reduction in plaque number and viral load. Concentrations > or = 100microM were associated with complete suppression of plaque formation. At low concentrations of A77, clusters of intracytoplasmic virus particles that appeared to lack tegument and an external membrane were detected. Treatment of uninfected CRFK cell monolayers with A77 was associated with reduction in mitochondrial function with minimal evidence of apoptosis.


CONCLUSIONS
Leflunomide may be an alternative to current calcineurin-based immunosuppressive protocols used in feline organ transplantation because of its antiherpesviral activity.
Williams
Courtney R
CR
Comparative Transplantation Laboratory, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, CA 95616-8745, USA.
Sykes
Jane E
JE
Mehl
Margo
M
MacLeod
Jennifer S
JS
Lindsay
LeAnn L
LL
Poland
Amy M
AM
Chen
Yi-Je
YJ
Kyles
Andrew E
AE
Waldman
W James
WJ
Gregory
Clare R
CR
D000814:Aniline Compounds / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002371:Cat Diseases / Q000188:drug therapy / Q000821:virology*; D002415:Cats; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003437:Crotonates; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D006564:Herpesviridae / Q000187:drug effects* / Q000502:physiology; D006566:Herpesviridae Infections / Q000188:drug therapy* / Q000821:virology; D006885:Hydroxybutyrates / Q000378:metabolism / Q000494:pharmacology*; D007555:Isoxazoles / Q000378:metabolism / Q000494:pharmacology*; D000077339:Leflunomide; D046529:Microscopy, Electron, Transmission / Q000662:veterinary; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009570:Nitriles; D016133:Polymerase Chain Reaction / Q000662:veterinary; D018709:Statistics, Nonparametric; D014052:Toluidines; D019562:Viral Load / Q000662:veterinary; D014779:Virus Replication / Q000187:drug effects
D000814:Aniline Compounds; D000998:Antiviral Agents; D003437:Crotonates; D004279:DNA, Viral; D006885:Hydroxybutyrates; D007555:Isoxazoles; D009570:Nitriles; D014052:Toluidines; C527525:teriflunomide; D000077339:Leflunomide