EFFECTS OF TOLL-LIKE RECEPTOR ACTIVATION BY SINTETIC AGONISTS ON BOVINE HERPESVIRUS REPLICATION

MARIN, M; PÉREZ, S; LEUNDA, M; FAVERÍN, C; ODEÓN, A

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General.; 2012

SAMIGE

Introduction. Upon ligand recognition, Toll-like receptors (TLRs) stimulate the strong production of a wide variety of cytokines. Studies on the role of TLR signaling on BoHV replication have not been reported. The aim of this work was to study the in vitro effects of the immunological effectors associated with the stimulation of viral TLRs on BoHV-1 and -5 replication. Materials and Methods. Peripheral blood leukocytes (PBLs) from BoHV-seronegative calves were stimulated for 6 or 24 h with the following TLR agonists: Poly(I:C) (TLR3), Imiquimod, CL075 and ssPolyU/LyoVec (TLR7/8) and ODN 2006 (TLR9), or a combination of them. Supernatants from stimulated PBLs were harvested and added to MDBK cells previously infected with BoHV-1 or BoHV-5 strains for 0, 4, 6 or 24 h (MOI 0.1). At 24 h, virus titres in supernatants were determined by end-point titration. Three replicas of each experiment were performed. As negative control, supernatants from non-stimulated PBL were added to infected MDBK cells. Results. When TLR ligands were individually tested, only the supernatants obtained after PBL stimulation with Imiquimod (TLR7/8 agonist) demonstrated antiviral activity. All other agonists had a positive effect on BoHV replication. Imiquimod significantly decreased (P<0.05) the extracellular BoHV yields during the first hours of infection. In contrast, treatment at 24 h after the initiation of infection completely failed to execute a protective effect. When Poly(I:C), a TLR3 agonist, was evaluated, an increase in virus titers was detected when treatments with supernatants from stimulated PBLs for 6 or 24 h were performed during the first hours of infection or at later stages of the viral replication cycle, respectively. In general, CL075, a TLR7/8 agonist, induced an increase in the extracellular BoHV-1 and -5 titers only at the time of MDBK infection. In contrast, another TLR7/8 agonist, ssPolyU/LyoVec, induced a significant increase (P<0.05) in the replication at all stimulation and infection times. Similar results were obtained with the TLR9 agonist. When a combination of agonists was evaluated, BoHV-1 titers in the extracellular media were significantly higher (P<0.05). However, a detrimental effect was observed on BoHV-5 replication when supernatants were added at the time of infection. Discussion.The nature and/or magnitude of changes in BoHV titers observed in the present work were dependent on the ligand as well as on the time of stimulation and the stage of the viral cycle at which the immunological effectors were added. This is the first evidence that timely activation of TLR7/8 signaling is effective in impairing BoHV replication. Our findings on the different TLRs analyzed contribute to the understanding of BoHV pathogenesis. Furthermore, gaining knowledge on the immune mechanisms involved in the response to viral infection is important for antiviral and vaccine development. Further studies on TLR signaling and the antiviral mechanism they trigger will greatly contribute to the prevention of herpesvirus infections.