When cells have been primed for six h with IFN prior to virus infection, CHIKV production was decreased in an IFN concentration dependent manner. IFN was most successful, followed by IFN and IFN. While pretreatment with 10,000 U/ml of IFN could minimize virus production around 25 fold, viral titers had been not reduced further than 6. 7 107 PFU/ml, indicating that CHIKV was rather insensitive to IFN pretreatment under the experimental conditions applied and nevertheless replicated to somewhat higher titers. When IFN was applied four h p. i., viral titers have been not signi cantly decreased, indicating that virus production was not greatly affected by high concentrations of IFN when IFN was added after the establishment of infection. Subsequent, the impact of IFN remedy on CHIKV RNA replica tion, independently of virus production and/or secondary in fection, was tested.
A CHIKV replicon was constructed in which the structural genes had been replaced by a rey luciferase enhanced green uorescent protein fusion gene. In this way, transfected cells could be visualized by uorescence microscopy and rep lication measured by luminometry. In vitro transcribed, capped CHIKrep FlucEGFP replicon RNA was transfected into Vero cells. Directly after transfection hop over to this site or 24 h posttransfection,Vtype I/II IFNs were added towards the wells in growing concen trations, and luciferase expression was measured 2 days following transfection. In benefits related to those obtained with CHIKV infection, when IFN was added directly immediately after RNA transfection, CHIKV replication was negatively affected inside a concentration dependent manner. In the concen trations applied, IFN was most effective, followed by IFN and IFN.
This can be similar to what was reported for SINV, an additional Old World alphavirus. When IFN was added 24 h p. t., nonetheless, Fluc expression could not be decreased further than roughly 50%, even with all the highest IFN concentrations. Col lectively, these final results recommend that CHIKV is insensitive to IFN once viral RNA replication has been established. CHIKV infection inhibits kind I/II inhibitor b-AP15 IFN signaling. Given that CHIKV replication is partially sensitive towards the priming of cells with variety I IFNs but is largely resistant to IFN treatment after viral RNA replica tion is effectively below way, it’s most likely that CHIKV blocks down stream IFN signaling and expression of IFN stimulated genes with antiviral activity. To test this hypothesis, the impact of CHIKV RNA replication on downstream IFN induced gene transcription was investigated.
Vero cells have been transfected with variety I IFN responsive or kind II IFN responsive Fluc reporter plasmids and were subsequently infected with CHIKV. Fluc expression was induced by stimulation with kind I/II IFNs at 4, 8, and 12 hpi and was normalized to Renilla luciferase activity expressed from a constitutive pro moter on a cotransfected pRL TK plasmid.