2’-Fluoro-2’-deoxycytidine inhibits murine norovirus replication and synergizes MPA, ribavirin and T705

Figures

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  Fig. 1. 2’-FdC exerts anti-MNV activity in RAW264.7 cells. RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h, and the culture medium was replaced by medium containing different concentrations of 2’-FdC for 20 h. (A) The viral RNA level and (B) NS1/2 protein expression were analyzed by qRT-PCR (n = 6) and western blotting, respectively. (C) RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h, and the culture medium was replaced by medium containing different concentrations of 2’-FdC for 20 h. Viral NS1/2 protein expression was analyzed by confocal assay. (D) RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h and then left untreated or treated with 100 µM 2’-FdC for 20 h. The viral titer was determined by TCID50 assay (n = 6). RAW264.7 cells were infected with (E) MNVCW3 or (F) MNVCR6 at a MOI of 1 for 1 h and then left untreated or treated with 2’-FdC (10 µM and 100 µM, respectively) for 20 h. The viral RNA level and NS1/2 protein expression were analyzed by qRT-PCR (n = 6) and western blotting, respectively. (G) RAW264.7 cells were left uninfected or infected with MNV-1 at an MOI of 1 for 1 h and then left untreated or treated with different…

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  Fig. 2. CTP, but not GTP, reverses 2’-FdC-mediated inhibition of MNV replication. RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h and then either left untreated or treated with 2’-FdC, CTP or combinations thereof at the indicated concentrations for 20 h. (A) Viral RNA and (B) NS1/2 protein expression were analyzed by qRT-PCR (n = 6) and western blotting, respectively. RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h and then left untreated or treated with 2’-FdC, GTP or combinations thereof at the indicated concentrations for 20 h. (C) Viral RNA and (D) NS1/2 protein expression were analyzed by qRT-PCR (n = 6) and western blotting, respectively. (E) RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h and then left untreated or treated with 2’-FdC (100 µM), CTP (100 µM), GTP (100 µM) or combinations thereof for 20 h. The viral titers were determined by TCID50 assay (n = 4). Data were normalized to the untreated control (set as 1). *, P < 0.05; **, P < 0.01; ns, not significant. β-actin was used as a loading control. For immunoblot results (B and D), the band intensity of the NS1/2 protein in each lane was quantified using Odyssey software, and the…

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  Fig. 3. Synergistic anti-MNV effects of 2’-FdC with MPA, ribavirin or T-705. (A) RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h and then left untreated or treated with 2’-FdC and MPA at the indicated concentrations for 20 h, alone or in combination. The combined effect of 2’-FdC and MPA on viral replication was analyzed by using qRT-PCR assay (n = 4) and mathematical modeling using MacSynergy. (B) RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h and then left untreated or treated with 2’-FdC and ribavirin with the indicated concentrations for 20 h, alone or in combination. The combined effect of 2’-FdC and ribavirin on viral replication was analyzed using a qRT-PCR assay (n = 2-4) and mathematical modeling using MacSynergy. (C) RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h and then left untreated or treated at the 2’-FdC and T705 with indicated concentrations for 20 h, alone or in combination. The combined effect of 2’-FdC and T705 on viral replication was analyzed using a qRT-PCR assay (n = 4) and mathematical modeling using MacSynergy. The three-dimensional surface plot represents the differences (within 95% confidence interval) between…