Article Figures & Data
Figures
- FIG 1
Effect of favipiravir treatment on viral yields in cell culture. Vero cells were pretreated overnight with the indicated concentrations of drug and then infected with RVFV 56/74 at a MOI of 0.1 PFU/cell. Infection proceeded for 72 hpi in the presence of the same concentration of favipiravir. Results are shown either as 50% tissue culture infective dose (TCID50) per milliliter (continuous line; left y axis) or as percentages of RVFV titer with respect to the one obtained in untreated cells (dashed line; right y axis). Values under the limit of sensitivity of the assay (estimated to be 10, indicated with a grid line) were arbitrarily represented as 2. Red line indicates 50%. Error bars denote standard deviation (SD).
- FIG 2
Serial passages of RVFV in Vero cells in the absence or presence of favipiravir. (A) Viral titers of RVFV after each passage in the absence or presence of different concentrations of favipiravir (T-705) as indicated. For concentrations higher than 80 μM, only 3 passages were performed. Viral titers in the supernatant of infected cells were determined at 72 hpi. Error bars denote SD. (B) Comparison of the viral titers obtained from virus recovered after passage 8 (previously propagated or not with 40 μM favipiravir) and those of parental RVFV 56/74 in the presence of a 5 to 80 μM range of favipiravir concentrations. Titers are represented as percentages with respect to those obtained without drug. Red line indicates 50%. Error bars denote SD. Asterisks denote a P value ≤ 0.0001 (p8-no Fav or RVFV 56/74 versus p8-40μM Fav; Student's t test). (C) Lack of detectable infectivity after blind passages as a criterion for RVFV extinction. Cell culture supernatants from passages carried out in the presence of favipiravir where no cytopathic effect (CPE) was observed (Fig. 2A; samples whose titers were below the limit of sensitivity of the assay) were subjected to additional passages (up to five) in the absence of drug. Original samples are named according to their passage number in the presence of the indicated favipiravir concentration (in μM). Flattened columns indicate no CPE detection; lifted columns indicate evident CPE.
- FIG 3
Effect of favipiravir in the specific activity of RVFV. (A) Viral titers of RVFV after each passage (n = 4) in the absence (white columns) or presence (black columns) of 40 μM favipiravir (redrawn from data shown in Fig. 2A). (B) Quantification by qRT-PCR (in triplicate) of RNA extracted from the supernatants of untreated (white columns) or 40 μM favipiravir-treated (black columns) Vero cells after each passage (n = 4). (C) Specific infectivity upon passage of RVFV in the absence (open symbols) or presence (closed symbols) of 40 μM favipiravir. Values correspond to the ratio between infectivity (A) and the amount of viral RNA (B). Error bars denote SD. Statistically significant differences are indicated by asterisks (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005; ****, P ≤ 0.0001; Student's t test).
- FIG 4
Distribution of mutations among individual clones. Plot of the distribution of the number of mutations per molecular clone in the RVFV populations passaged in the absence or presence of 40 μM favipiravir.
- FIG 5
Mutational spectrum induced by favipiravir on RVFV. Matrices of mutation types found in the glycoprotein Gc-coding region of RVFV passaged 4 times in Vero cells in the absence or presence of 40 μM favipiravir. The boxes below each matrix quantify the mutational bias according to the ratio shown on the left.
Tables
- TABLE 1
Mutant spectrum analysis by molecular cloning and Sanger sequencing of the glycoprotein Gc-coding region of RVFV passaged 4 times in Vero cells
RVFV populationa No. of nucleotides analyzed (clones/haplotypes)b No. different (total) mutationsc Minimum mutation frequencyd Maximum mutation frequencye No. different synonymous (nonsynonymous) mutations Total no. synonymous (nonsynonymous) mutations 56/74 p4, no drug 31,610 (29/21) 53 (375) 1.7 × 10−3 1.2 × 10−2 48 (5) 354 (21) 56/74 p4 + favipiravir 31,610 (29/29) 138 (348) 4.4 × 10−3 1.1 × 10−2 85 (53) 284 (64) ↵a The origin and passage history of the viral populations analyzed is described in Fig. 2 and in Materials and Methods.
↵b The genomic region analyzed spans residues 2110 to 3199 of the glycoprotein Gc-coding region; the residue numbering is that of the SA-75 genome. The values in parenthesis indicate the number of clones analyzed followed by the number of haplotypes (number of different RNA sequences).
↵c The number of different and total mutations were counted relative to the consensus sequence of the corresponding population. Different and total mutations were used to calculate the minimum and maximum mutation frequency, respectively.
↵d Data represent the average number of different mutations per nucleotide in the mutant spectrum relative to the consensus sequence of the corresponding population. The results indicate a significant 2.5-fold increase of minimum mutation frequency associated with replication in the presence of favipiravir (P < 0.0001; χ2 test).
↵e Data represent the average number of total mutations per nucleotide in the mutant spectrum relative to the consensus sequence of the corresponding population.
Supplemental material
- Supplemental file 1 -
Table S1
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- Supplemental file 1 -
