Fig. S1

Fig. S1. Virus growth in Neuro-2a cells. The cells were inoculated with 1088 (WT) or 1088/RFLuc at an MOI of 0.01 and incubated at 37˚C. The culture supernatant was collected at the indicated time points, and the virus titer in each supernatant was determined using the focus assay. The data represent the mean (n = 3) and standard deviation (the error bars are hidden by symbols).

0

1

2

3

4

5

6

24 48 72 96

Log1

0 (F

FU/m

L)

hours post-inoculation

1088 (WT)

1088/RFLuc

Fig. S2

Fig. S2. Kaplan–Meier survival curves of mice inoculated with 1088 (WT) or 1088/RFLuc. Groups of 10 hairless mice were inoculated with each virus (105 FFU) in the right hindlimb muscle and were observed daily. All mice that exhibited severe neurological signs were euthanized humanely. The log-rank (Mantel–Cox) test was performed using GraphPad Prism software (version 6.0), but no significant difference was detected (P ! 0.05).

1088 (WT)

Sur

viva

l rat

e (%

)

0 7 140

50

100

dpi

1088/RFLuc

Fig. S3

1088/RFLuc infection

favipiravir 0–6 dpi no treatment no infection

no treatment

1 dpi

- !10000

- 600

rela

tive

sign

al in

tens

ity

-

-

-

favipiravir 2–8 dpi

Fig. S3. (A) In vivo bioluminescence imaging of 1088/RFLuc-infected mice treated with favipiravir at 1 dpi. As described in Fig. 3, groups of eight hairless mice were inoculated with 1088/RFLuc (105 FFU) in the right hindlimb muscle and orally administered favipiravir (300 mg/kg/day) daily for 7 days, beginning 1 hpi (0–6 dpi) or 2 dpi (2–8 dpi). Uninfected control mice (n = 4) and infected mice without treatment (n = 4) were also included. All bioluminescence images that were obtained at 1 dpi are shown. (B) Kaplan–Meier survival curves of the mice. Log-rank (Mantel–Cox) tests were performed using GraphPad Prism software (version 6.0) and an asterisk indicates a significant difference (P < 0.0167, Bonferroni-corrected level).

A

B

Sur

viva

l rat

e (%

)

0 7 14 210

50

100

dpi

no infection, no treatment 1088/RFLuc infection, no treatment 1088/RFLuc infection, favipiravir (0–6 dpi) 1088/RFLuc infection, favipiravir (2–8 dpi)

*

Fig. S4

Fig. S4. Kaplan–Meier survival curves of 1088/RFLuc-infected mice with higher-dose favipiravir treatment. As described in Fig. 4, groups of seven or eight hairless mice were inoculated with 1088/RFLuc (105 FFU) in the right hindlimb muscle and administered favipiravir orally (300, 600, or 900 mg/kg/day) daily for 6–7 days, beginning at 2 dpi. Uninfected control mice (n = 4) and infected mice without treatment (n = 4) were also included in the analysis. Log-rank (Mantel–Cox) tests were performed among the three treatment groups using GraphPad Prism software (version 6.0). Significance was set at P < 0.0167 (Bonferroni-corrected level); however, no significant difference was detected.

a: no infection, no treatment b: 1088/RFLuc infection, no treatment c: 1088/RFLuc infection, favipiravir (300 mg/kg/day, 2–8 dpi) d: 1088/RFLuc infection, favipiravir (600 mg/kg/day, 2–8 dpi) e: 1088/RFLuc infection, favipiravir (900 mg/kg/day, 2–7 dpi)

Sur

viva

l rat

e (%

)

0 5 10 150

50

100

dpi