Fig 1.
Treatment with NHC inhibits LACV replications more potently than RBV or FAV in multiple cell types.
(A-C) Dose-dependent antiviral activity of RBV, FAV and NHC. Cells were treated with different concentrations of either RBV or FAV or NHC or DMSO (vehicle control/VC) immediately after virus adsorption to the cells. At 24 hr post-infection (hpi), virus titer was measured in the cell supernatant by plaque assay. Each line graph represents the mean ± s.d. of infectious viruses present in two individual wells in each of two independent experiments (four wells total) per cell type. (A) Titration of virus produced from drug-treated Vero cells; (B) Titration of virus produced from drug-treated N2a cells; (C) Titration of virus produced from drug-treated hNSC cells; (D) The EC50 (median effective concentration) was determined by extrapolating the dose-response curve of either RBV or FAV or NHC in Vero, N2a cells, and hNSCs.
Fig 2.
Molnupiravir (MOV) treatment increases survival of LACV-infected mice by inducing less fit and mutated virus in brain tissue.
(A) Male and female C57BL/6 mice aged 23 to 24 days were infected IP with LACV (1 x 103 pfu/mouse). Mice were treated orally with MOV (300 mg/kg) or vehicle (n = 13) twice daily, starting at day 0 dpi (2 hr prior to infection) in one group (n = 13) and 3 dpi in another group (n = 14). Kaplar-Meier analysis was used to calculate the difference of clinical disease onset out to day 28–30. (B-F) To assess virus replication and fitness we infected another cohort of mice IP with 1x103 pfu with and without MOV treatment started on the day of infection as above. On day five the mice were sacrificed and brain and lymph node tissues were harvested. (B) Infectious virus was quantified in brain homogenate using a plaque assay (n = 8 for vehicle control and n = 9 for MOV treatment), and (C, D) for viral-RNA by qRT-PCR from (C) brain (n = 8 for vehicle control and n = 9 for MOV treatment) and (D) lymph node (n = 8 for vehicle control and n = 8 for MOV treatment). Each symbol is shown as an individual animal from two different independent experiments. Lines and error bars for each set indicate mean ± s.d. (E) Smaller plaque sizes were observed in plaque assays of virus from brain tissue in the mice receiving MOV treatment compared to mice from the vehicle control group. (F) To quantify the size of plaques (surface area), wells with clearly isolated plaques were digitized and imported into FIJI for segmentation and analysis. Lines and error bars for each set indicate the mean ± s.d. Statistical comparison in (B-D) and (F) were done using the Mann–Whitney U test. For C and D the Ct values for viral RNA and GAPDH were subtracted from each other then used as the exponent of 2 to determine the fold difference, which was multiplied by 100 to give the percent difference, and this number was used in the statistical analysis.
Fig 3.
MPID-NGS of the mechanism of action of molnupiravir (MOV in LACV mouse model.
(A) Mutation rate of LACV in the brain tissues after MOV (n = 9) or vehicle control (VC, n = 5) treatment. On the boxplots, the middle lines indicate the medians of the values, top and bottom parts of the boxes indicate the inter-quartile rage (IQR), the lines extending to the lowest and highest values indicate the range of 1.5*IQR, and the “*” symbols indicate the mean values of the mutation rate. Statistical comparisons were performed using Mann-Whitney U test. (B) Correlation of the overall mutation rate and LACV copy numbers in the brain tissues measured by qRT-PCR.
Fig 4.
Molnupiravir (MOV) treatment increases survival after infection via an IN route.
(A-C) A mix of both male and female C57BL/6 mice at the age of day 23 to 25 were infected with LACV (1x102 pfu/mouse) through an IN route. Mice were treated orally with MOV (300 mg/kg, n = 13) or DMSO (n = 13) twice daily until the onset of clinical signs or 10 dpi, starting at day 0 (2 hrs prior to infection) and followed for development of clinical disease. Kaplar-Meier analysis was used to calculate the difference of clinical disease and statistical analysis (A). Brain tissue was harvested after animals showed clinical signs of the disease from the experiments (A). Out of 13 mice, 7 mice showed clinical signs in vehicle treated group, whereas 4 mice were clinical in MOV treated group. To quantify the virus in brain tissue we used (B) a plaque assay for quantifying infectious virus titer and (C) qRT-PCR for viral RNA (n = 7 for vehicle control and n = 4 for MOV treatment). Lines and error bars for each set indicate mean ± S.D. (D) Mutation density of LACV in brain tissue in presence (n = 4) or absence (vehicle, n = 7) of MOV treatment was measured by MPID-NGS in two sequenced regions (L7 and M4). On the boxplots, the middle lines indicate the medians of the values, top and bottom parts of the boxes indicate the inter-quartile rage (IQR), the lines extending to the lowest and highest values indicate the range of 1.5*IQR, and the “*” symbols indicate the mean values of the mutation rate. Statistical comparisons in panel (B-D) were performed using Mann-Whitney U test.
Fig 5.
Serial passage of LACV in the presence of increasing NHC concentrations to select for resistance.
(A) Study protocol of serial passaging of LACV in presence of DMSO or increasing concentrations of NHC (0.1 to 0.8 μM), with virus being passaged five times at each concentration. (B) Dose-dependent antiviral activity of NHC tested against the serially passaged LACV. Passaged viruses were used to infect Vero cells treated with different concentrations of NHC or with DMSO (VC). At 24 hr post infection (hpi), virus titer was measured from cell supernatant using a plaque assay. Each line graph represents the mean number of infectious viruses present in three individual wells of an independent experiment with an error bar indicating ± s.d. (C) After 20 passages the virus pools from passage with (n = 207) or without (n = 122) NHC were used to form plaques on Vero cells. Plaque sizes were imaged and their sizes measured; the distributions of plaque sizes for each virus group with the size distributions significantly different assessed as in Fig 2.
Fig 6.
Treatment with NHC inhibits the replication of other orthobunyaviruses (JCV and CVV).
Virus-infected cells were treated with different concentrations of NHC or DMSO (VC). At 24 hr post infection (hpi), virus titer was measured in the cell supernatant by plaque assay. Line graph represents the mean virus titer present in individual wells combined from three independent experiments with an error bar indicating ± s.d.