Norovirus Polymerase Fidelity Contributes to Viral Transmission In Vivo

Figures

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  FIG 1. Substitution I391L in the MNV NS7 RNA polymerase leads to restricted genetic diversity in viral populations. (A to C) Sensitivity of the WT strain and mutants I391L and S313T to different mutagenic compounds: 5-fluorouracil (A), favipiravir (B), and ribavirin (C). Sensitivity to each mutagen is expressed as the change in virus titer relative to the value for infections in the absence of mutagens. Each value is the average of results of three independent biological replicates (n = 3), with the error bars representing standard errors of the means (SEM). An apparent absence of SEM bars indicates that the bar is smaller than the respective symbol for these values. Sensitivity values for each MNV NS7 mutant were compared with WT values. Hence, those mutants with titer change curves above the WT curve were less sensitive to drug treatment, whereas those with curves below the WT curve showed greater sensitivity. Statistical analysis of relative levels of sensitivity in each NS7 mutant compared to the WT was performed by two-way analysis of variance (ANOVA) (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Mutation frequency analysis of MNV populations obtained after 8 serial passages in…

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  FIG 2. The MNV NS7 RNA polymerase mutant I391L shows WT-like replication kinetics during infection in cell culture. (A) Virus yield after reverse genetics recovery of full-length cDNA constructs containing either wild-type (WT) MNV or the polymerase mutants S313T and I391L. The values shown are the averages of results of at least three independent virus titer determinations for up to 4 independent cDNA transfections. Error bars represent SEM, with the statistical significance determined by one-way ANOVA (ns, not significant; ***, P < 0.001). (B to D) Replication kinetics of WT and mutant MNVs in RAW264.7 cells infected at the following different MOI’s: 0.01 TCID50/cell (B), 5 TCID50/cell (C), and 10 TCID50/cell (D). The values represent the averages of results of three independent biological replicates (n = 3), with the error bars representing SEM. Data were analyzed by two-way ANOVA (*, P < 0.05). The dashed line in panels A to C represents the limit of detection.

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  FIG 3. MNV NS7 RNA polymerase mutant I391L shows delayed replication kinetics in mice. (A and B) Virus titers (A) and viral RNA levels (B) shed in the feces of animals inoculated with 3 × 102 TCID50 MNV (n = 5). Mice were inoculated with recombinant viruses generated following recovery of MNV NS7 variants in BHK-21 cells by reverse genetics followed by 4 serial infections in RAW264.7 cells, as previously described (39). The presence of the desired mutations in the viral genome was confirmed by sequencing. Data were analyzed by two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). gRNA eq, genomic RNA equivalents. (C) Animals were infected as described for panel A, but the mice were inoculated with a dose of 3 × 103 TCID50 MNV per mouse (n = 5). Data represent results from a two-way ANOVA test (***, P < 0.001). (D) Viral RNA levels detected in various tissues collected from mice inoculated with 3 × 102 TCID50 of WT MNV or the NS7 polymerase mutants at 48 h postinfection. MLN, mesenteric lymph node (two-way ANOVA test results were nonsignificant). In all panels, the dashed line represents the detection limit. The apparent absence of SEM bars for some values in panels B and C…

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  FIG 4. MNV NS7 RNA polymerase mutant I391L is less sensitive to favipiravir treatment in vivo. Six animals were infected with 3 × 102 TCID50 MNV (n = 6). After 7 days, persistently infected mice were subjected to favipiravir treatment twice a day over a period of 4 days. Mouse feces samples were collected, and the titers (TCID50 per gram) were determined as previously described (10). The virus titer values represented are relative to the average virus titer at the day 0 time point, collected immediately before the beginning of treatment. Absolute virus titer values at day 0 were 5.80 and 5.51 log10 TCID50/g stool for the WT and the I391L mutant, respectively. Error bars represent SEM from a two-way ANOVA test (ns, not significant; *, P < 0.05; **, P < 0.01).

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  FIG 5. MNV NS7 RNA polymerase mutant I391L shows delayed transmission in susceptible hosts. Virus transmission from infected to uninfected mice was determined as detailed in Materials and Methods. In brief, five naive mice in each cage were housed with one mouse inoculated by oral gavage with 3 × 102 TCID50 MNV-3. Every mutant was tested in 4 independent cages (n = 20). MNV transmission was determined by qPCR detection of MNV in the feces of naive mice along a series of time points. (A and B) Graphs showing the percentages of noninfected animals along a series of time points (the log rank test was used for statistical analysis). (A) WT and S313T showed no significant (ns) difference in their transmission kinetics between mice. (B) The WT strain was transmitted significantly faster than the I391L mutant (**; P < 0.01). S313T also showed significantly increased transmission compared to I391L (not shown in the figure; P < 0.05). Animals directly infected by oral-gavage inoculation are not represented. (C) Viral RNA levels found in the feces of noninoculated animals after 4 days in contact with infected mice (n = 20). The dashed line represents the limit of detection, and the numbers above…