Favipiravir elicits antiviral mutagenesis during virus replication in vivo

1Due to elevated error frequencies during the replication of their genetic material, RNA virus populations exist as complex distributions of mutant genomes also known as quasispecies. Genetic variability confers viral populations the flexibility to rapidly adapt to the environment, typically the host, and respond to different selective constraints such as the immune system or antiviral compounds. As a consequence, changes in the replication fidelity of a virus can affect its virulence and transmission during natural infections. Recent evidence suggests that RNA virus replication error rates are finely balanced to generate ample diversity while maintaining sufficient accuracy in the transmission of genetic information. Pioneering theoretical studies on self-replicating genomes suggested that any live organism has a maximum error rate tolerated to copy its genome. Given their elevated mutation rates, it was anticipated that RNA viruses exist close to their corresponding tolerated threshold. Hence, slight increases in virus mutation frequencies might result in the extinction of the replicating population. These predictions led to the proposal of lethal mutagenesis of viruses as a new therapeutic approach based on reducing the fidelity of genome replication.

2Several nucleoside analogues (i.e., ribavirin, 5-fluorouracil, 5-azacytidine) and non-nucleoside compounds (amiloride) display antiviral activities in cell culture against a wide range of RNA viruses and appear to act via increased mutagenesis. Increased mutation frequencies are accompanied with decreased virus progeny, infectivity and fitness, leading in some cases to the complete extinction of the virus population.

3Despite all the evidence in cell culture, data confirming lethal mutagenesis as a plausible therapeutic approach in vivo remains limited. Although some of the antiviral compounds with mutagenic activity in cell culture are also reported to have antiviral activity in vivo, controversy exists regarding the therapeutic mechanism. To date, the most successful antiviral compound with mutagenic activity in cell culture is purine analogue ribavirin. Ribavirin is commonly used in combination with pegylated interferon in the treatment of hepatitis C virus (HCV) infections. There are contradictory results on whether the mode of action of ribavirin on HCV in vivo is due to mutagenesis, immunomodulatory activities, or other antiviral mechanisms. Although increased HCV mutation frequencies have been reported in several cases, some studies have not found increased mutagenesis as a factor contributing to the associated antiviral activity. Therefore there remains an open question as to whether or not ribavirin-mediated antiviral activity in vivo is as a result of mutagenesis or one of the other reported mechanisms. Hence, further investigations are needed to demonstrate that lethal mutagenesis is a conceivable approach for the general treatment of RNA virus infections in vivo.

4Several novel compounds eliciting antiviral mutagenesis in cell culture were recently identified. Among them, favipiravir is a novel broad spectrum nucleoside analogue which is effective in the control of a vast number of RNA viruses in vivo, although its therapeutic mechanism of action is still under study. Favipiravir was initially identified as an antiviral compound for the treatment of influenza virus infection whose activity correlates with increased mutagenesis in cell culture. A recent study demonstrated that favipiravir-triphosphate can be used as a substrate by the virus polymerase and incorporated ambiguously into RNA opposite C and U in the template molecule.

5With the aim of demonstrating lethal mutagenesis as a conceivable approach to treat viral infections in vivo, we investigated whether ribavirin and favipiravir elicit antiviral activities in a mouse model of persistent norovirus infection. Favipiravir caused significant decreases in virus titres and viral RNA levels and led to the clearance of infectious virus in the faeces of seven out of nine animals (only two out of ten animals did not shed detectable infectious virus in control group). Blind passaging of faecal and tissue samples in cell culture confirmed that favipiravir treatment led to viral extinction in some animals. In contrast, ribavirin showed limited efficacy in vivo which correlated with lower antiviral activity in cell culture when compared to favipiravir. Favipiravir antiviral activity in vivo was associated with a significant increase in viral mutation frequencies. Viral RNA isolated from faeces of treated animals showed decreased specific infectivity, and infectious virus from faeces showed decreased fitness in tissue culture. These data suggest that favipiravir causes increased mutagenesis leading to decreased infectivity and fitness of viral genomes, both features of viral populations approaching extinction during mutagenesis. These results constitute a proof of concept for lethal mutagenesis in vivo and support antiviral therapies based on mutagenic compounds at the clinical level. The data also support the use of favipiravir in the treatment of norovirus infections for which there are not yet licenced antivirals or vaccines available and highlight a need for further studies on favipiravir and other improved derivatives as possible broad-range antiviral strategies.

6To investigate whether ribavirin and favipiravir elicit any antiviral activity on norovirus replication, we employed two different murine norovirus (MNV) strains, MNV-1 and MNV-3. MNV-1 was the first MNV strain isolated as the causative agent of a fatal infection in Stat1−/− mice. Later studies identified many other MNV strains, including MNV-3, wide spread in different mice laboratory colonies. MNV is used as a model to study norovirus replication and pathogenesis and has facilitated a better understanding of the norovirus life cycle. MNV-1 efficiently replicates in cell culture although it shows limited virulence in wild-type mice. In contrast, MNV-3 typically produces lower yields in tissue culture, yet establishes long-term persistent infections in wild-type mice, being detected for at least 8 months after inoculation. Previous studies demonstrated that ribavirin and favipiravir elicit antiviral activity against MNV-1 and human norovirus (HuNoV) replicon in cell culture. However, the mechanism of action and their utility in vivo have not been described.

7The treatment of RAW264.7 cells infected with MNV (MOI of 0.01 TCID50/cell) with ribavirin or favipiravir resulted in significant reductions in virus yields, reaching up to a 3-log10 decrease for ribavirin and a 4-log10 for favipiravir (Figure 1). A decrease in cell viability was observed when cells were treated with high concentration of ribavirin, although this reduction never exceeded 50%. In contrast, no significant decrease in cell viability was observed in cells treated with favipiravir (Figure 1—figure supplement 1).

8To further investigate the mechanism of inhibition, we determined the effect of ribavirin and favipiravir upon MNV replication in single-cycle replication kinetics carried out by infecting RAW264.7 cells at high MOI. Favipiravir inhibition occurred in a gradual manner, with the greatest reduction in virus titre being observed at later time points (Figure 1C,D). This result is suggestive of a cumulative antiviral activity often observed with increasing number of mutations in viral genomes during successive rounds of replication. In contrast, ribavirin inhibited norovirus replication from an early time point (8 hr), suggesting a different mechanism of action, an additional antiviral activity relative to favipiravir, or potentially due to a different mutational spectrum activity elicited by these two compounds. However, it is alternatively possible that the delayed inhibition shown by favipiravir compared to ribavirin reflects the fact that it takes several more steps to convert favipiravir to the active form. To investigate this possibility, we repeated the kinetics by incubating the cells overnight with favipiravir before infection to facilitate favipiravir conversion into favipiravir-triphosphate. No major differences were observed with cells pre-incubated during 1 hr or overnight with favipiravir (data not shown), which suggests that ribavirin might be a more potent inhibitor of viral RNA replication. To confirm this possibility, we determined the viral RNA synthesis kinetics for MNV-3 in the presence of both compounds and confirmed that ribavirin inhibits viral RNA synthesis from an early time post-infection, while the effects of favipiravir are only observed at later time points (data not shown).

9Although ribavirin and favipiravir cause increased mutagenesis in several RNA viruses, the mechanism of antiviral activity against noroviruses is not known. To determine whether ribavirin and favipiravir treatment resulted in greater mutation frequencies, we carried out sequence analysis of individual molecular clones isolated from populations subjected to 4 passages in the presence of either 200 μM ribavirin or favipiravir. We found that both compounds caused significant increases in the mutation frequencies of replicating virus. Ribavirin treatment resulted in a ∼threefold increase while favipiravir led to a five to sixfold increase in the number of mutations per nucleotide (Figure 2). Importantly, alterations in the transition frequency patterns were also observed. Ribavirin treatment resulted in greater proportion of G to A and C to U transitions than in untreated virus populations, while favipiravir led to a slight increase in A to G and U to C transition rates (Table 1). These alterations are in agreement with those observed for other viruses treated with the same compounds.

10A signature feature for error catastrophe in virus populations subjected to mutagenesis is a decrease in virus specific infectivity. To investigate whether these compounds reduced the infectivity of treated norovirus populations, we serially passaged MNV in the presence of either ribavirin or favipiravir (Figure 3, Figure 4, respectively). In both cases, lower virus titres and encapsidated viral RNA levels were observed during passage in cell culture. After 5 passages, virus titres for MNV-1 treated with 400 μM favipiravir, and MNV-3 treated with either 400 μM ribavirin or favipiravir were close to or below the detection limit, indicating that ribavirin and favipiravir can cause lethal mutagenesis of MNV. We confirmed that MNV-3 was extinguished after 5 passages in the presence of 400 μM favipiravir, by three consecutive blind passages in RAW264.7 cells in the absence of the drug. The specific infectivity, a measure of the ratio of infectious virus per encapsidated genome, was consistently reduced during passages in the presence of favipiravir for both MNV strains. However, treatment with ribavirin resulted in decreased specific infectivity only for MNV-3 treated with the highest concentration (400 μM). This suggests that the lower mutagenic activity exhibited by ribavirin (Figure 2) is responsible for this limited effect on specific infectivity. MNV-3 was more sensitive to favipiravir and ribavirin than MNV-1 (Figures 3 and 4), although this difference could not be associated with significant variations in the mutation frequency values between these strains during infections in treated or untreated cells (Figure 2). This different behaviour may be related to the observation that MNV-3 has lower fitness than MNV-1 (compare virus titre yields for untreated infections in Figures 1, 3 and 4) which can result in a greater sensitivity to mutagenesis, as previously reported for other viruses. Confirming this possibility, a tissue culture-adapted MNV-3 population (18 serial passages in cell culture) responded similarly to MNV-1 when treated with 400 μM ribavirin (data not shown).

11To further investigate the effect of mutagenesis on virus fitness, RAW264.7 cells were infected with virus obtained after 5 passages in the presence of ribavirin or favipiravir (Figures 3 and 4) at the same MOI (0.01 TCID50/cell). Viral populations previously subjected to either favipiravir or ribavirin mutagenic treatment resulted in decreased virus titres, in agreement with a loss of infectivity as a consequence of mutagenesis (Figure 3—figure supplement 1, Figure 4—figure supplement 1).

12The efficacy of ribavirin and favipiravir as antiviral compounds in vivo was next investigated in C57BL/6 mice persistently infected with MNV-3. Animals were infected 4 weeks before the beginning of the treatment to allow the establishment of a persistent infection. In a preliminary experiment (Figure 5—figure supplement 1), we found that all the animals treated with favipiravir had lower virus titres (5/5) in their faeces while only 2 out of 5 animals treated with ribavirin had consistently lower titres (Figure 5—figure supplement 1). This suggests that favipiravir is more efficient than ribavirin in the control of norovirus replication in vivo, in agreement with data obtained in tissue culture. Hence, we conducted further experiments to determine whether favipiravir can drive lethal mutagenesis of MNV-3 during persistent infection in mice. With this aim, we treated mice persistently infected with MNV-3 with either placebo or 600 mg/kg/day of favipiravir for 8 weeks (Figure 5). Favipiravir was effective in the control of persistent norovirus replication in vivo with decreased virus titres and RNA levels in faeces observed (Figures 5 and 6) at very beginning of the treatment (day 1) and throughout the entire treatment period (day 53). A predicted half-life of 31 days is calculated for virus clearance in favipiravir-treated mice in contrast with 122 days in the untreated group (p = 0.0064, log-rank test; Figure 5B).

13After 53 days of treatment with favipiravir, infectious virus titres were undetectable in the faeces of 7 out of 9 animals, while only 2 out of 10 animals had undetectable titres in the control group, suggesting that the infections were cleared as a result of favipiravir treatment (Figure 5A,C). Similar results were obtained when homogenates of caecum and colon collected at the end-point (day 53) were analysed for the presence of MNV (Figure 5B). To confirm whether these samples were negative, we carried out a blind passage in RAW264.7 cells. We confirmed that three treated animals were negative for all the samples analysed (faeces, caecum, and colon) while the remaining treated and untreated animals (6 and 10 respectively) were positive for MNV (Figure 5—figure supplement 2). All these three animal samples remained negative by titration and qPCR after 3 passages in the absence of favipiravir. These results suggest that favipiravir has assisted in clearing the infection in 33% of treated mice.

14To investigate whether favipiravir treatment in vivo resulted in the selection of adapted MNV-3 variants, we carried out infections in RAW264.7 cells with virus samples recovered from mice. We did not identify differences in the sensitivity to favipiravir between samples isolated from treated and untreated animals, suggesting no adaptation to the treatment (Figure 5—figure supplement 3).

15Quantification of MNV RNA in faecal samples also showed lower viral levels in treated mice than in control animals for the duration of the study (Figure 6). The same three animals that were negative for MNV-3 above showed viral RNA levels in faeces below the detection limit after 53 days of treatment, determined both by RT-qPCR (Figure 6), and RT-PCR followed by agarose gel analysis (Figure 6—figure supplement 1), while all placebo-treated animals showed high levels of viral RNA. Quantification of viral RNA extracted from caecum and colon, the major tissues for virus replication during persistent infections, also confirmed that viral RNA could not be detected in these three treated mice that contain no amplifiable infectious virus in faeces and tissues, further supporting that virus infection was cleared in these animals (Figure 6).

16To clarify if favipiravir antiviral activity observed in vivo was associated with mutagenesis, we examined the mutation frequency of viral populations shed in the faeces of five different animals in each group. The mutation frequencies found in virus samples from favipiravir-treated animals were greater than in placebo-treated mice with an average of a 2.9-fold increase relative to control animals being observed (Figure 7A). We also determined the mutation frequencies in three samples isolated from ribavirin-treated animals in the preliminary experiment, and they were similar to those observed for placebo-treated animals (4.0 ± 2.2 vs 3.6 ± 1.4 substitutions per 10,000 nucleotides sequenced, respectively). These results suggest that the antiviral activity observed for favipiravir in vivo is linked to mutagenesis, and the clearance of infection in some of these animals is the consequence of lethal mutagenesis of persistently replicating virus.

17To investigate whether favipiravir-induced mutagenesis in vivo resulted in decreased infectivity of viral populations, we firstly isolated and quantified viral RNA from faeces at various times during the treatment of animals with favipiravir and used an equivalent genome copy number to transfect BHK-21 cells (Figure 7B). BHK-21 cells support MNV replication but not virus (re)infection, resulting in a single round of replication for the transfected genomes and a better correlation between infectious genome units and virus titre. Thus, this approach provides an indirect measure of viral genome specific infectivity, as only viable genomes will result in the recovery of infectious virus. Viral titres recovered were consistently lower in samples obtained from favipiravir-treated animals than in placebo-treated animals (Figure 7B), suggesting decreased specific infectivity as a consequence of increased mutagenesis. Virus titres for genomes isolated from ribavirin-treated animals showed no significant difference with untreated animals (Figure 7—figure supplement 1).

18To compare whether mutagenesis in treated animals resulted in lower fitness of virus samples recovered, we determined the relative replication rates of virus populations recovered from mice after 18 days of treatment. To this aim, we carried out MOI controlled infections (0.01 TCID50/cell) in RAW264.7 cells using virus previously isolated from faecal samples and propagated once in cell culture. Virus yields obtained using virus from favipiravir-treated animal samples were lower on average than those isolated from placebo-treated animal samples, suggesting that mutagenesis induced by favipiravir results in a viral fitness cost in vivo (Figure 7C). Thus, favipiravir causes increased mutagenesis and decreases the specific infectivity and fitness of norovirus in vivo which supports an antiviral activity mediated by enhanced mutagenesis.

19Lethal mutagenesis has been the subject of numerous studies in cell culture in the last few years as an alternative approach to classical antiviral therapies. Due to the elevated mutation frequencies in RNA viruses, it was predicted that an increase in the replication error rate might result in the extinction of the viral population. However, several in vivo studies carried out up to date have resulted in insufficient evidence to support lethal mutagenesis as a practical approach at the clinical level. In this study, we show that favipiravir, a purine nucleoside analogue, can cause the extinction of an RNA virus during replication in its natural host. The antiviral activity observed in vivo is associated with increased mutation frequencies and, importantly, reduced infectivity in virus samples isolated from the treated animals. These are features typically observed in viral quasispecies approaching extinction during lethal mutagenesis which constitutes a proof-of-principle for this antiviral strategy.

20Favipiravir was initially identified as an antiviral compound for the treatment of influenza virus infection and currently is being tested in a phase 3 clinical trial. Previous studies on mice and data from clinical trials in humans show that favipiravir is well tolerated in vivo with much less toxicity exhibited than ribavirin. In this study, we observed no major side effects in the mice treated during the 8 weeks. The only apparent side-effect is that treated mice show a modest reduced rate of weight gain compared to the control group (Figure 5—figure supplement 4).

21In addition to its activity in vivo against influenza virus and data shown here on MNV, favipiravir is efficient in the control and clearance of a broad number of RNA viruses including picornavirus, parmyxovirus, bunyavirus, arenavirus, and togavirus. Importantly, favipiravir has shown efficient antiviral activity in mouse models for Ebola virus infections, which has led some African countries to consider using this drug for the control of the current outbreak. Further studies are needed to elucidate if the mechanism of action of favipiravir against these other viruses is lethal mutagenesis, and if it constitutes a possible universal antiviral mutagen for the clinical treatment of viral diseases. In particular, an attractive possibility would be to study the effect of favipiravir in the control of HCV, particularly given the lower toxicity displayed by favipiravir.

22These results are also relevant for the development of antiviral strategies to control human norovirus (HuNoV) infections for which there are currently no licenced vaccines or antiviral therapies. Due to the absence of cell culture systems to recover and propagate infectious HuNoVs, MNV has been suggested as a potential surrogate system. HuNoVs are a significant cause of non-bacterial gastroenteritis with large economic losses typically associated with frequent outbreaks in contained environments (>$160 million in UK hospitals alone). In addition, they have been linked to other important disorders such as ulcerative colitis and persistent diarrhoea. Chronic norovirus infections constitute a major health problem in immunocompromised patients with no treatment yet available. Favipiravir or other derivatives with improved pharmacokinetic properties may constitute an attractive candidate for the treatment of these patients. The rapid evolution and large mutation frequencies of norovirus replicating in immunocompromised patients suggest that antiviral mutagenesis could be an effective approach. Favipiravir could also be considered as prophylactic treatment to reduce virus dissemination during the course of an outbreak, especially in contained environments such as hospitals or nurseries. The data obtained for persistently infected mice support this possibility with lower virus yields shed by treated animals since an early time during treatment (Figure 5).

23Recent evidence suggests that the combination of a mutagenic compound with a classical antiviral molecule can be more efficient in the extinction of a virus than the use of the compound alone or the combination of classical inhibitors only. Inhibitors with antiviral activity in vivo have been identified for multiple RNA viruses, including norovirus, encouraging further studies in this direction. Given its significant efficacy in the control of different RNA viruses, favipiravir or other derivatives with improved pharmacokinetics constitute attractive candidates to become universal antiviral compounds against viral diseases via lethal mutagenesis.

24Studies with mice were performed in the Department of Pathology BSU Unit (PCD 80/2802) after ethical review by the University of Cambridge Review Panel and subsequent approval by the UK Home Office (PPL70/7689). All animal procedures and care conformed strictly to the UK Home Office Guidelines under The Animals (Scientific Procedures) Act 1986.

25Procedures for the cultivation of cells and MNV infections have been previously described. Murine leukaemia macrophage cells RAW264.7 were used for the propagation and titration (TCID50 assay) of murine norovirus 1 and 3 (MNV-1 and MNV-3) used in this study. Baby hamster kidney cells (BHK-21) were used for the determination of infectivity in viral genomes isolated from animal samples. All the different cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin (complete DMEM) and maintained at 37°C with 10% CO2. MNV-1 and MNV-3 strains used in this study were obtained after reverse genetics recovery of infectious virus as previously described. Recovered viruses were then subjected to two consecutive passages in RAW264.7 cells. The resulting population was titrated and used as a passage 0 stock.

26RAW264.7 cells were grown until they formed confluent monolayers (∼2 × 106 cells in 35 mm diameter dish). Supernatant was then removed and replaced by 1 ml of complete DMEM containing either 200, 400, or 800 μM ribavirin or favipiravir and the cells were incubated for two additional hours at 37°C and 10% CO2. Cells were inoculated with 200 μl of virus at the multiplicity of infection (MOI) indicated and incubated for 1 hr at 37°C and 10% CO2. Supernatants were removed, cells washed to eliminate unattached virus, and 2 ml of fresh media containing ribavirin or favipiravir were added to each well. Cell cultures were collected at 24 hr post-infection and virus released through two consecutive freeze–thaw cycles.

27For experiments involving serial passage of virus populations in the presence of favipiravir or ribavirin, passage 1 cells were infected at an MOI of 0.01 TCID50/cell with MNV-1 or MNV-3. In subsequent passages, 200 μl of neat virus from the previous passage (1/10 of total virus) were applied to a new monolayer of cells.

28Viral RNA was extracted from 100 μl of viral samples, either supernatant from lysed infected cultures or PBS-resuspended faeces from animals, using EconoSpin columns (Epoch, Missouri City, TX), following protocols provided by the manufacturer. Viral RNA was quantified using a two-step qPCR approach following protocols described previously.

29For the calculation of mutation frequency in any virus population, we have followed protocols previously described. Briefly, 4 μl of purified RNA were reverse-transcribed in 20 μl final volume using SuperScript III (Roche, Switzerland) as indicated by the manufacturer. 3 μl of cDNA were then PCR amplified using high fidelity KOD polymerase (Toyobo) using primers spanning genomic positions 3734 to 3770 and 6074 to 6034 in MNV-1 and 3734 to 3770 and 5738 to 5711 in MNV-3. PCR products were purified with EconoSpin columns (Epoch) and directly ligated in plasmid PCR Blunt using Zero Blunt PCR cloning kit (Life Technologies, Carlsbad, CA). Positive Escherichia coli colonies were identified by PCR screening with primers flanking the vector-cloning site and GoTaq polymerase (Promega, Madison, WI). The resultant PCR products corresponding to individual MNV cDNA clones were sequenced and the mutation frequency in each population calculated.

304–5-week old male C57BL/6 mice were orally infected with 104 TCID50 units of MNV-3 as previously described. After 4 weeks, persistently infected animals were subjected once or twice daily to oral gavage treatment with ribavirin, favipiravir, or placebo. Ribavirin was dissolved in PBS before inoculation into animals, while favipiravir was resuspended in 0.5% carboxyl methyl cellulose (CMC) in PBS. For the preliminary experiment (Figure 5—figure supplement 1), animals were treated once or twice a day with 8 mg of ribavirin or favipiravir (∼300 or 600 mg/kg animal/day) for 18 days. For the larger experiment (10 mice per group) (Figures 5 and 6), animals were treated with 300 mg/kg animal favipiravir twice a day (600 mg/kg animal/day) for 8 weeks. Control animals were treated with 0.5% CMC in PBS. Faecal samples were collected at different time points along the treatment period and the presence of infectious particles and viral RNA determined. Animals were sacrificed after the 8-week treatment period and caecum and colon tissues collected to analyse the presence of viral RNA.

31To confirm the absence of infectious virus in those faecal and tissue samples that showed negative infectivity by TCID50 assays, 100 μl of samples homogenates were used to infect 105 cells. Infections were collected at 24 hr and subjected to two freeze-thawing cycles to release virus. The resulting virus was analysed by TCID50 assays. Those samples that remained negative after blind passage amplification were subjected to two additional blind passages in RAW264.7 cells as explained above, and the absence of infectious MNV was confirmed by TCID50 assays and qPCR.

32To determine whether virus replicating in vivo has acquired resistance to favipiravir, virus samples obtained from animal faeces at treatment day 53 were blind passaged in RAW264.7 cells as mentioned above. The amplified virus samples were applied to 105 RAW264.7 cell monolayers at an MOI of 0.01, and infections were allowed to proceed for 48 hr in the absence (DMEM) or presence of 200 μM favipiravir.