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Displaying 10 papers, 7 pages, start at 1, 28 Hits
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Inhibition of Virus Replication

The single-stranded RNA genome encodes a polyprotein, which is proteolytically cleaved into three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS5 protein, an RNA-dependent RNA polymerase, plays an important role in viral RNA synthesis and inhibits IFN signaling by binding to STAT2 (Grant et al., 2016) . ZIKV NS3 protein exhibits helicase activity that is essential for viral replication. The helicase domain of NS3 is activated by GTPγS (triphosphate), which facilitates the unwinding and translocation of RNA at the time of replication. The ZIKV helicase, along with NS5, is an attractive target for ZIKV drug development. Small membrane-associated interferon-inducible transmembrane proteins (IFITMs) are intrinsic immune system defenses that are able to inhibit replication of several pathogenic viruses. Both IFITM1 and IFITM3 have been reported to inhibit early stages of infection and replication of ZIKV in HeLa cells with the predominant role played by IFITM3 (Savidis et al., 2016) . Cavinafungin, an alaninal-containing lipopeptide of fungal origin, has recently been found to inhibit ZIKV polyprotein processing and cleavage of host protein signal peptides through inhibition of host endoplasmic reticulum signal peptidase in in vitro model (Estoppey et al., 2017) . Synthetic 25-hydroxycholesterol has been shown to inhibit ZIKV entry into the host in an in vivo assay using mouse and rhesus macaque models (Li et al., 2017 ). An in vitro study conducted in Vero cells using compounds such as ribavirin, CMX001, T-705, and T-1105 showed that T-705 (favipiravir) and T-1105 were able to reduce cell death caused by ZIKV (Cai et al., 2017) . Thus, these compounds that inhibit ZIKV replication in cell culture need to be explored further so that they can be used safely against ZIKV.
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CURRENT ANTIVIRAL OPTIONS FOR TREATING INFLUENZA INFECTIONS

In addition to M2 ion channel blockers and NA inhibitors, two small molecules that target the viral RNA-dependent RNA polymerase, favipiravir and baloxavir marboxil, are undergoing clinical evaluation in the US and Europe but already obtained approval by Japanese Health authorities. Favipiravir is a nucleoside analog that acts as a competitive inhibitor of viral polymerase substrate, approved since 2014 for the treatment of influenza infections with newly emerging strains and/or resistant to other antiviral agents. However, despite the apparent high threshold for drug resistance (24) and broad-spectrum antiviral potential notably validated in the context of recent Ebola virus outbreaks (25) , recent results of Phase II/III randomized trials on its therapeutic efficacy against uncomplicated influenza were not completely conclusive (26) . Baloxavir marboxil is a selective inhibitor of the cap-dependent endonuclease activity of the influenza viral PA polymerase subunit (27) , therefore interfering with the cap-snatching activity of the viral polymerase complex. In that regard, a very recent report disclosed for the first time the results of two randomized (Phases II and III) clinical trials evaluating the efficacy of a single-dose oral treatment with baloxavir marboxil in otherwise healthy outpatients with acute uncomplicated influenza, compared with placebo and a regular 5-day treatment with oseltamivir (28) . Overall, baloxavir marboxil and oseltamivir moderately reduced the time to symptom alleviation compared to placebo, while the former outperformed the two others in reducing viral loads. These results prompted the US Food and Drug Administration (FDA) to approve Xofluza R (baloxavir marboxil) for the treatment of acute uncomplicated influenza in patients 12 years of age and older who have been symptomatic for no more than 48 h (29). Nevertheless, this first antiviral flu treatment with a novel mechanism of action approved by the FDA in nearly 20 years does not seem to escape the problem of all other virus-targeted anti-influenza agents. The emergence of virus variants (mostly due to the I38T/M PA amino acid substitutions) conferring significant levels of reduced susceptibility to baloxavir marboxil was observed in up to 9.7% of the patients receiving the drug (28, 30) .

Target-Based Repurposing

Although serendipitous observation has historically proved its usefulness, the intrinsic necessity of the casual observation of an unintended and usually infrequent second benefit poses a significant hurdle for exploiting the full potential of drug repurposing, for which more controlled, systematic methodologies are needed. Target-based repurposing relies on having previous knowledge of the specific molecular or cellular determinant/function target recognized by the drug intended to be repurposed. If new research finds out that target is plays an important role in a condition or disease other than the original indication, there is a potential for repurposing. Of note, the target might but not necessarily has to play the same role in both conditions. For example, in the case of the previously mentioned favipiravir, the drug plays the same role as viral RNA polymerase inhibitor against both influenza and Ebola viruses. On the other hand, the Abelson tyrosine-protein kinase 2 (Abl2), target of the anticancer drug imatinib, has been found to be required for efficient fusion and release of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudovirions into the cytoplasm of the infected cell, a key step for viral replication (47) . An alternative scenario of target-based repurposing can happen when a particular drug of known mechanism of action is found to have a new molecular/cellular target, and this previously unrecognized second target is associated with a different disease. The molecule is therefore said to present polypharmacologyrelated features, meaning the capacity to act on multiple targets (48, 49) . Polypharmacological phenomena includes both a single drug acting on multiple targets of a unique disease pathway, or a single drug acting on multiple targets pertaining to multiple disease pathways (50) . In fact, polypharmacology is usually responsible for treatment toxicity or other undesirable adverse events, but some of these "side-effects" might also lead to drug repurposing, as further exemplified in the next sections. During the last decade, an increasing number of studies converged on proposing that many drugs, initially designed for a unique therapeutics target, are in fact expected to hit on average between 6 and 13 different targets (51, 52) .

THE EMERGENCE OF DRUG REPURPOSING APPROACHES IN THE FIELD OF ANTIVIRAL DRUG DISCOVERY

These last 10 years, there has been a remarkable growing interest for drug repurposing in the field of antiviral drug discovery, fueled by the incontestable reality of many known viral infections still lacking specific treatment. This interest is inversely correlated with the very low number of classic antiviral molecules that have been market-approved these last 5 years, mostly for the treatment of hepatitis C virus or HIV-related pathologies (72) . The best example of antiviral drug repurposing approaches are emerging viruses such as Ebola, Zika virus or MERS-CoV, for which there is an urgent and cost-effective need for therapeutics solutions. Indeed, to rapidly propose a solution in the context of a viral outbreak, one interesting approach consists to look at the available pharmacopeia used to treat pathogens. For example, chloroquine, a major antimalarial drug, has been proposed for the treatment of filoviral infections, and more largely for the treatment of other emerging pathogens, as it targets endosomal acidification, a pivotal step in the replication cycle of a large number of viruses (73, 74) . Another interesting illustration is the previously cited example of favipiravir, which proved its repurposing potential for the treatment of Zika or Ebola viral infections (25, 43, 75) .
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Introduction

Despite the clinical and public health importance of SFTSV, treatment options remain very limited. Ribavirin exhibits some inhibitory effects on SFTSV replication in vitro and in a type I interferon receptor-deficient mouse model, but it was not found to be effective in a retrospective cohort of patients [2, 7] . Comparatively, favipiravir (T-705), an RNA-dependent RNA polymerase inhibitor with broad-spectrum antiviral activities, demonstrated higher in vitro and in vivo antiviral effects against SFTSV. However, favipiravir is not clinically approved or readily available in many countries affected by SFTSV, including China. To identify drug compounds that can be used to treat patients or reduce the transmission of SFTSV, especially in the hospital setting, we conducted a drug repurposing program by screening a Food and Drug Administration (FDA)-approved drug library consisting of 1528 drug compounds. We first established a robust two-tier (ELISA followed by viral load reduction assay) screening platform for SFTSV. Using this drug screening platform, we identified five drug compounds with anti-SFTSV activity in vitro, and characterized the anti-SFTSV mechanism of the most potent drug, hexachlorophene, as an entry inhibitor of SFTSV.

Cell Viability Assay and Cytopathic Effect (CPE) Inhibition Assay

To explore a suitable assay for FDA-approved drug library screening, the cytopathic effect (CPE) inhibition assay was also performed as previously described with slight modifications [13] . Briefly, Vero cells seeded in 96-well plates were infected with SFTSV for 1 h with different multiplicities of infection (MOI) of 1.00, 0.10, or 0.01, followed by phosphate buffered saline (PBS) wash and replacement of fresh DMEM medium containing 0.1% DMSO as the negative control or favipiravir (50 µg/mL) as the positive control. The cell viability of each well was determined on day 1, 3, and 5 post-infection (dpi) by the CellTiterGlo luminescent assay.

Establishment of a Robust Antiviral Screening Platform for SFTSV

To establish a sensitive assay for anti-SFTSV drug screening, we compared the signal dynamic range and screening window of three assays (i.e., CPE inhibition assay, ELISA, and viral load reduction assay). The cell viability, SFTSV-NP protein expression, and the S-segment viral genome copy number in the culture supernatant were monitored at 1, 3, and 5 dpi, respectively. Favipiravir was used as a positive control in all three assays [7] . As shown in Figure 1a , differences of cell viability between the favipiravir-treated and the DMSO-treated groups were less than 20%, indicating that the CPE inhibition assay was not a sensitive assay for drug screening when favipiravir was used as a positive control. Next, the culture supernatants were subjected to ELISA and qRT-PCR analyses. As shown in Figure 1b , >10-fold and >15-fold read-outs between the favipiravir-treated and the DMSO-treated groups were detected with 0.01 MOI at 3 dpi and 5 dpi, respectively, by ELISA. In the viral load reduction assay, there was approximately 4-log 10 difference in SFTSV RNA load between the two groups at the 3 dpi and 5 dpi ( Figure 1c ). These data suggested that both the ELISA and the viral load reduction assay were sensitive drug screening assays for SFTSV. Considering the cost-effectiveness of the two assays and the need to balance between maximum drug efficacy and drug half-life (t 1/2 ), drug compound library screening was then performed using ELISA with 0.01 MOI of SFTSV as virus inoculum and the culture supernatant being collected at 3 dpi.

Hexachlorophene Interferes with SFTSV Entry into Cells

To differentiate whether the entry or the post-entry phases of the SFTSV replication cycle were interrupted by hexachlorophene, we performed a virus entry assay by exposing SFTSV-infected cells to hexachlorophene during the virus entry step, followed by quantification of the intracellular SFTSV viral RNA load at 2 hpi. As shown in Figure 4a , hexachlorophene or DMSO was co-mixed with SFTSV (5 MOI) and incubated with Vero cells for 2 h. Significantly lower viral RNA load was detected in the cells co-mixed with hexachlorophene (p = 0.0173) than those co-mixed with DMSO. Expectedly, there was no statistically significant difference between the DMSO and favipiravir groups, as the latter is a viral polymerase inhibitor (Figure 4a ). The result indicated that hexachlorophene treatment interfered with SFTSV entry. To further dissect the anti-SFTSV mechanism of hexachlorophene, we performed virus attachment and inactivation assays to investigate whether the drug inhibited virus attachment to the host cell surface or directly inactivated the viral particles by binding to the viral envelope. As shown in Figure 4b , no significant (p = 0.7806) inhibitory activity was observed when hexachlorophene was added to Vero cells pretreated with the drug compound (−4 to 0 hpi), which suggested that virus attachment to the host cell surface was not affected. Virus infectivity was also not significantly (p = 0.3335) impaired when SFTSV was pre-incubated with hexachlorophene for 2 h, followed by the detection of plaque formation when hexachlorophene concentrations were <0.1 µM (Figure 4c) .
To differentiate whether the entry or the post-entry phases of the SFTSV replication cycle were interrupted by hexachlorophene, we performed a virus entry assay by exposing SFTSV-infected cells to hexachlorophene during the virus entry step, followed by quantification of the intracellular SFTSV viral RNA load at 2 hpi. As shown in Figure 4a , hexachlorophene or DMSO was co-mixed with SFTSV (5 MOI) and incubated with Vero cells for 2 h. Significantly lower viral RNA load was detected in the cells co-mixed with hexachlorophene (p = 0.0173) than those co-mixed with DMSO. Expectedly, there was no statistically significant difference between the DMSO and favipiravir groups, as the latter is a viral polymerase inhibitor (Figure 4a ). The result indicated that hexachlorophene treatment interfered with SFTSV entry. To further dissect the anti-SFTSV mechanism of hexachlorophene, we performed virus attachment and inactivation assays to investigate whether the drug inhibited virus attachment to the host cell surface or directly inactivated the viral particles by binding to the viral envelope. As shown in Figure 4b , no significant (p = 0.7806) inhibitory activity was observed when hexachlorophene was added to Vero cells pretreated with the drug compound (−4 to 0 hpi), which suggested that virus attachment to the host cell surface was not affected. Virus infectivity was also not significantly (p = 0.3335) impaired when SFTSV was pre-incubated with hexachlorophene for 2 h, followed by the detection of plaque formation when hexachlorophene concentrations were <0.1 µM (Figure 4c ). assay. Vero cells were pre-treated by hexachlorophene for 4 h, followed by intensive wash and shift to 4 °C incubate with SFTSV (MOI = 5.0). After 2 h, the infectious inoculum was removed, cells were washed, and the intracellular viral RNA load was determined by qRT-PCR. (c) SFTSV inactivation assay. SFTSV was incubated with 10 µM hexachlorophene for 2 h, followed by standard plaque assay from diluting the mixture for 1000 fold (i.e., the remaining concentration of hexachlorophene was below its IC50). All experiments were performed in triplicates. Data are presented as mean values ± standard deviations. P value was calculated by Student's t-test (compared with the DMSO group).

Discussion

In the present study, we further expanded the spectrum of antiviral activity and described a novel antiviral mechanism of hexachlorophene. We showed that hexachlorophene potently inhibited SFTSV replication, as evidenced by significant (>2-log 10 ) reduction of viral RNA load and 100% plaque reduction at a drug concentration of 10 µM, which was below its CC 50 . The IC 50 of hexachlorophene against SFTSV (1.3-2.6 µM) was lower than that the other anti-SFTSV drug compounds reported thus far (caffeic acid, 180 µM; ribavirin, 40.1 µM; favipiravir, 25.0 µM; amodiaquine, 19.1 µM; and 2 -fluoro-2 -deoxycytidine, 3.7 µM) [38] [39] [40] . Using a combination of virus entry, attachment, inactivation, and membrane fusion assays, we showed that hexachlorophene interfered with virus entry and virus-induced cell fusion without affecting virus attachment to host cells or inactivating the virions. Molecular docking predicted that hexachlorophene bound stably with the deep hydrophobic pocket between domain I and domain III of the SFTSV Gc glycoprotein, providing insights into the structural interactions between hexachlorophene and SFTSV that explain the drug's in vitro antiviral activity.
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Abstract

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by SFTS virus (SFTSV), which is a novel bunyavirus. SFTSV was first isolated from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction in China. Subsequently, it was found to be widely distributed in Southeast Asia (Korea, Japan, and Vietnam). SFTSV can be transmitted not only from ticks but also from domestic animals, companion animals, and humans. Because the case fatality rate of SFTS is high (6-30%), development of specific and effective treatment for SFTS is required. Studies of potential antiviral drugs for SFTS-specific therapy have been conducted on existing or newly discovered agents in vitro and in vivo, with ribavirin and favipiravir being the most promising candidates. While animal experiments and retrospective studies have demonstrated the limited efficacy of ribavirin, it was also speculated that ribavirin would be effective in patients with a viral load <1 × 10 6 copies/mL. Favipiravir showed higher efficacy than ribavirin against SFTSV in in vitro assays and greater efficacy in animal models, even administrated 3 days after the virus inoculation. Although clinical trials evaluating the efficacy of favipiravir in SFTS patients in Japan are underway, this has yet to be confirmed. Other drugs, including hexachlorophene, calcium channel blockers, 2 ′ -fluoro-2 ′ -deoxycytidine, caffeic acid, amodiaquine, and interferons, have also been evaluated for their inhibitory efficacy against SFTSV. Among them, calcium channel blockers are promising because in addition to their efficacy in vitro and in vivo, retrospective clinical data have indicated that nifedipine, one of the calcium channel blockers, reduced the case fatality rate by >5-fold. Although further research is necessary to develop SFTS-specific therapy, considerable progress has been achieved in this area. Here we summarize and discuss recent advances in antiviral drugs against SFTSV.

Calcium Channel Blockers

2 ′ -Fluoro-2 ′ -deoxycytidine 2 ′ -Fluoro-2 ′ -deoxycytidine (2 ′ -FdC) is a nucleoside inhibitor used in anticancer drugs. It inhibits various RNA and DNA viruses in vitro, such as Borna virus (Bajramovic et al., 2004) , Lassa virus (Welch et al., 2016) , Crimean-Congo hemorrhagic fever virus (Welch et al., 2017) , influenza virus (Kumaki et al., 2011) , and herpesviruses (Wohlrab et al., 1985) . Smee et al. (2018) has shown the antiviral activity of 2 ′ -FdC against various bunyaviruses, such as La Crosse virus, Maporal virus, Punta Toro virus, Rift Valley fever virus, San Angelo virus, Heartland virus, and SFTSV. The IC 90 of 2 ′ -FdC against SFTSV was 3.7 µM in an in vitro assay ( Table 1) . This value was much lower than that of ribavirin (49.7 µM) in the same study and favipiravir (22 µM) in the study conducted by Tani et al. (2016) . In an in vivo study using IFNAR −/− mice, a 100 mg/kg/day treatment with 2 ′ -FdC was 100% protective against death caused by SFTSV (Table 2) . However, all the mice treated with 2 ′ -FdC experienced substantial weight loss after SFTSV inoculation, whereas the favipiravir-treated mice displayed very little weight loss, suggesting that favipiravir was more effective than 2 ′ -FdC in controlling morbidity during the infection (Smee et al., 2018) . It was also found that treatments with 100 mg/kg/day of either 2 ′ -FdC or favipiravir significantly reduced the viral titers in the serum. Furthermore, there was a slight discrepancy both in the survival rates and virus titers between mice treated with 100 mg/kg/day of 2 ′ -FdC and those with 200 mg/kg/day of 2 ′ -FdC. The survival rate was 80 vs. 100% for 200-and 100-mg/kg/day treatments, respectively; and the virus titer in the serum of 200 mg/kg/day-treated mice was higher than that of mice receiving the 100-mg/kg/day treatment. It was speculated that this was caused by the limited sample size (n = 4 or 5).

Amodiaquine

Amodiaquine is a novel compound that is routinely prescribed as an antimalarial drug is reported to show antiviral effects against ebolavirus (Gignoux et al., 2016; Sakurai et al., 2018) , dengue virus (Boonyasuppayakorn et al., 2014) , and zika virus (Balasubramanian et al., 2017) . The mechanism of inhibitory activity of amodiaquine against malaria and those viruses remains unclear. Baba et al. (2017) investigated the effect of amodiaquine and other halogen molecules (fluorine, bromine, and iodine) against the replication of SFTSV in vitro. All the derivatives also displayed anti-SFTSV activity, and the IC 50 was 36.6, 31.1, and 15.6 µM for fluorine bromine, and iodine, respectively ( Table 1) . Among the compounds tested, amodiaquine was identified as a selective inhibitor against SFTSV replication. The CC 50 and the IC 50 of amodiaquine was >100 and 19.1 µM, respectively. The IC 50 of amodiaquine was lower than those of ribavirin (40.1 µM) and favipiravir (25.0 µM).

DISCUSSION

Favipiravir exhibited higher effectiveness than ribavirin in in vitro and in vivo studies (Tani et al., 2016 (Tani et al., , 2018 . Meanwhile, favipiravir remained effective when it was used following SFTSV infection in animal models (Tani et al., 2016 (Tani et al., , 2018 indicating its potential as an effective drug for treating SFTS patient. Currently, clinical trials are underway to evaluate the efficacy of favipiravir for treating patients with SFTS in Japan (Cyranoski, 2018; Spengler et al., 2018) . Besides, it would be desirable to use intravenous administration because SFTS patients with severe symptoms could have difficulty in taking drugs orally.
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OTHER VIRUSES Ebola Virus

To capture the Ebola infection dynamics, Nguyen et al. (2015) used the target cell-limited model and compared EBOV to pandemic IAV. EBOV infection time is significantly slower than IAV infection time (9.5 h vs. 30-80 min) (Holder et al., 2011; Pinilla et al., 2012; Nguyen et al., 2015) . Furthermore, the viral replication rate has been estimated as ∼63 ffu/mL day −1 cell −1 , EBOV is hence highly efficient with a virion half-live of ∼23 h (c = 1.05 day −1 ) . Unfortunately, these results are uncertain due to parameter identifiability problems. Nonetheless, the target cell-limited model confirmed the viral growth seen in experimental data, starting at day 3 post infection with a complete target cell depletion at day 6. Madelain et al. (2015) extended the target cell-limited model by an eclipse phase (non-/virus-producing infected cells) and found a half-life for virus-producing infected cells of 6.4 h and a basic reproductive ratio of R 0 ∼ 9. The authors furthermore studied the antiviral effect in mice treated with Favipiravir, an antiviral drug that blocks the RNA-dependent RNA polymerase in a broad spectrum of RNA viruses (Furuta et al., 2013) . By inhibiting the virus production rate p, they found a sharp decrease in viral load that was associated with an increasing drug efficacy of 95, 98.5, and 99.6% at days 2, 3, and 6 after the onset of treatment. Since Favipiravir achieves its maximal efficacy after 3 days, an early treatment initiation is suggested (Madelain et al., 2015) . With patient data of survivors and fatalities from the Uganda Ebola disease outbreak in /2001 , Martyushev et al. (2016 studied the relationship between virus replication and disease severity. For this purpose, they extended the target cell-limited model by two target cell populations: potential target cells (T 2 ), that are recruited via proinflammatory cytokines (e.g., recruited macrophages, hepatocytes, splenocytes, and endotheliocytes), which become susceptible target cells (T 1 ), that are the primary target for viral replication (e.g., macrophages and dendritic cells). Ebola disease severity is described by a 2 log(10) higher plasma viral load, that is correlated with an extensive recruitment of potential target cells and a 2.2-fold higher basic reproductive ratio; R 0 ∼ 6 for fatal cases and R 0 ∼ 2.8 for nonfatal cases. Hence, the higher viral load in fatal cases and a massive infection/hypersecretion of cytokines by active virus-producing replication cells is associated with the potential severity of the Ebola disease (Wauquier et al., 2010; Martyushev et al., 2016) . Additionally, antiviral intervention of (i) an antibody-based therapy that affects the de novo infection (k), (ii) a siRNA-based treatment that blocks viral production (p), and (iii) a nucleoside analog-based therapy (e.g., Favipiravir) have been evaluated in mono-and combination therapy. The combination of nucleoside analog-based therapy and siRNA-based turned out to be most efficient if initiated 4 days post symptom onset, while the antibody-based therapy seemed insufficient (Martyushev et al., 2016) . The authors then demonstrated that a critical inhibition rate of 80.5% in fatal cases and 58.5% in nonfatal cases is needed to prevent fatal outcomes of the Ebola virus disease.

Zika Virus

Recently, Best et al. (2017) developed a series of models with and without incorporation of the immune response and fitted those to plasma viral load data of ZIKV-infected nonhuman primates. Within that model series, the target cell-limited model only extended by an eclipse phase that distinguishes between non-actively and actively virus-producing infected cells was the best-suited model to reproduce the data. Furthermore, the incorporation of key players of the IIR or AIR, e.g., by IFN or natural killer cells, respectively, did not improve the model fitting and thus has been neglected. The simple eclipse phase model estimated an eclipse phase of ∼4 h (already observed via modeling in Osuna et al., 2016) and a basic reproductive ratio of R 0 ∼ 10.7. The degradation rate of productively infected cells was estimated with δ = 4.5 day −1 , corresponding to a lifetime of ∼5 h. The authors furthermore included the effect of antiviral therapy by inhibition of the viral production rate. With the broad spectrum RNA polymerase inhibitor Favipiravir, the time to undetectable plasma viremia could be reduced by 2 days if the initiation of therapy starts at the time point of infection (t = 0 days post infection). The therapy initiation at day 2 post infection led to the same result compared to no drug treatment, leading to undetectable plasma viral load after 5 days post infection (Best et al., 2017) . By integrating the immune response via IFN and neutralizing antibodies into the eclipse phase model, Aid et al. (2017) found a positive effect of both in controlling the viral infection in the periphery. The overall best fit was achieved by initiating IFN response at day 1.5 while the activity of neutralizing antibodies started at day 6 (Aid et al., 2017) .
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Polymerase inhibitors

A historical compound is moroxydine [231] [232] [233] . It is also active against HSV and VZV. The most thoroughly studied of these molecules is Favipiravir (T-705). In vitro studies have demonstrated the high antiviral potency of the drug and mouse studies have demonstrated its protective efficacy against a wide range of influenza viruses A and B. This molecule also seems to be effective against other viruses [234] [235] [236] [237] [238] .
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Currently, influenza vaccination and two classes of antiviral drugs-M2 ion channel blockers (amantadine and rimantadine) and neuraminidase (NA) inhibitor (oseltamivir, zanamivir, and peramivir)-and the novel treatment option using polymerase inhibitor (favipiravir) are considered as mainstays in influenza infection treatment and control. The use of influenza vaccinations remains challenging due to antigenic drifts and shifts, with seasonal variation of new circulating species. Production of vaccine is time consuming with efficacy concerns, especially in the case of pandemic. Variations in vaccine efficacy caused by age should be aware, with studies suggesting that vaccineconferred protection may not be optimal in certain age groups (3) .
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PA endonuclease inhibitors:

Ribavirin (Figure 14 ) (187) and Favipiravir (188, 189) (also named T-705, Figure 14 ) are RdRp inhibitors that both have a nucleoside fragment. Ribavirin was approved as a broad-spectrum antiviral drug for years (190, 191) and Favipiravir has advanced to phase II clinical trials (USA) and phase III clinical trials (Japan). Ribavirin can influence the DNA/RNA synthesis of the host cell (192, 193) through a combination of several different mechanisms (147) . Further research reveals that ribavirin and its analogues (5-azacytidine and 5-fluorouracil) are lethal mutagens of influenza virus (194) . As a nucleobase mimetic, Favipiravir and its analogues showed to be effective against strains that are resistant to NA inhibitors and M2 ion channel protein inhibitors (188, (195) (196) (197) (198) (199) .
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Introduction

In the present study, we aimed at characterizing the pathological changes in the liver of CCHFV-infected IFNAR 2/2 mice in more detail. Furthermore, we employed this model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 (favipiravir) against CCHFV in vivo. These drugs are either in clinical use or in an advanced stage of clinical testing. Ribavirin inhibits CCHFV replication in cell culture [15] and is administered to CCHF patients, though its clinical benefit is not proven and discussed controversially [16] [17] [18] [19] . It shows beneficial effects in the neonatal and STAT1 2/2 mouse models [9, 10] . Ribavirin currently is the only drug available for treatment of CCHF. Arbidol is a broadspectrum antiviral showing activity against a range of RNA viruses in vitro and in vivo, most notably influenza A virus [20] [21] [22] [23] [24] . In Russia and China, the drug is in clinical use primarily for prophylaxis and treatment of acute respiratory infections including influenza. Arbidol is assumed to act via hydrophobic interactions with membranes and virus proteins, thus inhibiting viral fusion and entry [25] [26] [27] . T-705 is a potent inhibitor in vitro and in animal models of influenza virus, phleboviruses, hantaviruses, arenaviruses, alphaviruses, picornaviruses, and norovirus [28] [29] [30] [31] [32] [33] [34] [35] . Following conversion to T-705-ribofuranosyl-59-triphosphate, it presumably acts as a nucleotide analog that selectively inhibits the viral RNA-dependent RNA polymerase or causes lethal mutagenesis upon incorporation into the virus RNA [36] [37] [38] [39] [40] . T-705 (favipiravir) is currently in late stage clinical development for the treatment of influenza virus infection.
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Proposed prophylactic and therapeutic regimen against the Ebola virus infection

The EBOV has undergone a rapid mutation during its spread through humans [224] [225] [226] . The EBOV is an RNA virus the replication of which is highly error prone with nearly one viral mutation occurring during each cycle of replication. This extremely high mutation rate leads to significant genetic and antigenic diversity that allows the EBOV population to evolve resistance to antiviral medications and vaccines [227, 228] . A combination therapy has been used in the treatment of RNA virus infections, such as the human immunodeficiency virus (HIV) [229, 230] and hepatitis C [231, 232] to minimize the development of drug resistance. Given the broad cell tropism and high replication rate of the EBOV due to the potent suppression of both innate and adaptive immune responses of the host, patients with the EBOV infection have an extremely high viral load. The selective pressure in the presence of the high mutation rate and viral load during the human EBOV infection make the evolution of the EBOV viral strains resistant to a single drug inevitable. The currently available medications in the proposed regimen-which is a treatment regimen containing a cocktail of antiviral medications targeting the different steps of the EBOV replication in order to achieve maximal suppression of viral replication and to prevent the rapid development of resistance to favipiravir, the only drug in the regimen that is directed against a mutable target of the EBOV-has been shown to reduce the replication of the EBOV. [233] [234] [235] .
The primary target of the EBOV is the mononuclear phagocytic system. The spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells during the advanced stage of the disease [6, 236, 237] . The EBOV may produce a viral load of up to 10 10 virions per ml serum in terminally ill patients [80] . Oral amiodarone prophylaxis, by inducing a Niemann-Pick C-like phenotype in the cells of the mononuclear phagocytic system, may prevent viral entry into these cells during needle stick injury. Through protection of the mononuclear system by our prophylaxis and cocktail therapy, we hope to offer a better chance of survival to these patients by allowing them to develop a natural body immune defense against the EBOV infection. The liver, containing the largest number of fixed tissue macrophages (Kupffer cells), as part of the reticuloendothelial immune defense system of the body, is a major target for the EBOV infection [238, 239] . The EBOV replicates to high titer in the liver [240] . Hepatic apoptosis may play a role in the pathogenesis of the EBOV infection [88] . Toremifene is added to the treatment regimen for hepatic protection because amiodarone does not exert inhibitory action against the EBOV in hepatocyte. However, both amiodarone and toremifene can increase QTc and the risk of Torsades de pointes. Therefore electrocardiogram should be carefully monitored if both drugs are to be used. Amiodarone, favipiravir, and toremifene are available and stockpileable in oral preparations. These properties are advantageous in outbreak situations and contingency planning of a potential EBOV epidemic or pandemic. The avoidance of intravenous administration will prevent needle stick injury in healthcare workers caring for the infected patients.
IFN-β may have potential as an adjunctive postexposure therapy for high-risk exposure, such as needle stick injury, by inducing IFITM1 to limit entry of the EBOV. Post-exposure IFN-β treatment was associated with a trend towards lower plasma and tissue viral burden and pro-inflammatory cytokines production [56] . The reduction in viral load and cytokine dysregulation coupled with optimal supportive therapy may improve the chance of survival of the host to allow the development of natural immunity to control the underlying EBOV infection. IFITM1 is active against multiple viruses, including the EBOV [185, 188] and hepatitis C 1 ml of blood may contain 10 9 to 10 10 virions in terminally ill patient. Prophylactic amiodarone therapy may protect macrophage, monocyte and endothelial cells immediately from EBOV during needle stick injury and accidental exposure and allow time for the consideration of IFN-β, toremifene, favipiravir and convalescent blood serum therapy. 2 Amiodarone is unable to protect hepatocyte from EBOV infection. 3 Both amiodarone and toremifene can increase the risk of QT prolongation and Torsades de pointes. 4 The recommended dosage for treatment of human EBOV infection may be 2 to 5 times higher than influenza studies. Please confirm the recommended dose with the drug company. 5 N-acetylcysteine intravenous infusion at 100 mg/kg/day to control cytokine dysregulation (e.g. add 5 g of intravenous preparation of N-acetylcysteine into each liter of intravenous replacement fluid). [186, 187, 241, 242] . Interferon induced IFITM1 plays an important role in the treatment of human HCV infection by inhibiting entry of HCV into the host cell [243] . Six million international units (MIU) of IFN-β intravenous administration is as effective as a three MIU twice-daily regimen for treatment of the HCV infection [244] , but has lesser side effects that require discontinuation of the medication [245, 246] . As the aim of IFN-β therapy in our regimen for post needle stick prophylaxis against the EBOV infection is to induce IFITM1 to limit viral entry, the dose of IFN-β for the post needle stick prophylaxis [247, 248] or induction therapy [249, 250] for HCV infection in humans is chosen. Once infection is fully established, IFN-β are replaced by convalescent blood serum and high-dose NAC infusion for providing passive humoral immunity and for the control of ROS-dependent NF-κB-induced cytokine dysregulation respectively.