key: cord-0767047-vviu1yp4 authors: Bugert, J. J.; Hucke, F.; Zanetta, P.; Bassetto, M.; Brancale, A. title: Antivirals in medical biodefense date: 2020-02-19 journal: Virus Genes DOI: 10.1007/s11262-020-01737-5 sha: 343601b32bd8b9b5c2617ee533ad50884dee0615 doc_id: 767047 cord_uid: vviu1yp4 The viruses historically implicated or currently considered as candidates for misuse in bioterrorist events are poxviruses, filoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses and a number of arboviruses causing encephalitis, including alpha- and flaviviruses. All these viruses are of concern for public health services when they occur in natural outbreaks or emerge in unvaccinated populations. Recent events and intelligence reports point to a growing risk of dangerous biological agents being used for nefarious purposes. Public health responses effective in natural outbreaks of infectious disease may not be sufficient to deal with the severe consequences of a deliberate release of such agents. One important aspect of countermeasures against viral biothreat agents are the antiviral treatment options available for use in post-exposure prophylaxis. These issues were adressed by the organizers of the 16th Medical Biodefense Conference, held in Munich in 2018, in a special session on the development of drugs to treat infections with viruses currently perceived as a threat to societies or associated with a potential for misuse as biothreat agents. This review will outline the state-of-the-art methods in antivirals research discussed and provide an overview of antiviral compounds in the pipeline that are already approved for use or still under development. Antiviral compounds effective in infections caused by tropical and vector-borne viruses were a neglected topic of international antivirals research until very recently. A number of compounds are now in clinical trials, and very few have received regulatory approval, or have made it to the market. While infections with arthropod-borne and tropical viruses are fairly common in nature, severe outcomes are, with a few exceptions, very rare. Therefore, countermeasures against such unlikely events, especially in the developed world, are regarded as giving little or no return on investments and are sidelined by grant driven research and manufacturers. While this is a legitimate point of view for academia and the pharmaceutical industry, governments have to consider countermeasures against rare agents released, or threatened to be released deliberately by individuals or groups aiming to cause maximum societal disruption and chaos. For such events governments have to prepare credible countermeasures in order to be able to provide prophylaxis, isolation, and treatment for large numbers of exposed and infected individuals. The basis of all considerations on countermeasures and biothreat preparedness is an agent-related risk assessment, which includes numerous criteria like availability of stocks or samples for potential perpetrators, ease of handling, pathogenicity, transmission pathways, tenacity, availability of vaccines, antivirals, and others. This requires research into these countermeasures, including the development, testing This review refers to a presentation given by the authors at the 16th Medical Biodefense Conference held in Munich in 2018 and is intended to widen the view on the thematic issue with a special focus on biodefense-related aspects. Edited by Lothar Zöller. and stockpiling of vaccines and antiviral drugs, particularly for dangerous biological agents. This review will focus on viral agents that fit into this category, briefly discussing their relevance for public health and biodefense, mode of action, and give an overview of treatment options available or in the pipeline. Viral hemorrhagic fevers (VHFs) cause the highest mortality in human hosts among all known viral agents. Encephalitides and severe respiratory infections caused by a range of viruses are other diseases with often severe clinical outcomes. The recent emergence of such infections from geographical hotspots is mainly a consequence of the rapid development of ground and air transport. Vector-borne infections are also affected by climate change. Large-scale outbreaks were first described for Monkeypox virus in central Africa in the 1970s [117] , while outbreaks of mosquitoborne Chikungunya virus [85] and Dengue virus infections in the Indian Ocean islands were seen mostly in the twentyfirst century [124] . The historic Ebola outbreak in West Africa in 2013-2014, followed by a more recent one in the Republic of Congo with 1891 fatalities [37] , has attracted extensive media attention. The rapid and uncontrolled spread of Ebola fever in Africa has been considered as a threat for the national security of developed countries with regard to the risk of imported cases but also for economic reasons. The Bundeswehr Institute of Microbiology (IMB) was involved in the international effort to contain Ebola fever in West Africa during the 2014-2016 outbreak [120] . The institute also runs a research program for antiviral drug development and hosts the biennial Medical Biodefense Conference (MBDC). Antiviral compounds and their possible role in biodefense were a special theme during the MBDC in 2018. The selection of topics with a focus on pox-, alpha-and flaviviruses was guided by the NATO AMedP-6 'Handbook on the medical aspects of nuclear, biological and chemical (NBC) defensive operations-Part II.' Smallpox, albeit eradicated in nature, continues to be perceived as a threat for several reasons, one of them being the risk that variola virus might be brought back with the methods of synthetic biology. Military forces and first responders in many countries were revaccinated in the early 2000s for fear that Iraq might have weaponized smallpox virus (which it had not, as was revealed later on). Emergency plans were developed to deal with a deliberate release. While no licensed drug was available at the time to treat infections with variola virus, a drug effective against orthopoxviruses, tecovirimat, has recently been approved by the United States Federal Drug Administration [55] . Smallpox as an exclusively human infection was eradicated by vaccination, but this is impossible for zoonoses like yellow fever, which has a number of non-human reservoir hosts. This is an important distinction, and in the case of an acute zoonotic viral infection, post-exposure antiviral treatment of the unvaccinated is a potentially lifesaving option in need of further development. Unfortunately, the public health repository of antiviral countermeasures for such infections is woefully small. VHFs are caused by infection with RNA viruses. The standard of treatment for RNA virus infections where it shows efficacy, is ribavirin, developed in 1963 [32] . Where possible, early start of treatment of acute virus infections gives the best results and, in this context, accurate and rapid virus diagnosis is essential. The crucial role of a well-organized public health system and classic quarantine approaches was demonstrated in the recent Ebola outbreaks in West-and Central Africa. However, the need for new antiviral agents had generally been recognized and been reviewed by David Freestone as early as 1985 [47] . While many virus infections are asymptomatic, new or improved antiviral drugs are needed for the prevention and/or treatment of a number of significant conditions caused by viruses which at present cannot be controlled by alternative measures, including vector control, immunization and treatment with existing antiviral drugs. The need for specialized BSL-3/BSL-4 facilities with trained personnel for experiments with life viruses, and animal challenge, has further restricted research to a few high-security sites worldwide. As a result, there are no FDAapproved antivirals for Ebola or the causative viral agents of many other viral hemorrhagic fevers, viral encephalitides, and respiratory infections. Few therapeutic interventions are available except for supportive therapy. In the following sections we will give a summary of the antivirals session held during the MBDC 2018, as well as an overview of antiviral drug development methodologies and selected experimental antivirals designed for potential biothreat agents. After an introduction on the chances and challenges encountered in the development of novel antivirals (Brancale-MBDC-2018-GO1), a discussion on the current conditions in UK/EU research networks, obstacles at the interface between research and industry, and preparedness for the treatment of infections with biodefense-related viruses followed. Further contributions outlined the methodical approach to antivirals design and biological evaluation (Fig. 1) . Using examples from chemists present at the meeting, the structural approach (Step 1; Bassetto-MBDC-2018-GO1), based on in silico dynamic models of antivirals targets, i.e., small-molecule inhibitors of polymerases, proteases, methyltransferases, and ProTide-based improvements of antiviral nucleosides [98, 132] , were explained in detail. The dynamic models are based on solved NMR structures of protein targets. The preselection of virtual candidate antiviral compounds in in silico models against viral protein targets reduces the number of compounds by four magnitudes (10 6 library → 10 2 selected candidates). The compounds are then synthesized, shipped and compared at a standard concentration (10 μM at IMB) for comparative effectiveness and toxicity in organotypical cell lines against a panel of viruses of interest for the biodefense community, including alpha-, bunya-, filo-, flavi-, ortho-/paramyxo-, and poxviruses. Hit compounds with high efficacy and low toxicity are identified (Step 2). This is followed by IC 50 /CC 50 evaluation (Step 3) of emerging hit to lead compounds, aiming for selective indices > 30 in sensitive (e.g., Huh-7 hepatoma cells) and organotypical cell lines selected for the pathogenic traits of the viruses of interest (e.g., U138 glioblastoma cells for encephalitis viruses). This usually results in another reduction of candidate numbers by one to two magnitudes. To confirm drug targets, target validation is then carried out, either by the use of enzymatic assays for viral enzyme targets (Silvestri-MBDC-2018-GO3) or by induction of resistant virus strains showing resistance mutations in the antiviral target areas, as shown with tecovirimat (ST-246) for orthopoxviruses. This concludes the classical in vitro evaluation of antiviral drug candidates. The winnowing process up to this point leads to a reduction ratio of six magnitudes (10 6 to 1). If in vitro toxicity is minimal, the compounds go straight into pharmacokinetics testing (rodent models), and into animal models of viral infections (Step 5). Here a dramatic rate of attrition leads to only one out of ten compounds tested in animal models making it into phase I clinical studies [79] . To further select compounds prior to animal testing, complex infection models, including in vitro 3D models, are currently the focus of much research in the antivirals field [77] . Functional models of virus infection at barriers, and the effect of antivirals on the virus passing the barrier, give an indication of antiviral effects on typical viral pathogenesis, e.g., encephalitis viruses that are being tested on models of the blood brain barrier (Step 4; Hurler-MBDC-2018-GP1). A successful prediction by in vitro functional models of antivirals efficacy in vivo, particularly using primary human organotypic cells, would also result in a significant reduction of unsuccessful drug testing in animal models. The evaluation cycle described above follows the general considerations as outlined by Huggins et al. for Ebolavirus (EBOV) in 1999 [64] , with the addition of in silico design with dynamic models for compound preselection, which had not yet been available at that time, and represents a methodical approach to antivirals design and development. This approach is used by groups active in the field and is also the basis of the 'Antivirals Platform' collaboration between Cardiff University and IMB into prophylaxis and treatment of infections caused by viral biothreat agents, which is funded by SER CYMRU/ MRC and IMB's basic funding. The platform established comprises all steps from molecular design to in vitro testing in complex infection models. Talks Complementing the recent review by De Clercq and Li [32] this section will focus on small-molecule antiviral compounds and discuss a selection of compounds that are either FDA approved or lately proved effective against viruses associated with a biothreat risk in in vitro experiments, animal or phase I-III clinical studies. Subsections give a brief overview of the viral agents in the order of relevance for biodefense, the FDA-approved treatment options, and antivirals in development, with top candidates highlighted in yellow in Table 1 , which lists virus-specific compounds in the same order of relevance, detailing compound class, target and stage of development. Variola virus (smallpox virus), a member of the orthopoxvirus (OPV) genus of the family poxviridae, was used in the 18th century as a biological warfare agent by British and American forces in North America [36] , and remains on the top of the list of biological threat agents for warfare or bioterrorism [35, 107] . Effective vaccines and FDAapproved antivirals exist and could be used to control a deliberate release. Variola virus (VariolaV), which only infects humans, was declared eradicated in 1980, after a global vaccination campaign. Handling of VariolaV requires BSL-4 containment. Virus stocks are officially kept in only two designated laboratories in Russia and the US. Monkeypox virus (BSL-3), a zoonotic agent causing sequelae similar to smallpox but less fatal, is endemic in central Africa (Democratic Republic of Congo; DRC); recent introductions to the UK were travel-related. Poxviruses are transmitted by contact infection and via the respiratory tract, causing a systemic infection in humans and animals. Smallpox virus infection leads to a fatal multiorgan failure syndrome within 7-14 days, in complicated cases with a hemorrhagic syndrome and CNS involvement. Smallpox has played a role in large-scale epidemics in history and its causative agent continues to be considered a potential biological warfare agent [35] . Orthopoxviruses (OPV) are ovoid-shaped enveloped viruses with Group I double-stranded (ds) DNA genomes, replicating via a virus-encoded DNA polymerase, an antivirals target, in the cytoplasm of infected cells [42] . Poxviruses enter cells by macropinocytosis, but a poxvirusspecific receptor is still elusive [100] . Anti-poxvirus drugs. One of the first effective drugs in clinical use as a parenteral treatment in severe OPV infections was cidofovir, a bisphosphonate developed at REGA, in Belgium [31, 35] ) and FDA approved against human cytomegalovirus (HCMV). The ether lipid analogue brincidofovir (CMX001), a prodrug of cidofovir, has shown efficacy in small animal models and is awaiting FDA approval [26, 44, 56, 115, 116, 118, 139] . The F13L virus egress inhibitor tecovirimat (ST-246, TPOXX®) has been independently developed to treat smallpox infections and has been FDA approved since 2018. Tecovirimat has recently been used to treat non-human primates infected with variola, and humans exposed to OPV [55, 104, 118, 146] . Tecovirimat (TPOXX®) is currently stockpiled in the US and production for similar stockpiles in Europe is planned. Anti-poxvirus drugs effective in animal models are reviewed in more detail elsewhere [133] . Further candidate anti-poxvirus drugs include kinase inhibitors imatinib (Gleevec/STI-571; [122, 123] ) and olomoucine [61] , terameprocol [119] , mitoxandrone [5] , the membrane targeting ddBCNA cf2642 [99] , bisbenzimide derivatives [151] , FC-6407, a OPV D4 processivity factor mimic [111] , and a number of natural extracts that have shown interesting antiviral activity against OPV in in vitro infection models [27, 153] (Table 1) . Filoviruses are category A select agents, World Health Organization risk group 4 pathogens, high on the list of potential biological threat agents [107] and their handling requires BSL-4 containment. In nature they infect primates, pigs and bats (free-tailed and fruit bats) and are transmitted to human hosts by exposure to infected bush meat and body fluids of human patients. Ebola and Marburg viruses (EBOV/MARV) cause severe viral hemorrhagic fevers with hematemesis, bloody diarrhea, prostration and case fatality rates of up to 90% within three days of infection. The EBOV envelope glycoprotein has been used in the VSV-EBOV vaccine, which is 70-100% effective preventing disease in exposed and vaccinated [15, 16] , defense against the use of filoviruses as biological weapons would benefit from an effective virus-targeting therapy. There are currently no licensed antiviral drug treatments for filoviruses. However, in a recent multi-outbreak, multi-country study (PALM-"Together save lives") started in November 2018 in the DRC, two monoclonal antibodies (Mabs) emerged as giving the greatest chance to survive Ebolavirus infection. Zmapp, mAb114 and REGN-EB3 were compared to the small-molecule drug remdesivir [150] . The trial was stopped early with REGN-EB3 and mAb114 giving the greatest chance to survive Ebolavirus infection. The WHO recommends using these two Mabs for all further treatments [150] . Remdesivir (GS-5734;1-cyano-substituted adenosine nucleotide analogue), a nucleoside-analogue prodrug and lead compound of the small-molecule antivirals class, has been shown to inhibit EBOV in cell culture and in non-human primates likely by chain termination [144] , but showed lower efficacy in the clinical trial compared to monoclonal antibody-based therapeutics. A good alternative, albeit not tested in the DRC clinical trial, may be T705 (favipiravir; [48] ), a repurposed drug synthesized by FUJIFILM-Toyam Chemical Co., licensed for use against influenza virus in Japan, and since found to be a broad-spectrum inhibitor of viral RNA polymerases [34, 49] . T705 and the related pyrazinecarboxamide compounds T-1105 and T-1106 have similar antiviral properties-see also section "Alphaviridae." FDA approval for use of favipiravir to treat filovirus infections is pending. Several animal pilot studies, most recently in non-human primates (NHP), have shown the efficacy of favipiravir [13, 14] ). While extensively tested, ribavirin is not FDA approved for EBOV [65] . Other promising candidates (Table 1) are the FGI-106 entry inhibitor [8] , CM-10-18-type glycan processing inhibitors, active against Marburg virus and Ebola virus in mouse models [25] , a number of kinase inhibitors, including AR-12 (OSU-03012; [24, 102] ), and K11777, a protease inhibitor developed for Chagas disease, which has additional activity against SARS-CoV and Ebola virus [154] . Alphaviruses are mosquito-borne viruses, but some can be effectively transmitted via the aerosol route from contaminated rodent feces. Rodents, birds and possibly marine species are maintenance reservoirs [43] . Alphaviruses can cause a number of diseases in humans, including Chikungunya fever, Eastern, Western and Venezuelan equine encephalitis. The handling of the respective viruses requires BLS-3 containment. Two type species, Venezuelan and Eastern Equine Encephalitis viruses (VEEV and EEEV), are considered potential biological threat agents [107] with up to 70% mortality in unprotected populations [142] and represent category B select agents. While human infections with VEEV and EEEV are rare, sporadic and unpredictable but explosive epidemics caused by Chikungunya virus (CHIKV) have occurred in the last decade mainly in South-East Asia and in South America, Central America and the Caribbean, globally amounting to millions of cases. Autochthonous cases of Chikungunya fever have been reported in Italy [93] . Viremia with rashes and fever usually lead to death of cells lining joints, causing arthritis and joint pain. CHIKV infections of neurons can result in potentially fatal encephalitis. Fatal infections, mainly seen in human infants, are rare, but longlasting polyarthralgia and encephalitis cause significant morbidity [95] . Alphaviruses are enveloped viruses with positive-sense ss-RNA genomes. Most experimental antivirals target the viral RNA polymerase. There are no licensed antiviral drugs against alphaviruses causing arthritis and encephalitis, and the treatment of infections is mainly supportive (anti-inflammatory drugs, glucocorticoids). Anti-alphavirus drugs. While pox-and filoviruses are highly lethal biological agents, alphavirus infections are rarely fatal, but can lead to large numbers of incapacitated individuals, due to severe arthralgias and headaches. In this sense, alphaviruses might be effective biological threat agents where incapacitation and saturation of medical care facilities are the goal of a perpetrator (incapacitating agents). Specific antivirals should be able to pass the blood brain barrier (BBB) to control postexposure encephalitis. Intravenous Ribavirin, which is FDA approved for HCV and respiratory syncytial virus (RSV) infection, does not pass the BBB, thus alleviating peripheral symptoms but not providing cure [1, 3] . Intranasal ribavirin may be more effective. Ribavirin resolves joint swelling in CHIKV [121] , but has no activity against VEEV in vitro [45] . Sofosbuvir, an FDA-approved antiviral drug against HCV, which has been suggested for repurposing against various viruses, has been evaluated for in vitro activity against CHIKV [40] . Among the most promising novel compounds is the broad-spectrum antiviral candidate favipiravir (T-705), initially developed to treat human influenza, which shows a potent antiviral effect in small animal models. The drug is licensed in Japan, while FDA approval is pending [49] . An in vitro comparison between ribavirin and favipiravir revealed that efficacy is cell-type dependent [45] . Efficacy was also shown in a mouse model [2] . Other compounds of interest (Table 1) include drugs approved for other medical conditions and tested for repurposing. Those are the old antiparasitic suramin, which shows ameliorating effects against CHIKV infection in mice [82] and the anthelmintic ivermectin, which shows in vitro activity against a range of alphaviruses [141] . Compounds with known cellular targets include the cancer drugs mefenamic acid and sorafenib, inhibiting replication of CHIKV and other alphaviruses via eIF4E dephosphorylation in vivo [90, 126] , and halofuginone, a prolyl t-RNA synthetase inhibitor in veterinary use that is active in vitro against both alpha-and flaviviruses [66] . Also promising is the virus-specific antiviral ML336 that inhibits Nsp4 of VEEV and EEEV in vivo [72] . Less well-described compounds are LL-37 peptide, an alphavirus entry inhibitor in vitro [4] , compound 25 that was identified in silico and optimized to inhibit CHIKV replication in vitro [9] , Prest-37 and -392, with in vitro activity against VEEV nsP1 capping enzyme [41] , and baicalin, which inhibits CHIKV replication in vitro by interfering with a cellular target [113] . [97, 112] , while recently favipiravir was evaluated and found to enhance survival in cynomolgus (crab-eating) macaques [125] . A further interesting compound is LHF 535, an entry inhibitor targeting arenaviral GP2 [91] . Human pathogenic bunyaviruses, particularly Hantaviruses and Crimean-Congo Hemorrhagic Fever Virus (CCHFV), can cause hemorrhagic fevers, and CCHFV is on the list of potential biological threat agents (NATO AMed P-6 [107] . The focus towards the identification of antiviral agents has mostly been on CCHFV infections, which are common in endemic areas, but are either asymptomatic or cause a nonspecific febrile illness that does not require hospitalization or specific treatment. Few patients develop hypotension and hemorrhage, and medical management is then largely supportive, with volume replacement, and prevention of edema and inflammation [68] . Ribavirin has been used to treat CCHF patients under compassionate use protocols with some success since 1985 [140] , especially if given early in the course of the infection, but many studies with apparently beneficial results lack controls. Recent randomized clinical trials were unable to show significant beneficial effects of ribavirin versus CCHFV [70, 78] . Further interesting candidates for virus-specific treatment (Table 1) include favipiravir (T-705), which has been evaluated against a number of phleboviruses (PhleboV) and to treat CCHFV infection in rodent models [53, 54, 59] , galidesivir (BCX4430), effective against Rift Valley fever virus (RVFV) infection in a hamster model and investigated for use by the FDA [145] , 2′-fluoro-2′-deoxycytidine (2FdC), which showed protective effects against infections with PhleboV in a rodent model [134] , and the FGI-106 entry inhibitor [135] . Flaviviruses causing hemorrhagic fever or severe encephalitis (Omsk hemorrhagic fever, Dengue and Yellow fever, Russian spring-summer encephalitis/Tick Borne Encephalitis (TBEV)) are listed as potential biological threat agents (NATO AMed P-6 [107] ) and handling requires BSL-3/ BSL-4 containment. Flaviviruses are arthropod-borne viruses that are endemic worldwide with virus/vector specific geographical distributions, causing regular outbreaks and fatalities, with 30.000 cases/year through yellow fever in Africa alone [51, 149] . Infections with flaviviruses can lead to hemorrhagic fevers (Omsk hemorrhagic fever, yellow fever (YF) and dengue fever with case fatality rates of up to 30%) or affect the CNS, causing encephalitis (e.g., Japanese encephalitis, tick borne encephalitis with case fatality rates up to 20%, Zika and West Nile encephalitis). Human-tohuman transmission is not effective. Live vaccines against yellow fever (17D) and Japanese Encephalitis (JE), a number of inactivated TBEV vaccines, and most recently a live Dengue virus vaccine are available. Flaviviruses are a large family of mosquito-or tick-transmitted enveloped viruses with a Group IV positive-sense single-strand RNA genome, using G-protein coupled receptors for entry into host cells [42] . Anti-flavivirus drugs. Ribavirin is an effective early treatment for yellow fever under compassionate use protocols, but fails to improve survival of dengue infections in nonhuman primates (NHP; [92, 103] . Out of a quite large number of drugs investigated for repurposing against flaviviruses by the FDA (Table 1) , the most promising candidate is sofosbuvir [18] . Sofosbuvir was initially developed and approved by FDA for treatment of hepatitis C. It shows activity against a number of flaviviruses in vitro and in the mouse model [33, 105] . Further interesting candidates (13 compounds listed in Table 1 ) inhibit the viral polymerase [39, 128] ), NS2B/NS3 protease and kinases [23, 24] ), cell entry and membrane trafficking [20, 109] , and other flavivirus targets. The action and the efficacy of most of these compounds in vivo are yet to be determined. The major shortcoming of all candidates so far tested in animal models for the treatment of infections with Usutu (UsutuV), Dengue (DENV) and Zika viruses (ZikaV) is their rather low efficacy [24, 101] . Orthomyxoviruses, in particular influenza viruses, although not on top of the list of potential biological threat agents, are fast-moving airborne pathogens capable of causing pandemics with significant mortality. Recombinant influenza viruses could be considered as potential biological threat agents. Handling of avian influenza viruses and other influenza viruses with high pathogenic potential requires BSL-3 containment. Pandemic influenza viruses type A are transmitted by the respiratory route to birds and mammals, type B only from human to human, as well as via saliva, nasal secretions, feces and blood, causing acute respiratory distress with potentially fatal outcomes in humans. In humans, infection of the respiratory tract can lead to pneumonia, secondary pneumonia and overwhelming immune responses, followed by multiorgan failure in rare cases. Orthomyxoviruses are globally endemic, and cause sporadic outbreaks, rarely pandemics. Orthomyxoviruses are enveloped viruses with a negative-sense segmented ss-RNA genome. The viral RNA polymerase has a high error rate of 1/10,000. Vaccines are composed of HA/NA subunits (purified from inactivated virions), purified subunits from recombinant sources, or live/ attenuated strains of the endemic strains/subtypes of influenza A virus (currently H1N1 and H3N2), as well as those of influenza B viruses [42] . Anti-orthomyxovirus drugs. FDAapproved neuraminidase inhibitors oseltamivir (Tamiflu®), zanamivir (Relenza®), laninamivir (Inavir®), and peramivir have marginal clinical benefits only when given early but may be useful in severe infections requiring hospitalization/ mechanical ventilation [57] . In 2018 baloxavir-marboxil (Xofluza®), an inhibitor of the viral cap-dependent endonuclease (CEN; influenza virus polymerase PA subunit), was approved by the FDA for the treatment of acute, uncomplicated influenza among patients aged 12 years or older [80, 110] . Favipiravir developed and approved in Japan specifically for treatment of influenza virus infections, and its combination with neuraminidase inhibitors was shown to be effective in a mouse model [10, 48] . Further interesting candidates are haloxanide/nitazoxanide, thiazolide compounds that were originally developed as antiparasitic agents, but were shown to inhibit influenza virus hemagglutinin maturation and intracellular trafficking of viral components in infected cells and that are now in clinical trials [83, 138] as well as cycloheptathiophene-3-carboxamide, which interferes with the polymerase PA-PB1 subunits of influenza virus [106] . Paramyxoviridae are fast-moving airborne pathogens infecting animals and humans. Hendra (HeV) and Nipah (NiV) viruses, in the genus Henipavirus, are considered zoonotic agents in Australia (horses) and South-East Asia (pigs), respectively. Both viruses may be able to infect other domesticated mammals, and there is a real concern in the veterinary and biodefense communities about spill-over infections and the high fatality rate in humans (632 human NiVcases: 59% case fatality [7, 131] . Henipaviruses have so far not caused global epidemics, but due to a high percentage of severe outcomes, as well as lack of vaccines or treatments, HeV and NiV are designated biosafety level (BSL-4) agents [106] . They are currently not on the NATO AMed P-6 list of biological threat agents but their potential as agents for bioterrorism has been discussed [84, 89] . [62] . However, anti-vaccine movements have led to the loss of herd immunity and the reemergence of measles in many developed countries [28, 46] . Paramyxoviruses are a family of enveloped viruses with a negative-sense ss-RNA genome (mononegavirales) replicating in the cytoplasm [42] . Anti-paramyxovirus drugs. Ribavirin administered with cyclodextrin has been shown to be effective in a mouse model for measles encephalitis [69] . A very promising candidate antiviral against measles is ERDRP-0519, which has been shown effective against canine distemper virus in a ferret model [81] ; however, early resistance development has been described [74] . Favipiravir has a protective effect against Nipah virus infections in the hamster model [29] , and remdesivir inhibits a number of paramyxoviruses in vitro [88] . ddBCNAs (see sections "Poxviridae "and "Flaviviridae"; [99] ) and the plant extract naphthoquinone droserone have anti-measles activities in vitro [87] . The nucleoside-analogue 4′-azidocytidine (R1479; balapiravir) was developed to inhibit HCV [108] , paramyxoviruses, and filoviruses in vitro [63] , but showed low efficacy and high toxicity in hepatitis C patients in early clinical trials [108] . Combination treatments with antiviral compounds using different modes of action (MoA) are further increasing efficacy and, by means of individual dose reduction, allow for lower toxicity of the individual compounds. This exploits possible synergies between synthetic small molecules and natural extracts, virus-specific and broad-spectrum agents, and celltargeting compounds. The use and potential benefits of multidrug cocktails, mainly reduction of resistance mutation and toxicity through dose reduction, have been pointed out by many authors, including in the context of yellow fever treatment [103] . Examples for synergistic effects in combinations of antiviral compounds with similar or different MoA are ribavirin with vitamin A in measles infections [12] , ribavirin with favipiravir in Zika virus infections [75] , and ribavirin with mefenamic acid in infections with Chikungunya virus [126] . Antiviral drug combinations may also be a way to deal with emerging antiviral drug resistance [74] . Broad-spectrum antivirals on the other hand show significant activity against several members of the same or distinct virus families, allowing the empirical treatment of severe viral infections prior to positive diagnosis of the viral agent. Leading examples are at his point the pyrazinecarboxamide compounds T-705 (favipiravir; [2, 7, 48] ), T-1105 and T-1106, which are broad-spectrum viral RNA polymerase inhibitors, initially developed for the treatment of influenza virus, and found effective against bunyaviruses [21, 54, 59] , alphaviruses [1] , filoviruses [13] arenaviruses [125] , paramyxoviruses [29] , and flaviviruses [128] . A favipiravir resistance mechanism in influenza virus has been described [52] . Other potential broad-spectrum agents are remdesivir (GS-5734), another RNA polymerase inhibitor [137] active against filo-, corona-, and paramyxoviruses [88, 129, 130] , FGI-106 with inhibitory activity against filo-, bunya-, and flaviviruses [8] , galidesivir (BCX4430) with activity against filo-, bunya-, and flaviviruses [39, 143, 145] , N 4 -hydroxycytidine (NHC) inhibiting influenza-, paramyxo-, flavi-, corona-, as well as alphaviruses [152] , and 2′fluoro-2′-deoxycytidine (2′-FdC), which was reported to inhibit various viruses in vitro, including Borna virus, HCV, Lassa virus, certain herpes viruses, and which also inhibits influenza viruses in mice [134] . Previously thought as a onefamily-broad-spectrum compound, sofosbuvir (Sovaldi™, Soforal™) has in vitro and in vivo activity against several members of the family flaviviridae, and has most recently been shown to be effective against Chikungunya virus [40] . Natural product antivirals are single molecule natural compounds or complex mixtures of organic molecules (e.g., plant extracts) with antiviral activity. Natural product antivirals frequently exhibit broad-spectrum antiviral activity and often a single active compound cannot be identified in extracts [27] . Viral hemorrhagic fevers (VHFs) cause the highest mortality in human hosts of all known viral agents and treatment options are a serious concern both in public health and in biodefense scenarios [67] . If specific antiviral treatment options are not available, supportive care is the mainstay of clinical interventions in VHF, including hemodynamic, hematological, pulmonary and neurological support treatments. Treatment with corticosteroids, vasoactive substances, hemodialysis, and mechanical ventilation saves the patients with the worst clinical symptoms. The only currently widely available antiviral drug, ribavirin, is not approved by the FDA for intravenous application in VHF and is used under compassionate use protocols only. Intravenous ribavirin reduces mortality of HFRS if combined with hemodialysis and both morbidity and mortality in the case of Lassa fever (LassaF). Ribavirin (Copegus™, Rebetol™, Virazole® ICN/Valeant (IND)) is used for the treatment of infections with African arenaviruses (Lujo-and Lassa fever) and bunyaviruses (HFRS, Crimean-Congo fever, and Rift Valley fever). However, intravenous ribavirin does not show any benefits for the treatment of any of the VHFs caused by filoviruses, or in infections with RNA viruses causing severe encephalitis [15, 67] . Antiviral drug development is determined by the virus life cycle, both the steps of viral replication per se and the cellular processes supporting viral replication. The action of antivirals targeting a viral replication step may be augmented by an antiviral hitting a different viral target or a cell process, or secondary effects via drug metabolism, resulting in synergy. Most antivirals in the experimental pipeline are either small molecules designed from scaffolds, mostly nucleoside analogues, or natural extracts/complex organic active compounds derived from extracts. The stages of antiviral drug development begin with in silico design and go via testing in single cell types (organotypic cell lines or primary cells) to determine IC50/CC50 = SI, and complex infection models to animal models, clinical trials, and eventually regulatory approval/market. A major hindrance to antivirals development is that of many compounds that show activity in vitro only very few are effective in animal models. Development may also stop for lack of interest and funding. Human organoids/complex in vitro infection models (e.g., barrier models) may provide a bridge to predict activity in clinical trials. There are only a small number of antivirals with regulatory approval to treat virus infections, some of which have already been described to select for drug resistant strains. A number of drugs with antiviral activities which are approved for other conditions are being evaluated for repurposing, but the number of compounds currently in the experimental pipeline for clinical testing is small. Consequently, while there are treatment options, they may not be available in sufficient quantity in a biological threat situation. Therefore, research in identification, development, clinical testing and the stockpiling of approved antivirals in sufficient quantities must be a priority for the government actors put in charge of a credible response to deliberate releases of some of the biological agents discussed here. It is well known that even the threat of a biological attack would cause mass hysteria with concomitant economic disruption. Only timely preparation underlined by visible infrastructure, stockpiles of drugs and vaccines, and well-considered emergency plans will allow governments to give the necessary assurances when needed, to avoid negative outcomes [58] . Ideally, research on novel antivirals should also be a priority for research funding and pharmaceutical companies. As long as this is not the case, government funding and research in governmentfunded laboratories in collaboration with specialized university research groups organized in antiviral platforms have to step into the breach, when considerations of market performance and public health priorities are focusing resources elsewhere. 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Dissertation for the Master of Protease inhibitors targeting coronavirus and filovirus entry Acknowledgements We thank Dr Romano Silvestri for thoughtful comments and suggestions, and members of the Brancale/Bugert antiviral platform for critical reading of the manuscript. In this review article, research involving human participants and/or animals is reported and cited.Informed consent Informed consent was required as per instructions to authors of the respective publishing journals. Conflict of interest The authors declare no conflict of interest, particularly, no recommendations regarding priority development of drugs or preferred use are made, except in the context of regulatory approval.