• [...]
• From patent : FIELD OF THE INVENTION
• The invention relates generally to compounds with antiviral activity, more particularly nucleosides active against Flaviviridae infections and most particularly to inhibitors of hepatitis C virus RNA-dependent RNA polymerase.
• BACKGROUND OF THE INVENTION
  • • Viruses comprising the Flaviviridae family comprise at least three distinguishable genera including pestiviruses, flaviviruses, and hepaciviruses (Calisher, et al., J. Gen. Virol., 1993, 70, 37-43). While pestiviruses cause many economically important animal diseases such as bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV, hog cholera) and border disease of sheep (BDV), their importance in human disease is less well characterized (Moennig, V., et al., Adv. Vir. Res. 1992, 48, 53-98). Flaviviruses are responsible for important human diseases such as dengue fever and yellow fever while hepaciviruses cause hepatitis C virus infections in humans. Other important viral infections caused by the Flaviviridae family include West Nile virus (WNV) Japanese encephalitis virus (JEV), tick-borne encephalitis virus, Junjin virus, Murray Valley encephalitis, St Louis enchaplitis, Omsk hemorrhagic fever virus and Zika virus. Combined, infections from the Flaviviridae virus family cause significant mortality, morbidity and economic losses throughout the world. Therefore, there is a need to develop effective treatments for Flaviviridae virus infections.
    • The hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide (Boyer, N. et al. J Hepatol. 32:98-112, 2000) so a significant focus of current antiviral research is directed toward the development of improved methods of treatment of chronic HCV infections in humans (Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 80-85, (1999); Gordon, C. P., et al., J. Med. Chem. 2005, 48, 1-20; Maradpour, D.; et al., Nat. Rev. Micro. 2007, 5(6), 453-463). A number of HCV treatments are reviewed by Bymock et al. in Antiviral Chemistry & Chemotherapy, 11:2; 79-95 (2000).
    • RNA-dependent RNA polymerase (RdRp) is one of the best studied targets for the development of novel HCV therapeutic agents. The NS5B polymerase is a target for inhibitors in early human clinical trials (Sommadossi, J., WO 01/90121 A2, US 2004/0006002 A1). These enzymes have been extensively characterized at the biochemical and structural level, with screening assays for identifying selective inhibitors (De Clercq, E. (2001) J. Pharmacol. Exp. Ther. 297:1-10; De Clercq, E. (2001) J. Clin. Virol. 22:73-89). Biochemical targets such as NS5B are important in developing HCV therapies since HCV does not replicate in the laboratory and there are difficulties in developing cell-based assays and preclinical animal systems.
    • Currently, there are primarily two antiviral compounds, ribavirin, a nucleoside analog, and interferon-alpha (α) (IFN), which are used for the treatment of chronic HCV infections in humans. Ribavirin alone is not effective in reducing viral RNA levels, has significant toxicity, and is known to induce anemia. The combination of IFN and ribavirin has been reported to be effective in the management of chronic hepatitis C (Scott, L. J., et al. Drugs 2002, 62, 507-556) but less than half the patients given this treatment show a persistent benefit. Other patent applications disclosing the use of nucleoside analogs to treat hepatitis C virus include WO 01/32153, WO 01/60315, WO 02/057425, WO 02/057287, WO 02/032920, WO 02/18404, WO 04/046331, WO2008/089105 and WO2008/141079 but additional treatments for HCV infections have not yet become available for patients. Therefore, drugs having improved antiviral and pharmacokinetic properties with enhanced activity against development of HCV resistance, improved oral bioavailability, greater efficacy, fewer undesirable side effects and extended effective half-life in vivo (De Francesco, R. et al. (2003) Antiviral Research 58:1-16) are urgently needed.
    • Certain ribosides of the nucleobases pyrrolo[1,2-f][1,2,4]triazine, imidazo[1,5-f][1,2,4]triazine, imidazo[1,2-f][1,2,4]triazine, and [1,2,4]triazolo[4,3-f][1,2,4]triazine have been disclosed in Carbohydrate Research 2001, 331(1), 77-82; Nucleosides & Nucleotides (1996), 15(1-3), 793-807; Tetrahedron Letters (1994), 35(30), 5339-42; Heterocycles (1992), 34(3), 569-74; J. Chem. Soc. Perkin Trans. 1 1985, 3, 621-30; J. Chem. Soc. Perkin Trans. 1 1984, 2, 229-38; WO 2000056734; Organic Letters (2001), 3(6), 839-842; J. Chem. Soc. Perkin Trans. 1 1999, 20, 2929-2936; and J. Med. Chem. 1986, 29(11), 2231-5. However, these compounds have not been disclosed as useful for the treatment of HCV. Babu, Y. S., WO2008/089105 and WO2008/141079, discloses ribosides of pyrrolo[1,2-f][1,2,4]triazine nucleobases with antiviral, anti-HCV, and anti-RdRp activity.
• [...]

A "small molecule" antiviral compound has been shown to protect rhesus monkeys against lethal Ebolavirus disease, even when given up to three days after virus inoculation.

The compound, called GS-5734, is a nucleoside analog. After uptake into cells, GS-5734 is converted to a nucleoside triphosphate (illustrated, bottom panel) which is incorporated by the viral RNA dependent RNA polymerase as it copies the viral genome. However, the nucleoside is chemically different from ATP (illustrated, top) and no further nucleotides can be incorporated into the growing RNA strand. RNA synthesis ceases, blocking production of infectious virus particles.

In cell culture GS-5734 inhibits viral replication at micromolar concentrations, in a variety of human cell types including monocyte-derived macrophages, primary macrophages, endothelial cells, and a liver cell line. The drug inhibits replication of several strains of Zaire ebolavirus, including Kikwit and Makona (from the West African outbreak); Bundibugyo ebolavirus, and Sudan ebolavirus. It also inhibits replication of another filovirus, Marburg virus, as well as viruses of different families, including respiratory syncytial virus, Junin virus, Lassa fever virus, and MERS-coronavirus, but not chikungunya virus, Venezuelan equine encephalitis virus, or HIV-1.

The RNA dependent RNA polymerase of Ebolaviruses has not yet been produced in active form, so the authors determined whether GS-5734 inhibits a related polymerase from respiratory syncytial virus. As predicted, the compound was incorporated into growing RNA chains by the enzyme, and caused premature termination.

Typically tests of antiviral candidates begin in a small animal, and if the results are promising, proceed to nonhuman primates. While a mouse model of Ebolavirus infection is available, the serum from these animals degrades GS-5374. Consequently a rhesus monkey model of infection was used to test the compound.

After intravenous administration of GS-5374, the NTP derived from it was detected in peripheral blood mononuclear cells, testes, epididymis, eyes, and brain within 4 hours. All 12 monkeys inoculated intramuscularly with Zaire ebolavirus died by 9 days post-infection. In contrast, all animals survived after administration of GS-5374 2 or 3 days after virus inoculation. These animals also had reduced virus associated pathology as measured by liver enzymes in the blood and blod clotting. Viral RNA in serum reaches 109 copies per milliliter on days 5 and 7 in untreated animals, and was undetectable in 4 of 6 treated animals.

It is likely that resistant viruses can be obtained by passage in the presence of GS-5734; whether such mutant viruses emerge early in infection, and at high frequency, is an important question that will impact clinical efficacy of the drug. The authors did not detect changes in the viral RNA polymerase gene that might be assoicated with resistance, but further work is needed to address how readily such mutants arise.

These promising results have lead to the initiation of a phase I clinical trial to determine whether GS-5734 is safe to administer to humans, and if the drug reaches sites where Ebolaviruses are known to replicate. However, determining the efficacy of the compound requires treatment of acutely Ebolavirus infected humans, of which there are none. It might be of interest to determine the ability of GS-5734 to clear persistent virus from previously infected individuals.

You can bet that GS-5734 has already been tested for activity against Zika virus.

Broad-spectrum coronavirus antiviral drug discovery

Allison L. Totura ORCID Icon & Sina Bavari

Pages 397-412 | Received 16 Aug 2018, Accepted 07 Feb 2019, Published online: 08 Mar 2019

1. Introduction

• 1.1. Coronaviridae phylogeny and emergence
  • • Highly pathogenic coronaviruses SARS-CoV and MERS-CoV recently emerged into human populations, but other human coronaviruses (HCoVs) including HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 are estimated to have circulated in human populations for hundreds of years, causing mild respiratory illness to which approximately 5–30% of ‘common colds’ are attributed [7,8]. Within the Coronaviridae family (order Nidovirales) four genera are recognized: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. The six HCoVs (Table 1) currently identified belong to the genera alphacoronavirus (HCoV-229E and HCoV-NL63) and betacoronavirus (SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-HKU1). Gammacoronaviruses and deltacoronaviruses have no known viruses that infect humans, but contain important agricultural pathogens of livestock. Epizootic coronaviruses in animals cause a wide range of disease signs resulting from respiratory, enteric, and neurological tissue tropism. Although HCoVs cause primarily respiratory symptoms, the potential for a wide range of severe disease symptoms in humans caused by infection by future emergent coronaviruses cannot be excluded. Despite the severity and diversity of coronavirus disease signs and symptoms affecting a large number of important livestock species as well as humans, there are no proven therapies that specifically target CoVs.
    • In addition to CoVs known to cause disease in humans and livestock, a large number of highly diverse coronaviruses have been identified based on sequences collected from sampling bat species. Bat coronavirus (BatCoV) sequences recovered from sampling sites on different continents (Asia, Europe, Africa, North America) over the last decade contain putative BatCoVs from diverse branches of the betacoronavirus and alphacoronavirus phylogenetic tree [9–12]. Importantly, the two coronaviruses that cause the most severe disease in humans, SARS-CoV and MERS-CoV, emerged from BatCoVs that were not previously recognized to infect humans or animals other than bats [12–14]. Recent studies suggest that BatCoV-SHC014 and BatCoV-WIV1 are genetically similar to SARS-CoV and enter cells using human receptors [10,15,16]. Similarly, BatCoV-HKU4 and BatCoV-HKU5 are MERS-like BatCoVs that may also be circulating in bat populations, and some MERS-like BatCoVs may also be able to recognize human host cell receptors [17–19]. Such BatCoVs are now called ‘pre-emergent’, because they may have the potential to emerge into human populations. Importantly, therapeutics that rely on host memory responses to target CoV infection are often not effective against pre-emergent BatCoVs that differ antigenically from known HCoVs, highlighting the need for pan-coronavirus therapeutics that target conserved mechanisms utilized by HCoVs and BatCoVs [15].
    • SARS-CoV and MERS-CoV likely evolved from BatCoVs that infected other intermediate host animals in closer proximity to humans, resulting in SARS and MERS outbreaks (Figure 1) [20,21]. SARS-CoV was detected in small animals like civets and raccoon dogs that were present in live-animal markets [20]. Evolution of SARS-CoV evidenced by genomic sequence differences between zoonotic SARS-CoV strains infecting civets and epidemic SARS-CoV isolates likely resulted from viral adaptation, which is thought to be required for emergent CoVs to become transmissible from person-to-person [22,23]. MERS-CoV has been identified in dromedary camels, and is now known to be endemic in camel populations in the Middle East and Sub-Saharan Africa.
    • Emergence of coronaviruses into human populations, including highly pathogenic viruses like SARS-CoV and MERS-CoV, has occurred by spillover from bat reservoir hosts into intermediate hosts. The intermediate hosts during the 2003 SARS-CoV epidemic included civets and other small carnivore species located in wet animal markets. MERS-CoV has been identified in dromedary camels, and is particularly associated with active infection of juvenile camels. Novel emerging CoVs may occur in the future via infection from bat populations into other intermediate animal hosts. Additional evidence from BatCoVs indicates that pre-emergent CoVs with the ability to directly infect human cells may be poised for emergence into human populations. Based on prior research from SARS and MERS outbreaks, animal workers that have contact with intermediate animal host species and health-care workers that are exposed to nosocomial CoV infections are at increased risk of highly pathogenic coronavirus transmission. More severe disease in SARS and MERS cases resulted in patients that were over the age of 65 or had comorbidities such as obesity, heart disease, diabetes, renal disease, or hypertension.
• 1.2. Epidemiological features of CoV outbreaks
  • • Research on coronavirus-specific antiviral drugs has focused primarily on highly pathogenic coronaviruses SARS-CoV and MERS-CoV due to the major potential consequences of pandemics resulting from these pathogens. SARS-CoV and MERS-CoV did not transmit as efficiently from person-to-person compared to other respiratory pathogens like seasonal influenza, but mortality in patients of SARS (approximately 10%) and MERS (approximately 35%) greatly exceeded typical seasonal influenza case-fatality rates (2.4 deaths per 100,000 cases) [24]. Air travel facilitated these CoVs in seeding outbreaks in regions distant from initial localized viral spread: SARS-CoV emerged in the Guangdong province of southeastern China in late 2002, and then spread rapidly to other parts of the world, with outbreaks in major cities including Beijing, Hong Kong, Singapore, and Toronto, resulting in one of the first pandemics of the twenty-first century [5]. Since MERS-CoV emerged in 2012, MERS cases have been exported from the Middle East to Europe, North America, Africa, and South East Asia, including a major outbreak of 186 people in the Republic of Korea in 2015 [25]. Superspreaders (individuals that transmit SARS-CoV or MERS-CoV to a large number of people) played an important role in initiating and perpetuating CoV outbreaks: the 2015 MERS-CoV outbreak in the Republic of Korea started from a single traveler case, and just five cases were responsible for more than 80% of the transmission events [25]. SARS-CoV and MERS-CoV were transmitted by close contact, with known outbreaks occurring in hotels, apartment buildings, and hospitals or health-care centers. Health-care workers, in particular, were at risk for infection by SARS-CoV and MERS-CoV at high rates [5,26]. In addition, animal workers were more likely to come into contact with CoV infected animals, and a large percentage of MERS patients had contact with intermediate host camels [26,27]. Analysis of severe SARS or MERS disease identified disproportionately high case-fatality rates in elderly patients (age > 65 years) and patients with pre-existing comorbidities including diabetes, heart disease, hypertension, and renal disease [4,26,28]. Based on these epidemiological considerations, pan-coronavirus therapeutics are needed to i) protect populations with occupational risk for transmission of CoVs, ii) protect populations with susceptibility to severe disease from CoVs, iii) work in concert with public health measures like quarantine and contact tracing, and iv) be rapidly deployable to geographically distant regions from local HCoV epidemics.

2. In vitro systems for pan-coronavirus drug discovery

• 2.1. Reverse genetics systems
  • • Advances in the study of highly pathogenic coronaviruses and potential pan-coronavirus drug candidates partially depend on the technology to genetically manipulate CoVs to probe mechanisms of viral pathogenesis and antiviral drug activity. Reverse genetics systems synthetically generate viruses from known viral sequences [29]. In situations where clinical isolates of infectious material are unavailable due to restriction for collecting patient samples, shipping infectious materials, or availability of containment laboratories, reverse genetics systems provide essential research materials for studies on viral pathogenesis and model development. Prior to the SARS pandemic, robust reverse genetics systems to manipulate the genomes of CoVs had already been developed by systematic assembly of cDNA cassettes into full-length infectious clones, allowing precise and targeted genetic manipulation of viral genes [30,31]. Infectious clones allow the creation of near-homogenous viral stocks, whereas traditional viral stocks are prepared by amplification of infectious material in cell culture over many passages. Strategies to build reverse genetics systems were rapidly applied to both SARS-CoV and MERS-CoV within the first year of identification of these viruses [32,33].
    • In addition to reconstructing epidemic strains of CoVs, reverse genetic systems allow targeting of mutations to specific viral genes and assembly of viruses when infectious material is not available. As an example, the ability to isolate mutations in particular genes was applied to studies of the spike (S) glycoprotein of SARS-CoV, while maintaining the isogenic background of the viral replicase and other structural proteins. Mutations from zoonotic, early, middle, and late epidemic strains of the SARS-CoV outbreak were inserted into the S glycoprotein of the epidemic strain of SARS-CoV (Urbani) to determine the effect of evolution on viral entry into human cells as well as viral pathogenesis in rodent and primate models of disease [34–36]. By targeting mutations to a specific viral gene, reverse genetics systems allow researchers to probe cause-and-effect relationships of host pathogenic responses to viral genetic changes. In addition, reverse genetics techniques were utilized to study pre-emergent BatCoV strains: recombinant versions of BatCoV-HKU3, BatCoV-WIV1, and BatCoV-SHC014 (SARS-CoV-like), as well as BatCoV-HKU5 (MERS-CoV-like) viruses, were generated and used for in vitro and in vivo models of emerging coronavirus disease [15,16,37,38]. Panels of zoonotic, epidemic, and pre-emergent viruses synthesized by reverse genetics techniques encompass a diverse array for use in high-throughput platforms for the discovery of countermeasures that are effective against the broadest range of CoVs without being reliant on procuring clinical isolates.
• 2.2. Cell-based systems
  • • Like all other viruses, coronaviruses require host cell machinery to replicate their genomes, produce progeny virions, and cause disease. Cell lines require expression of the host cell receptor as well as expression of necessary proteases to facilitate viral entry, although additional host factors may also be important for infection. The S glycoprotein of coronaviruses, the main determinant of host cell attachment and viral entry, is not well conserved between HCoVs. Most human coronaviruses use different host cell receptors for viral entry, and may also require different host cell proteases that allow fusion of viral and cellular membranes (Table 1) [39]. Although all known HCoVs have viral tropism targeted at the human respiratory tract, lung cell lines infected by a broad range of HCoVs have not been defined. A key feature of SARS-CoV and MERS-CoV is that highly pathogenic coronaviruses grow to higher viral titer on a wider range of cell lines than the other mildly pathogenic coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 [40–44]. High throughput approaches to screen compound libraries for targeted activity against coronaviruses have been underdeveloped and limited in the number of viral strains used [45–52]. Infection of panels of cell lines from various animal species with HCoVs and BatCoVs informs on the potential host range of the pathogen, and may help to identify susceptible mammalian host involved in viral spread. However, productive infection of cell lines does not always translate to recapitulation of pathogenesis in the same animal model that the cell lines are derived from, which may be due to receptor availability in live animals or other biological and immunological factors during infection.
    • Infection of pseudostratified airway epithelium cultures from primary cells of the lung provides a cell culture model that simulates infection of cells in a more complex environment more closely resembling the human respiratory tract. Known as Human Airway Epithelia (HAE) or Normal Human Bronchial Epithelia (NHBE) cells, these cultures can be infected with all of the HCoVs identified thus far, including SARS-CoV and MERS-CoV, providing a potential platform to screen novel CoVs for emergence into human populations [42,53–56]. However, several limitations are associated with HAEs including difficulty in collection due to the scarcity of donors and difficulty in maintenance because of limited capacity for cell divisions. HAEs may be sourced from donors with a preexisting disease state, which could influence viral pathogenesis. In addition, because of the genetic variability of donors, HAEs cultures often differ in expression levels of genes crucial to infectivity, including the various host receptors for HCoVs, which leads to high variability in the infectivity of these cultures. Importantly, these in vitro methods fail to capture more complex viral interactions that occur with an intact immune system including infiltration of proinflammatory cells that may promote and contribute to ARDS in the most severe forms of SARS and MERS. Organ-on-a-chip models in development may provide the next generation of in vitro models that could capture these critical interactions between respiratory cells and immune cells, but infection of these novel culture systems has not been reported with coronaviruses [57].

3. In vivo systems for pan-coronavirus drug discovery

• 3.1. Small animal models for pan-coronavirus drug discovery
  • • Following the emergence of SARS-CoV in 2003, small animal model development was initiated by inoculating animals with human epidemic isolates of SARS-CoV that replicated in mice, hamsters, guinea pigs, and ferrets, but only ferrets exhibited disease signs resulting from infection (Table 2) [60–63]. SARS-CoV replicated in laboratory strains of mice, but did not cause disease signs, and virus was rapidly cleared from the lung in these models [60]. Serial passage of SARS-CoV in the lungs of mice by multiple research groups resulted in mouse-adapted SARS-CoV strains that caused lethal lung disease in wild-type mouse intranasal (IN) infection models [64,65]. Mouse-adapted SARS-CoV MA15 is the best characterized small animal model of CoV infection, and has been used to test several pan-coronavirus drug candidates [66,67]. The benefit of mouse-adapted models of SARS-CoV in wild-type inbred mouse strains includes reproducible susceptibility to disease assayed by survival, weight loss, and whole body plethysmography of individual mice as well as quantification of infiltrating cells, viral titers, histopathology, and transcriptomics and proteomics changes in target organs. To evaluate pathogenesis of emergent viruses in vivo, zoonotic SARS-CoV and pre-emergent BatCoV mutations have been incorporated into the SARS-CoV MA15 backbone, providing novel animal models for viruses that have the potential to emerge into humans [15,35]. Additional valuable avenues of research on variables known to impact severe CoV disease in the MA15 models of SARS-CoV include age, dose, and host genetic contributions to disease phenotypes [68,69]. The greatest limitations of SARS-CoV mouse-adapted models for drug discovery are the incorporation of mutations in the SARS-CoV genome (particularly for testing antiviral drugs that target viral genes with mutations) and acknowledged differences between mouse and human immune responses.
    • Unlike SARS-CoV, human clinical strains of MERS-CoV (Table 3) did not replicate in mice, hamsters, or ferrets, and further studies of the host receptor identified critical amino acid residue differences between the MERS-CoV receptor, DPP4, in laboratory animal model species that prevented entry into cells compared to human DPP4 [70–72]. MERS-CoV infection of rabbits resulted in viral replication in the upper respiratory tract, but no clinical disease signs that reflect more severe MERS-CoV disease symptoms were reported, although the model has been used for limited testing of MERS-CoV antiviral therapeutics [73,74]. However, due to the utility of the mouse-adapted SARS-CoV model, a mouse model continued to be pursued, and adenovirus-vectored transient expression of the human DPP4 receptor in mice and subsequent replication of MERS-CoV in these mice determined that MERS-CoV replication was dependent on human DPP4 expression in rodents [75]. Transgenic expression of human DPP4 in mice allowed MERS-CoV replication in mice, but resulted in lethal brain disease not representative of MERS-CoV infection in humans [76,77]. Replacing mouse DPP4 with the expression of human DPP4 in mice resulted in a humanized DPP4 mouse model that allows MERS-CoV replication within the lung and some MERS-associated lung pathology, but no lethal disease [78]. Similarly, knock-in expression of the human DPP4 exons 10–12 in mice allowed viral replication but no overt MERS disease signs [79]. Serial passage of MERS-CoV in the lungs of these mice resulted in a MERS-MA virus that caused lethal disease in mice, including weight loss and severe lung pathology [79]. Identification of amino acids in mouse DPP4 that prevent entry of MERS-CoV into mouse cells led to the rational design of the mouse DPP4 gene edited by CRISPR/Cas9 to express two human DPP4 mutations (288/330 DPP4) [80]. 288/330 DPP4 mice supported viral replication without severe disease or lethality, but serial passage of MERS-CoV (generating a mouse-adapted virus called MERS-15) produced lethal disease in the 288/330 DPP4 mice [80]. Although many of the same metrics of disease to MA15-SARS-CoV are available in the MERS-15 or MERS-MA models for drug discovery of coronavirus antivirals, an additional drawback is the requirement for both mouse-adapted virus and a modified rodent host. Despite these limitations, mouse models of adapted SARS-CoV and MERS-CoV are currently the best-developed models of highly pathogenic coronavirus infection available for pan-coronavirus drug discovery.
• 3.2. Primate models for pan-coronavirus discovery
  • • Small animal models have been more thoroughly developed as models of SARS-CoV and MERS-CoV infection, due to ease of manipulation with rodents and increased costs and ethical concerns associated with nonhuman primates (NHPs). However, NHP model development of highly pathogenic coronavirus infections is pivotal in the evaluation of pan-coronavirus therapeutics, because host immune responses from NHPs share greater homology with humans compared to rodents, and may more accurately indicate immunological biomarkers of severe disease needed to evaluate pan-coronavirus therapeutics. Disease signs are observed in NHP models of infection without adaptation of CoVs required in rodent models of SARS-CoV and MERS-CoV. Both SARS-CoV and MERS-CoV isolates from humans replicate in NHPs, indicating conservation of important aspects to coronavirus-induced diseases including respiratory tract biology, receptor homology, and pattern of expression of host receptor and proteases.
    • SARS-CoV infection of common laboratory primate species by the IT route including African green monkeys, rhesus macaques, cynomolgus macaques, and common marmosets, resulted in disease signs with differing degrees of severity, but none were reflective of the lethal SARS disease seen in humans (Table 2) [81–85]. Commonly reported disease signs included lethargy and increased respiratory rates following SARS-CoV infection in multiple NHP models of infection, but other acute signs of illness including fever or dyspnea were infrequently reported. The most severe disease phenotypes were observed in the histopathology of the lungs at acute times post-infection (3–6 days) with typical findings of pulmonary lesions and pneumonitis and occasional observations of diffuse alveolar damage [86]. Although none of the NHP species that were infected with SARS-CoV developed lethal respiratory disease reflective of SARS patients, NHP models did recapitulate enhanced disease in aged NHPs, including aberrant innate immune signaling programs [87]. However, lack of emulation of human SARS disease was never resolved in an NHP model that could be used for consistent evaluation of therapeutic candidates against SARS-CoV.
    • MERS-CoV infection of nonhuman primate models was reported, with the best characterized NHP models of MERS-CoV infection in rhesus macaques and common marmosets (Table 3) [88–91]. Administering MERS-CoV via the IT route to either rhesus macaques or common marmosets resulted in mild disease with very few observable disease phenotypes [89,91]. However, infecting rhesus macaques or common marmosets by multiple concurrent routes (IN, IT, oral, and ocular) resulted in moderate disease in rhesus macaques, but more severe disease in marmosets [88,90]. Infecting NHPs by multiple routes likely caused a systemic infection potentially not representative of human MERS-CoV infection. Although the marmoset is currently the best developed NHP model of MERS-CoV disease, discrepancies in disease severity of marmosets infected by multiple routes and marmosets infected by the IT route illuminate potential difficulties with using this model for drug development studies. Small size and fragility of marmosets precluded serial blood draws on multiple days following infection, and may confound experimental outcomes resulting from MERS-CoV infection or treatment [92]. Absence of reproducible clinical disease signs like fever, respiratory distress, or lethality that recapitulated human symptoms of SARS or MERS indicates that currently developed models present significant challenges to testing of pan-coronavirus antivirals in NHP models of infection.

4. Pan-coronavirus antivirals

• 4.1. Targeting CoV nonstructural proteins
  • • Coronavirus nsps are highly conserved components of the coronavirus lifecycle that mediate viral replication including 3C-like protease (3CLpro), papain-like Protease (PLpro), and RNA-dependent RNA polymerase (RdRp). The CoV RdRp replicates the viral RNA genome and generates viral RNA transcripts, essential functions that cannot be performed by cellular polymerases. Another essential element of the CoV lifecycle is proteolytic processing of viral polyproteins into functional nsps by two viral proteases, the 3CLpro and PLpro. In addition to polymerase and protease functions, other essential functions performed by the nsps of CoVs include immune antagonism, double membrane vesicle organization, scaffolding for replication complex formation, nucleic acid binding, helicase activity, and viral RNA proofreading which may be future targets of coronavirus specific antiviral drug discovery [93].
    • 4.1.1. GS-5734
      • • GS-5734 is a small molecule nucleoside analog that has demonstrated antiviral activity in vitro against several viral families of emerging infectious diseases including Filoviridae, Pneumoviridae, Paramyxoviridae, and Coronaviridae [45,66,94]. Efficacy of GS-5734 in post-exposure treatment of Ebola virus-infected nonhuman primates led to GS-5734 inclusion in an experimental therapy for an infant survivor of Ebola virus disease [45,95]. These encouraging results demonstrated that GS-5734 may be an acceptable therapeutic intervention to lethal viral disease, even days after viral exposure, and tolerated by patients with viral diseases that were previously treated primarily with supportive care. Based on activity against MERS-CoV within a larger panel targeting lethal viruses from multiple viral families, additional studies demonstrated that GS-5734 decreased viral titers and viral RNA in in vitro models of both SARS-CoV and MERS-CoV infection of HAEs [66]. Additionally, GS-5734 had similar effects against other diverse CoVs including HCoV-NL63 and Mouse Hepatitis Virus (MHV, betacoronavirus group 2a) [66,96]. Importantly, GS-5734 inhibited replication of pre-emergent BatCoVs including BatCoV-HKU5, BatCoV-HKU3, BatCoV-SHC014, and BatCoV-WIV1 [66]. Activity in vivo against CoVs was supported by ameliorated disease signs (weight loss, lung viral titers) in MA15 SARS-CoV infected mice treated prophylactically or therapeutically with GS-5734 [66]. Although viral resistance to GS-5734 was shown experimentally in vitro, mutations to conserved motifs in SARS-CoV and MHV resulted in decreased viral fitness in vitro and in vivo [96]. Altogether, in vitro and in vivo data support GS-5734 development as a potential pan-coronavirus antiviral based on results against several CoVs, including highly pathogenic CoVs and potentially emergent BatCoVs.
    • 4.1.2. Lopinavir–Ritonavir
      • • Lopinavir–ritonavir was initially developed as an HIV-1 protease inhibitor but in vitro activity also targeted SARS-CoV nonstructural protein 3CLpro [97]. During the SARS-CoV epidemic, lopinavir–ritonavir combination therapy with ribavirin in SARS patients was associated with decreased viral load and decreased adverse clinical outcomes of death or ARDS when compared with historical control cases [98]. Shortly after the emergence of MERS, high throughput screening approaches of known antiviral compounds identified lopinavir activity against MERS-CoV in vitro [51]. Oral treatment with lopinavir-ritonavir in the marmoset model of MERS-CoV infection resulted in modest improvements in MERS disease signs, including decreased pulmonary infiltrates identified by chest x-ray, decreased interstitial pneumonia, and decreased weight loss [92]. MERS patient case reports of treatment regimens including lopinavir-ritonavir were associated with positive disease outcomes including defervescence, viral clearance from serum and sputum, and survival [99,100]. Based on in vitro and in vivo activity against MERS-CoV, a clinical trial has been designed using combination of lopinavir-ritonavir and IFN-β1b therapies in hospitalized MERS patients in Saudi Arabia [101].
    • 4.1.3. Ribavirin
      • • Ribavirin is a guanosine analog with in vitro activity against a large number of highly lethal emerging viruses. Mechanistically, ribavirin inhibits RNA synthesis by viral RdRp as well as inhibits mRNA capping. However, studies demonstrated that while SARS-CoV, MERS-CoV, and HCoV-OC43 were sensitive to ribavirin in vitro, doses that significantly inhibited CoV replication exceeded ribavirin concentrations attainable by typical human regimens [46,102–104]. Recently, it was demonstrated that excision of ribavirin nucleoside analogs by conserved coronavirus proofreading mechanisms likely accounted for decreased in vitro efficacy of ribavirin than expected [105]. Additional in vivo testing of ribavirin in mouse models found limited activity against MA15 SARS-CoV by ribavirin alone, and suggested that ribavirin treatment enhanced SARS disease signs [65,106]. However, combination treatment of ribavirin and type I Interferons in primate models improved MERS disease signs [107]. Ribavirin has been given as part of treatment regimens for SARS and MERS patients, but meta-analyses of case studies have found limited (if any) efficacy of ribavirin in treating patients with highly pathogenic coronavirus respiratory syndromes [108,109].
• 4.2. Targeting CoV structural and accessory proteins
  • • Coronavirus structural proteins compose the virion, including the Spike (S) glycoprotein, Envelope (E) protein, Membrane (M) protein, and the Nucleopcapsid (N) protein (Figure 2). These proteins also perform important functions in the viral life cycle: S is the main determinant of cell tropism, host range, and viral entry; E facilitates viral assembly and release, and has viroporin activity; M maintains the membrane structure of the virion; and N encapsidates the viral RNA genome. Although most of these functions are essential to viral infection, CoVs tolerated E deletion and remained replication competent, but viral fitness was impaired [110]. Unfortunately, while structural protein functions are similar between CoVs, protein identity is less conserved than with nsps, making the development of pan-coronavirus therapeutics directly targeting structural proteins problematic. Genes encoding structural and accessory proteins are interspersed at the 3ʹ end of the coronavirus RNA genome (Figure 2). Deletion of accessory protein genes using reverse genetics systems demonstrated that these proteins were not essential for viral replication, but impacted viral replication or viral fitness in vitro and in vivo [111,112]. However, unlike nonstructural proteins or structural proteins, significant variation in number, function, and sequence of accessory proteins between closely related viruses makes accessory proteins poor targets for pan-coronavirus therapeutic approaches.
    • 4.2.1. Monoclonal antibody therapeutics
      • • Monoclonal antibodies (mAbs) have potential utility in combating highly pathogenic viral diseases, by prophylactic and therapeutic neutralization of structural proteins on virions. In vitro and in vivo approaches by multiple groups identified mAbs targeting either SARS-CoV or MERS-CoV that inhibited viral replication and ameliorated SARS and MERS disease in animal models [74,78,89,113]. As an example, antibodies generated against the S glycoprotein of MERS-CoV inhibited viral replication when administered 24 h prior to infection, as well as 24 h postinfection in a humanized DPP4 mouse model [78]. In general, mAbs that were effective against CoV infection in animal models targeted the highly variable S glycoprotein, but these mAbs lack cross-protection against other related CoVs [114]. Monoclonal antibodies developed against SARS-CoV, MERS-CoV, or other emerging CoVs may require separate formulations for each virus due to differences in the targeted antigen. For example, mAbs targeted against S from 2003 SARS-CoV isolates failed to neutralize closely related BatCoV-SHC014 and only some mAbs neutralized BatCoV-WIV1 [15,16]. In general, mAbs target specific epitopes, and viruses avoid neutralization by accruing mutations in the targeted epitope that allow viral escape from mAb therapy. Pre-clinical and clinical mAb formulations may include a cocktail of multiple mAbs that target different epitopes to ensure that viruses cannot escape neutralization. However, SARS-CoV S tolerated mutations in multiple epitopes allowing escape from neutralization from multiple mAbs, and the introduction of mAb escape mutations enhanced pathogenesis of the virus in some animal models [115]. In sum, efficacious monoclonal antibody therapy against highly pathogenic coronaviruses may require several mAbs targeting conserved epitopes and rigorous testing is required to demonstrate that viral evasion of mAbs does not result in enhanced virulence.
• 4.2.2. Coronavirus vaccines
  • • Vaccines have long been considered the gold standard for infectious disease prevention and eradication targeted at human populations as well as conferring the benefits of long-lived immune protection for the individual. Zoonotic pathogens like coronaviruses emerge from animal reservoir species, thus vaccination strategies are unlikely to lead to eradication while the virus continues to circulate in reservoir hosts. One Health approaches to solving the problems of emerging infectious diseases consider the environment and animal health, as well as human health [116]. For example, vaccination strategies targeting the camel intermediate host of MERS-CoV have been developed, which may work to repress viral replication in camels, preventing MERS-CoV transmission to humans [117].
    • In human infections of highly pathogenic coronaviruses SARS-CoV and MERS-CoV, the most vulnerable populations are patients over the age of 65 and patients with comorbidities, and design of efficacious vaccines for patients in these groups is difficult. Vaccine formulations that have been developed against SARS-CoV not only fail to protect animal models of aged populations, but also result in immunopathology in younger populations, where SARS disease is enhanced in vaccinated groups that are subsequently challenged with SARS-CoV [118,119]. In addition, vaccines generate memory immune responses to specific pathogens, and no vaccine formulations have been developed that are effective against multiple CoVs. Due to the diversity of BatCoVs, it seems unlikely that current therapeutic strategies targeting specific SARS-CoV or MERS-CoV antigens will be efficacious against future coronaviruses that emerge into the human population. Vaccines formulated against the SARS-CoV epidemic antigens do not offer effective protection against SARS-like BatCoVs that are currently circulating in bat populations [15]. Rather, a modular vaccine platform that can be rapidly adjusted for newly emergent viral antigens in potentially pandemic CoVs may be able to provide emergency vaccine coverage against emergent viral strains.
• 4.3. Targeting host factors essential for CoV infection
  • • 4.3.1. Host factor modulation
      • • Antiviral compounds that specifically target viral proteins may result in viral escape by mutation in the targeted viral proteins, as has been described with monoclonal antibodies and GS-5734 [96,115]. However, targeting conserved host mechanisms utilized by multiple coronavirus as an essential part of the viral life cycle is an approach to pan-coronavirus drug development where viral escape by mutation is less likely. Several groups have attempted to inhibit host proteases (including furin, cathepsins, and TMPRSS2) that process viral S glycoproteins at the cell surface during viral entry [50,120–122]. However, due to variation in viral S glycoproteins among different CoVs and variation in the host proteases required for viral entry (Table 1), combinations of protease inhibitors would be required for pan-coronavirus treatment regimens, particularly for emergent novel CoVs where host protease requirements have not been evaluated. Additional host targets with less established mechanisms of activity include Cyclosporins, a class of cyclophilin inhibitors with antiviral activity against coronaviruses in addition to immunosuppressive properties [123]. Non-immunosuppressive derivatives of cyclosporins like alisporivir retained antiviral properties in vitro against coronaviruses including SARS-CoV, MERS-CoV, but were not effective against SARS-CoV in mouse models of infection [124].
    • 4.3.2. Host immune modulation
      • • Interferons (IFNs) are a class of immunomodulatory compounds produced by host cells in response to detection of pathogen-specific motifs, resulting in IFN secretion that affects not only the stimulated cell, but also neighboring cells. Early in infection, IFN stimulation results in altered cellular transcriptional programs, leading to an antiviral state characterized by the activation of a large set of host genes with partially defined antiviral functions [125]. Based on these potentially beneficial immunomodulatory properties in the context of infections, IFNs have been used for the treatment of emerging viral infections where no specific antiviral drugs yet exist, with the greatest benefits resulting from administration very early following infection. For SARS-CoV and MERS-CoV, Type I IFNs were effective at decreasing viral replication in vitro and showed additional benefits in in vivo primate models of infection [103,104,107,126,127]. IFNs used in the treatment of SARS and MERS patients often occurred in combinatory therapies with other drugs including ribavirin and lopinavir-ritonavir, although potential beneficial effects of therapies were limited, potentially due to administration at later times postinfection [108]. Upstream stimulants of IFN induction, including polyI:C resulted in IFN signaling cascade activation, with demonstrated effectivity in vitro and in vivo against SARS-CoV and MERS-CoV [67,75]. Additional immunomodulatory compounds that regulate the expression of innate immune genes have been suggested as potential therapeutics for highly pathogenic coronaviruses; however, compounds that modulate the host response require significant testing in the most rigorous animal models before therapeutic applications could be pursued. For example, corticosteroids (methylprednisolone) were given as treatment during the SARS and MERS epidemics due to immunomodulatory effects that suppress inflammatory responses with no perceived benefit and possible deleterious effects [108,109].

5. Expert opinion

• In response to outbreaks of previously unrecognized respiratory syndromes characterized by atypical pneumonia in 2003 and 2012, collaborative new research programs were quickly established that identified the etiologic agents involved as highly pathogenic coronaviruses SARS-CoV and MERS-CoV. These coronaviruses may have the potential to cause devastating pandemics due to unique features in virus biology including rapid viral replication, broad host range, cross-species transmission, person-to-person transmission, and lack of herd immunity in human populations. SARS-CoV and MERS-CoV were contained by diligent enforcement of public health measures that limited viral spread to approximately 10,000 cases total for both SARS and MERS since 2003. However, the threat of SARS-CoV, MERS-CoV, or an as-yet unknown BatCoV that causes severe disease in humans makes antiviral therapeutics that broadly target coronaviruses a highly desirable commodity to ensure global public health. The current challenge is to produce medical countermeasures that can protect vulnerable populations against known coronaviruses like SARS-CoV and MERS-CoV, but that are also effective against novel highly pathogenic coronaviruses that may emerge from animal reservoir hosts.
• While SARS-CoV and MERS-CoV were rapidly identified following clinical reports of novel atypical pneumonia, progress in developing effective antivirals for SARS-CoV and MERS-CoV has been impeded by several factors. A key finding from our review of the literature is that current animal models for highly pathogenic coronaviruses SARS-CoV and MERS-CoV are not adequate to support advanced development of antiviral therapeutics. MERS-CoV continues to circulate on the Arabian Peninsula, providing the opportunity to investigate some treatments of MERS in clinical trials. However, future emerging coronaviruses may require use of the FDA Animal Efficacy Rule for Investigational New Drugs (INDs). INDs must fulfill the Animal Efficacy Rule criteria of i) reasonable safety for initial use in humans, ii) pharmacological data that support reasonably well-understood mechanism of activity against the pathogen, and iii) efficacy in animal models with disease signs representative of clinical illness in humans (including one non-rodent model). While the vigorous pursuit of small animal models has been successful in generating rodent models that recapitulate severe SARS and MERS disease signs (including morbidity and mortality), progress in generating additional animal models has lagged, particularly in primate models of SARS-CoV or MERS-CoV infection. Past strategies for experimental treatment regimens primarily relied on combination therapies with approved drugs known to have acceptable safety profiles and broad-spectrum antiviral activity including IFNs, ribavirin, and corticosteroids. However, analyses of data returned from these treatments indicated that most regimens were not effective in treating SARS and MERS patients. With sufficient investment in the development of drug discovery pipeline model systems, pan-coronavirus targets based on supportive in vitro and in vivo evidence for effective treatments during the current MERS outbreaks and future outbreaks of emergent CoVs (Figure 3).
• Currently, the state of pan-coronavirus drug discovery is not structured to provide adequate pre-clinical therapeutics to combat emerging CoV pathogens. A diverse array of coronaviruses is needed that includes epidemic isolates of SARS-CoV and MERS-CoV, zoonotic viruses isolated from intermediate reservoir hosts, pre-emergent CoVs from bats, and clinical isolates of mildly pathogenic HCoVs. In vitro testing in compatible cell lines uses high throughput screening to identify novel targets that mitigate replication of coronaviruses. Targets identified by in vitro methods can be confirmed using human airway epithelial cultures. Based on these results, lead targets will be tested in small animal models and nonhuman primate models of highly pathogenic coronavirus infections that recapitulate signs of human SARS or MERS patients. Our analysis identified several key weaknesses in both in vitro and in vivo models of highly pathogenic coronavirus virus infection impeding the identification of pan-coronavirus antiviral drugs.
• In addition, logistical challenges to drug development have hindered discovery of pan-coronavirus therapeutics. Availability of diverse coronavirus clinical isolates for building in vitro and in vivo systems of drug discovery is limited. Most of the emphasis of the discovery of coronavirus antivirals has been targeted toward the genus betacoronavirus, which includes SARS-CoV and MERS-CoV. However, while therapeutics that target known coronavirus threats to public health are of paramount importance, it is critical to note that future outbreaks of emerging highly pathogenic coronaviruses in humans could result from other coronavirus genera with unique tropism to different tissues, different clinical signs and symptoms, and altered transmission profiles that cannot be captured by limiting drug discovery studies to the very few viral strains of SARS-CoV and MERS-CoV currently available to researchers. Currently, Biodefense and Emerging Infections Research Resources Repository (BEI Resources) is a source for a limited number of strains of SARS-CoV, MERS-CoV, and HCoV-NL63. Laboratory strains of HCoV-OC43 and HCoV-229E are available through the American Type Culture Collection, but strains of HCoV-HKU1 are not available through either resource. High throughput surrogate systems at biosafety level 2 are not well-developed and do not yet capture the phylogenetic diversity within the family Coronaviridae. Biosafety level 3 conditions are required for working with SARS-CoV, MERS-CoV, or pre-emergent zoonotic strains due to the severe disease these viruses cause, which restricts the number of laboratories that can safely perform screening for pan-coronavirus therapeutics.
• In the last 15 years, two outbreaks of previously unknown highly pathogenic coronaviruses, SARS-CoV and MERS-CoV, have demonstrated that CoVs will continue to spill over into human populations, likely facilitated by interaction between infected animals and humans. Reverse genetics approaches have generated pre-emergent BatCoVs from sequence, especially those related to SARS-CoV and MERS-CoV. These particularly novel avenues of research have identified that BatCoV strains with similar pathogenic profiles to SARS-CoV or MERS-CoV continue to circulate within bat populations, indicating a continued vulnerability to highly pathogenic coronavirus emergence. While the next emerging coronavirus may be symptomatically or antigenically similar to SARS-CoV or MERS-CoV, the possibility exists that novel highly pathogenic coronaviruses may be poised for spillover into human populations, with potentially disastrous consequences. Currently, public health measures have been adequate to stymie the spread of SARS-CoV and MERS-CoV primarily due to disease surveillance coupled with viruses with limited person-to-person transmission. However, biological factors that increase cross-species transmission or facilitate person-to-person spread may lead to future coronavirus strains not capable of being contained by timely quarantine of infected individuals. Any increase in highly pathogenic CoV virulence, pathogenesis, or transmission would likely require a targeted medical countermeasure. Without strategic research programs that fill the gaps identified in our literature review, medical countermeasures that target highly pathogenic coronaviruses cannot be brought to market, leaving global public health vulnerable to this emerging threat.

Article Highlights

• Broad-spectrum drugs targeting coronaviruses must have efficacy against known highly pathogenic human coronaviruses SARS-CoV and MERS-CoV, but also have activity against additional novel coronaviruses that may emerge in the future.
• Conventional approaches identifying adaptive-based therapeutics like vaccines and monoclonal antibodies against coronaviruses target antigens that are not conserved and are unlikely to retain therapeutic efficacy against diverse coronavirus pathogens.
• Reverse genetics approaches that generate novel coronaviruses currently circulating in bats are an innovative but under-utilized resource to provide additional zoonotic and pre-emergent virus diversity to in vitro and in vivo drug discovery platforms.
• Many of the treatments used in SARS or MERS patients in outbreak situations were not based on clear in vitro and in vivo model evidence of efficacy, and meta-analyses of treatments failed to show effective therapeutic regimens.
• Development of a drug discovery pipeline consisting of in vitro and in vivo models of coronavirus infection is needed to identify antivirals targeting essential mechanisms of infection.

 All research at a Fort Detrick laboratory that handles high-level disease-causing material, such as Ebola, is on hold indefinitely after the Centers for Disease Control and Prevention found the organization failed to meet biosafety standards.

No infectious pathogens, or disease-causing material, have been found outside authorized areas at the U.S. Army Medical Research Institute of Infectious Diseases.

The CDC inspected the military research institute in June and inspectors found several areas of concern in standard operating procedures, which are in place to protect workers in biosafety level 3 and 4 laboratories, spokeswoman Caree Vander Linden confirmed in an email Friday.

The CDC sent a cease and desist order in July.

After USAMRIID received the order from the CDC, its registration with the Federal Select Agent Program, which oversees disease-causing material use and possession, was suspended. That suspension effectively halted all biological select agents and toxin research at USAMRIID, Vander Linden said in her email.

The Federal Select Agent Program does not comment on whether a program such as USAMRIID is registered and cannot comment on action taken to enforce regulations, Kathryn Harben, a spokeswoman for the CDC, wrote in an email.

“As situations warrant, [Federal Select Agent Program] will take whatever appropriate action is necessary to resolve any departures from regulatory compliance in order to help ensure the safety and security of work with select agents and toxins,” Harben said in the email.

The suspension was due to multiple causes, including failure to follow local procedures and a lack of periodic recertification training for workers in the biocontainment laboratories, according to Vander Linden. The wastewater decontamination system also failed to meet standards set by the Federal Select Agent Program, Vander Linden said in a follow-up email.

“To maximize the safety of our employees, there are multiple layers of protective equipment and validated processes,” she said.

Vander Linden could not say when the laboratory would be able to continue research.

“USAMRIID will return to fully operational status upon meeting benchmark requirements for biosafety,” she said in an email. “We will resume operations when the Army and the CDC are satisfied that USAMRIID can safely and consistently meet all standards.”

USAMRIID has been working on modified biosafety level 3 procedures and a new decontamination system since flooding in May 2018. This “increased the operational complexity of bio-containment laboratory research activities within the Institute,” she said.

At the time of the cease and desist order, USAMRIID scientists were working with agents known to cause tularemia, also called deer fly or rabbit fever, the plague and Venezuelan equine encephalitis, all of which were worked on in a biosafety level 3 laboratory. Researchers were also working with the Ebola virus in a biosafety level 4 lab, Vander Linden said.

Of the pathogens, Ebola, bacteria Yersinia pestis (plague), and bacterium Francisella tularensis (tularemia) are on the list of the Health and Human Services select agents and toxins. The three are considered Tier 1 agents, which pose a severe public health and safety threat.

Venezuelan equine encephalitis also falls under the Federal Select Agent Program, according to the Code of Federal Regulations.

The military research institute is looking at each of its contracts to see what will be affected by the shutdown. USARMIID work outside the lab is not expected to be affected, including on Ebola, Vander Linden said.

“We are coordinating closely with the CDC to ensure that critical, ongoing studies within bio-containment laboratories are completed under appropriate oversight and that research animals will continue to be cared for in accordance with all regulations,” she said in an email. “Although much of USAMRIID’s research is currently on hold, the Institute will continue its critical clinical diagnostic mission and will still be able to provide medical and subject matter expertise as needed to support the response to an infectious disease threat or other contingency.”

According to the Code of Federal Regulations, which also lists required training, records and biosafety plans, Federal Select Agents Program registration can be suspended to protect public health and safety. It is not clear if this is why the USAMRIID registration was suspended.

The code also gives the Department of Health and Human Services, under which the CDC falls, the right to inspect any site and records, without prior notifications. Vander Linden said in the email that the CDC inspected USAMRIID several times over the past year, both unannounced and on a regularly scheduled basis.

USAMRIID will work to meet requirements set by the Army and the CDC and have its suspension lifted, Vander Linden said.

“While the Institute’s research mission is critical, the safety of the workforce and community is paramount,” she said. “USAMRIID is taking the opportunity to correct deficiencies, build upon strengths, and create a stronger and safer foundation for the future.”

BEIJING, Jan. 30 (Xinhua) -- Chinese researchers have found three existing drugs with fairly good inhibitory effects on the novel coronavirus (2019-nCoV) at the cellular level, a local newspaper has reported.

The three drugs are Remdesivir, Chloroquine and Ritonavir. They are now under relevant procedures to gain approval for clinical use, said Hubei Daily on Wednesday.

The discovery was jointly made by researchers from the Academy of Military Medical Sciences and the Wuhan Institute of Virology (WIV) under the Chinese Academy of Sciences (CAS).

Previously, researchers from the Shanghai Institute of Materia Medica under the CAS and ShanghaiTech University jointly selected 30 existing drug candidates, biologically active natural products and traditional Chinese medicines which may have therapeutic effects on the novel coronavirus.

The candidates included 12 anti-HIV drugs like Indinavir, Saquinavir, Lopinavir and Carfilzomib, two anti-respiratory syncytial virus drugs, an anti-schizophrenia drug, an immunosuppressant, as well as traditional Chinese medicines including Polygonum cuspidatum.

Since the outbreak of 2019-nCoV, several research teams led by the WIV have carried out research on five aspects, including rapid detection products, antiviral drugs and vaccines, animal traceability research, as well as etiology and epidemiology research.

A team led by renowned virologist Shi Zhengli in the WIV said on Wednesday 2019-nCoV may originate in bats.

The genome sequencing of the novel coronavirus is as high as 96 percent identical with a type of coronavirus from bats, the team said, adding that the new coronavirus enters the receptor using the same cells with SARS virus.

SARS virus was responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002 and 2003.

The latest finding provides critical evidence to study the pathology and origin of the virus, it said.

On Jan. 2, researchers from the WIV confirmed the whole genome sequence of 2019-nCoV and successfully isolated the virus strain on Jan. 5.

On Jan. 11, the institute submitted the genome sequence information of the coronavirus to the World Health Organization, to share the information globally.

The WIV researchers have also developed an antibody test paper for further research to combat the novel coronavirus, which has claimed more than 130 lives and infected nearly 6,000 people as of the end of Tuesday.

Dear Editor,

In December 2019, a novel pneumonia caused by a previously unknown pathogen emerged in Wuhan, a city of 11 million people in central China. The initial cases were linked to exposures in a seafood market in Wuhan.1 As of January 27, 2020, the Chinese authorities reported 2835 confirmed cases in mainland China, including 81 deaths. Additionally, 19 confirmed cases were identified in Hong Kong, Macao and Taiwan, and 39 imported cases were identified in Thailand, Japan, South Korea, United States, Vietnam, Singapore, Nepal, France, Australia and Canada. The pathogen was soon identified as a novel coronavirus (2019-nCoV), which is closely related to sever acute respiratory syndrome CoV (SARS-CoV).2 Currently, there is no specific treatment against the new virus. Therefore, identifying effective antiviral agents to combat the disease is urgently needed.

An efficient approach to drug discovery is to test whether the existing antiviral drugs are effective in treating related viral infections. The 2019-nCoV belongs to Betacoronavirus which also contains SARS-CoV and Middle East respiratory syndrome CoV (MERS-CoV). Several drugs, such as ribavirin, interferon, lopinavir-ritonavir, corticosteroids, have been used in patients with SARS or MERS, although the efficacy of some drugs remains controversial.3 In this study, we evaluated the antiviral efficiency of five FAD-approved drugs including ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine and two well-known broad-spectrum antiviral drugs remdesivir (GS-5734) and favipiravir (T-705) against a clinical isolate of 2019-nCoV in vitro.    [,...]

Remdesivir has been recently recognized as a promising antiviral drug against a wide array of RNA viruses (including SARS/MERS-CoV5) infection in cultured cells, mice and nonhuman primate (NHP) models. It is currently under clinical development for the treatment of Ebola virus infection.6 Remdesivir is an adenosine analogue, which incorporates into nascent viral RNA chains and results in pre-mature termination.7 Our time-of-addition assay showed remdesivir functioned at a stage post virus entry [...], which is in agreement with its putative anti-viral mechanism as a nucleotide analogue. Warren et al. showed that in NHP model, intravenous administration of 10 mg/kg dose of remdesivir resulted in concomitant persistent levels of its active form in the blood (10 μM) and conferred 100% protection against Ebola virus infection.7 Our data showed that EC90 value of remdesivir against 2019-nCoV in Vero E6 cells was 1.76 μM, suggesting its working concentration is likely to be achieved in NHP. Our preliminary data (Supplementary information, Fig. S2) showed that remdesivir also inhibited virus infection efficiently in a human cell line (human liver cancer Huh-7 cells), which is sensitive to 2019-nCoV.2

Chloroquine, a widely-used anti-malarial and autoimmune disease drug, has recently been reported as a potential broad-spectrum antiviral drug.8,9 Chloroquine is known to block virus infection by increasing endosomal pH required for virus/cell fusion, as well as interfering with the glycosylation of cellular receptors of SARS-CoV.10 Our time-of-addition assay demonstrated that chloroquine functioned at both entry, and at post-entry stages of the 2019-nCoV infection in Vero E6 cells [...]. Besides its antiviral activity, chloroquine has an immune-modulating activity, which may synergistically enhance its antiviral effect in vivo. Chloroquine is widely distributed in the whole body, including lung, after oral administration. The EC90 value of chloroquine against the 2019-nCoV in Vero E6 cells was 6.90 μM, which can be clinically achievable as demonstrated in the plasma of rheumatoid arthritis patients who received 500 mg administration.11 Chloroquine is a cheap and a safe drug that has been used for more than 70 years and, therefore, it is potentially clinically applicable against the 2019-nCoV.

Our findings reveal that remdesivir and chloroquine are highly effective in the control of 2019-nCoV infection in vitro. Since these compounds have been used in human patients with a safety track record and shown to be effective against various ailments, we suggest that they should be assessed in human patients suffering from the novel coronavirus disease.

The Wuhan Institute of Virology, part of the China Academy of Sciences, has applied to patent the use of Gilead Sciences’ remdesivir to treat the current coronavirus outbreak.

The company has partnered with Chinese health authorities to run a Phase III clinical trial to assess remdesivir for treatment of the virus. The drug was originally developed to treat the Ebola virus, but wasn’t effective. Preclinical assays have suggested that the drug might be effective against the coronavirus, 2019-nCoV, as was published in the New England Journal of Medicine (NEJM). The drug was given to a U.S. patient for compassionate use on day seven of the disease and their condition improved on day eight.

The new clinical trial will be conducted at Friendship Hospital in Beijing, China. The trial will enroll 270 patients with mild and moderate pneumonia caused by the virus.

“Gilead is working closely with global health authorities to respond to the novel coronavirus (2019-nCoV) outbreak through the appropriate experimental use of our investigational compound remdesivir. While there are no antiviral data for remdesivir that show activity against 2019-nCoV at this time, available data in other coronaviruses give us hope,” the company stated.

The Wuhan Institute submitted the patent application jointly with the Military Medicine Institute of the People’s Liberation Army Academy of Military Science. Researchers with both organizations noted in a paper published in Nature’s Cell Research this week that both remdesivir and chloroquine, used to treat malaria, may be effective in stalling the coronavirus.

“Even if the Wuhan Institute’s application gets authorized, the role is very limited because Gilead still owns the fundamental patent of the drug,” said Zhao Youbin, a Shanghai-based intellectual property attorney at Purplevine IP Service Co. “Any exploitation of the patent must seek approval from Gilead.”

The Wuhan Institute indicated it filed the patent application on January 21, but also noted it would temporarily drop the patent claims if it had the opportunity to collaborate with foreign biopharma companies to battle the epidemic.

The World Health Organization (WHO), however, is trying to downplay media reports of any drug breakthroughs against the outbreak, stating there are “no known” drugs against the virus. “There are no known effective therapeutics against this 2019-nCoV and WHO recommends enrollment into a randomized controlled trial to test efficacy and safety,” the organization stated today. “A master global clinical trial protocol for research and prioritization of therapeutics is ongoing at the WHO.”

To date, the coronavirus has infected almost 25,000 and killed almost 500. The coronavirus, which comes from the same family of viruses as the common cold, SARS and MERS, began in the city of Wuhan, China. It is believed to have originated in bats and made the jump to human beings, possibly at a seafood market in the city. The virus’s genome is very similar to the SARS virus. It is an airborne virus, although it does not appear to survive long outside the body or on surfaces and remain infectious. It seems to require close contact or exposure to droplets, such as coughing or sneezing, from someone who is infected. However, there are some signs that it can be transmitted prior to symptoms occurring. It causes flu-like symptoms that in some cases become pneumonia.

Gilead’s remdesivir is an experimental drug that isn’t licensed or approved anywhere in the world. It is being rushed into clinical trials in China. Gilead’s chief medical officer, Merdad Parsey, told Bloomberg that the drug could enter clinical trials in China as early as next week in patients with moderate and severe symptoms.

China can manufacture chloroquine and currently wants access to remdesivir. Bloomberg points out that the country’s decision to seek a patent “instead of invoking the heavy-handed ‘compulsory license’ option that lets nations override drug patents in national emergencies, underscores the delicate balancing act before China as it signals commitment toward intellectual property rights alongside curbing the virus outbreak.”

“The fact that they have applied for a patent means there’s growing awareness about this in the country,” said Wang Yanhu, a senior partner at Albright Law Offices in Beijing. “The government is compelled to avoid using the compulsory license because it has been making efforts to show China respects intellectual property rights and the abuse of compulsory licensing will draw international criticism.”

Gilead is presently shipping enough doses of the drug to China to treat 500 patients and is increasing its supply in case the clinical trials are effective.

China is forging ahead in the search for treatments for people sickened by the new coronavirus that has infected more than 28,000 people in a countrywide epidemic, killed more than 500 and seeded smaller outbreaks in 24 other nations.

The need is urgent: There are no approved treatments for illnesses caused by coronaviruses.

On Thursday, China began enrolling patients in a clinical trial of remdesivir, an antiviral medicine made by Gilead, the American pharmaceutical giant.

The drug has to be given intravenously, is experimental and not yet approved for any use, and has not been studied in patients with any coronavirus disease. But studies of infected mice and monkeys have suggested that remdesivir can fight coronaviruses.

And it appears to be safe. It was tested without ill effects in Ebola patients, although it did not work well against that virus, which is in a different family from coronaviruses.

Doctors in Washington State gave remdesivir to the first coronavirus patient in the United States last week after his condition worsened and pneumonia developed when he’d been in the hospital for a week. His symptoms improved the next day..

A single case cannot determine whether a drug works, but a report on the Washington patient, in The New England Journal of Medicine, has nonetheless sparked excitement about the drug.

Another report published on Tuesday by scientists in China added to the enthusiasm, showing that remdesivir blocked the new coronavirus, officially known as 2019-nCoV, from infecting cells grown in the lab.

“It is important to keep in mind that this is an experimental medicine that has only been used in a small number of patients with 2019-nCoV to date, so we do not have an appropriately robust understanding of the effect of this drug to warrant broad use at this time,” Ryan McKeel, a Gilead spokesman, said in an email.

Two clinical trials will take place in Wuhan, China, the center of the outbreak; 500 patients will receive the drug, and comparison groups will get a placebo, Mr. McKeel said.

One trial, which began enrolling patients on Thursday, includes people who are severely ill with symptoms such as needing oxygen. The other trial will involve patients who are hospitalized but not as sick.

The patients will be given the drug intravenously for 10 days, and then assessed 28 days after the treatment to see how they fared compared to the placebo groups.

If the drug works, will Gilead be able to provide enough for everyone who needs it?  “There are currently limited available clinical supplies of remdesivir, but we are working to increase our available supply as rapidly as possible,” Mr. McKeel said.

Gilead had stockpiled the drug, as well as the materials used to make it, for use against Ebola. The company is now using that stockpile for the trials in China and for individual patients like the one in Washington State, whose doctors sought special permission from the Food and Drug Administration for “compassionate use” so that they could give him an unapproved drug.

The company plans to speed production and is looking for “manufacturing partners in multiple geographies,” Mr. McKeel said, adding that Gilead was going ahead with these preparations without knowing yet whether the drug works against the new coronavirus.

In the meantime, the Wuhan Institute of Virology has applied for a patent in China to use remdesivir to treat the coronavirus, according to a statement on the institute’s website.

Gilead already has patents for the drug in China and other parts of the world, and in 2016 filed additional patent applications to use it against coronaviruses. But the company’s application for coronavirus use is still pending, Mr. McKeel said.

“Gilead has no influence over whether a patent office issues a patent to the Chinese researchers,” he added.

In its statement, the virology institute said it would not exercise its patent rights “if relevant foreign companies intend to contribute to the prevention and control of China’s epidemic.”

The report from China published on Tuesday about remdesivir also found that chloroquine, a cheap drug used for decades to treat malaria, could also fight the new coronavirus. Researchers are recommending that it also be studied, along with various antiviral medications, including some of the ones used to treat H.I.V.

Richard Whitley, M.D., Distinguished Professor at UAB and principal investigator of the U19 grant

The investigational drug remdesivir, developed through research conducted through the Antiviral Drug Discovery and Development Center, or AD3C, and centered at the University of Alabama at Birmingham, is being used to treat select infected patients in the United States and in China who have been affected by the outbreak of novel coronavirus (2019-nCoV). 

UAB was awarded a $37.5 million, five-year U19 grant from the National Institute of Allergy and Infectious Diseases Centers of Excellence for Translational Research to study and develop treatment for high-priority emerging infections. Work has been taking place in earnest to develop drugs for emerging influenza, flaviviruses (dengue, West Nile virus and Zika), coronaviruses that cause SARS and MERS, and alphaviruses such as Venezuelan equine encephalitis virus and chikungunya. The grant is a multi-institutional collaboration to accelerate drug discovery for these emerging infections and is a public-private partnership between academic institutions and Gilead Sciences.

Remdesivir, developed to treat the coronavirus causing MERS, was found to have significant activity against the 2019-nCoV strain when the outbreak began in the Chinese city of Wuhan. Importantly, remdesivir had demonstrated efficacy in treating other medically important coronaviruses MERS and SARS in cell culture and animal models. Based on the compassionate plea requests of treating physicians in the United States, Gilead Sciences released remdesivir for use in a few patients, although the drug has not yet been tested for safety or efficacy in these diseases. “The release of remdesivir for safety and efficacy studies is a major accomplishment for the AD3C – namely the U19 grant – as it shows significant and swift advance of antiviral drugs to help treat and respond to emerging infectious disease outbreaks on an international scale and, importantly, to anticipate the introduction of these infections in the United States,” said Richard Whitley, M.D., Distinguished Professor at UAB and principal investigator of the U19 grant.

Gilead Sciences and supporting researchers and clinicians are working with health authorities from the World Health Organization and in China to establish a placebo-controlled study to determine whether remdesivir is safe and effective in treating 2019-nCoV.

• “This is a prime example of how the research we are conducting at UAB plays a critical role in treating patients on a global scale and our contribution of substantial scientific advances.”   – Richard Whitley, M.D., UAB Distinguished Professor

“The collaboration between UAB, our colleagues at Southern Research, Vanderbilt University and the University of North Carolina, along with our pharmaceutical partner Gilead Sciences, is indicative of our collaborative approach to respond to outbreaks in real time, and in helping communities worldwide fight 2019-nCoV. This is a prime example of how the research we are conducting at UAB plays a critical role in treating patients on a global scale and our contribution of substantial scientific advances,” Whitley continued.  

Whitley expressed that the potential for mutation of 2019-nCoV means that UAB’s AD3C and partners will need to build backup molecules for potential testing and treatment in the near future. [...]

UAB is the lead institution for AD3C and research conducted; but the team unifies scientists experienced in virology, viral immunology, pathogenesis, medicinal chemistry and translation to human disease from UAB, University of North Carolina, Vanderbilt University, Emory University, Washington University, The University of Texas Medical Branch, Southern Research, the Emory Institute of Drug Discovery, the University of Colorado, Denver, and Oregon Health & Science University.

Amid the ongoing coronavirus outbreak, Gilead Sciences’ experimental remdesivir has emerged as the most promising candidate against the deadly pathogen. But its patent in China has also drawn some unexpected confusion.

The Chinese pharma BrightGene has successfully copied remdesivir, the company said in a disclosure (PDF, Chinese) to China’s Nasdaq-style Star market on Wednesday.

What's more, the Suzhou-based firm said it has already mass-produced remdesivir's active ingredient and is in the process of turning it into finished doses. The company’s stock jumped 20% at the news, hitting the daily price move cap allowed on the exchange.

BrightGene doesn’t seem to plan to bypass Gilead entirely, though. The company made clear the generic version is still in an R&D phase, and that its final marketing requires permission from the patent holder, Gilead.

In an interview with China’s business news publication Jiemian, BrightGene's board secretary explained that there isn’t any patent infringement issue at this point because it’s not selling the product. But manufacturing a copycat to a patent-protected med at scale without any license is an unusual move that could revive concerns about the protection of intellectual property in the country.

IP protection in China has long been criticized by the Western world and was cited as a reason behind the latest trade war President Donald Trump waged against the country. As part of the first phase of a broader trade deal (PDF), the White House recently signed with Beijing to resolve the dispute, China has promised to implement some American-style enforcement of drug patent rights. These include allowing for a preliminary injunction against a generic maker amid a patent fight.

The BrightGene incident represents the latest twist to the patent controversy around remdesivir in China. A few days ago, researchers at the Chinese Academy of Sciences’ Wuhan Institute of Virology—based at the center of the outbreak—said they had applied for a patent on the use of remdesivir to treat the novel coronavirus disease, now officially named by the World Health Organization as COVID-19.

The research institute said it filed the application “from the perspective of protecting national interests,” but the move drew criticism.

At a recent internal company conference, Gilead CEO Daniel O’Day said the company owns all patents around remdesivir, including for coronaviruses. But he also stressed that the priority for the company is to examine the drug’s use in clinical trials and to ramp up production if its efficacy is confirmed. Gilead “will not get into a patent dispute,” he said.

Last week, two phase 3 trials kicked off in Wuhan to test remdesivir in adult patients with mild-to-moderate or severe respiratory disease caused by the novel coronavirus. Together, investigators aim to enroll 760 patients, with readouts expected as early as April.

Gilead is providing the drug for free for the studies. BrightGene also said it will provide its version “mainly through donations” during the epidemic if it’s granted the marketing go-ahead.

On top of all those controversies, Chinese IP law does provide for compulsory licenses for eligible companies to produce generic versions of patented drugs during a state of emergency or other unusual circumstances, or in the interests of the public. However, authorities will not likely apply it to remdesivir, especially now that Gilead’s offering it free of charge and as drug IP remains a sensitive topic on the international community’s radar.

On February 5, 2020, it was reported that clinical trials of Gilead Sciences’ remdesivir had launched in Wuhan, China. The experimental antiviral is to be evaluated as a treatment for 2019-nCoV, or what is now being called Covid-19.

But there are now reports that the company and health authorities in China are having difficulties recruiting eligible patients. The trial’s goal is to test more than 700 patients infected with the coronavirus, but at this point, there have been fewer than 200 people recruited. At a Saturday press conference, Zhang Xinmin, an official from China’s Ministry of Science and Technology, said they had recruited 168 patients with severe symptoms and 17 with mild and moderate symptoms at 11 sites across Wuhan, Zhang Xinmin.

The clinical trial plan is to recruit 761 patients infected with the coronavirus, including 308 with mild and moderate symptoms and 452 with severe infections. The severely ill patients are required to be within 12 days of disease onset and can’t have taken other treatments in the last 30 days. Mild and moderate patients interested in the trial had to be within eight days of disease onset. All candidates are required to have positive lab results.

Most patients started taking therapies at home, either one recommended by China’s media or based on online anecdotes. There have also been false-negative test results because some of the lab assays appear to be flawed.

Meanwhile, state-owned pharmaceutical companies China Resources Pharmaceutical Group and China Medicine Health Industry Co. are speeding production of chloroquine. This drug appears to be effective in treating the coronavirus with no severe side effects. It has been in clinical use for more than 70 years.

Another study is ongoing of favipiravir compared to AbbVie’s Kaletra, an antiretroviral for HIV. China health officials have recommended broader clinical use of the drug based on 80 coronavirus patients. Zhejiang Hisun Pharmaceutical Co. manufactures favipiravir.

The outbreak appears to be slowing, although the number of infections in China continues to rise. It was reported yesterday that a neurologist, who was director of Wuchang Hospital in Wuhan, died from the infection despite a “full-effort rescue.” Liu Zhiming, 51, was the most prominent victim of the disease after whistleblower Li Wenliang died Feb. 7.

Current numbers indicate a global total of more than 73,000 confirmed infections. The number of cases in the U.S. is at least 29, after 14 infected American evacuees arrived from a cruise ship in Japan. The death toll is at least 1,874 people, all but five in mainland China.

Secretary-General Antonio Guterres, of the United Nations, said the outbreak poses “a very dangerous situation” for the world, but “is not out of control.” He added that “the risks are enormous and we need to be prepared worldwide for that.”

On Friday, Egypt reported its first case of coronavirus.

There is quite a bit of concern that there are unreported cases, which will make controlling the spread of the disease more difficult. Singapore’s prime minister Lee Hsien Loong recently posted a video to his Facebook channel, saying, “If the virus is widespread it is futile to try to trace every contact. If we still hospitalize and isolate every suspect case our hospitals will be overwhelmed.”

The World Health Organization, however, continues to say there is no evidence of “efficient community transmission outside of China.”

[...]

U.S. tests experimental treatment and watches for disruptions of Chinese drug supplies. 

A clinical trial has begun in Nebraska to test whether an experimental drug can treat the new coronavirus, starting with an American who was quarantined on the Diamond Princess cruise ship in Japan, the National Institutes of Health said on Tuesday.

Meanwhile, the Food and Drug Administration said it was closely watching the supplies of 20 unrelated drugs that are either made in China, where the epidemic has drastically reduced manufacturing, or contain ingredients from China. The agency did not say which drugs, but the world relies heavily on China for supplies of many essential medications, like aspirin and penicillin.

In the trial, the patient is being treated with the drug remdesivir, an antiviral developed by Gilead Sciences. 

The test is taking place at the University of Nebraska Medical Center in Omaha, which has a special biocontainment unit, according to the National Institute of Allergy and Infectious Diseases, part of the N.I.H. Thirteen people from the cruise ship have been taken there for treatment.

There are no approved treatments for illnesses caused by coronaviruses, including the new one, known as Covid-19. Remdesivir is already being tested in two clinical trials in China, but efforts to enroll patients there have faltered. 

“We urgently need a safe and effective treatment for Covid-19,” said Dr. Anthony S. Fauci, the director of the allergy and infectious diseases institute,  at a briefing at the Department of Health and Human Services.

Several companies are also working to develop a vaccine for the virus. One of them, Moderna, said Monday it had delivered an experimental vaccine to the N.I.H. for early testing in humans, a record-setting pace. 

But “even at rocket speed,” releasing a vaccine would take at least a year, Dr. Fauci cautioned. 

He projected that initial human trials would begin in a month and a half, with about 45 people, and last three to four months. Then it would have to be expanded to “hundreds, if not thousands” of subjects in countries with active disease transmission, which would take six to eight months, he said.  [...]