Antiviral Research

Efforts to mitigate COVID-19 include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often exhibit decreased activity in these cells due to inefficient metabolization. Limited SARS-CoV-2 replication and propagation occurs in human cells, which are the most relevant testing platforms. By performing serial passages of a SARS-CoV-2 isolate in the human hepatoma cell line clone Huh7.5, we selected viral populations with improved viability in human cells. Culture adaptation led to the emergence of a significant number of high frequency changes (>90% of the viral population) in the region coding for the spike glycoprotein, including a deletion of nine amino acids in the N-terminal domain and 3 amino acid changes (E484D, P812R, and Q954H). We demonstrated that the Huh7.5-adapted virus exhibited a >3-Log10 increase in infectivity titers (TCID50) in Huh7.5 cells, with titers of ~8 Log10TCID50/mL, and >2-Log10 increase in the human lung cancer cell line Calu-1, with titers of ~6 Log10TCID50/mL. Culture adaptation in Huh7.5 cells further permitted efficient infection of the otherwise SARS-CoV-2 refractory human lung cancer cell line A549, with titers of ~6 Log10TCID50/mL. The enhanced ability of the virus to replicate and propagate in human cells permitted screening of a panel of nine nucs, including broad-spectrum compounds. Remdesivir, EIDD-2801 and to a limited extent galidesivir showed antiviral effect across these human cell lines, whereas sofosbuvir, uprifosbuvir, valopicitabine, mericitabine, ribavirin, and favipiravir had no apparent activity. ImportanceThe cell culture adapted variant of the SARS-CoV-2 virus obtained in the present study, showed significantly enhanced replication and propagation in various human cell lines, including lung derived cells otherwise refractory for infection with the original virus. This SARS-CoV-2 variant will be a valuable tool permitting investigations across human cell types, and studies of identified mutations could contribute to our understanding of viral pathogenesis. In particular, the adapted virus can be a good model for investigations of viral entry and cell tropism for SARS-CoV-2, in which the spike glycoprotein plays a central role. Further, as shown here with the use of remdesivir and EIDD-2801, two nucs with significant inhibitory effect against SARS-CoV-2, large differences in the antiviral activity are observed depending on the cell line. Thus, it is essential to select the most relevant target cells for pre-clinical screenings of antiviral compounds, facilitated by using a virus with broader tropism.

Antiviral interventions are urgently required to support vaccination programmes and reduce the global burden of COVID-19. Prior to initiation of large-scale clinical trials, robust preclinical data in support of candidate plausibility are required. The speed at which preclinical models have been developed during the pandemic are unprecedented but there is a vital need for standardisation and assessment of the Critical Quality Attributes. This work provides cross-validation for the recent report demonstrating potent antiviral activity of probenecid against SARS-CoV-2 in preclinical models (1). Vero E6 cells were pre-incubated with probenecid, across a 7-point concentration range, or control media for 2 hours before infection with SARS-CoV-2 (SARS-CoV-2/Human/Liverpool/REMRQ0001/2020, Pango B; MOI 0.05). Probenecid or control media was then reapplied and plates incubated for 48 hours. Cells were fixed with 4% v/v paraformaldehyde, stained with crystal violet and cytopathic activity quantified by spectrophotometry at 590 nm. Syrian golden hamsters (n=5 per group) were intranasally inoculated with virus (SARS-CoV-2 Delta variant B.1.617.2; 103 PFU/hamster) for 24 hours prior to treatment. Hamsters were treated with probenecid or vehicle for 4 doses. Hamsters were ethically euthanised before quantification of total and sub-genomic pulmonary viral RNAs. No inhibition of cytopathic activity was observed for probenecid at any concentration in Vero E6 cells. Furthermore, no reduction in either total or sub-genomic RNA was observed in terminal lung samples from hamsters on day 3 (P > 0.05). Body weight of uninfected hamsters remained stable throughout the course of the experiment whereas both probenecid- (6 - 9% over 3 days) and vehicle-treated (5 - 10% over 3 days) infected hamsters lost body weight which was comparable in magnitude (P > 0.5). The presented data do not support probenecid as a SARS-CoV-2 antiviral. These data do not support use of probenecid in COVID-19 and further analysis is required prior to initiation of clinical trials to investigate the potential utility of this drug.

The need for antiviral drugs is real and relevant. Broad spectrum antiviral drugs have a particular advantage when dealing with rapid disease outbreaks, such as the current COVID-19 pandemic. Since viruses are completely dependent on internal cell mechanisms, they must cross cell membranes during their lifecycle, creating a dependence on processes involving membrane dynamics. Thus, in this study we examined whether the synthesis of glycosphingolipids, biologically active components of cell membranes, can serve as an antiviral therapeutic target. We examined the antiviral effect of two specific inhibitors of GlucosylCeramide synthase (GCS); (i) Genz-123346, an analogue of the FDA-approved drug Cerdelga(R), (ii) GENZ-667161, an analogue of venglustat which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit the replication of four different enveloped RNA viruses of different genus, organ-target and transmission route: (i) Neuroinvasive Sindbis virus (SVNI), (ii) West Nile virus (WNV), (iii) Influenza A virus, and (iv) SARS-CoV-2. Moreover, GCS inhibitors significantly increase the survival rate of SVNI-infected mice. Our data suggest that GCS inhibitors can potentially serve as a broad-spectrum antiviral therapy and should be further examined in preclinical and clinical trial. Analogues of the specific compounds tested have already been studied clinically, implying they can be fast-tracked for public use. With the current COVID-19 pandemic, this may be particularly relevant to SARS-CoV-2 infection. One Sentence SummaryAn analogue of Cerdelga(R), an FDA-approved drug, is effective against a broad range of RNA-viruses including the newly emerging SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models. ImportanceThe arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

{beta}-L-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (L-BHDU) prevents varicella-zoster virus (VZV) replication in cultured cells and in vivo. Its mechanism of action was investigated by evaluating its activity against related herpesviruses and by analyzing resistant VZV strains. L-BHDU was effective against herpes simplex virus type 1 (HSV-1) with an EC50 of 0.22 {micro}M in human foreskin fibroblast (HFF) cells. L-BHDU also inhibited HSV-2 and simian varicella virus (SVV) to a lesser extent. VZV mutants resistant to L-BHDU and other antiviral compounds were obtained by serial passage of the wild type VZV pOka and VZV Ellen strains in the presence of increasing drug concentrations. VZV strains resistant to L-BHDU (L-BHDUR) were cross-resistant to acyclovir (ACV) and brivudin (BVdU) but not to foscarnet (PFA) and cidofovir (CDV). Conversely, ACV-resistant strains were also resistant to L-BHDU. Whole genome sequencing of L-BHDUR strains identified mutations in ATP-binding (G22R) and nucleoside binding (R130Q) domains of VZV thymidine kinase (TK). The wild type and mutant forms of VZV TK were cloned as GST fusion proteins and expressed in E. coli. The partially purified TKG22R-GST and TKR130Q- GST proteins failed to convert thymidine to thymidine monophosphate whereas the wild type TK-GST protein was enzymatically active. Similarly, L-BHDUR virus TK did not phosphorylate the drug. As expected, wild type VZV converted L-BHDU to L-BHDU monophosphate and diphosphate forms. In conclusion, L-BHDU effectiveness against VZV and HSV-1 depends on thymidine kinase activity.

Current approaches of drugs repurposing against 2019 coronavirus disease (COVID-19) have not proven overwhelmingly successful and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to cause major global mortality. Daclatasvir (DCV) and sofosbuvir (SFV) are clinically approved against hepatitis C virus (HCV), with satisfactory safety profile. DCV and SFV target the HCV enzymes NS5A and NS5B, respectively. NS5A is endowed with pleotropic activities, which overlap with several proteins from SARS-CoV-2. HCV NS5B and SARS-CoV-2 nsp12 are RNA polymerases that share homology in the nucleotide uptake channel. We thus tested whether SARS-COV-2 would be susceptible these anti-HCV drugs. DCV consistently inhibited the production of infectious SARS-CoV-2 in Vero cells, in the hepatoma cell line (HuH-7) and in type II pneumocytes (Calu-3), with potencies of 0.8, 0.6 and 1.1 M, respectively. Although less potent than DCV, SFV and its nucleoside metabolite inhibited replication in Calu-3 cells. Moreover, SFV/DCV combination (1:0.15 ratio) inhibited SARS-CoV-2 with EC50 of 0.7:0.1 M in Calu-3 cells. SFV and DCV prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Both drugs inhibited independent events during RNA synthesis and this was particularly the case for DCV, which also targeted secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial DCV in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans. Doses higher than those approved may ultimately be required, but these data provide a basis to further explore these agents as COVID-19 antiviral candidates.

Although a potent Yellow fever vaccine is available since 1937, up to 200.000 severe cases are reported per year, which indicates that virus vaccines require additional support by antiviral therapies. Direct-acting antiviral drugs against severe and widespread diseases, such as DENV and Yellow fever infections with more than millions of diagnosed diseases per year, are still not available. Since antivirals development against neglected diseases is uneconomical, a broadspectrum antiviral compound would be of public benefit. Here, we show that IMP-1088, a recently published myristoyltransferase-1/2 inhibitor suppressing Rhino- and Polioviruses, inhibits replication of HIV-1, Yellow fever virus, Dengue virus, Vaccinia virus, CMV, and human Herpesvirus 8 in the low nanomolar range, indicating that IMP-1088 has broad-range activity against different pathogenic virus families. The inhibition relies on virally encoded myristoylation signals since Zika, Chikungunya, and Enterovirus 71 are not affected by IMP-1088. Furthermore, we show that the Yellow fever NS5 protein is myristoylated and IMP-1088 treatment of Dengue and Yellow fever infected cells leads to a re-localisation of the viral NS5 proteins. Author SummaryTreatment of viral diseases requires the development of tailored drugs specific to inhibit certain virus families. This specificity results in missing treatment options for important human pathogens such as Yellow fever and Dengue virus infection since the development is laborious and costly. Substances acting on various virus families could solve this problem. Here, we describe that IMP-1088, an inhibitor of the cellular myristoyltransferase, inhibits HIV-1, Dengue virus, Yellow fever viruses, Vaccinia virus, and Herpesviruses at low concentrations, which do not affect cell proliferation. Viruses without predicated myristoylation sites, such as Zika viruses, were not inhibited by IMP-1088. Since no experimental evidence was provided that Yellow fever virus proteins are myristoylated, we analysed the post-translational modification of Yellow fever NS5 protein. We determined the subcellular localisation to understand the mechanism of the IMP-1088 mediated suppression and could show that both the Dengue and the Yellow fever NS5 proteins are re-localised by IMP-1088 treatment.

Effective therapeutics to inhibit the replication of SARS-CoV-2 in infected individuals are still under development. The nucleoside analogue N4-hydroxycytidine (NHC), also known as EIDD-1931, interferes with SARS-CoV-2 replication in cell culture. It is the active metabolite of the prodrug Molnupiravir (MK-4482), which is currently being evaluated for the treatment of COVID-19 in advanced clinical studies. Meanwhile, inhibitors of dihydroorotate dehydrogenase (DHODH), by reducing the cellular synthesis of pyrimidines, counteract virus replication and are also being clinically evaluated for COVID-19 therapy. Here we show that the combination of NHC and DHODH inhibitors such as teriflunomide, IMU-838/vidofludimus, and BAY2402234, strongly synergizes to inhibit SARS-CoV-2 replication. While single drug treatment only mildly impaired virus replication, combination treatments reduced virus yields by at least two orders of magnitude. We determined this by RT-PCR, TCID50, immunoblot and immunofluorescence assays in Vero E6 and Calu-3 cells infected with wildtype and the Alpha and Beta variants of SARS-CoV-2. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC in nascent viral RNA, thus precluding the correct synthesis of the viral genome in subsequent rounds of replication, thereby inhibiting the production of replication competent virus particles. This concept was further supported by the rescue of replicating virus after addition of pyrimidine nucleosides to the media. Based on our results, we suggest combining these drug candidates, which are currently both tested in clinical studies, to counteract the replication of SARS-CoV-2, the progression of COVID-19, and the transmission of the disease within the population. SIGNIFICANCEO_LIThe strong synergy displayed by DHODH inhibitors and the active compound of Molnupiravir might enable lower concentrations of each drug to antagonize virus replication, with less toxicity. C_LIO_LIBoth Molnupiravir and DHODH inhibitors are currently being tested in advanced clinical trials or are FDA-approved for different purposes, raising the perspective of rapidly testing their combinatory efficacy in clinical studies. C_LIO_LIMolnupiravir is currently a promising candidate for treating early stages of COVID-19, under phase II/III clinical evaluation. However, like Remdesivir, it appears only moderately useful in treating severe COVID-19. Since the combination inhibits virus replication far more strongly, and since DHODH inhibitors may also suppress excessive immune responses, the combined clinical application bears the potential of alleviating the disease burden even at later stages. C_LI

Upon HSV-1 infection, viral protein 16 (VP16), supported by Host Cell Factor C1 (HCFC1), is rapidly transported into the nucleus, and help to express a series of HSV-1 immediate-early proteins to begin its lytic replication. However, no direct evidence has shown if the HCFC1 deficiency can affect the proliferation of HSV-1 so far. Here, we showed that the HCFC1 deficiency led to a strong resistance to HSV-1 infection. Moreover, we identified Host Cell Factor C1 Regulator 1 (HCFC1R1) as a new host factor acting early in HSV infection for the transport of the HSV-1 capsid to the nucleus. The HCFC1R1 deficiency also led to a strong resistance to HSV-1 infection. The HCFC1R1 deficiency did not affect the attachment of HSV-1 to host cells but act early in HSV-1 infection by perturbing the formation of HCFC1/VP16 complex. Remarkably, in addition to wild-type HSV-1 infection, the host cells in the absence of either HCFC1 or HCFC1R1 showed strong resistant to the infection of TK-deficient HSV-1, which strain can course severe symptoms and tolerate to the current anti-HSV drug Acyclovir. Our data suggest that HCFC1 or HCFC1R1 may be used as the novel target for developing anti-HSV-1 therapies. IMPORTANCEHerpes simplex virus-1 (HSV-1) is widely spread in the human population and can cause a variety of herpetic diseases. Acyclovir, a guanosine analogue that targets the TK protein of HSV-1, is the first specific and selective anti-HSV-1 drug. However, the rapid emergence of resistant HSV-1 strains is occurring worldwide, endangering the efficacy of Acyclovir. Alternatively, targeting host factors is another strategy to stop HSV-1 infection. Unfortunately, although the HSV-1s receptor, Nectin-1, was discovered in 1998, no effective antiviral drug to date has been developed by targeting Nectin-1. Targeting multiple pathways is the ultimate choice to prevent HSV-1 infection. Here we demonstrated that the deletion of HCFC1 or HCFC1R1 exhibits a strong inhibitory effect on both wild-type and TK-deficient HSV-1. Overall, we present evidence that HCFC1 or HCFC1R1 may be used as the novel target for developing anti-HSV-1 therapies with a defined mechanism of action.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is critical to develop and evaluate antiviral compounds that either directly target CoV functions or modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar concentrations of 6',6'-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is used by two CoV methyltransferases involved in the capping of the 5 end of the viral mRNAs. Passaging of wild-type MERS-CoV in the presence of DFA selected a virus population with a [~]100-fold decreased DFA sensitivity, which carried various amino acid substitutions in viral nonstructural proteins (nsps). Specifically, mutations were present in the RNA polymerase subunit (nsp12) and in nsp13, the helicase subunit containing a nucleoside triphosphate hydrolase activity that has been implicated in CoV capping. We hypothesize that DFA directly or indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other betacoronaviruses, but found it to have limited impact on their replication, while being quite cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further characterization of DFA derivatives as an inhibitor of MERS-CoV replication. ImportanceCurrently, there is a lack of antiviral drugs with proven efficacy against human CoV infections including the MERS-CoV that is endemic in the Middle East, the pandemic SARS-CoV-2 and potential future zoonotic CoV. This highlights the importance to investigate new drug targets and identify compounds that can be used to inhibit CoV replication. In this study, we characterize the inhibitory effect of DFA on MERS-CoV replication by phenotypic studies, time-of-addition studies, and the generation and genotyping of a DFA-resistant virus population. Our results revealed that DFA needs further improvement to reduce its cytotoxic side-effects and potentially enhance its broad-spectrum activity. Despite this observation, we think that DFA can be used to understand the function and metabolic interactions of the CoV RNA-synthesizing machinery, or as a starting point for the design of new compounds of the same class.

The SARS-CoV-2 main protease (3CLpro) is one of the promising therapeutic target for the treatment of COVID-19. Nirmatrelvir is the only the 3CLpro inhibitor authorized for treatment of COVID-19 patients at high risk of hospitalization; other 3Lpro inhibitors are in development. We recently repored on the in vitro selection of a SARS-CoV2 3CLpro (L50F-E166A-L167F; short 3CLprores) virus that is cross-resistant with nirmatrelvir and yet other 3CLpro inhibitors. Here, we demonstrate that the resistant virus replicates efficiently in the lungs of intranassaly infected hamsters and that it causes a lung pathology that is comparable to that caused by the WT virus. Moreover, 3CLprores infected hamsters transmit the virus efficiently to co-housed non-infected contact hamsters. Fortunately, resistance to Nirmatrelvir does not readily develop (in the clinical setting) since the drug has a relatively high barrier to resistance. Yet, as we demonstrate, in case resistant viruses emerge, they may easily spread and impact therapeutic options for others. Therefore, the use of SARS-CoV-2 3CLpro protease inhibitors in combinations with drugs that have a different mechanism of action, may be considered to avoid the development of drug-resistant viruses in the future.

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6x to 72x). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. Abstract ImportancePaxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug resistant viruses. In order to guide the use of novel antivirals it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next generation SARS-CoV-2 3CLpro inhibitors.

Influenza viruses are important respiratory pathogens that cause substantial morbidity and mortality annually. In addition to seasonal influenza outbreaks, new emerging influenza A viruses (IAV) can cause pandemic influenza outbreaks. Apart from effective vaccines, there is a need for better treatment options to combat infections with these viruses when vaccines are not available or show reduced efficacy (e.g., in immmunocompromised patients). The limited range of licensed antiviral drugs and emergence of drug-resistance mutations highlight the need for novel intervention strategies like host-targeted antivirals. Repurposing FDA-approved kinase inhibitors may offer a fast-track for a new generation of host-targeted antivirals. Small molecule kinase inhibitors (SMKIs) can inhibit replication of viruses and improve survival in vivo; however, no SMKI has been approved for clinical use against IAV infections. In the present study, we tested eight non-receptor tyrosine kinase-inhibitors (NRTKIs) used to treat cancer and autoimmune diseases for their antiviral potential. Six of those potently inhibited virus replication ([≥]1,000-fold) in A549 cells infected with either A(H1N1)pdm09 or seasonal A(H3N2) strains. These compounds were validated in a biologically relevant ex vivo model of human precision-cut lung slices (hPCLS) to provide proof of principle and show efficacy against contemporary seasonal and pandemic IAVs. We identified the steps of the virus infection cycle affected by these inhibitors and assessed the effect of these NRTKIs on the host response. Considering their established safety profiles, our studies show that the use of these NRTKI shows promise and warrants further development as an alternative strategy to treat influenza virus infections.

The geographical distribution and incidence of tick-borne encephalitis (TBE) have steeply increased over the past decades, raising to represent a major health concern in Asia and Europe. Symptoms of TBE, caused by infections with tick-borne encephalitis virus (TBEV), range from mild, flu-like symptoms to severe neurological disease, often accompanied by long-term sequelae persisting for several years following pathogen encounter. While effective vaccines against TBEV are available, no antiviral drugs are currently approved and therapeutic options for patients suffering from TBE are limited to supportive measures. Compounds able to disrupt viral nucleic acid synthesis bear the potential of effectively limiting viral replication and spread. Seeking to fill the therapeutic gap, we evaluated the efficacy of a panel of approved and investigational antiviral compounds in containing TBEV infection. Combining several cell lines, human neural organoids and organotypic rat brain slice cultures, we found that the nucleoside analogs remdesivir and bemnifosbuvir efficiently limit viral replication. Through infectious virus quantification, immunofluorescence analysis and flow cytometry, we report significant, dose-dependent reduction of viral loads across all models used, with inhibition observed at low doses for both drugs. Notably, while we observed bemnifosbuvir to be well tolerated, we report important cytotoxicity of remdesivir when applied to human neural organoids. Our findings identify bemnifosbuvir and remdesivir as novel treatment strategies for TBE, providing an accessible and timely response to a clinical challenge of pressing concern.

The recent Mpox virus (MPXV) outbreak was caused by a novel and more pathogenic clade 1b virus. We compared the antiviral efficacy of Tecovirimat in cell culture, against the two ancestral clades 1a and 2a, the clade 2b that circulated in 2022, and the recent clade 1b virus. We report that Tecovirimat similarly inhibits the replication of all four MPXV clades, at nanomolar concentrations (nM). Our results suggest that Tecovirimat remains a therapeutic option against the latest clade 1b virus.

The emergence of SARS-CoV-2 variants has necessitated continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 {Delta}3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging {Delta}3a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro, encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 {Delta}3a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2. IMPORTANCESmall-molecule antiviral drugs have been used for the treatment of SARS-CoV-2 infections. However, drug-resistant SARS-CoV-2 mutants to currently US FDA-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remain limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins ({Delta}3a7b-Nluc) to identify nirmatrelvir resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using {Delta}3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 {Delta}3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

Mpox virus (MPXV) is an orthopoxvirus that causes the human disease mpox, which is characterized by fever, myalgia, and formation of rashes and lesions, and which garnered worldwide attention due to a global outbreak in 2022. In response to the outbreak, the antivirals tecovirimat, cidofovir, and brincidofovir have been used as emergency treatment for mpox. However, because of drug resistance and toxicity risks with those compounds, there is still a need for additional antivirals to treat orthopoxvirus diseases. Since cidofovir is a nucleoside analogue, we investigated a selection of other such compounds for antiviral activity against orthopoxviruses. We developed in vitro screening assays using fluorescent strains of vaccinia virus (VACV) and modified vaccinia Ankara (MVA) to measure the antiviral potency of test compounds. We found that tenofovir alafenamide and adefovir dipixovil, both acyclic phosphonates, had strong potential combinations of anti-orthopoxvirus activity and low toxicity after testing them against MVA and VACV, with EC50 values in the single digit micromolar and nanomolar range, while other potential hits included trifluridine and two arabinosides. We then recapitulated the results with MPXV using a luciferase-based assay. These data reinforce the interest of repurposing nucleoside analogues as antivirals to treat poxvirus infections and provide a basis for high throughput screening and mechanistic and antiviral resistance studies.

Many poxviruses are significant human and animal pathogens, including viruses that cause smallpox and mpox. Identification of inhibitors of poxvirus replication is critical for drug development to manage poxvirus threats. Here we tested two compounds, nucleoside trifluridine and nucleotide adefovir dipivoxil, for antiviral activities against vaccinia virus (VACV) and mpox virus (MPXV) in physiologically relevant primary human fibroblasts. Both trifluridine and adefovir dipivoxil potently inhibited replication of VACV and MPXV (MA001 2022 isolate) in a plaque assay. Upon further characterization, they both conferred high potency in inhibiting VACV replication with half maximal effective concentrations (EC50) at low nanomolar levels in our recently developed assay based on a recombinant VACV secreted Gaussia luciferase. Our results further validated that the recombinant VACV with Gaussia luciferase secretion is a highly reliable, rapid, non-disruptive, and simple reporter tool for identification and chracterization of poxvirus inhibitors. Both compounds inhibited VACV DNA replication and downstream viral gene expression. Given that both compounds are FDA-approved drugs, and trifluridine is used to treat ocular vaccinia in medical practice due to its antiviral activity, our results suggest that it holds great promise to further test trifluridine and adefovir dipivoxil for countering poxvirus infection, including mpox.

Hepatitis E virus (HEV) causes roughly 20 million yearly global infections, and is associated with chronic hepatitis, neurological sequelae and pregnancy-related adverse outcomes that require antiviral therapeutic intervention. While there are currently no approved HEV-specific therapeutics, ribavirin and pegylated interferon, prescribed off-label, are the current standard of care. However, ribavirin resistance and toxicity highlight the unmet clinical need to identify safer, HEV-specific antivirals. Auranofin, an FDA-approved anti-rheumatic drug, displays antiviral activity against several viruses. Therefore, we investigated auranofins potential as an antiviral and its mechanism of action against HEV. We demonstrated that auranofin displays dose-dependent antiviral activity against two genotypes of HEV that cause a significant proportion of human disease, as well as against a ribavirin treatment failure-associated mutant. Because auranofin is known to increase reactive oxygen species (ROS), we investigated the antiviral mechanism of action via treatment with ROS inhibitors. ROS inhibitors reversed auranofin-mediated ROS promotion and antiviral activity, suggesting the observed antiviral effects are mediated by ROS. Furthermore, treatment with a different ROS promotor, D-amino acid oxidase (DAAO), also displays antiviral activity against HEV, which was also reversed by treatment with a ROS inhibitor, suggesting that ROS accumulation alone is antiviral. We also demonstrated that combined treatment with auranofin and ribavirin exhibits synergistic antiviral activity in vitro, which supports repurposing auranofin as an antiviral against HEV, potentially in combination with ribavirin. Overall, this study has important implications in repurposing auranofin as an antiviral against HEV and in delineating the mechanism of action against HEV via ROS. ImportanceHepatitis E virus (HEV) lacks approved antiviral therapies, and off-label treatments are limited by toxicity and emerging resistance. This study identifies the FDA-approved drug auranofin as an effective in vitro inhibitor of HEV, including two globally relevant human-associated genotypes and a ribavirin treatment failure-associated mutant. Auranofins activity highlights the therapeutic potential of host-targeting antivirals, particularly those that promote the generation of reactive oxygen species, in treating HEV infection. These findings support further in vivo investigations of auranofin as a treatment for HEV and suggest that modulating host redox pathways by promoting reactive oxygen species may represent a promising strategy for broad-spectrum antiviral development.

N4-hydroxycytidine (NHC), the active metabolite of Molnupiravir, is incorporated into nascent RNA of SARS-CoV-2 and interferes with subsequent virus replication. We have previously described synergy between NHC and inhibitors of dehydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine synthesis. Upon DHODH inhibition, the lack of endogenous pyrimidines conceivably enhances NHC incorporation. However, the question remains whether preventing the synthesis of just one pyrimidine base, cytidine, might as well augment the antiviral efficacy of NHC. We tested this by inhibiting CTP synthetases (CTPSs), the cellular enzymes that directly catalyze the synthesis of a cytidine nucleotide. We observed that inhibitors of CTP synthetase (CTPSis), namely cyclopentenyl cytosine (CPEC) as well as STP938 and STP720, display a strong synergy with NHC for diminishing SARS-CoV-2 replication in cell culture, as shown earlier for DHODH inhibitors. NHC and CTPSis in combination prevented the cytopathic effect of SARS-CoV-2 and strongly reduced the release of viral RNA and infectious particles, as well as the synthesis of viral proteins. This combination was also active against an Omicron variant of SARS-CoV-2. Addition of cytidine, but not uridine, rescued virus growth under these conditions. Of note, treating SARS-CoV-2-infected hamsters with the CTPS1 inhibitor STP938 strongly diminished COVID pathology. We propose that CTPS inhibition has the potential to increase the efficacy of antiviral cytidine analogues and to treat coronavirus infections. HIGHLIGHTSO_LIThe efficacy of NHC against SARS-CoV-2 replication in cell culture models is intensified by several orders of magnitude through targeting cellular CTP-Synthetase. C_LIO_LIThe drug combination still displays its effect against SARS-CoV-2 replication in the presence of uridine, suggesting that serum uridine cannot counteract its efficacy. C_LIO_LICTPS inhibition diminishes COVID-19-like pathology in an established animal model. C_LI